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Sample records for cell cultures facilitates

  1. Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

    Science.gov (United States)

    Ou, Dong-Bo; He, Yong; Chen, Rui; Teng, Ji-Wei; Wang, Hong-Tao; Zeng, Di; Liu, Xiong-Tao; Ding, Lu; Huang, Jin-Yan; Zheng, Qiang-Sun

    2011-12-01

    The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells. Copyright © 2011 Wiley Periodicals, Inc.

  2. Hydrogel/poly-dimethylsiloxane hybrid bioreactor facilitating 3D cell culturing

    NARCIS (Netherlands)

    Schurink, B.; Luttge, R.

    2013-01-01

    The authors present a hydrogel/poly-dimethylsiloxane (PDMS) hybrid bioreactor. The bioreactor enables a low shear stress 3D culture by integrating a hydrogel as a barrier into a PDMS casing. The use of PDMS allows the reversible adhesion of the device to a commercially available microelectrode

  3. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Science.gov (United States)

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  4. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  5. Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

    OpenAIRE

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To ove...

  6. Cultural hegemony? Educators’ perspectives on facilitating cross-cultural dialogue

    Science.gov (United States)

    Zaidi, Zareen; Verstegen, Daniëlle; Vyas, Rashmi; Hamed, Omayma; Dornan, Tim; Morahan, Page

    2016-01-01

    Background We live in an age when education is being internationalized. This can confront students with ‘cultural hegemony’ that can result from the unequal distribution of power and privilege in global society. The name that is given to awareness of social inequality is ‘critical consciousness’. Cross-cultural dialogue provides an opportunity for learners to develop critical consciousness to counter cultural hegemony. The purpose of this research was to understand how learners engage with cross-cultural dialogue, so we can help them do so more effectively in the future. Method The setting for this research was an online discussion in an international health professions educator fellowship program. We introduced scenarios with cultural references to study the reaction of participants to cultural conversation cues. We used an inductive thematic analysis to explore power and hegemony issues. Results Participants reflected that personally they were more likely to take part in cross-cultural discussions if they recognized the context discussed or had prior exposure to educational settings with cultural diversity. They identified barriers as lack of skills in facilitating cross-cultural discussions and fear of offending others. They suggested deliberately introducing cultural issues throughout the curriculum. Conclusion Our results indicate that developing critical consciousness and cross-cultural competency will require instructional design to identify longitudinal opportunities to bring up cross-cultural issues, and training facilitators to foster cross-cultural discussions by asking clarifying questions and navigating crucial/sensitive conversations. PMID:27890048

  7. Cultural hegemony? Educators’ perspectives on facilitating cross-cultural dialogue

    Directory of Open Access Journals (Sweden)

    Zareen Zaidi

    2016-11-01

    Full Text Available Background: We live in an age when education is being internationalized. This can confront students with ‘cultural hegemony’ that can result from the unequal distribution of power and privilege in global society. The name that is given to awareness of social inequality is ‘critical consciousness’. Cross-cultural dialogue provides an opportunity for learners to develop critical consciousness to counter cultural hegemony. The purpose of this research was to understand how learners engage with cross-cultural dialogue, so we can help them do so more effectively in the future. Method: The setting for this research was an online discussion in an international health professions educator fellowship program. We introduced scenarios with cultural references to study the reaction of participants to cultural conversation cues. We used an inductive thematic analysis to explore power and hegemony issues. Results: Participants reflected that personally they were more likely to take part in cross-cultural discussions if they recognized the context discussed or had prior exposure to educational settings with cultural diversity. They identified barriers as lack of skills in facilitating cross-cultural discussions and fear of offending others. They suggested deliberately introducing cultural issues throughout the curriculum. Conclusion: Our results indicate that developing critical consciousness and cross-cultural competency will require instructional design to identify longitudinal opportunities to bring up cross-cultural issues, and training facilitators to foster cross-cultural discussions by asking clarifying questions and navigating crucial/sensitive conversations.

  8. Cultural hegemony? Educators' perspectives on facilitating cross-cultural dialogue.

    Science.gov (United States)

    Zaidi, Zareen; Verstegen, Daniëlle; Vyas, Rashmi; Hamed, Omayma; Dornan, Tim; Morahan, Page

    2016-01-01

    We live in an age when education is being internationalized. This can confront students with 'cultural hegemony' that can result from the unequal distribution of power and privilege in global society. The name that is given to awareness of social inequality is 'critical consciousness'. Cross-cultural dialogue provides an opportunity for learners to develop critical consciousness to counter cultural hegemony. The purpose of this research was to understand how learners engage with cross-cultural dialogue, so we can help them do so more effectively in the future. The setting for this research was an online discussion in an international health professions educator fellowship program. We introduced scenarios with cultural references to study the reaction of participants to cultural conversation cues. We used an inductive thematic analysis to explore power and hegemony issues. Participants reflected that personally they were more likely to take part in cross-cultural discussions if they recognized the context discussed or had prior exposure to educational settings with cultural diversity. They identified barriers as lack of skills in facilitating cross-cultural discussions and fear of offending others. They suggested deliberately introducing cultural issues throughout the curriculum. Our results indicate that developing critical consciousness and cross-cultural competency will require instructional design to identify longitudinal opportunities to bring up cross-cultural issues, and training facilitators to foster cross-cultural discussions by asking clarifying questions and navigating crucial/sensitive conversations.

  9. Facilitating a just and trusting culture.

    Science.gov (United States)

    Pattison, Jill; Kline, Theresa

    2015-01-01

    The purpose of this paper is to identify managerial and organizational characteristics and behaviors that facilitate the fostering of a just and trusting culture within the healthcare system. Two studies were conducted. The initial qualitative one was used to identify themes based on interviews with health care workers that facilitate a just and trusting culture. The quantitative one used a policy-capturing design to determine which factors were most likely to predict outcomes of manager and organizational trust. The factors of violation type (ability vs integrity), providing an explanation or not, blame vs no blame by manager, and blame vs no blame by organization were all significant predictors of perceptions of trust. Limitations to the generalizability of findings included both a small and non-representative sample from one health care region. The present findings can be useful in developing training systems for managers and organizational executive teams for managing medical error events in a manner that will help develop a just and trusting culture. A just and trusting culture should enhance the likelihood of reporting medical errors. Improved reporting, in turn, should enhance patient safety. This is the first field study experimentally manipulating aspects of organizational trust within the health care sector. The use of policy-capturing is a unique feature that sheds light into the decision-making of health care workers as to the efficaciousness of particular managerial and organizational characteristics that impact a just and trusting culture.

  10. Cyclodextrin-facilitated bioconversion of 17 beta-estradiol by a phenoloxidase from Mucuna pruriens cell cultures

    NARCIS (Netherlands)

    Woerdenbag, H.J.; Pras, N.; Frijlink, H.W.; Lerk, C.F.; Malingré, T.M.

    1990-01-01

    After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased

  11. African Journal of Cross-Cultural Psychology and Sport Facilitation ...

    African Journals Online (AJOL)

    African Journal of Cross-Cultural Psychology and Sport Facilitation: Advanced Search. Journal Home > African Journal of Cross-Cultural Psychology and Sport Facilitation: Advanced Search. Log in or Register to get access to full text downloads.

  12. Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

    Science.gov (United States)

    Zhou, Haibo; Shi, Jianmin; Zhang, Chao; Li, Pei

    2018-02-28

    Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis. © 2018 The Author(s).

  13. Language as a Facilitator of Cultural Connection

    Science.gov (United States)

    Gonzalez, Miigis B.; Aronson, Benjamin D.; Kellar, Sidnee; Walls, Melissa L.; Greenfield, Brenna L.

    2018-01-01

    Understanding culture as a means of preventing or treating health concerns is growing in popularity among social behavioral health scientists. Language is one component of culture and therefore may be a means to improve health among Indigenous populations. This study explores language as a unique aspect of culture through its relationship to other demographic and cultural variables. Participants (n = 218) were adults who self-identified as American Indian, had a type 2 diabetes diagnosis, and were drawn from two Ojibwe communities using health clinic records. We used chi-squared tests to compare language proficiency by demographic groups and ANOVA tests to examine relationships between language and culture. A higher proportion of those living on reservation lands could use the Ojibwe language, and fluent speakers were most notably sixty-five years of age and older. Regarding culture, those with greater participation and value belief in cultural activities reported greater language proficiency. PMID:29782622

  14. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from

  15. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  16. Cultural hegemony? Educators? perspectives on facilitating cross-cultural dialogue

    OpenAIRE

    Zaidi, Zareen; Verstegen, Dani?lle; Vyas, Rashmi; Hamed, Omayma; Dornan, Tim; Morahan, Page

    2016-01-01

    Background: We live in an age when education is being internationalized. This can confront students with ‘cultural hegemony’ that can result from the unequal distribution of power and privilege in global society. The name that is given to awareness of social inequality is ‘critical consciousness’. Cross-cultural dialogue provides an opportunity for learners to develop critical consciousness to counter cultural hegemony. The purpose of this research was to understand how learners engage with c...

  17. African Journal of Cross-Cultural Psychology and Sport Facilitation ...

    African Journals Online (AJOL)

    Focus and Scope. The African Journal of Cross-Cultural Psychology and Sport Facilitation is a professional journal of the Association of Psychology in Sport and Human Behaviour. It publishes a wide variety of original articles and reports relevant to cultural and sport behaviour, theoretical propositions, research outcomes ...

  18. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  19. Youth Culture and Cell Phone

    Directory of Open Access Journals (Sweden)

    mohammad saeed zokaei

    2009-11-01

    Full Text Available Iranian youth’s leisure culture has been immediately affected by the digital media culture. As a communicative media, cell phone has crossed borders of youth norms and identity; and in addition to facilitating their communication, has changed its patterns. Applying Bourdieu’s concepts of habitus and field, and relied on the qualitative and quantitative data gathered from the mobile youth users, the present study argues that mobile has produced a new field in which youth’s opportunities for leisure, entertainment, communication, and independence have extended. In addition, cell phone has facilitated and compensated for some defects in public sphere, and therefore empowered youth agency, individuality, and power. Despite this strengthening, cell phone does not cross borders of gender and class differences, or the levels of social capital.

  20. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  1. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  2. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  3. LITERATURE AS A FACILITATOR OF TARGET CULTURE LEARNING

    Directory of Open Access Journals (Sweden)

    Onur TOPALOĞLU

    2016-04-01

    Full Text Available The purpose of this study is to investigate how literature courses, involved in the curriculum of the department of English Language Teaching from the second year to the fourth year, help students to acquire the target culture in EFL classes. Since learning a language does not mean only learning the lexical structures of any language, culture holds an important place in internalizing the way of thinking and appropriate use of target language. This study has been designed in a naturalistic environment, thus interview and observation were used as the main data collection techniques. The study was designed as a descriptive qualitative research. Participants were chosen by the help of an experienced colleague working in the above mentioned department for three years, and most of the participants were under her supervision, thus this case facilitated the process of interviewing participants. The findings showed that the literature courses contribute much to gain the target culture due the very nature of literature reflecting the society and traditions of society in which it was written. In addition, leaarners may have the chance of comparing thier own culture with target culture and this facilitates their learning. However, some missing points and misapplications were reported by students in providing a more appropriate environment for reflecting the target culture.

  4. Liver Cell Culture Devices

    NARCIS (Netherlands)

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver

  5. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  6. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  7. Plant cell culture initiation

    NARCIS (Netherlands)

    Hall, R.D.

    2000-01-01

    The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10-15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our

  8. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  9. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...... and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers....

  10. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  11. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

    Science.gov (United States)

    Ali, Niwa; Zirak, Bahar; Rodriguez, Robert Sanchez; Pauli, Mariela L; Truong, Hong-An; Lai, Kevin; Ahn, Richard; Corbin, Kaitlin; Lowe, Margaret M; Scharschmidt, Tiffany C; Taravati, Keyon; Tan, Madeleine R; Ricardo-Gonzalez, Roberto R; Nosbaum, Audrey; Bertolini, Marta; Liao, Wilson; Nestle, Frank O; Paus, Ralf; Cotsarelis, George; Abbas, Abul K; Rosenblum, Michael D

    2017-06-01

    The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of T regs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Exploring Strategies in Facilitating Cultural Diversity: A Freirean Approach

    Science.gov (United States)

    Jamal, Zenobia; Guo, Shibao

    2008-01-01

    The student population in Canada's higher education institutions is becoming increasingly racially and culturally diverse. Canadian higher education has the obligation to build inclusive teaching and learning environments where the needs and aspirations of students from diverse cultures and backgrounds can be addressed in an equitable manner.…

  13. Principles of cancer cell culture.

    Science.gov (United States)

    Cree, Ian A

    2011-01-01

    The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory.

  14. Cross Cultural Validation Of Perceived Workfamily Facilitation Scale ...

    African Journals Online (AJOL)

    The work family interface contains four unique factors based on studies from western countries. However, some of these studies have questioned the cross cultural adoption of psychological concept, and called for a re-validation prior to adoption. The main purpose of this study is to re-validate the four factor structure that ...

  15. White cells facilitate opposite- and same-sex mating of opaque cells in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Li Tao

    2014-10-01

    Full Text Available Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1 impair the promoting role of white cells (MTLa in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.

  16. Facilitating cross-cultural adjustment : the case of North European expatriates in China

    OpenAIRE

    Reegård, Kine

    2011-01-01

    Cross-cultural adjustment is considered crucial for expatriate success. Such adjustment may be enhanced by providing the expatriates with the knowledge and awareness of norms and appropriate behaviours of the host country by means of cross-cultural training. Language training may also facilitate interaction with host nationals, thereby providing the expatriate with insight into the host country’s culture increase their understanding of the new environment. Further, cross-cultural training and...

  17. Publications in academic medical centers: technology-facilitated culture clash.

    Science.gov (United States)

    Berner, Eta S

    2014-05-01

    Academic culture has a set of norms, expectations, and values that are sometimes tacit and sometimes very explicit. In medical school and other health professions educational settings, probably the most common norm includes placing a high value on peer-reviewed research publications, which are seen as the major evidence of scholarly productivity. Other features of academic culture include encouraging junior faculty and graduate students to share their research results at professional conferences and lecturing with slides as a major way to convey information. Major values that faculty share with journal editors include responsible conduct of research and proper attribution of others' words and ideas. Medical school faculty also value technology and are often quick to embrace technological advances that can assist them in their teaching and research. This article addresses the effects of technology on three aspects of academic culture: education, presentations at professional meetings, and research publications.The technologies discussed include online instruction, dissemination of conference proceedings on the Internet, plagiarism-detection software, and new technologies deployed by the National Center for Biotechnology Information, the home of PubMed. The author describes how the ease of deploying new technologies without faculty changing their norms and behavior in the areas of teaching and research can lead to conflicts of values among key stakeholders in the academic medical community, including faculty, journal editors, and professional associations. The implications of these conflicts and strategies for managing them are discussed.

  18. Experiences in Broker-Facilitated Participatory Cross-Cultural Research

    Directory of Open Access Journals (Sweden)

    Stephanie P. Kowal

    2017-05-01

    Full Text Available Health researchers are increasingly using community-based participatory research approaches because of the benefits accrued through ongoing community engagement. The documentation of our research partnership highlights key ethical and analytical challenges researchers face in participatory research, particularly in projects partnering with service providers or cultural brokers in cross-cultural settings. In this article, we describe how choices made to accommodate a participatory research approach in the examination of vaccination behavior impacted the process and outcomes of our qualitative inquiries. First, we found that employing multiple interviewers influenced the breadth of discussion topics, thus reducing the ability to achieve saturation in small study populations. This was mitigated by (a having two people at each interview and (b using convergent interviewing, a technique in which multiple interviewers discuss and include concepts raised in interviews in subsequent interviews to test the validity of interview topics. Second, participants were less engaged during the informed consent process if they knew the interviewer before the interview commenced. Finally, exposing identity traits, such as age or immigration status, before the interview affected knowledge cocreation, as the focus of the conversation then mirrored those traits. For future research, we provide recommendations to reduce ethical and analytical concerns that arise with qualitative interview methods in participatory research. Specifically, we provide guidance to ensure ethical informed consent processes and rigorous interview techniques.

  19. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  20. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  1. Nanoparticle Facilitated Extracellular Electron Transfer in Microbial Fuel Cells

    Science.gov (United States)

    2014-10-13

    harvestingelectrical power directly from waste and renewable biomass and thus represent a promising technology for sustainable energy production.1−5 Central...cell membrane (Figure 3e), serving as a porous semiconducting “ shell ” to facilitate the charge transport at bacteria/electrode or bacteria/bacteria

  2. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue cultures...

  3. Concave microwell plate facilitates chondrogenesis from mesenchymal stem cells.

    Science.gov (United States)

    Ko, Ji-Yun; Im, Gun-Il

    2016-11-01

    To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes. Pellets cultured in concave microwell plates had a significantly higher level of GAG per DNA content and greater proteoglycan content than those cultured in tubes at day 7 and 14. Three chondrogenic markers, SOX-9, COL2A1 and aggrecan, showed significantly higher expression in pellets cultured in concave microwell plates than those cultured in tubes at day 7 and 14. At day 21, there was not a significant difference in the expression of these markers. COL10A1, the typical hypertrophy marker, was significantly lower in concave microwell plates during the whole culture period. Runx-2, a marker of hypertrophy and osteogenesis, was significantly lower at day 7 in pellets cultured in concave microwell plates than those cultured in tubes. Concave microwell plates provide a convenient and effective tool for the study of in vitro chondrogenesis and may replace the use of propylene culture tube.

  4. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...... with mass production. The last part of this thesis also includes perspectives on how to expand the latest designed device to facilitate culturing of tissue and co-culturing of cells....

  5. Autophagy Facilitates Salmonella Replication in HeLa Cells

    Science.gov (United States)

    Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

    2014-01-01

    ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

  6. Lewis lung carcinoma progression is facilitated by TIG-3 fibroblast cells.

    Science.gov (United States)

    Yamauchi, Yoshikane; Izumi, Yotaro; Asakura, Keisuke; Kawai, Kenji; Wakui, Masatoshi; Ohmura, Mitsuyo; Suematsu, Makoto; Nomori, Hiroaki

    2013-09-01

    The interactions of tumor cells with stromal fibroblasts influence tumor biology, but the exact mechanisms involved are still unclear. In the present study, we evaluated the effects of a human lung fibroblast cell line, TIG-3, on Lewis lung carcinoma (LLC) cells both in vitro and in vivo. LLC and TIG-3 cells were co-cultured/co-implanted in vitro and in vivo. Cell invasion was assayed. Local tumor growth, as well as lung metastasis, were evaluated after subcutaneous cell co-implantation into NOD/SCID/γ-null (NOG) mice. LLC, and TIG-3 cells were pre-treated with either SB431542, a small molecule TGF-β receptor antagonist, or siRNA for transforming growth factor (TGF)-β before co-culture or co-implantation, and the effects of pre-treatments were compared both in cell culture and in mice. Subcutaneous LLC tumor growth (L group) in NOG mice was significantly increased by co-implantation of TIG-3 cells (L+T group) at four weeks. The number of macroscopic lung metastases was also significantly increased in the L+T group in comparison to the L group. In vitro cell invasion was significantly increased in the L+T group in comparison to the L group. In vitro expression of phosphorylated-SMAD3 was significantly increased in the L+T group in comparison to the L group. Furthermore, pre-treatment with either SB431542 or siRNA for TGF-β reduced the invasiveness both in culture and in mice. This study suggested that in vitro as well as in vivo progression of LLC was facilitated by co-culture/co-implantation with TIG-3 cells, and that this process was at least in part dependent on TGF-β-mediated interactions.

  7. Polymer microfiber meshes facilitate cardiac differentiation of c-kit{sup +} human cardiac stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kan, Lijuan [Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA (United States); Thayer, Patrick [Department of Chemical Engineering, School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA (United States); Fan, Huimin [Research Institute of Heart Failure, Shanghai East Hospital of Tongji University, Shanghai (China); Ledford, Benjamin; Chen, Miao [Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA (United States); Goldstein, Aaron [Department of Chemical Engineering, School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA (United States); Cao, Guohua [School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA (United States); He, Jia-Qiang, E-mail: jiahe@vt.edu [Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA (United States)

    2016-09-10

    Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit{sup +} human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5 µm) elastomeric meshes consisting of aligned 1.7 µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases. - Highlights: • First study to characterize c-kit{sup +} human cardiac stem cells on microfiber meshes. • Microfiber meshes seem reducing cell proliferation, but no effect on cell viability. • Microfiber meshes facilitate the elongation of human cardiac stem cells in culture. • Cardiac but not smooth muscle differentiation were enhanced on microfiber meshes. • Microfiber meshes may be used as cardiac patches in cell-based cardiac therapy.

  8. Multistage carcinogenesis in cell culture.

    Science.gov (United States)

    Rubin, H

    2001-01-01

    Rodent fibroblasts explanted from embryos to culture undergo a period of declining growth rate in serial passages leading to crisis, followed by the appearance of variants which can multiply indefinitely. If the "immortal" cell line was established by low density passage, i.e., 3T3 cells, it has a low saturation density and is non-tumorigenic. If it was established by high density passage, it has a high saturation density and is tumorigenic. The establishment of cells goes through successive stages, including increased capacity to multiply in low serum concentration, growth to high saturation density, growth in suspension, assisted tumour formation in susceptible hosts and unassisted tumour formation. Chromosome aberrations and aneuploidy occur long before the capacity to produce tumours appears. Contrary to conventional belief, human fibroblast populations also undergo a continuous loss of capacity to multiply from the time of explantation, with only the longest surviving clone reaching the Hayflick limit. Neoplastic transformation of rodent cells is strongly favoured by maintaining them in a quiescent state at confluence for prolonged periods, which results in genetic damage to the cells. It also produces a large variety of chromosomal aberrations in human cells and extends their replicative lifespan. Individual clones are more susceptible to spontaneous transformation than their heterogeneous parental cultures. The implications of these results for tumour development in vivo are that oncogenic genetic changes may be common under stressful conditions which restrict replication, and that such changes are maximized when a rogue clone reaches a critical size that reduces stabilizing interactions with neighbouring clones. An alternative explanation, described in the Addendum, which we retrospectively favor is that the easily transformed clones are a minority in the uncloned parental population. The reason they transform before the parental population is that when

  9. Barriers and facilitators to cultural competence in rehabilitation services: a scoping review.

    Science.gov (United States)

    Grandpierre, Viviane; Milloy, Victoria; Sikora, Lindsey; Fitzpatrick, Elizabeth; Thomas, Roanne; Potter, Beth

    2018-01-15

    There is an important need to evaluate whether rehabilitation services effectively address the needs of minority culture populations with North America's increasingly diverse population. The objective of this paper was therefore to review and assess the state of knowledge of barriers and facilitators to cultural competence in rehabilitation services. Our scoping review focused on cultural competence in rehabilitation services. Rehabilitation services included in this review were: audiology, speech-language pathology, physiotherapy, and occupational therapy. A search strategy was developed to identify relevant articles published from inception of databases until April 2015. Titles and abstracts were screened by two independent reviewers according to specific eligibility criteria with the use of a liberal-accelerated approach. Full-text articles meeting inclusion criteria were then screened. Key study characteristics were abstracted by the first reviewer, and findings were verified by the second reviewer. After duplicates were removed, 4303 citations were screened. Included articles suggest that studies on cultural competence occur most frequently in occupational therapy (n = 17), followed by speech language pathology (n = 11), physiotherapy (n = 6), and finally audiology (n = 1). Primary barriers in rehabilitation services include language barriers, limited resources, and cultural barriers. Primary facilitators include cultural awareness amongst practitioners, cultural awareness in services, and explanations of health care systems. To our knowledge, this review is the first to summarize barriers and facilitators to cultural competence in rehabilitation fields. Insufficient studies were found to draw any conclusions with regards to audiological services. Minimal perspectives based on patient/caregiver experiences in all rehabilitation fields underscore a research gap. Future studies should aim to explore both patient/caregiver and practitioner

  10. Target cell cyclophilins facilitate human papillomavirus type 16 infection.

    Science.gov (United States)

    Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

    2009-07-01

    Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

  11. Target cell cyclophilins facilitate human papillomavirus type 16 infection.

    Directory of Open Access Journals (Sweden)

    Malgorzata Bienkowska-Haba

    2009-07-01

    Full Text Available Following attachment to primary receptor heparan sulfate proteoglycans (HSPG, human papillomavirus type 16 (HPV16 particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

  12. A Cultural-Historical Model to Understand and Facilitate Children's Development

    Science.gov (United States)

    Wong, Pui Ling

    2015-01-01

    Parents and educators strive to help their children to develop optimally. Given the diversity of values and practices among dynamic modern populations it is important to understand all the dimensions that affect the development of children in their communities. A cultural-historical lens facilitates such a holistic understanding. Taking this lens,…

  13. What Facilitates Cultural Entrepreneurship?--A Study of Indian Cultural Entrepreneurs

    Science.gov (United States)

    Sardana, Deepak

    2018-01-01

    The importance of a 'cultural industry' to a national economy and the development of a "creative society" has grown increasingly. However, research on cultural entrepreneurship is in its early stages and is yet to catch the attention of mainstream entrepreneurship and creativity scholars. In this article the objective is to enhance our…

  14. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  15. Establishment of automated culture system for murine induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Koike Hiroyuki

    2012-11-01

    Full Text Available Abstract Background Induced pluripotent stem (iPS cells can differentiate into any cell type, which makes them an attractive resource in fields such as regenerative medicine, drug screening, or in vitro toxicology. The most important prerequisite for these industrial applications is stable supply and uniform quality of iPS cells. Variation in quality largely results from differences in handling skills between operators in laboratories. To minimize these differences, establishment of an automated iPS cell culture system is necessary. Results We developed a standardized mouse iPS cell maintenance culture, using an automated cell culture system housed in a CO2 incubator commonly used in many laboratories. The iPS cells propagated in a chamber uniquely designed for automated culture and showed specific colony morphology, as for manual culture. A cell detachment device in the system passaged iPS cells automatically by dispersing colonies to single cells. In addition, iPS cells were passaged without any change in colony morphology or expression of undifferentiated stem cell markers during the 4 weeks of automated culture. Conclusions Our results show that use of this compact, automated cell culture system facilitates stable iPS cell culture without obvious effects on iPS cell pluripotency or colony-forming ability. The feasibility of iPS cell culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

  16. Microfluidic cell culture systems for drug research.

    Science.gov (United States)

    Wu, Min-Hsien; Huang, Song-Bin; Lee, Gwo-Bin

    2010-04-21

    In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

  17. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

    Directory of Open Access Journals (Sweden)

    Cecilia González

    Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

  18. Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

    Directory of Open Access Journals (Sweden)

    Yoshinobu Uchihara

    2015-01-01

    Full Text Available Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

  19. The disaccharide moiety of bleomycin facilitates uptake by cancer cells.

    Science.gov (United States)

    Schroeder, Benjamin R; Ghare, M Imran; Bhattacharya, Chandrabali; Paul, Rakesh; Yu, Zhiqiang; Zaleski, Paul A; Bozeman, Trevor C; Rishel, Michael J; Hecht, Sidney M

    2014-10-01

    The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

  20. Cell culture techniques in honey bee research

    Science.gov (United States)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  1. Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

    Science.gov (United States)

    Barba-Gutierrez, D Alonso; Daneri-Navarro, A; Villagomez-Mendez, J Jesus Alejandro; Kanamune, J; Robles-Murillo, A Karina; Sanchez-Enriquez, S; Villafan-Bernal, J Rafael; Rivas-Carrillo, J D

    2016-03-01

    Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  3. Good Cell Culture Practice for stem cells and stem-cell-derived models.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

    2017-01-01

    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  4. Improving safety culture in hospitals: Facilitators and barriers to implementation of Systemic Falls Investigative Method (SFIM).

    Science.gov (United States)

    Zecevic, Aleksandra A; Li, Alvin Ho-Ting; Ngo, Charity; Halligan, Michelle; Kothari, Anita

    2017-06-01

    The purpose of this study was to assess the facilitators and barriers to implementation of the Systemic Falls Investigative Method (SFIM) on selected hospital units. A cross-sectional explanatory mixed methods design was used to converge results from a standardized safety culture survey with themes that emerged from interviews and focus groups. Findings were organized by six elements of the Ottawa Model of Research Use framework. A geriatric rehabilitation unit of an acute care hospital and a neurological unit of a rehabilitation hospital were selected purposefully due to the high frequency of falls. Hospital staff who took part in: surveys (n = 39), interviews (n = 10) and focus groups (n = 12), and 38 people who were interviewed during falls investigations: fallers, family, unit staff and hospital management. Implementation of the SFIM to investigate fall occurrences. Percent of positive responses on the Modified Stanford Patient Safety Culture Survey Instrument converged with qualitative themes on facilitators and barriers for intervention implementation. Both hospital units had an overall poor safety culture which hindered intervention implementation. Facilitators were hospital accreditation, strong emphasis on patient safety, infrastructure and dedicated champions. Barriers included heavy workloads, lack of time, lack of resources and poor communication. Successful implementation of SFIM requires regulatory and organizational support, committed frontline staff and allocation of resources to identify active causes and latent contributing factors to falls. System-wide adjustments show promise for promotion of safety culture in hospitals where falls happen regularly. © The Author 2017. Published by Oxford University Press in association with the International Society for Quality in Health Care. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  5. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

  6. The Evolution of Polystyrene as a Cell Culture Material.

    Science.gov (United States)

    Lerman, Max J; Lembong, Josephine; Muramoto, Shin; Gillen, Greg; Fisher, John P

    2018-04-10

    Polystyrene (PS) has brought in vitro cell culture from its humble beginnings to the modern era, propelling dozens of research fields along the way. This review discusses the development of the material, fabrication, and treatment approaches to create the culture material. However, native PS surfaces poorly facilitate cell adhesion and growthin vitro. To overcome this, liquid surface deposition, energetic plasma activation, and emerging functionalization methods transform the surface chemistry. This review seeks to highlight the many potential applications of the first widely accepted polymer growth surface. Although the majority of in vitro research occurs on 2D surfaces, the importance of 3D culture models cannot be overlooked. Here the methods to transition PS to specialized 3D culture surfaces are also reviewed. Specifically, casting, electrospinning, 3D printing, and microcarrier approaches to shift PS to a 3D culture surface are highlighted. The breadth of applications of the material makes it impossible to highlight every use, but the aim remains to demonstrate the versatility and potential as both a general and custom cell culture surface. The review concludes with emerging scaffolding approaches and, based on the findings, presents our insights on the future steps for PS as a tissue culture platform.

  7. Animal-cell culture media: History, characteristics, and current issues.

    Science.gov (United States)

    Yao, Tatsuma; Asayama, Yuta

    2017-04-01

    Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

  8. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  9. Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell-based immunotherapies.

    Science.gov (United States)

    Fekete, Natalie; Béland, Ariane V; Campbell, Katie; Clark, Sarah L; Hoesli, Corinne A

    2018-04-19

    In recent years, cell-based therapies targeting the immune system have emerged as promising strategies for cancer treatment. This review summarizes manufacturing challenges related to production of antigen presenting cells as a patient-tailored cancer therapy. Understanding cell-material interactions is essential because in vitro cell culture manipulations to obtain mature antigen-producing cells can significantly alter their in vivo performance. Traditional antigen-producing cell culture protocols often rely on cell adhesion to surface-treated hydrophilic polystyrene flasks. More recent commercial and investigational cancer immunotherapy products were manufactured using suspension cell culture in closed hydrophobic fluoropolymer bags. The shift to closed cell culture systems can decrease risks of contamination by individual operators, as well as facilitate scale-up and automation. Selecting closed cell culture bags over traditional open culture systems entails different handling procedures and processing controls, which can affect product quality. Changes in culture vessels also entail changes in vessel materials and geometry, which may alter the cell microenvironment and resulting cell fate decisions. Strategically designed culture systems will pave the way for the generation of more sophisticated and highly potent cell-based cancer vaccines. As an increasing number of cell-based therapies enter the clinic, the selection of appropriate cell culture vessels and materials becomes a critical consideration that can impact the therapeutic efficacy of the product, and hence clinical outcomes and patient quality of life. © 2018 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

  10. Advances in cell culture: anchorage dependence

    Science.gov (United States)

    Merten, Otto-Wilhelm

    2015-01-01

    Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

  11. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    Science.gov (United States)

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy. © 2015 AlphaMed Press.

  12. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Directory of Open Access Journals (Sweden)

    Tali Mass

    Full Text Available The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag~4, the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  13. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  14. Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

    Directory of Open Access Journals (Sweden)

    Coukos George

    2011-08-01

    Full Text Available Abstract Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.

  15. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  16. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.

    2003-01-01

    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  17. Understanding asexual identity as a means to facilitate culturally competent care: A systematic literature review.

    Science.gov (United States)

    Jones, Catriona; Hayter, Mark; Jomeen, Julie

    2017-12-01

    To provide a contemporary overview of asexuality and the implications this has for healthcare practice. Individuals belonging to sexual minority groups face many barriers in accessing appropriate health care. The term "sexual minority group" is usually used to refer to lesbian women, gay, bisexual and transgender individuals. Anecdotal and research evidence suggests that those who identify as asexual have similar poor experiences. Systematic review and qualitative analysis. This work uses a systematic review and qualitative analysis of the existing interview data from self-identified asexuals, to construct features of the asexual identity. The findings will help practitioners and health professionals develop an understanding of this poorly understood construct. Ultimately this work is aimed at facilitating culturally competent care in the context of asexuality. Qualitative analysis produced three themes, which can be used, not only to frame asexuality in a positive and normalising way, but also to provide greater understanding of asexuality, "romantic differences coupled with sexual indifference," "validation through engagement with asexual communities" and "a diversity of subasexual identities." Having some understanding of what it means to identify as asexual, and respecting the choices made by asexuals can markedly improve the experiences of those who embrace an asexual identity when engaging with health care. Anecdotal evidence, taken from one of the largest asexual online forums, suggests that a number of self-identified asexuals choose not to disclose their identity to healthcare professionals through fear of their asexual status being pathologised, problematised or judged. Given that asexuality is a poorly understood concept, this may be due to lack of understanding on behalf of healthcare providers. The review provides health professionals and practitioners working in clinical settings with some insights of the features of an asexual identity to facilitate

  18. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

    Science.gov (United States)

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  19. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Seagrave, J.C.; Valdez, J.G.; Tobey, R.A.; Gurley, L.R.

    1985-06-01

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC1 2 , one being increased in amount while the other was reduced to an insignificant level. In ZnC1 2 -treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC1 2 -treated cells. While ZnC1 2 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC1 2 -treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC1 2 -treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  20. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  1. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-01-01

    in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies

  2. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  3. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  4. In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

    Science.gov (United States)

    Schmid, Richard; Tarau, Ioana-Sandra; Rossi, Angela; Leonhardt, Stefan; Schwarz, Thomas; Schuerlein, Sebastian; Lotz, Christian; Hansmann, Jan

    2018-01-01

    The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  5. Introgression of genetic material from Zea mays ssp. Mexicana into cultivated maize was facilitated by tissue culture

    International Nuclear Information System (INIS)

    Wang, L.; Gu, X.; Qu, M.; Luan, J.; Zhang, J.

    2012-01-01

    Zea mays ssp. mexicana, a wild relative of cultivated maize (Z. mays ssp. mays), is a useful gene resource for maize breeding. In this study, two populations were generated by conventional breeding scheme (population I) or tissue culture regime (population II), respectively, to introgress genetic material of Z. mays ssp. mexicana into maize. Karyotype analysis showed that the arm ratios of 10 pairs of chromosomes in parent maize Ye515 and derivative lines from 2 different populations with 26% and 38% chromosome variation frequencies, respectively. Alien chromatin was detected in the root tip cells of progeny plants through genomic in situ hybridization (GISH). There were 3.3 chromosomes carrying alien chromatin on average in population I and 6.5 in population II. The hybridization signals were located mainly at the terminal or sub terminal regions of the chromosomes and the sizes were notably variant among lines. Based on those results, it is concluded that the introgression of genetic material from Z. mays ssp. mexicana into cultivated maize was facilitated by tissue culture, and subsequently some excellent materials for maize breeding were created. (author)

  6. Substrate utilisation by plant-cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, M W

    1982-01-01

    Plant cell cultures have been grown on a wide range of carbon sources in addition to the traditional ones of sucrose and glucose. Biomass yields and growth rates vary greatly between the different carbon sources and there is a variation in response between different cell cultures to individual carbon sources. Some attempts have been made to grow cell cultures on 'waste' and related carbon sources, such as lactose, maltose, starch, molasses and milk whey. Only maltose was found to support growth to anything near the levels observed with glucose and sucrose. In the case of molasses carbon source cell growth was either non-existent or only just measurable. All the data point to glucose as being the most suitable carbon source, principally on the grounds of biomass yield and growth rate. It should be noted, however, that other carbon sources do appear to have a major (positive) influence on natural product synthesis. Uptake into the cell is an important aspect of carbohydrate utilisation. There is strong evidence that from disaccharides upwards, major degradation to smaller units occurs before uptake. In some cases the necessary enzymes appear to be excreted into the culture broth, in others they may be located within the cell wall; invertase that hydrolyses sucrose is a good example. Once the products of carbohydrate degradation and mobilisation enter the cell they may suffer one of two fates, oxidation or utilisation for biosynthesis. The precise split between these two varies depending on such factors as cell growth rate, cell size, nutrient broth composition and carbohydrate status of the cells. In general rapidly growing cells have a high rate of oxidation, whereas cells growing more slowly tend to be more directed towards biosynthesis. Carbohydrate utilisation is a key area of study, underpinning as it does both biomass yield and natural product synthesis. (Refs. 13).

  7. Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

    International Nuclear Information System (INIS)

    Henderson, R.F.; Waide, J.J.; Scott, G.G.

    1994-01-01

    The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion

  8. Identification of molecular pathways facilitating glioma cell invasion in situ.

    Directory of Open Access Journals (Sweden)

    Ido Nevo

    Full Text Available Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

  9. Cell culture experiments planned for the space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  10. HAMLET binding to α-actinin facilitates tumor cell detachment.

    Science.gov (United States)

    Trulsson, Maria; Yu, Hao; Gisselsson, Lennart; Chao, Yinxia; Urbano, Alexander; Aits, Sonja; Mossberg, Ann-Kristin; Svanborg, Catharina

    2011-03-08

    Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

  11. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2016-08-05

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC

  12. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-01-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca 2+ (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia.

  13. Neurobasal media facilitates increased specificity of siRNA-mediated knockdown in primary cerebellar cultures

    DEFF Research Database (Denmark)

    Gustafsson, Julie Ry; Katsioudi, Georgia; Issazadeh-Navikas, Shohreh

    2016-01-01

    be effectively grown in Neurobasal™ media. NEW METHOD: We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer. RESULTS: We observed a more specific knockdown of target...... in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media. COMPARISON WITH EXISTING METHODS: Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared...... to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects. CONCLUSIONS: Specific knockdown of target can be achieved in primary...

  14. Angiotensin II facilitates breast cancer cell migration and metastasis.

    Directory of Open Access Journals (Sweden)

    Sylvie Rodrigues-Ferreira

    Full Text Available Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

  15. Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

    Science.gov (United States)

    Soto-Cerrato, Vanessa; Manuel-Manresa, Pilar; Hernando, Elsa; Calabuig-Fariñas, Silvia; Martínez-Romero, Alicia; Fernández-Dueñas, Víctor; Sahlholm, Kristoffer; Knöpfel, Thomas; García-Valverde, María; Rodilla, Ananda M; Jantus-Lewintre, Eloisa; Farràs, Rosa; Ciruela, Francisco; Pérez-Tomás, Ricardo; Quesada, Roberto

    2015-12-23

    Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

  16. Cell Culture in Microgravity: Opening the Door to Space Cell Biology

    Science.gov (United States)

    Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Adaptational response of human cell populations to microgravity is investigated using simulation, short-term Shuttle experiments, and long-term microgravity. Simulation consists of a clinostatically-rotated cell culture system. The system is a horizontally-rotated cylinder completely filled with culture medium. Low speed rotation results in continuous-fall of the cells through the fluid medium. In this setting, cells: 1) aggregate, 2) propagate in three dimensions, 3) synthesize matrix, 4) differentiate, and 5) form sinusoids that facilitate mass transfer. Space cell culture is conducted in flight bioreactors and in static incubators. Cells grown in microgravity are: bovine cartilage, promyelocytic leukemia, kidney proximal tubule cells, adrenal medulla, breast and colon cancer, and endothelium. Cells were cultured in space to test specific hypotheses. Cartilage cells were used to determine structural differences in cartilage grown in space compared to ground-based bioreactors. Results from a 130-day experiment on Mir revealed that cartilage grown in space was substantially more compressible due to insufficient glycosaminoglycan in the matrix. Interestingly, earth-grown cartilage conformed better to the dimensions of the scaffolding material, while the Mir specimens were spherical. The other cell populations are currently being analyzed for cell surface properties, gene expression, and differentiation. Results suggest that some cells spontaneously differentiate in microgravity. Additionally, vast changes in gene expression may occur in response to microgravity. In conclusion, the transition to microgravity may constitute a physical perturbation in cells resulting in unique gene expressions, the consequences of which may be useful in tissue engineering, disease modeling, and space cell biology.

  17. A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

    Science.gov (United States)

    Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2018-01-01

    Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

  18. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

  19. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  20. Transfection in Primary Cultured Neuronal Cells.

    Science.gov (United States)

    Marwick, Katie F M; Hardingham, Giles E

    2017-01-01

    Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

  1. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our focus...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....

  2. Plant Cell Culture Initiation: practical tips

    NARCIS (Netherlands)

    Hall, R.D.

    2001-01-01

    The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10-15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our

  3. Cell culture from sponges: pluripotency and immortality

    NARCIS (Netherlands)

    Caralt Bosch, de S.; Uriz, M.J.; Wijffels, R.H.

    2007-01-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a

  4. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  5. Isolation and culture of Celosia cristata L cell suspension protoplasts

    Directory of Open Access Journals (Sweden)

    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  6. A Pilot Study: Facilitating Cross-Cultural Understanding with Project-Based Collaborative Learning in an Online Environment

    Science.gov (United States)

    Shadiev, Rustam; Hwang, Wu-Yuin; Huang, Yueh-Min

    2015-01-01

    This study investigated three aspects: how project-based collaborative learning facilitates cross-cultural understanding; how students perceive project-based collaborative learning implementation in a collaborative cyber community (3C) online environment; and what types of communication among students are used. A qualitative case study approach…

  7. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  8. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  9. mCell: Facilitating Mobile Communication of Small Groups

    OpenAIRE

    Mikko T. Tarkiainen; Jonna Häkkilä; Jan Blom; Merja Haveri; Jyri Virtanen

    2008-01-01

    Mobile communication technology offers a potential platform for new types of communication applications. Here, we describe the development and experiences with a mobile group communication application, mCell, that runs on a mobile phone. We present the underlying design implications, the application implementation, and a user study, where three groups used the application for one month. The findings of the user study reveal general user experiences with the application and show different patt...

  10. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...... on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...

  11. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M

    2006-01-01

    culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...... on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...

  12. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  13. Light transfer in agar immobilized microalgae cell cultures

    Science.gov (United States)

    Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

    2017-09-01

    This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

  14. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  15. Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking.

    Science.gov (United States)

    Tan, Hong-Kee; Toh, Cheng-Xu Delon; Ma, Dongrui; Yang, Binxia; Liu, Tong Ming; Lu, Jun; Wong, Chee-Wai; Tan, Tze-Kai; Li, Hu; Syn, Christopher; Tan, Eng-Lee; Lim, Bing; Lim, Yoon-Pin; Cook, Stuart A; Loh, Yuin-Han

    2014-05-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a "do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.

  16. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

  17. OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Xiaoli Chen

    2015-09-01

    Full Text Available Generating engraftable hematopoietic stem cells (HSCs from pluripotent stem cells (PSCs is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2 accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo.

  18. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    Energy Technology Data Exchange (ETDEWEB)

    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Nakagawa, Shin [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takamura, Naoki [Pharmaceutical Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka (Japan); Kato, Akiko [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takebayashi, Minoru [Department of Psychiatry, National Hospital Organization Kure Medical Center, Kure (Japan); Hisaoka-Nakashima, Kazue [Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima (Japan); Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan)

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  19. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    International Nuclear Information System (INIS)

    Boku, Shuken; Nakagawa, Shin; Takamura, Naoki; Kato, Akiko; Takebayashi, Minoru; Hisaoka-Nakashima, Kazue; Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro

    2013-01-01

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis

  20. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  1. Studies on level of cytokines and expression of connexin43 in tumor and normal cells in culture conditions

    International Nuclear Information System (INIS)

    Asati, V.; Pandey, B.N.

    2016-01-01

    Factors secreted from the tumor cells in culture medium have been known to facilitate the growth of fresh cultures and also to affect the cellular radio-sensitivity. Moreover, expression of gap junction proteins like connexin-43 is known as a key player in cell survival and proliferation. The present study is aimed to evaluate the effects of conditioned medium on the growth of respective tumor/normal cells and the expression of connexin-43 in these cells

  2. Teacher Candidates' Perceptions of Debriefing Circles to Facilitate Self-Reflection during a Cultural Immersion Experience

    Science.gov (United States)

    Addleman, Rebecca A.; Brazo, Carol J.; Dixon, Kristin; Cevallos, Tatiana; Wortman, Shary

    2014-01-01

    This study followed 9 teacher candidates through a 3-week cultural immersion experience in which they volunteered in educational settings where they were not members of the majority culture. This learning experience was designed to help candidates better understand their culturally, ethnically, and linguistically diverse future students. A…

  3. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

    Science.gov (United States)

    Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

    2015-01-15

    Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    Science.gov (United States)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  5. Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

    Directory of Open Access Journals (Sweden)

    Md Amranul Haque, Masato Nagaoka, Bayar Hexig and Toshihiro Akaike

    2010-01-01

    Full Text Available Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs direct embryonic stem (ES cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

  6. Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

    International Nuclear Information System (INIS)

    Haque, Md Amranul; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

    2010-01-01

    Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types. (topical review)

  7. TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

    Science.gov (United States)

    Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

    2010-02-01

    Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

  8. In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

    Science.gov (United States)

    Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

    2017-07-14

    Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

  9. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    Science.gov (United States)

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  10. Facilitating cultural border crossing in urban secondary science classrooms: A study of inservice teachers

    Science.gov (United States)

    Monteiro, Anna Karina

    Research acknowledges that if students are to be successful science, they must learn to navigate and cross cultural borders that exist between their own cultures and the subculture of science. This dissertation utilized a mixed methods approach to explore how inservice science teachers working in urban schools construct their ideas of and apply the concepts about the culture of science and cultural border crossing as relevant to the teaching and learning of science. The study used the lenses of cultural capital, social constructivism, and cultural congruency in the design and analysis of each of the three phases of data collection. Phase I identified the perspectives of six inservice science teachers on science culture, cultural border crossing, and which border crossing methods, if any, they used during science teaching. Phase II took a dialectical approach as the teachers read about science culture and cultural border crossing during three informal professional learning community meetings. This phase explored how teachers constructed their understanding of cultural border crossing and how the concept applied to the teaching and learning of science. Phase III evaluated how teachers' perspectives changed from Phase I. In addition, classroom observations were used to determine whether teachers' practices in their science classrooms changed from Phase I to Phase III. All three phases collected data through qualitative (i.e., interviews, classroom observations, and surveys) and quantitative (Likert items) means. The findings indicated that teachers found great value in learning about the culture of science and cultural border crossing as it pertained to their teaching methods. This was not only evidenced by their interviews and surveys, but also in the methods they used in their classrooms. Final conclusions included how the use of student capital resources (prior experiences, understandings and knowledge, ideas an interests, and personal beliefs), if supported by

  11. How do culture media influence in vitro perivascular cell behavior?

    Science.gov (United States)

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

    2015-12-01

    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. © 2015 International Federation for Cell Biology.

  12. Comparing Facilitator Priorities of Suicide Survivor Support Groups: A Cross-Cultural Comparison Between Japanese and American Groups.

    Science.gov (United States)

    Feigelman, William; Feigelman, Beverly; Kawashima, Daisuke; Shiraga, Keisuke; Kawano, Kenji

    2017-08-01

    A total of 56 Japanese and 59 American survivor of suicide support group facilitators were asked to rank the mutual aid objectives of their groups following Shulman's scheme in terms of their frequency and importance. Both American and Japanese facilitators showed an emphasis on personal adaptation goals (such as helping bereaved feel less isolated in their grief or encouraging bereaved to share their coping with loss experiences) over collective goals (such as raising monies for more research on mental illness or trying to combat societal suicide stigma in their local communities). Differences were also noted with American facilitators evaluating helping with problem solving, sharing different ways of coping, viewing personal issues as societal problems, and advocating for promoting social change as significantly higher than the Japanese did. We believe some of these contrasts reflect differences in American and Japanese cultural values.

  13. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  14. How the Internet Facilitates the Activity within a Consumer Culture : - A Study of the Online Vinyl Record Network

    OpenAIRE

    Söderlindh, Stefan; Broman, David

    2009-01-01

    The purpose of this thesis is to describe and analyze how the online vinyl record network functions from both a consumer and retailer perspective, in order to gain an understanding of how the Internet facilitates the activity within a consumer culture. The vinyl record industry is experiencing a revival, with an upswing in sales and media attention and a significant increase in the amount of online trading. This inductive study contains data from qualitative interviews with ten vinyl record c...

  15. Traditional and Modern Cell Culture in Virus Diagnosis.

    Science.gov (United States)

    Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

    2016-04-01

    Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses.

  16. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  17. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

    Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

    2017-12-01

    Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2014-01-01

    An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

  19. Enhanced infectivity of bluetongue virus in cell culture by centrifugation.

    OpenAIRE

    Sundin, D R; Mecham, J O

    1989-01-01

    The effects of centrifugation of the infection of cell culture with bluetongue virus (BTV) were investigated. Baby hamster kidney cells were infected with BTV with or without centrifugation. Viral antigen was detected by immunofluorescence at 24 h in both centrifuged and noncentrifuged cultures. However, after 24 h of infection, the production of PFU in centrifuged cell cultures was 10- to 20-fold greater than that seen in cultures not centrifuged. In addition, centrifugation enhanced the dir...

  20. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  1. Facilitators, barriers, and components of a culturally tailored afterschool physical activity program in preadolescent African American girls and their mothers.

    Science.gov (United States)

    Alhassan, Sofiya; Greever, Cory; Nwaokelemeh, Ogechi; Mendoza, Albert; Barr-Anderson, Daheia J

    2014-01-01

    Traditional physical activity (PA) programs have not been effective in increasing PA in African American girls. Currently, there is limited information regarding the components of PA programs that drive participation in African American girls. The purpose of our investigation was to describe the facilitators, barriers, and components of a culturally tailored afterschool PA program that will potentially inspire the participation of African American mother-daughter dyads. Six focus groups (n=12 mother-daughter dyads; daughters, 7-10 yrs in age) were conducted between March and May 2012. Focus group semi-structured interviews were transcribed, coded, and systematically analyzed using NVivo. Mothers reported a preference for non-traditional (dancing, household chores) types of PA. While daughters preferred to participate in both dance-based and traditional types (walking, riding bikes) of PA. Participants felt that the use of a culturally tailored dance program would be appealing because it highlights the cultural and historical legacy of the African American culture. Mothers wanted programs that would allow them time to spend with their daughters. Top three dance styles that mothers wanted to participate in were African, hip-hop, and Salsa/samba, while daughters reported that they would enjoy participating in hip-hop, African, and jazz. The most common responses given for resources needed for participating in a culturally tailored afterschool dance program were the location of the program, transportation, and childcare for siblings. Our investigation highlights some cultural factors related to facilitators and barriers of PA that should be addressed in designing PA studies for African American girls and their mothers.

  2. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  3. Turbulent Dynamics of Epithelial Cell Cultures

    Science.gov (United States)

    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  4. Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment.

    Directory of Open Access Journals (Sweden)

    Al Shaimaa Hasan

    Full Text Available Cardiosphere-derived cells (CDCs, one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1 into a regulatory anti-inflammatory phenotype (M2. Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages, but decreased the expression of CD86 (a marker for M1 macrophages 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair.

  5. Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

    Science.gov (United States)

    Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

    2016-01-01

    Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

  6. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  7. Barriers and facilitators to childhood obesity prevention among culturally and linguistically diverse (CALD) communities in Victoria, Australia.

    Science.gov (United States)

    Cyril, Sheila; Nicholson, Jan M; Agho, Kingsley; Polonsky, Michael; Renzaho, Andre M

    2017-06-01

    Childhood obesity is rising among culturally and linguistically diverse (CALD) groups who show poor engagement in obesity prevention initiatives. We examined the barriers and facilitators to the engagement of CALD communities in obesity prevention initiatives. We used the nominal group technique to collect data from 39 participants from Vietnamese, Burmese, African, Afghani and Indian origins living in disadvantaged areas of Victoria, Australia. Data analysis revealed ranked priorities for barriers and facilitators for CALD community engagement in obesity prevention initiatives. CALD parents identified key barriers as being: competing priorities in the post-migration settlement phase; language, cultural and program accessibility barriers; low levels of food and health literacy; junk food advertisement targeting children; and lack of mandatory weight checks for schoolchildren. Key facilitators emerged as: bicultural playgroup leaders; ethnic community groups; and school-based healthy lunch box initiatives. This study has identified several policy recommendations including: the implementation of robust food taxation policies; consistent control of food advertising targeting children; improving CALD health literacy using bicultural workers; and matching health promotional materials with CALD community literacy levels. Implications for Public Health: These recommendations can directly influence public health policy to improve the engagement of CALD communities in obesity prevention services and ultimately reduce the widening obesity disparities in Australia. © 2017 The Authors.

  8. From a culture of caution to a culture of confidence: facilitating the good governance of administrative data in the UK

    Directory of Open Access Journals (Sweden)

    Leslie Stevens

    2017-04-01

    We conclude by focussing on how to overcome the culture of caution, to one of confidence. We suggest the adoption of our decision-making matrix to help data custodians distinguish between real versus perceived barriers to data sharing (i.e. dispelling legal myths and identifying areas where changes can be made. We also introduce strategic solutions in our public interest mandate which entails overt commitment to use public sector data when it is in the public interest to do so.

  9. Differential marker expression by cultures rich in mesenchymal stem cells

    Science.gov (United States)

    2013-01-01

    Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

  10. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  11. Protection of cultured mammalian cells by rebamipide

    International Nuclear Information System (INIS)

    Antoku, Shigetoshi; Aramaki, Ryoji; Tanaka, Hisashi; Kusumoto, Naotoshi.

    1997-01-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO 2 incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with 14 C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  12. Bioactive poly(ethylene glycol) hydrogels to recapitulate the HSC niche and facilitate HSC expansion in culture.

    Science.gov (United States)

    Cuchiara, Maude L; Coşkun, Süleyman; Banda, Omar A; Horter, Kelsey L; Hirschi, Karen K; West, Jennifer L

    2016-04-01

    Hematopoietic stem cells (HSCs) have been used therapeutically for decades, yet their widespread clinical use is hampered by the inability to expand HSCs successfully in vitro. In culture, HSCs rapidly differentiate and lose their ability to self-renew. We hypothesize that by mimicking aspects of the bone marrow microenvironment in vitro we can better control the expansion and differentiation of these cells. In this work, derivatives of poly(ethylene glycol) diacrylate hydrogels were used as a culture substrate for hematopoietic stem and progenitor cell (HSPC) populations. Key HSC cytokines, stem cell factor (SCF) and interferon-γ (IFNγ), as well as the cell adhesion ligands RGDS and connecting segment 1 were covalently immobilized onto the surface of the hydrogels. With the use of SCF and IFNγ, we observed significant expansion of HSPCs, ∼97 and ∼104 fold respectively, while maintaining c-kit(+) lin(-) and c-kit(+) Sca1(+) lin(-) (KSL) populations and the ability to form multilineage colonies after 14 days. HSPCs were also encapsulated within degradable poly(ethylene glycol) hydrogels for three-dimensional culture. After expansion in hydrogels, ∼60% of cells were c-kit(+), demonstrating no loss in the proportion of these cells over the 14 day culture period, and ∼50% of colonies formed were multilineage, indicating that the cells retained their differentiation potential. The ability to tailor and use this system to support HSC growth could have implications on the future use of HSCs and other blood cell types in a clinical setting. © 2015 Wiley Periodicals, Inc.

  13. The evolution of chicken stem cell culture methods.

    Science.gov (United States)

    Farzaneh, M; Attari, F; Mozdziak, P E; Khoshnam, S E

    2017-12-01

    1. The avian embryo is an excellent model for studying embryology and the production of pharmaceutical proteins in transgenic chickens. Furthermore, chicken stem cells have the potential for proliferation and differentiation and emerged as an attractive tool for various cell-based technologies. 2. The objective of these studies is the derivation and culture of these stem cells is the production of transgenic birds for recombinant biomaterials and vaccine manufacture, drug and cytotoxicity testing, as well as to gain insight into basic science, including cell tracking. 3. Despite similarities among the established chicken stem cell lines, fundamental differences have been reported between their culture conditions and applications. Recent conventional protocols used for expansion and culture of chicken stem cells mostly depend on feeder cells, serum-containing media and static culture. 4. Utilising chicken stem cells for generation of cell-based transgenic birds and a variety of vaccines requires large-scale cell production. However, scaling up the conventional adherent chicken stem cells is challenging and labour intensive. Development of a suspension cell culture process for chicken embryonic stem cells (cESCs), chicken primordial germ cells (PGCs) and chicken induced pluripotent stem cells (ciPSCs) will be an important advance for increasing the growth kinetics of these cells. 6. This review describes various approaches and suggestions to achieve optimal cell growth for defined chicken stem cells cultures and use in future manufacturing applications.

  14. The significance of 'facilitator as a change agent'--organisational learning culture in aged care home settings.

    Science.gov (United States)

    Grealish, Laurie; Henderson, Amanda; Quero, Fritz; Phillips, Roslyn; Surawski, May

    2015-04-01

    To explore the impact of an educational programme focused on social behaviours and relationships on organisational learning culture in the residential aged care context. The number of aged care homes will continue to rise as the frail older elderly live longer, requiring more formal care and support. As with other small- to medium-sized health services, aged care homes are faced with the challenge of continuous development of the workforce and depend upon registered nurses to lead staff development. A mixed-method evaluation research design was used to determine the impact of an educational programme focused on social aspects of learning on organisational learning culture. One hundred and fifty-nine (pre) and 143 (post) participants from three aged care homes completed the Clinical Learning Organisational Culture survey, and three participant-researcher registered nurse clinical educators provided regular journal entries for review. While each site received the same educational programme over a six-month period, the change in organisational learning culture at each site was notably different. Two aged care homes had significant improvements in affiliation, one in accomplishment and one in recognition. The educators' journals differed in the types of learning observed and interventions undertaken, with Eucalyptus focused on organisational change, Grevillea focused on group (student) change and the Wattle focused on individual or situational change. Clinical educator activities appear to have a significant effect on organisational learning culture, with a focus on the organisational level having the greatest positive effect on learning culture and on individual or situational level having a limited effect. Clinical educator facilitation that is focused on organisational rather than individual interests may offer a key to improving organisational learning culture. © 2014 John Wiley & Sons Ltd.

  15. Facilitating Occupational Therapy Student Learning to Enhance Cross-Cultural Effectiveness

    Science.gov (United States)

    Gilbertson, Barbara Carol Hooper

    2012-01-01

    Developing cultural awareness and effectiveness is critical to meaningful and successful occupational therapy practice and of concern to occupational therapists worldwide (World Federation of Occupational Therapists, 2010). Occupational therapy graduates, not fully representative of the demographics of the populations they will meet clinically,…

  16. Minimizing cross-cultural maladaptation: How minority status facilitates change in international acculturation.

    Science.gov (United States)

    Volpone, Sabrina D; Marquardt, Dennis J; Casper, Wendy J; Avery, Derek R

    2018-03-01

    Culturally savvy organizations recognize that selecting and developing people who can be effective in a global workforce is important in today's business environment. Nevertheless, many companies struggle to identify and develop talent who are happy and successful working and living outside their home country. We examine 1 factor that may foster success in a host country-minority status in 1's home country-as a predictor of change in acculturation over time. Specifically, we draw on the conservation of resources model to suggest that international students who have been a member of more minority groups in their home country have unique experiences working with dissimilar others that offer advantages when acculturating to new cultures and novel situations. Then, change in host country acculturation is explored as a mechanism to explain how minority status in the home country relates to intentions to leave the host country and psychological well-being 6 months after entry. Two moderators (cultural intelligence, perceived diversity climate of the host institution) of these relationships are also examined. Results revealed that the relationship between minority status in the home country and change in host country acculturation was positive and stronger for those with higher cultural intelligence. Further, the relationship between change in host country acculturation and psychological well-being was positive when perceived diversity climate of the host institution was high, but was not significant when perceived diversity climate was low. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

  17. Ligwomi Cult: A Facilitator of the Socio-Cultural Development of Igbo ...

    African Journals Online (AJOL)

    Ligwomi cult practiced among Igbo Imabana people of Abi Local Government Area of Cross River State is unlike other cults whose activities are regarded as nefarious. This cult has great socio-cultural significance and has brought some remarkable development in Igbo Imabana. We are poised to investigate such ...

  18. Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2018-02-01

    Full Text Available Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs, are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX. These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

  19. Differences in gene expression of cells growing in conventional 2D versus 3D cell culture

    International Nuclear Information System (INIS)

    Zschenker, Oliver; Cordes, Nils; Streichert, Thomas

    2009-01-01

    Full text: Telomeres are DNA protein complexes on the ends of chromosomes that distinguish the ends of chromosomes from double strand breaks and prevent degradation or fusion by nonhomologous end-joining. The loss of telomeres is associated with a loss of heterochromatic features leading to a less compact chromatin structure which allows e.g. DNA repair proteins to get better access to the site of the DNA damage and facilitate chromosome fusions. Telomerase is an enzyme that can counteract the loss of telomeres by adding telomeric repeats on the ends of chromosomes. Since telomerase is active in most tumor cells, telomerase is suggested to be the reason for the unlimited number of cell divisions of cancer cells. TRF2 is one of the most important proteins of the Shelterin complex protecting the telomeres from shortening by inhibiting ATM which is up-stream of the DNA repair mechanisms. Thus, we are concentrating on TRF2 and telomerase to investigate the differences in DNA repair in telomeric (heterochromatic) versus euchromatic regions. Human cancer cells with differences in status of p53 and telomerase like A549, UT-SCC15 and FaDu cells are used. Without any treatment, FaDu cells express high levels of telomerase and TRF2 in conventional 2D cell culture which is in contrast to e.g. A549. We found that telomerase is even higher expressed in 3D than in 2D cell culture. To connect telomere associated processes to both repair of radiogenic DNA damage/lesions and to cell-extracellular matrix interactions, we performed whole genome microarray analysis. By comparing the differential expression of genes associated with these three cell functions, we intend to yield new molecular insight into radiotherapy relevant tumor characteristics, particularly radioresistance and DNA damage response network processing. (author)

  20. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  1. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

    Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

    2000-01-01

    Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

  2. Electrospinning of microbial polyester for cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Oh Hyeong [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Lee, Ik Sang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Ko, Young-Gwang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Meng, Wan [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Jung, Kyung-Hye [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Kang, Inn-Kyu [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Ito, Yoshihiro [Kanagawa Academy of Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki 213-0012 (Japan)

    2007-03-01

    Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture.

  3. Designing an E-Learning Application to Facilitate Health Care Professionals' Cross-Cultural Communication.

    Science.gov (United States)

    Balasubramaniam, Nagadivya; Kujala, Sari; Ayzit, Dicle; Kauppinen, Marjo; Heponiemi, Tarja; Hietapakka, Laura; Kaihlanen, Anu

    2018-01-01

    In recent times, health care professionals (HCP) have come across a number of migrants as their patients. The cultural differences lead to communicational challenges between the migrant patients and health care professionals. Our project aimed to discover HCPs' attitudes, challenges and needs on cross-cultural communication, so that we can develop an e-learning solution that would be helpful for them. By conducting interviews with HCPs, we identified five crucial categories of problems and the current solutions that experienced professionals use to tackle those problems. These interviews also helped us in understanding the motivational factors of HCPs, when using e-learning application. Health care professionals prefer a focus on examples and themes such as death and pain that they face in their everyday work. Changing attitudes by e-learning application is challenging. However, e-learning was recognized as a flexible way for supporting traditional training with HCPs who are busy at work most of the time.

  4. Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Kito, Hiroaki; Yamazaki, Daiju; Ohya, Susumu; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2011-01-01

    Highlights: → We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. → The ER stress facilitated the expression of inward rectifier K + channel (K ir 2.1) and induced sustained membrane hyperpolarization. → The membrane hyperpolarization induced sustained Ca 2+ entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. → The K ir 2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K + channel (K ir 2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K ir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca 2+ concentration due to Ca 2+ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K ir 2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  5. Using The Culture Of Childhood To Facilitate Compassionate, Student Centered And Holistic Education

    Directory of Open Access Journals (Sweden)

    Shaun McGurgan

    2017-06-01

    Full Text Available Much work has been done on describing the faults of the education system and yet solutions are few and far between. What I have presented here is one solution to a possible range of solutions. By using the culture of childhood as the foundation for an authentic student centred education I hope to develop human beings who are integrated – because they have developed all their capacities – and self confident – because they have been affirmed.

  6. Retrovirally transduced NCAM140 facilitates neuronal fate choice of hippocampal progenitor cells.

    Science.gov (United States)

    Kim, Ju Hee; Lee, Jung-Ha; Park, Jin-Yong; Park, Chang-Hwan; Yun, Chae-Ok; Lee, Sang-Hun; Lee, Yong-Sung; Son, Hyeon

    2005-07-01

    Neural cell adhesion molecule (NCAM) influences proliferation and differentiation of neuronal cells. However, only a little is known about the downstream effects of NCAM signalling, such as alterations in gene transcription, which are associated with cell fate choice. To examine whether NCAM plays a role in cell fate choice during hippocampal neurogenesis, we performed a gain-of-function study, using a retroviral vector which contained full-length NCAM140 cDNA and the marker gene EGFP, and found that NCAM140 promoted neurogenesis by activating proneural transcription activators with concurrent inhibition of gliogenesis. The enhanced transcript levels of proneural transcription factors in NCAM140-transduced cells were down-regulated by treatment of the cells with mitogen-activated protein kinase kinase (MEK) inhibitor PD098059. Overall, these findings suggest that NCAM140 may facilitate hippocampal neurogenesis via regulation of proneurogenic transcription factors in an extracellular signal-regulated kinase (ERK)-dependent manner.

  7. Stimulation of the proliferation of hemopoietic stem cells in irradiated bone marrow cell culture

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, H.; Seto, A.

    1981-01-01

    Long-term hemopoiesis was established in bone marrow cell culture in vitro. This culture was shown to support the recovery proliferation of hemopoietic stem cells completely in vitro after irradiation. Hemopoietic stem cells were stimulated into proliferation in culture when normal bone marrow cells were overlayed on top of the irradiated adherent cell colonies. These results indicate that proliferation and differentiation of hemopoietic stem cells in vitro are also supported by stromahemopoietic cell interactions

  8. Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

    Science.gov (United States)

    Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

    2016-11-14

    Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

  9. Particle Trajectories in Rotating Wall Cell Culture Devices

    Science.gov (United States)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  10. Microfluidic engineered high cell density three-dimensional neural cultures

    Science.gov (United States)

    Cullen, D. Kacy; Vukasinovic, Jelena; Glezer, Ari; La Placa, Michelle C.

    2007-06-01

    Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 µm) 3D neural cultures supported by passive diffusion, cell densities =104 cells mm-3), continuous medium perfusion at 2.0-11.0 µL min-1 improved viability compared to non-perfused cultures (p death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 µL min-1 (p 90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

  11. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing......, these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...

  12. Barriers and Facilitators of Iranian Elderly in Use of ATM Machines: A Qualitative Research in the Way of Cultural Probes

    Directory of Open Access Journals (Sweden)

    Azade Mokhberi

    2013-10-01

    Full Text Available Objectives: Having considering to the rate of increasing of elder people population in Iran, is preadicted that in the next thirty years the number of elderly people who need to independently work with electronic devices such as ATMs machineswill be significantly increased while there is no sufficient studies in order to modification of these devices considering physical, mental and psychology abilities of the elder people. Methods & Materials: This article is part of a qualitative study with cultural Probes Methodology to explore the needs and barriers and facilitators in the work ATMs. This study collected data through observation, interview and documentation participants respectively. This qualitative study was conducted in two stages. In the first phase interviewed with 30 elderly people in Tehran.In the second Phase according to cultural probes method we designed a package to extract the needs and problems associated with ATM. purposive sample of 10 elderly people in Tehran, 6 participants were female and 4 males. Interviews continued until data saturation, data Were coded and categorized by content analysis method. Results: Six key factors required for the design of ATM data were extracted according to the Iranian people, that pleasant, unpleasant, wants and desires, problems and obstacles, banking and mishaps. that the classification of barriers and facilitating factors these factors were extracted for the elderly. The results showed a much higher barriers to Elderly people using ATMs in facilitating conditions are present. Conclusion: According to the Iranian people using ATMs barrier is recommended to attention to ATMs in the the environment, nature and location of the installation, ATMs appear particularly relevant in the context of software that can be tailored to the elderly should be done. Finally, it is suggested that future studies in different groups of people, especially older people with disabilities and low literacy are doing.

  13. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  14. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  15. Characterization of host lymphoid cells in antibody-facilitated bone marrow chimeras

    International Nuclear Information System (INIS)

    McCarthy, S.A.; Griffith, I.J.; Gambel, P.; Francescutti, L.H.; Wegmann, T.G.

    1985-01-01

    The authors have produced stable murine antibody-facilitated (AF) chimeras by the simultaneous injection of P1 bone marrow cells and anti-P2 monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These AF chimeras are healthy, long-lived, and exhibit no overt signs of graft-versus-host disease. They are immunocompetent and tolerant of host, P2-encoded alloantigens. Donor cell engraftment and takeover, monitored by glucosephosphate isomerase isozyme patterns, is usually complete (greater than 95%) in the peripheral blood, bone marrow, and hemopoietic stem cell compartments of long-term (greater than 3 months posttransplantation) AF chimeras. The authors report here, however, that splenic, lymph node, and thymic leukocytes of AF chimeras represent donor/host chimeric populations. Spleen cell populations of AF chimeras exhibit substantial chimera-to-chimera variation in the preponderant residual host cell type(s) present. Interpretations of the implications of these findings are discussed

  16. Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation.

    Science.gov (United States)

    Powers, Daryl E; Millman, Jeffrey R; Bonner-Weir, Susan; Rappel, Michael J; Colton, Clark K

    2010-01-01

    Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO(2gas)). Because the cells are consuming oxygen supplied by diffusion, a difference between pO(2gas) and that experienced by the cells (pO(2cell)) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen-permeable silicone rubber membranes can be used to control pO(2cell) during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte-like increases with decreasing pO(2) only when using oxygen-permeable silicone membrane-based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO(2cell) being equal to pO(2gas) at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO(2) distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO(2cell), and these results suggest they are a valuable tool for investigating pO(2) effects in many applications, such as stem cell differentiation. Copyright 2009 American Institute of Chemical Engineers

  17. A Non-Destructive Culturing and Cell Sorting Method for Cardiomyocytes and Neurons Using a Double Alginate Layer

    Science.gov (United States)

    Terazono, Hideyuki; Kim, Hyonchol; Hayashi, Masahito; Hattori, Akihiro; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-01-01

    A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture. PMID:22870332

  18. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Directory of Open Access Journals (Sweden)

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  19. Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

    Science.gov (United States)

    Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

    2016-05-15

    Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Dose response of tracheal epithelial cells to ionizing radiation in air-liquid interface cultures

    International Nuclear Information System (INIS)

    Fukutsu, K.; Yamada, Y.; Shimo, M.

    2002-01-01

    The dose-response relationships of tracheal epithelial cells to ionizing radiation was examined in air-liquid interface cultures, which were developed for the purpose of simulating in vivo conditions. The cultures investigated in this study were expected to be advantageous for the performance of irradiation experiments using short-range α rays. The level of dose response of air-liquid interface cultures to ionizing radiation proved to be the same as that for in vivo conditions. This result indicates that air-liquid interface cultures will prove most useful, to facilitate future studies for the investigation of the biological effects induced in tracheal epithelial cells by ionizing radiation, especially by α-rays. (orig.)

  1. Biogelx: Cell Culture on Self-Assembling Peptide Gels.

    Science.gov (United States)

    Harper, Mhairi M; Connolly, Michael L; Goldie, Laura; Irvine, Eleanore J; Shaw, Joshua E; Jayawarna, Vineetha; Richardson, Stephen M; Dalby, Matthew J; Lightbody, David; Ulijn, Rein V

    2018-01-01

    Aromatic peptide amphiphiles can form self-supporting nanostructured hydrogels with tunable mechanical properties and chemical compositions. These hydrogels are increasingly applied in two-dimensional (2D) and three-dimensional (3D) cell culture, where there is a rapidly growing need to store, grow, proliferate, and manipulate naturally derived cells within a hydrated, 3D matrix. Biogelx Limited is a biomaterials company, created to commercialize these bio-inspired hydrogels to cell biologists for a range of cell culture applications. This chapter describes methods of various characterization and cell culture techniques specifically optimized for compatibility with Biogelx products.

  2. Development of a microfluidic perfusion 3D cell culture system

    Science.gov (United States)

    Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

    2018-04-01

    Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

  3. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  4. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  5. A common basis for facilitated legitimate exchange of biological materials proposed by the European Culture Collections' Organisation

    Directory of Open Access Journals (Sweden)

    Dagmar Fritze

    2009-12-01

    Full Text Available Being charged with the task of accessioning and supplying of living microbiological material, microbial culture collections are institutions that play a central role between the interests of a variety of user communities. On the one side are the providers of living microbiological material, such as individual scientists, institutions and countries of origin and on the other side are the various kinds of recipients/users of cultures of microorganisms from academia and industry. Thus, providing access to high quality biological material and scientific services while at the same time observing donor countries' rights, intellectual property rights, biosafety and biosecurity aspects poses demanding challenges. E.g. donor countries rights relate to Article 15 of the Convention on Biological Diversity: "Contracting parties …. recognize the sovereign rights of states over their natural resources …. shall facilitate access to resources … and not impose restrictions that run counter to the aims of the Convention. Access to natural resources shall be by mutually agreed terms and subject to prior informed consent ..." The use of a proposed standard contract by culture collections is discussed as a way of contractually safeguarding the existing research commons, while observing the new rights established in the Convention on Biological Diversity as well as other existing and new legislation impacting on the accessibility of living microbial material.

  6. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  7. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  8. Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells

    DEFF Research Database (Denmark)

    Dong, Guoyi; Shang, Zhouchun; Liu, Longqi

    2017-01-01

    Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here......, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes...... such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus...

  9. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  10. A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

    Science.gov (United States)

    Maeda, Yumi; Tamura, Toshiki; Fukutomi, Yasuo; Mukai, Tetsu; Kai, Masanori; Makino, Masahiko

    2011-01-01

    Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells. PMID:22132248

  11. The impact of cell culture equipment on energy loss.

    Science.gov (United States)

    Davies, Lleucu B; Kiernan, Michael N; Bishop, Joanna C; Thornton, Catherine A; Morgan, Gareth

    2014-01-01

    Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels.

  12. Developing a Culture to Facilitate Research Capacity Building for Clinical Nurse Consultants in Generalist Paediatric Practice

    Directory of Open Access Journals (Sweden)

    Lesley Wilkes

    2013-01-01

    Full Text Available This paper reports a research capacity building exercise with a group of CNCs practicing in the speciality of paediatrics in New South Wales (NSW, Australia. It explores the first step in building a research culture, through identifying the research priorities of members of the NSW Child Health Networks Paediatric Clinical Nurse Consultant group, and this forms the major focus of this paper. A nominal group technique (NGT was utilised with sixteen members to identify research topics for investigation which were considered a priority for improving children's health care. The group reviewed and prioritised 43 research topics in children's health which were identified in the literature. As a result of conducting this research prioritisation exercise, the group chose two research topics to investigate: reasons for children representing to the Emergency Department and a comparison of the use of high-flow and low-flow nasal prongs in children with bronchiolitis. The research team will continue to mentor the nurses throughout their research projects which resulted from the NGT. One bridge to leadership development in enhancing patient care is translating knowledge to practice and policy development. This study leads the way for a group of CNCs in paediatric nursing to combine their research capacity and influence clinical knowledge.

  13. Biona-C Cell Culture pH Monitoring System

    Science.gov (United States)

    Friedericks, C.

    1999-01-01

    Sensors 2000! is developing a system to demonstrate the ability to perform accurate, real-time measurements of pH and CO2 in a cell culture media in Space. The BIONA-C Cell Culture pH Monitoring System consists of S2K! developed ion selective sensors and control electronics integrated with the fluidics of a cell culture system. The integrated system comprises a "rail" in the Cell Culture Module (CCM) of WRAIR (Space Biosciences of Walter Read Army Institute of Research). The CCM is a Space Shuttle mid-deck locker experiment payload. The BIONA-C is displayed along with associated graphics and text explanations. The presentation will stimulate interest in development of sensor technology for real-time cell culture measurements. The transfer of this technology to other applications will also be of interest. Additional information is contained in the original document.

  14. Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations

    Directory of Open Access Journals (Sweden)

    Elin eForslund

    2012-10-01

    Full Text Available Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are also transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended times. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells. This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g. conjugation, immune synapse formation and cytotoxic events. The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at

  15. Sequential cancer mutations in cultured human intestinal stem cells

    NARCIS (Netherlands)

    Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M.; Offerhaus, G. Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J.; Medema, Jan Paul; Kops, Geert J. P. L.; Clevers, Hans

    2015-01-01

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain

  16. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  17. Radiosensitivity of normal human epidermal cells in culture

    International Nuclear Information System (INIS)

    Dover, R.; Potten, C.S.

    1983-01-01

    Using an in vitro culture system the authors have derived #betta#-radiation survival curves over a dose range 0-8 Gy for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells. (author)

  18. Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures.

    Science.gov (United States)

    Gebhard, C; Gabriel, C; Walter, I

    2016-06-01

    Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. © 2015 The Authors. Anatomia, Histologia, Embryologia Published by Blackwell Verlag GmbH.

  19. 21st Century Cell Culture for 21st Century Toxicology.

    Science.gov (United States)

    Pamies, David; Hartung, Thomas

    2017-01-17

    There is no good science in bad models. Cell culture is especially prone to artifacts. A number of novel cell culture technologies have become more broadly available in the 21st century, which allow overcoming limitations of traditional culture and are more physiologically relevant. These include the use of stem-cell derived human cells, cocultures of different cell types, scaffolds and extracellular matrices, perfusion platforms (such as microfluidics), 3D culture, organ-on-chip technologies, tissue architecture, and organ functionality. The physiological relevance of such models is further enhanced by the measurement of biomarkers (e.g., key events of pathways), organ specific functionality, and more comprehensive assessment cell responses by high-content methods. These approaches are still rarely combined to create microphysiological systems. The complexity of the combination of these technologies can generate results closer to the in vivo situation but increases the number of parameters to control, bringing some new challenges. In fact, we do not argue that all cell culture needs to be that sophisticated. The efforts taken are determined by the purpose of our experiments and tests. If only a very specific molecular target to cell response is of interest, a very simple model, which reflects this, might be much more suited to allow standardization and high-throughput. However, the less defined the end point of interest and cellular response are, the better we should approximate organ- or tissue-like culture conditions to make physiological responses more probable. Besides these technologic advances, important progress in the quality assurance and reporting on cell cultures as well as the validation of cellular test systems brings the utility of cell cultures to a new level. The advancement and broader implementation of Good Cell Culture Practice (GCCP) is key here. In toxicology, this is a major prerequisite for meaningful and reliable results, ultimately

  20. The p75NTR tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    International Nuclear Information System (INIS)

    Khwaja, Fatima; Tabassum, Arshia; Allen, Jeff; Djakiew, Daniel

    2006-01-01

    The p75 neurotrophin receptor (p75 NTR ) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75 NTR retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (ΔDD) dominant-negative antagonist of p75 NTR showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75 NTR -dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75 NTR expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75 NTR rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75 NTR was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75 NTR -dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75 NTR expressing prostate cancer cells

  1. The p75{sup NTR} tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Khwaja, Fatima [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States); Tabassum, Arshia [Toronto Western Hospital, Toronto, ON, M5T258 (Canada); Allen, Jeff [National Center for Complementary and Alternative Medicine, N.I.H., Bethesda, MD 20892 (United States); Djakiew, Daniel [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057-1436 (United States) and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057-1436 (United States)

    2006-03-24

    The p75 neurotrophin receptor (p75{sup NTR}) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75{sup NTR} retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted ({delta}DD) dominant-negative antagonist of p75{sup NTR} showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75{sup NTR}-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75{sup NTR} expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75{sup NTR} rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75{sup NTR} was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75{sup NTR}-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75{sup NTR} expressing prostate cancer cells.

  2. Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

    Science.gov (United States)

    Aspord, Caroline; Pedroza-Gonzalez, Alexander; Gallegos, Mike; Tindle, Sasha; Burton, Elizabeth C.; Su, Dan; Marches, Florentina; Banchereau, Jacques; Palucka, A. Karolina

    2007-01-01

    We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development. PMID:17438063

  3. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    Science.gov (United States)

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  4. Bioreactor design for successive culture of anchorage-dependent cells operated in an automated manner.

    Science.gov (United States)

    Kino-Oka, Masahiro; Ogawa, Natsuki; Umegaki, Ryota; Taya, Masahito

    2005-01-01

    A novel bioreactor system was designed to perform a series of batchwise cultures of anchorage-dependent cells by means of automated operations of medium change and passage for cell transfer. The experimental data on contamination frequency ensured the biological cleanliness in the bioreactor system, which facilitated the operations in a closed environment, as compared with that in flask culture system with manual handlings. In addition, the tools for growth prediction (based on growth kinetics) and real-time growth monitoring by measurement of medium components (based on small-volume analyzing machinery) were installed into the bioreactor system to schedule the operations of medium change and passage and to confirm that culture proceeds as scheduled, respectively. The successive culture of anchorage-dependent cells was conducted with the bioreactor running in an automated way. The automated bioreactor gave a successful culture performance with fair accordance to preset scheduling based on the information in the latest subculture, realizing 79- fold cell expansion for 169 h. In addition, the correlation factor between experimental data and scheduled values through the bioreactor performance was 0.998. It was concluded that the proposed bioreactor with the integration of the prediction and monitoring tools could offer a feasible system for the manufacturing process of cultured tissue products.

  5. Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

    Science.gov (United States)

    Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

    2016-12-01

    The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

  6. A cortical attractor network with Martinotti cells driven by facilitating synapses.

    Directory of Open Access Journals (Sweden)

    Pradeep Krishnamurthy

    Full Text Available The population of pyramidal cells significantly outnumbers the inhibitory interneurons in the neocortex, while at the same time the diversity of interneuron types is much more pronounced. One acknowledged key role of inhibition is to control the rate and patterning of pyramidal cell firing via negative feedback, but most likely the diversity of inhibitory pathways is matched by a corresponding diversity of functional roles. An important distinguishing feature of cortical interneurons is the variability of the short-term plasticity properties of synapses received from pyramidal cells. The Martinotti cell type has recently come under scrutiny due to the distinctly facilitating nature of the synapses they receive from pyramidal cells. This distinguishes these neurons from basket cells and other inhibitory interneurons typically targeted by depressing synapses. A key aspect of the work reported here has been to pinpoint the role of this variability. We first set out to reproduce quantitatively based on in vitro data the di-synaptic inhibitory microcircuit connecting two pyramidal cells via one or a few Martinotti cells. In a second step, we embedded this microcircuit in a previously developed attractor memory network model of neocortical layers 2/3. This model network demonstrated that basket cells with their characteristic depressing synapses are the first to discharge when the network enters an attractor state and that Martinotti cells respond with a delay, thereby shifting the excitation-inhibition balance and acting to terminate the attractor state. A parameter sensitivity analysis suggested that Martinotti cells might, in fact, play a dominant role in setting the attractor dwell time and thus cortical speed of processing, with cellular adaptation and synaptic depression having a less prominent role than previously thought.

  7. Radiosensitivity of primary cultured fish cells with different ploidy

    International Nuclear Information System (INIS)

    Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

    1986-01-01

    The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

  8. Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

    Science.gov (United States)

    Spel, Lotte; Boelens, Jaap-Jan; van der Steen, Dirk M.; Blokland, Nina J.G.; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H.M.

    2015-01-01

    Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20–40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses. PMID:26452036

  9. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

    Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

    2011-01-01

    In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

  10. Arachidonic metabolism and radiation toxicity in cultures of vascular endothelial cells

    International Nuclear Information System (INIS)

    Eldor, A.; Vlodavsky, I.; Fuks, Z.; Matzner, Y.; Rubin, D.B.

    1989-01-01

    The authors conclude that the observed changes in eicosanoid production by vascular endothelial cells exposed to ionizing irradiation may be relevant to the pathogenesis of post-radiation injury in small and large blood vessels. Anomalies of PGI 2 production may lead to thrombosis and accelerated arteriosclerosis which are observed in irradiated vessels. The generation of potent cells may greatly facilitate inflammation in irradiated vessels. The model of irradiated cultured endothelial cells may also be useful for the study of various methods and agents aimed at reducing the radiation induced damage to blood vessels. Evaluation of the capacity of cultured endothelial cells to produce eicosanoids may serve as an appropriate index for the metabolic damage induced by radiation. (author)

  11. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    Science.gov (United States)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  12. Radiation adaptive response for the growth of cultured glial cells

    International Nuclear Information System (INIS)

    Suzuki, S.; Miura, Y.; Kano, M.; Toda, T.; Urano, S.

    2003-01-01

    Full text: To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of aging on the response, glial cells were cultured from young and aged rats (1 month and 24 months old). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signaling pathway factors in RAR was investigated using their inhibitors, activators and mutated glial cells. RAR was observed in cells cultured from young rats, but was not in cells from aged rats. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The activators of PKC instead of low dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxia-telangiectasia (AT) cells from AT patients showed no RAR. These results indicated that PKC, ATM, DNAPK and/or PI3K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with aging. Proteomics data of glial cells exposed to severe stress of H 2 O 2 or X-rays also will be presented in the conference since little or no difference has not been observed with slight stress yet

  13. Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro.

    Directory of Open Access Journals (Sweden)

    David Warrilow

    Full Text Available Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

  14. Enrichment of Adipose-Derived Stromal Cells for BMPR1A Facilitates Enhanced Adipogenesis.

    Science.gov (United States)

    Zielins, Elizabeth R; Paik, Kevin; Ransom, Ryan C; Brett, Elizabeth A; Blackshear, Charles P; Luan, Anna; Walmsley, Graham G; Atashroo, David A; Senarath-Yapa, Kshemendra; Momeni, Arash; Rennert, Robert; Sorkin, Michael; Seo, Eun Young; Chan, Charles K; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

    2016-02-01

    Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-γ) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity

  15. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  16. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions....

  17. Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, Hiroko; Seto, Akira

    1981-01-01

    A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

  18. Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture.

    Science.gov (United States)

    Lee, Jaslyn J L; Chen, Liwei; Cao, Bin; Chen, Wei Ning

    2016-01-01

    The oleaginous yeast Rhodosporidium toruloides has great biotechnological potential. It accumulates a high amount of lipids which can be used for biofuels and also produces carotenoids which are valuable in the food and pharmaceutical industry. However, the location of these two hydrophobic products in the cell membrane prohibits its efficient harvesting and separation. Here, the transporter Pdr10 was engineered into R. toruloides and cultured in two-phase media containing oil. This enabled the production and in situ export of carotenoids into the oil and concurrent separation from intracellular lipids in the cells. When Pdr10 strain was cultured in the two-phase media, carotenoids and fatty acids yield increased from 1.9 to 2.9 μg/mg and 0.07 to 0.09 mg/mg, respectively. A total of 1.8 μg/mg carotenoids was exported by Pdr10 strain, as compared to 0.3 μg/mg in the wild type. In the Pdr10 strain, the composition of carotenoids and fatty acid it produced also changed. Torulene became the major carotene produced instead of torularhodin. Also, the unsaturated fatty acid C18:2 became the dominant fatty acid produced instead of the saturated C16:0, which was similar to the grape seed oil used in the two-phase media. This indicated that oil was being consumed by the cells, which was supported by the increased intracellular glycerol levels detected by gas chromatography-mass spectrometry (GC-MS). Our approach represents an easy and greener extraction method which could serve to increase the yield and facilitate separation of carotenoids and fatty acids.

  19. Quantitative volumetric Raman imaging of three dimensional cell cultures

    Science.gov (United States)

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-03-01

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  20. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

    2016-08-15

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

  1. Growth of melanocytes in human epidermal cell cultures

    International Nuclear Information System (INIS)

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C.

    1990-01-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient

  2. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  3. Work System Assessment to Facilitate the Dissemination of a Quality Improvement Program for Optimizing Blood Culture Use: A Case Study Using a Human Factors Engineering Approach.

    Science.gov (United States)

    Xie, Anping; Woods-Hill, Charlotte Z; King, Anne F; Enos-Graves, Heather; Ascenzi, Judy; Gurses, Ayse P; Klaus, Sybil A; Fackler, James C; Milstone, Aaron M

    2017-11-20

    Work system assessments can facilitate successful implementation of quality improvement programs. Using a human factors engineering approach, we conducted a work system assessment to facilitate the dissemination of a quality improvement program for optimizing blood culture use in pediatric intensive care units at 2 hospitals. Semistructured face-to-face interviews were conducted with clinicians from Johns Hopkins All Children's Hospital and University of Virginia Medical Center. Interview data were analyzed using qualitative content analysis. Blood culture-ordering practices are influenced by various work system factors, including people, tasks, tools and technologies, the physical environment, organizational conditions, and the external environment. A clinical decision-support tool could facilitate implementation by (1) standardizing blood culture-ordering practices, (2) ensuring that prescribing clinicians review the patient's condition before ordering a blood culture, (3) facilitating critical thinking, and (4) empowering nurses to communicate with physicians and advocate for adherence to blood culture-ordering guidelines. The success of interventions for optimizing blood culture use relies heavily on the local context. A work system analysis using a human factors engineering approach can identify key areas to be addressed for the successful dissemination of quality improvement interventions. © The Author 2017. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding...

  5. Application of cell co-culture system to study fat and muscle cells.

    Science.gov (United States)

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  6. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

    Shah, Pranjul Jaykumar; Vedarethinam, Indumathi; Kwasny, Dorota

    2011-01-01

    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through...

  7. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  8. Developing resources to facilitate culturally-sensitive service planning and delivery - doing research inclusively with people with learning disabilities.

    Science.gov (United States)

    Unwin, Gemma; Larkin, Michael; Rose, John; Kroese, Biza Stenfert; Malcolm, Stephen

    2016-01-01

    (Please see www.Toolsfortalking.co.uk for an easy read summary of the project.) The Tools for Talking are a set of resources that were developed through collaboration between Black, Asian and minority ethnic people with learning disabilities and researchers at the University of Birmingham. The resources were designed to be used by people with learning disabilities and service providers to facilitate culturally-sensitive communication and information sharing, service planning and delivery. They comprise illustrative videos and exploratory activities relating to five topics, namely, culture, activities, support from staff, important people, choices and independence. These topics emerged as important to people with learning disabilities during the 'Access to Social Care-Learning Disabilities' (ASC-LD) study which involved interviews with 32 adults with learning disabilities from Black, Asian and minority ethnic communities. The results of the ASC-LD study were used to develop a set of draft resources which were then co-developed through collaboration with people with learning disabilities and service providers. A 'Partnership event' was convened to involve stakeholders in the development of the resources. This paper describes the refinement of these materials by people with learning disabilities from Black, Asian and minority ethnic backgrounds in cooperation with a range of other stakeholders. Background Black, Asian and minority ethnic people with learning disabilities face inequities in health and social care provision. Lower levels of service uptake and satisfaction with services have been reported, however, this is largely based on the views of carers. The 'Access to Social Care: Learning Disabilities (ASC-LD)' study sought to explore the views and experiences of social support services among adults with learning disabilities from Black, Asian and minority ethnic communities. Interviews with 32 Black, Asian and minority ethnic adults with learning disabilities

  9. Protein biosynthesis in cultured human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1980-10-31

    A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.

  10. Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.

    Directory of Open Access Journals (Sweden)

    Stefan Stich

    Full Text Available Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC including changes in their immunogenic properties after 3-dimensional (3D-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS, a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have

  11. Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies

    Czech Academy of Sciences Publication Activity Database

    Žižková, Martina; Suchá, Rita; Tylečková, Jiřina; Jarkovská, Karla; Mairychová, Kateřina; Kotrčová, Eva; Marsala, M.; Gadher, S. J.; Kovářová, Hana

    2015-01-01

    Roč. 12, č. 1 (2015), s. 83-95 ISSN 1478-9450 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell therapy * immunomodulation * neural stem cell differentiation * neural subpopulation * neurodegenerative disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.465, year: 2015

  12. Control of fibronectin synthesis by rat granulosa cells in culture

    International Nuclear Information System (INIS)

    Skinner, M.K.; Dorrington, J.H.

    1984-01-01

    The secreted and cellular [ 35 S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited

  13. Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

    Directory of Open Access Journals (Sweden)

    Song-I Chun

    Full Text Available A reference reagent, 3-(trimethylsilyl propionic-2, 2, 3, 3-d4 acid sodium (TSP, has been used frequently in nuclear magnetic resonance (NMR and magnetic resonance spectroscopy (MRS as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells.A human osteosarcoma cell line (MG-63 was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding.In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation and cell viability. High concentrations of TSP (from 10 to 30 mM reduced both cell proliferation and viability (to 30% of the control after one week of exposure, but no such effects were found using low concentrations of TSP (0-10 mM.This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

  14. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Flonia Levy-Adam

    2008-06-01

    Full Text Available Heparanase is a heparan sulfate (HS degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158-Asp(171, termed KKDC was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.

  15. Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells

    International Nuclear Information System (INIS)

    Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

    2015-01-01

    Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage

  16. Characterization of hemopoietic stem cell chimerism in antibody-facilitated bone marrow chimeras

    International Nuclear Information System (INIS)

    Francescutti, L.H.; Gambel, P.; Wegmann, T.G.

    1985-01-01

    The authors have previously described a model for bone marrow transplantation that involves preparation of the host with monoclonal antibody against class I or class II antigens instead of irradiation or cytotoxic drugs. This allows engraftment and subsequent repopulation of the host by donor tissue. They have previously reported on chimerism in the peripheral blood of P1----(P1 X P2)F1 animals. In this report, the authors describe the examination of the bone marrow and spleen stem cell chimerism of these antibody-facilitated (AF) chimeras, by determining, with an isozyme assay, the phenotype of methylcellulose colonies grown from stem cells. They have found a correlation between peripheral blood chimerism and the stem cell constitution of both spleen and bone marrow. The peripheral blood chimerism also correlates with the level of chimerism in macrophages derived from peritoneal exudate cells. These findings indicate that assaying the peripheral blood of such chimeras provides an excellent indication of the degree of chimerism at the stem cell level and stands in sharp contrast to the level of chimerism in certain lymphoid compartments

  17. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

    Science.gov (United States)

    van Montfoort, Nadine; Camps, Marcel G; Khan, Selina; Filippov, Dmitri V; Weterings, Jimmy J; Griffith, Janice M; Geuze, Hans J; van Hall, Thorbald; Verbeek, J Sjef; Melief, Cornelis J; Ossendorp, Ferry

    2009-04-21

    Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

  18. Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

    Science.gov (United States)

    Yoshimitsu, Ryosuke; Hattori, Koji; Sugiura, Shinji; Kondo, Yuki; Yamada, Rotaro; Tachikawa, Saoko; Satoh, Taku; Kurisaki, Akira; Ohnuma, Kiyoshi; Asashima, Makoto; Kanamori, Toshiyuki

    2014-05-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices. © 2013 Wiley Periodicals, Inc.

  19. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  20. Adherence of Moraxella bovis to cell cultures of bovine origin.

    Science.gov (United States)

    Annuar, B O; Wilcox, G E

    1985-09-01

    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  1. Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

    Science.gov (United States)

    Garba, Abubakar; Acar, Delphine D; Roukaerts, Inge D M; Desmarets, Lowiese M B; Devriendt, Bert; Nauwynck, Hans J

    2017-09-01

    Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell

  2. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  3. ATM facilitates mouse gammaherpesvirus reactivation from myeloid cells during chronic infection.

    Science.gov (United States)

    Kulinski, Joseph M; Darrah, Eric J; Broniowska, Katarzyna A; Mboko, Wadzanai P; Mounce, Bryan C; Malherbe, Laurent P; Corbett, John A; Gauld, Stephen B; Tarakanova, Vera L

    2015-09-01

    Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    proven effective in the control of agricultural pests in an environmentally ..... Prakash G and Srivastava A K 2005 Statistical media optimization for cell growth and ... Juss. suspension cultures; Process Biochemistry 40 3795–3800. Prakash G ...

  5. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... Full Length Research Paper. Establishment of the callus ... study provided an efficient way for E. angustifolia cell suspension culture to produce secondary metabolite. .... was also observed that in these treatments the stem.

  6. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    Enhancement of Diosgenin Production in Plantlet and Cell Cultures of Dioscorea zingiberensis by Palmarumycin C13 from the Endophytic fungus, Berkleasmium sp. Dzf12. Y Mou, K Zhou, D Xu, R Yu, J Li, C Yin, L Zhou ...

  7. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Additionally, sorghum cell suspension cultures have been initiated from the friable ... proteomics technologies. The field of proteomics is .... air dried at room temperature and resuspended in 2 ml of urea buffer [9 M urea, 2 M ...

  8. Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

    International Nuclear Information System (INIS)

    Tokita, N.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

  9. Immunocytochemical characterization of explant cultures of human prostatic stromal cells

    NARCIS (Netherlands)

    A. Kooistra (Anko); A.M.J. Elissen (Arianne ); J.J. Konig (Josee); M. Vermey; Th.H. van der Kwast (Theo); J.C. Romijn (Johannes); F.H. Schröder (Fritz)

    1995-01-01

    textabstractThe study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human

  10. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

    NARCIS (Netherlands)

    Huang, L.; van Loveren, C.; Ling, J.; Wei, X.; Crielaard, W.; Deng, D.M.

    2016-01-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated

  11. Free-energy carriers in human cultured muscle cells

    NARCIS (Netherlands)

    Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

    1985-01-01

    Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

  12. Radiation effects on cultured human lymphoid cells

    International Nuclear Information System (INIS)

    Johansson, L.; Nilsson, K.; Carlsson, J.; Larsson, B.; Jakobsson, P.

    1981-01-01

    The cloning efficiency of human normal and malignant lymphoid cells is usually low. Radiation effects in vitro on such cells can therefore not be analysed with conventional cloning. However, this problem can be circumscribed by using the growth extrapolation method. A panel of human leukemia-lymphoma cell-lines representing Epstein-Barr virus carrying lymphoblastoid cells of presumed non-neoplastic derivation and neoplastic T- and B-lymphocytes was used to test the efficiency of this method. The sensitivity to radiation could be determined for all these cell types. The growth extrapolation method gave generally the same result as conventional cloning demonstrated by comparison with one exceptional cell-line with capacity for cloning in agar. The sensitivity varied largely between the different cell types. A common feature was that none of the cell lines had a good capacity to accumulate sublethal radiation injury. (Auth.)

  13. Radiosensitivity of cultured insect cells: II. Diptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D 0 values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells

  14. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  15. 5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

    Science.gov (United States)

    Tong, D; Poot, M; Hu, D; Oda, D

    2000-03-01

    Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

  16. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-te...

  17. Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

    Science.gov (United States)

    Yin, Yancun; Chen, Chen; Chen, Jinliang; Zhan, Renhui; Zhang, Qiang; Xu, Xiaoyan; Li, Defang; Li, Minjing

    2017-07-01

    The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Mitochondrial respiratory modifiers confer survival advantage by facilitating DNA repair in cancer cells

    International Nuclear Information System (INIS)

    Chauhan, Ankit; Khanna, Suchit; Singh, Saurabh; Rai, Yogesh; Soni, Ravi; Kalra, Namita; Dwarakanath, B.S.; Bhatt, Anant Narayan

    2014-01-01

    High rate of aerobic glycolysis (Warburg effect), one of the primary hallmarks of cancer cells, acquired during the multistep development of tumors is also responsible for therapeutic resistance. Underlying this hallmark is the compromised respiratory metabolism that contributes to the acquisition of the glycolytic phenotype for sustained ATP production and cell proliferation. Nevertheless, the exact mechanisms underlying the glycolysis-linked radio-resistance in cancer cells remain elusive. In this study, we transiently elevated glycolysis by treating human cell lines (HEK293, BMG-1 and OCT-1) with mitochondrial respiratory modifiers (MRMs) viz. 2,4-dinitrophenol, Photosan-3, and Methylene blue to examine if transient stimulation of glycolysis before irradiation using MRMs is sufficient to confer radioresistance. Treatment with MRMs led to a significant (two-fold) increase in glucose consumption and lactate production together with a robust increase in the protein levels of two key regulators of glucose metabolism, i.e. GLUT-1 and HK-II. MRMs also enhanced the clonogenic survival and facilitated DNA repair by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. Inhibition of glucose uptake by inhibitors like 2-deoxy-D-glucose (2-DG), 3-bromo pyruvate (3-BP) and fasentin under conditions of stimulated glycolysis not only reversed the effect but also sensitized the cells to radiation more profoundly. The inhibition of glycolysis using 2-DG also reduced the levels of Ku 70 (NHEJ) and Rad-51 (HR) proteins. Thus, our results suggest that enhanced glycolysis in cancer cells may confer radio-resistance and offers survival advantage partly by enhancing the repair of DNA damage. (author)

  19. Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

    NARCIS (Netherlands)

    Spel, Lotte; Boelens, Jaap Jan; Van Der Steen, Dirk M.; Blokland, Nina J G; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H M; Boes, Marianne; Nierkens, Stefan

    2015-01-01

    Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20-40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for

  20. Cell proliferation and radiosensitivity of cow lymphocytes in culture

    International Nuclear Information System (INIS)

    Modave, C.; Fabry, L.; Leonard, A.

    1982-01-01

    The harlequin-staining technique has been used to study, after PHA-stimulation, the cell proliferation of cow lymphocytes in culture and to assess the radiosensitivity in first mitosis cells. At the 48 h fixation time, only 34% of the cells are in first mitosis whereas 55% are already in second and 11% in third mitosis. The exposure of cow lymphocytes to 200 rad X-rays result in the production of 16% dicentric chromosomes in first mitosis cells [fr

  1. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  2. Characterization of glucocerebrosidase in peripheral blood cells and cultured blastoid cells

    NARCIS (Netherlands)

    Aerts, J. M.; Heikoop, J.; van Weely, S.; Donker-Koopman, W. E.; Barranger, J. A.; Tager, J. M.; Schram, A. W.

    1988-01-01

    We have characterized glucocerebrosidase in various cell types of peripheral blood of control subjects and in cultured human blastoid cells. The intracellular level of glucocerebrosidase in cultured blastoid cells (10-30 nmol substrate hydrolyzed/h.mg protein) resembles closely values observed for

  3. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of

  4. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  5. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

  6. Nanoscaled red blood cells facilitate breast cancer treatment by combining photothermal/photodynamic therapy and chemotherapy.

    Science.gov (United States)

    Wan, Guoyun; Chen, Bowei; Li, Ling; Wang, Dan; Shi, Shurui; Zhang, Tao; Wang, Yue; Zhang, Lianyun; Wang, Yinsong

    2018-02-01

    Red blood cells (RBCs)-based vesicles have been widely used for drug delivery due to their unique advantages. Intact RBCs contain a large amount of oxyhemoglobin (oxyHb), which can assist with photodynamic therapy (PDT). Indocyanine green (ICG), a photosensitizer both for photothermal therapy (PTT) and PDT, shows potent anticancer efficacy when combined with chemotherapeutic drug doxorubicin (DOX). In this study, we prepared nanoscaled RBCs (RAs) containing oxyHb and gas-generating agent ammonium bicarbonate (ABC) for co-loading and controlled release of ICG and DOX, thus hoping to achieve synergistic effects of PTT/PDT and chemotherapy against breast cancer. Compared to free ICG, ICG and DOX co-loaded RAs (DIRAs) exhibited nearly identical PTT efficiency both in vitro and in vivo, but meanwhile their PDT efficiency was enhanced significantly. In mouse breast cancer cells, DIRAs significantly inhibited cell growth and induced cell apoptosis after laser irradiation. In breast tumor-bearing mice, intratumoral injection of DIRAs and followed by local laser irradiation almost completely ablated breast tumor and further suppressed tumor recurrence and metastasis. In conclusion, this biomimetic multifunctional nanosystem can facilitate breast cancer treatment by combining PTT/PDT and chemotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

    Directory of Open Access Journals (Sweden)

    Catherine Creppe

    2014-12-01

    Full Text Available Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq. Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.

  8. Aromatase inhibitor (anastrozole) affects growth of endometrioma cells in culture.

    Science.gov (United States)

    Badawy, Shawky Z A; Brown, Shereene; Kaufman, Lydia; Wojtowycz, Martha A

    2015-05-01

    To study the effects of aromatase inhibitor (anastrozole) on the growth and estradiol secretion of endometrioma cells in culture. Endometrioma cells are grown in vitro until maximum growth before used in this study. This was done in the research laboratory for tissue culture, in an academic hospital. Testosterone at a concentration of 10 μg/mL was added as a substrate for the intracellular aromatase. In addition, aromatase inhibitor was added at a concentration of 200 and 300 μg/mL. The effect on cell growth and estradiol secretion is evaluated using Student's t-test. The use of testosterone increased estradiol secretion by endometrioma cells in culture. The use of aromatase inhibitor significantly inhibited the growth of endometrioma cells, and estradiol secretion. Aromatase inhibitor (anastrozole) may be an effective treatment for endometriosis due to inhibition of cellular aromatase. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  10. Determination of thymidine in serum used for cell culture media

    International Nuclear Information System (INIS)

    Schaer, J.C.; Maurer, U.; Schindler, R.

    1978-01-01

    Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

  11. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations...

  12. Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

    Science.gov (United States)

    Wolf, Moritz K F; Closet, Aurélie; Bzowska, Monika; Bielser, Jean-Marc; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

    2018-05-21

    Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Cell death in Tetrahymena thermophila: new observations on culture conditions.

    Science.gov (United States)

    Christensen, S T; Sørensen, H; Beyer, N H; Kristiansen, K; Rasmussen, L; Rasmussen, M I

    2001-01-01

    We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems. Copyright 2001 Academic Press.

  14. Contributions of 3D Cell Cultures for Cancer Research.

    Science.gov (United States)

    Ravi, Maddaly; Ramesh, Aarthi; Pattabhi, Aishwarya

    2017-10-01

    Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilised with ease and flexibility. Also, the number of assays and end-points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional (3D) cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilised for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the 'absolute in vitro' and 'true in vivo'. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. J. Cell. Physiol. 232: 2679-2697, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  16. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

    OpenAIRE

    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  17. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  18. Automation of 3D cell culture using chemically defined hydrogels.

    Science.gov (United States)

    Rimann, Markus; Angres, Brigitte; Patocchi-Tenzer, Isabel; Braum, Susanne; Graf-Hausner, Ursula

    2014-04-01

    Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

  19. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture........ The contact angle of SU-8 surface was significantly reduced from 90° to 25° after the surface modification. The treated SU-8 surfaces provided a cell culture environment that was comparable with cell culture flask surface in terms of generation time and morphology....

  20. Cell culture supernatants for detection perforin ELISA

    African Journals Online (AJOL)

    Najwa

    2014-02-19

    Feb 19, 2014 ... Leukemia is a cancer originating in any of hematopoietic cell that tends to ... treatment of children (Borek and Jaskolski, 2001). The current study was .... which led to the best results at 48 h of exposure than after 72 h of cells ...

  1. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  2. Isolation and Characterization of Poliovirus in Cell Culture Systems.

    Science.gov (United States)

    Thorley, Bruce R; Roberts, Jason A

    2016-01-01

    The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

  3. Animal-cell culture in aqueous two-phase systems

    NARCIS (Netherlands)

    Zijlstra, G.M.

    1998-01-01

    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in

  4. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  5. Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.

    Science.gov (United States)

    Erickson, Andrea K; Jesudhasan, Palmy R; Mayer, Melinda J; Narbad, Arjan; Winter, Sebastian E; Pfeiffer, Julie K

    2018-01-10

    RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

    2017-01-01

    Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

  7. Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation.

    Science.gov (United States)

    Ahirwar, Dinesh K; Nasser, Mohd W; Ouseph, Madhu M; Elbaz, Mohamad; Cuitiño, Maria C; Kladney, Raleigh D; Varikuti, Sanjay; Kaul, Kirti; Satoskar, Abhay R; Ramaswamy, Bhuvaneswari; Zhang, Xiaoli; Ostrowski, Michael C; Leone, Gustavo; Ganju, Ramesh K

    2018-05-03

    The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

  8. Radiosensitivity of cultured insect cells: I. Lepidoptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D 0 , d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D 0 of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects

  9. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

  10. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

  11. Microbially-reduced graphene scaffolds to facilitate extracellular electron transfer in microbial fuel cells.

    Science.gov (United States)

    Yuan, Yong; Zhou, Shungui; Zhao, Bo; Zhuang, Li; Wang, Yueqiang

    2012-07-01

    A one-pot method is exploited by adding graphene oxide (GO) and acetate into an microbial fuel cell (MFC) in which GO is microbially reduced, leading to in situ construction of a bacteria/graphene network in the anode. The obtained microbially reduced graphene (MRG) exhibits comparable conductivity and physical characteristics to the chemically reduced graphene. Electrochemical measurements reveal that the number of exoelectrogens involved in extracellular electron transfer (EET) to the solid electrode, increases due to the presence of graphene scaffolds, and the EET is facilitated in terms of electron transfer kinetics. As a result, the maximum power density of the MFC is enhanced by 32% (from 1440 to 1905 mW m(-2)) and the coulombic efficiency is improved by 80% (from 30 to 54%). The results demonstrate that the construction of the bacteria/graphene network is an effective alternative to improve the MFC performance. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. A solution-processed binary cathode interfacial layer facilitates electron extraction for inverted polymer solar cells.

    Science.gov (United States)

    Zhang, Xinyuan; Li, Zhiqi; Liu, Chunyu; Guo, Jiaxin; Shen, Liang; Guo, Wenbin

    2018-03-15

    The charge transfer and separation are significantly affected by the electron properties of the interface between the electron-donor layer and the carrier-transporting layer in polymer solar cells (PSCs). In this study, we investigate the electron extraction mechanism of PSCs with a low temperature solution-processed ZnO/PEI as electron transport layer. The incorporation of PEI layer can decrease the work function of ZnO and reduce interfacial barrier, which facilitates electron extraction and suppresses bimolecular recombination, leading to a significant performance enhancement. Furthermore, PEI layer can induce phase separation and passivite inorganic surface trap states as well as shift the interfacial energy offset between metal oxide and organic materials. This work offers a simple and effective way to improve the charge transporting property of organic photovoltaic devices. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

    Science.gov (United States)

    Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

    2015-07-01

    Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p cell level correlated positively with the number of patient colonies (r = 0.762, p Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research

  14. Duchenne muscular dystrophy: normal ATP turnover in cultured cells

    International Nuclear Information System (INIS)

    Fox, I.H.; Bertorini, T.; Palmieri, G.M.A.; Shefner, R.

    1986-01-01

    This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

  15. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  16. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Garrison, Brian S; Rybak, Adrian P; Beerman, Isabel; Heesters, Balthasar; Mercier, Francois E; Scadden, David T; Bryder, David; Baron, Roland; Rossi, Derrick J

    2017-08-03

    The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521 / Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521 -deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

  17. Cloning higher plants from aseptically cultured tissues and cells

    Science.gov (United States)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  18. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  19. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  20. A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

    Science.gov (United States)

    Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

    2018-02-01

    Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J.

    1990-01-01

    The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells

  2. Acrylic acid surface-modified contact lens for the culture of limbal stem cells.

    Science.gov (United States)

    Zhang, Hong; Brown, Karl David; Lowe, Sue Peng; Liu, Guei-Sheung; Steele, David; Abberton, Keren; Daniell, Mark

    2014-06-01

    Surface treatment to a biomaterial surface has been shown to modify and help cell growth. Our aim was to determine the best surface-modified system for the treatment of limbal stem cell deficiency (LSCD), which would facilitate expansion of autologous limbal epithelial cells, while maintaining cultivated epithelial cells in a less differentiated state. Commercially available contact lenses (CLs) were variously surface modified by plasma polymerization with ratios of acrylic acid to octadiene tested at 100% acrylic acid, 50:50% acrylic acid:octadiene, and 100% octadiene to produce high-, mid-, and no-acid. X-ray photoelectron spectroscopy was used to analyze the chemical composition of the plasma polymer deposited layer. Limbal explants cultured on high acid-modified CLs outgrew more cells. Immunofluorescence and RT2-PCR array results indicated that a higher acrylic acid content can also help maintain progenitor cells during ex vivo expansion of epithelial cells. This study provides the first evidence for the ability of high acid-modified CLs to preserve the stemness and to be used as substrates for the culture of limbal cells in the treatment of LSCD.

  3. Dose verification by OSLDs in the irradiation of cell cultures

    International Nuclear Information System (INIS)

    Meca C, E. A.; Bourel, V.; Notcovich, C.; Duran, H.

    2015-10-01

    The determination of value of irradiation dose presents difficulties when targets are irradiated located in regions where electronic equilibrium of charged particle is not reached, as in the case of irradiation -in vitro- of cell lines monolayer-cultured, in culture dishes or flasks covered with culture medium. The present study aimed to implement a methodology for dose verification in irradiation of cells in culture media by optically stimulated luminescence dosimetry (OSLD). For the determination of the absorbed dose in terms of cell proliferation OSL dosimeters of aluminum oxide doped with carbon (Al 2 O 3 :C) were used, which were calibrated to the irradiation conditions of culture medium and at doses that ranged from 0.1 to 15 Gy obtained with a linear accelerator of 6 MV photons. Intercomparison measurements were performed with an ionization chamber of 6 cm 3 . Different geometries were evaluated by varying the thicknesses of solid water, air and cell culture medium. The results showed deviations below 2.2% when compared with the obtained doses of OSLDs and planning system used. Also deviations were observed below 3.4% by eccentric points of the irradiation plane, finding homogeneous dose distribution. Uncertainty in the readings was less than 2%. The proposed methodology contributes a contribution in the dose verification in this type of irradiations, eliminating from the calculation uncertainties, potential errors in settling irradiation or possible equipment failure with which is radiating. It also provides certainty about the survival curves to be plotted with the experimental data. (Author)

  4. Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

    Science.gov (United States)

    Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

    2014-01-01

    Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

  5. A microwell cell culture platform for the aggregation of pancreatic β-cells.

    Science.gov (United States)

    Bernard, Abigail B; Lin, Chien-Chi; Anseth, Kristi S

    2012-08-01

    Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered.

  6. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

    OpenAIRE

    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

    2014-01-01

    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  7. Formation and action of oxygen activated species in cell cultures

    International Nuclear Information System (INIS)

    Hoffmann, M.E.; Meneghini, R.

    1982-01-01

    The differences of hydrogen peroxide sensibility of mammal cell lineages (man, mouse, chinese hamster) in culture are studied. The cellular survival and the frequency of DNA induced breaks by hydrogen peroxide are analysed. The efficiency of elimination of DNA breaks by cells is determined. The possible relation between the cell capacity of repair and its survival to hydrogen peroxide action is also discussed. (M.A.) [pt

  8. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

  9. Induction of pluripotent stem cells from fibroblast cultures.

    Science.gov (United States)

    Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

    2007-01-01

    Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

  10. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

    Science.gov (United States)

    Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

    2012-09-10

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977

    International Nuclear Information System (INIS)

    Freed, J.J.

    1977-07-01

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature

  12. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Freed, J.J.

    1977-07-01

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature.

  13. Implementing oxygen control in chip-based cell and tissue culture systems.

    Science.gov (United States)

    Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

    2016-09-21

    Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

  14. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Science.gov (United States)

    2010-07-01

    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed to...

  15. Cell sources for in vitro human liver cell culture models

    Science.gov (United States)

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  16. HUMAN CELLS IN CULTURE: REVISlTED*

    African Journals Online (AJOL)

    advantages, e.g. the generation time is reduced to about. 1/10000 that of the ... or less reflects the cellular biology of the donor tissut:'Y .... X-linked. Autosomal recessive. Autosomal recessive. Autosomal recessive mothers of affected males, however, show that only 50% of the cell population is defective, which furnishes an.

  17. Cultural relativism: maintenance of genomic imprints in pluripotent stem cell culture systems.

    Science.gov (United States)

    Greenberg, Maxim Vc; Bourc'his, Déborah

    2015-04-01

    Pluripotent stem cells (PSCs) in culture have become a widely used model for studying events occurring during mammalian development; they also present an exciting avenue for therapeutics. However, compared to their in vivo counterparts, cultured PSC derivatives have unique properties, and it is well established that their epigenome is sensitive to medium composition. Here we review the specific effects on genomic imprints in various PSC types and culture systems. Imprinted gene regulation is developmentally important, and imprinting defects have been associated with several human diseases. Therefore, imprint abnormalities in PSCs may have considerable consequences for downstream applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

    Science.gov (United States)

    Gmunder, F. K.; Nordau, C. G.; Tschopp, A.; Huber, B.; Cogoli, A.

    1988-01-01

    The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

  19. Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

    Science.gov (United States)

    Palmini, Gaia; Zonefrati, Roberto; Mavilia, Carmelo; Aldinucci, Alessandra; Luzi, Ettore; Marini, Francesca; Franchi, Alessandro; Capanna, Rodolfo; Tanini, Annalisa; Brandi, Maria Luisa

    2016-10-14

    The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

  20. Cytotoxicity of extracts of spices to cultured cells.

    Science.gov (United States)

    Unnikrishnan, M C; Kuttan, R

    1988-01-01

    The cytotoxicity of the extracts from eight different spices used in the Indian diet was determined using Dalton's lymphoma ascites tumor cells and human lymphocytes in vitro and Chinese Hamster Ovary cells and Vero cells in tissue culture. Alcoholic extracts of the spices were found to be more cytotoxic to these cells than their aqueous extracts. Alcoholic extracts of several spices inhibited cell growth at concentrations of 0.2-1 mg/ml in vitro and 0.12-0.3 mg/ml in tissue culture. Ginger, pippali (native to India; also called dried catkins), pepper, and garlic showed the highest activity followed by asafetida, mustard, and horse-gram (native to India). These extracts also inhibited the thymidine uptake into DNA.

  1. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  2. Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

    Science.gov (United States)

    Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

    2013-05-03

    Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial

  3. A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

    Science.gov (United States)

    Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

    2017-06-01

    Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  5. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  6. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.

    2003-01-01

    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed...... and introducing the observed difference between batches as one of the factors in an expanded three-dimensional ANOVA, we were able to overcome an otherwisecrucial lack of sufficiently reproducible duplicate values. We could thereby show that the effect of changing the apical medium was much more marked when...... the radioactive lipid precursors were added on the apical, rather than on the basolateral, side. Theinsert cell cultures were obviously polarized. We argue that it is not reasonable to reject troublesome experimental results, when we do not know a priori that something went wrong. The ANOVA is a very useful...

  7. Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture.

    Science.gov (United States)

    Kuss, Mitchell A; Wu, Shaohua; Wang, Ying; Untrauer, Jason B; Li, Wenlong; Lim, Jung Yul; Duan, Bin

    2017-09-13

    Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  8. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  9. Low-intensity pulsed ultrasound stimulation facilitates osteogenic differentiation of human periodontal ligament cells.

    Directory of Open Access Journals (Sweden)

    Bo Hu

    Full Text Available Human periodontal ligament cells (hPDLCs possess stem cell properties, which play a key role in periodontal regeneration. Physical stimulation at appropriate intensities such as low-intensity pulsed ultrasound (LIPUS enhances cell proliferation and osteogenic differentiation of mesechymal stem cells. However, the impacts of LIPUS on osteogenic differentiation of hPDLCs in vitro and its molecular mechanism are unknown. This study was undertaken to investigate the effects of LIPUS on osteogenic differentiation of hPDLCs. HPDLCs were isolated from premolars of adolescents for orthodontic reasons, and exposed to LIPUS at different intensities to determine an optimal LIPUS treatment dosage. Dynamic changes of alkaline phosphatase (ALP activities in the cultured cells and supernatants, and osteocalcin production in the supernatants after treatment were analyzed. Runx2 and integrin β1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrinβ1 was used to assess the effects of integrinβ1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm2 with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPUS-treated cells started to increase at days 3 and 7, respectively, and peaked at day 11. LIPUS treatment significantly augmented the production of osteocalcin after day 5. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin β1, while a significant decline when the integrinβ1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrinβ1 antibody were less than those in the LIPUS-treated group. In conclusion, LIPUS promotes osteogenic differentiation of hPDLCs, which is associated with upregulation of Runx2 and

  10. Managing treatment for end-stage renal disease--a qualitative study exploring cultural perspectives on facilitators and barriers to treatment adherence.

    Science.gov (United States)

    Griva, K; Ng, H J; Loei, J; Mooppil, N; McBain, H; Newman, S P

    2013-01-01

    Although adherence to hemodialysis (HD) regimes is important to maximise good clinical outcomes, it remains suboptimal and not well understood, particularly for those in non-Western settings and patients from Asian cultures. This qualitative study sought to explore cultural perspectives on facilitators and barriers to treatment adherence in HD patients. A descriptive exploratory design was used for the study, incorporating individual semi-structured interviews (n = 17) and three focus groups (n = 20). Each interview/focus group was audio-taped and transcribed verbatim, and coding was conducted by two coders using an iterative process. Study participants identified personal and social/contextual factors as major barriers or facilitators of treatment adherence. Barriers include time consumption, forgetfulness, concerns about safety, poor knowledge/understanding, poor communication and lack of control/social pressure. Participants also identified facilitators, both internal (self-initiated) and external (initiated by family, health care professional and peers) to ensure treatment adherence. These included support from family members and social obligation towards others, risk perception, establishment of routines and peer support. Internal and external factors can hinder or facilitate adherence to diet, fluid and medications in the context of dialysis. Several of these barriers/facilitators can be effectively addressed in the context of interventions and psycho-educational programmes.

  11. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    International Nuclear Information System (INIS)

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-01

    Research highlights: → The proliferation of dramatic increased by co-cultured with Sertoli cells. → VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. → The MHC expression of ECs induced by INF-γ and IL-6, IL-8 and sICAM induced by TNF-α decreased respectively after co-cultured with Sertoli cells. → ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10 3 , 1 x 10 4 or 1 x 10 5 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10 4 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P 4 cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single

  12. Identification of differences in gene expression in primary cell cultures of human endometrial epithelial cells and trophoblast cells following their interaction

    DEFF Research Database (Denmark)

    Høgh, Mette; Islin, Henrik; Møller, Charlotte

    2006-01-01

    The interaction between the cell types was simulated in vitro by growing primary cell cultures of human endometrial epithelial cells and trophoblast cells together (co-culture) and separately (control cultures). Gene expression in the cell cultures was compared using the Differential Display method and confirmed...

  13. High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

    Science.gov (United States)

    Chen, Yu-Chih; Yoon, Euisik

    2017-01-01

    Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

  14. Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

    Science.gov (United States)

    Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

    2010-12-01

    Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

  15. A study of chromosomal aberrations in amniotic fluid cell cultures.

    Science.gov (United States)

    Wolstenholme, J; Crocker, M; Jonasson, J

    1988-06-01

    This paper represents the analysis of 1916 routine amniotic fluid specimens harvested by an in situ fixation technique in a prospective study with regard to cultural chromosome anomalies. Excluding constitutional abnormalities, 2.9 per cent of 19,432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 per cent) and chromatid/chromosome breaks and gaps (18 per cent) being the most frequent, followed by interchange aberrations (13 per cent) and trisomy (5 per cent). No case was found of more than one colony from the same culture showing the same anomaly without it being present in other cultures from the same fluid. The wholly abnormal colonies had a surplus of trisomies and from the mathematical considerations presented one may infer that these are likely to reflect the presence of abnormal cells in the amniotic fluid. Partly abnormal colonies appeared at a frequency that would correspond to virtual absence of selection against chromosomally abnormal cells when cultured in vitro. The aberrations found were similar to those seen as single cell anomalies, except for chromatid breaks and exchanges. The data suggest a basic preferential induction of trisomy for chromosomes 2, 18, 21, and the Y-chromosome. Structural aberrations showed a marked clustering of breakpoints around the centromeres. The frequency of mutant cells was low (1.4 X 10(-3)) before culture was initiated. At harvest, the frequency of abnormal cells was much higher (3 X 10(-2)) corresponding to 3 X 10(-3) mutations per cell per generation accumulating over approximately ten generations in vitro.

  16. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin

    2012-01-01

    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  17. Arsenic exposure induces the Warburg effect in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.edu

    2013-08-15

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect.

  18. Transfection in perfused microfluidic cell culture devices: A case study.

    Science.gov (United States)

    Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

    2017-08-01

    Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

  19. Cell culture media impact on drug product solution stability.

    Science.gov (United States)

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

    2016-07-08

    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

  20. Arsenic exposure induces the Warburg effect in cultured human cells

    International Nuclear Information System (INIS)

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T.

    2013-01-01

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect

  1. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings......, in addition to isolating the neural tissue from nutrient delivery and to creating unwanted gradients (2). This means that typical techniques used to investigate neurodegenerative diseases cannot mimic in vivo conditions, as closely as desired. We have developed a novel microfluidic system for culturing PC12...... cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro culturing...

  2. Production of betalaines by Myrtillocactus cell cultures. Passage from heterotrophic state to autotrophic state with Asparagus cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Bulard, C; Mary, J; Chaumont, D; Gudin, C

    1982-11-01

    Myrtillocactus tissue cultures are grown from the epicotyl of young plantlets. With an appropriate growing medium it is possible, after transfer of fragments of these cultures to a liquid environment, to obtain dissociation and proliferation of cells. The production of betalaic pigments is induced in solid surroundings by adjustement of the growing medium composition and can be maintained in a liquid environment. The multiplication of pigmented cells in suspension may thus be obtained. The conversion of Asparagus cell suspensions from the heterotrophic state (use of lactose as source of carbon) to the autotrophic state (carbon supplied by CO/sub 2/) is obtained by a gradual reduction in the sugar concentration of the medium combined with a rise in the CO/sub 2/ content of the gas mixture atmosphere injected into the cultivator. The passage to the autotrophic state of a Myrtillocactus suspension would enable the production conditions of a metabolite (Betalaine) to be studied by micro-algae culture techniques.

  3. Biocompatibility of Tygon® tubing in microfluidic cell culture.

    Science.gov (United States)

    Jiang, Xiao; Jeffries, Rex E; Acosta, Miguel A; Tikunov, Andrey P; Macdonald, Jeffrey M; Walker, Glenn M; Gamcsik, Michael P

    2015-02-01

    Growth of the MDA-MB-231 breast cancer cell line in microfluidic channels was inhibited when culture media was delivered to the channels via microbore Tygon® tubing. Culture media incubated within this tubing also inhibited growth of these cells in conventional 96-well plates. These detrimental effects were not due to depletion of critical nutrients due to adsorption of media components onto the tubing surface. A pH change was also ruled out as a cause. Nuclear magnetic resonance spectroscopy of the cell growth media before and after incubation in the tubing confirmed no detectable loss of media components but did detect the presence of additional unidentified signals in the aliphatic region of the spectrum. These results indicate leaching of a chemical species from microbore Tygon® tubing that can affect cell growth in microfluidic devices.

  4. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Directory of Open Access Journals (Sweden)

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  5. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    International Nuclear Information System (INIS)

    Kim, Jae Sung; Lee, Young Keun; Chung, Byung Yeoup; Lee, Young Bok; Hwang Hye Yeon

    2004-10-01

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark, and was increased by adding 1 μM Cu 2+ and 100 μM methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of γ radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS (β-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs

  6. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

    Science.gov (United States)

    Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When

  7. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity.

    Science.gov (United States)

    Bradley, Jillian H; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P; Gregg, Randal K

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter

  8. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

  9. A Novel Counter Sheet-flow Sandwich Cell Culture Device for Mammalian Cell Growth in Space

    Science.gov (United States)

    Sun, Shujin; Gao, Yuxin; Shu, Nanjiang; Tang, Zemei; Tao, Zulai; Long, Mian

    2008-08-01

    Cell culture and growth in space is crucial to understand the cellular responses under microgravity. The effects of microgravity were coupled with such environment restrictions as medium perfusion, in which the underlying mechanism has been poorly understood. In the present work, a customer-made counter sheet-flow sandwich cell culture device was developed upon a biomechanical concept from fish gill breathing. The sandwich culture unit consists of two side chambers where the medium flow is counter-directional, a central chamber where the cells are cultured, and two porous polycarbonate membranes between side and central chambers. Flow dynamics analysis revealed the symmetrical velocity profile and uniform low shear rate distribution of flowing medium inside the central culture chamber, which promotes sufficient mass transport and nutrient supply for mammalian cell growth. An on-orbit experiment performed on a recovery satellite was used to validate the availability of the device.

  10. Cultured meat from stem cells: challenges and prospects.

    Science.gov (United States)

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.

    Directory of Open Access Journals (Sweden)

    Sanne Hindriksen

    Full Text Available The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC. We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.

  12. Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.

    Science.gov (United States)

    Hindriksen, Sanne; Bramer, Arne J; Truong, My Anh; Vromans, Martijn J M; Post, Jasmin B; Verlaan-Klink, Ingrid; Snippert, Hugo J; Lens, Susanne M A; Hadders, Michael A

    2017-01-01

    The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.

  13. Oxidative Stress Induces Senescence in Cultured RPE Cells.

    Science.gov (United States)

    Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

  14. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  15. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    Science.gov (United States)

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  16. Mesenchymal stem cells enhance the metastasis of 3D-cultured hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Chang; Liu, Yang; Xu, Xiao-xi; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

    2016-01-01

    Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined. In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated. It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system. Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β. The online version of this article (doi:10.1186/s12885-016-2595-4) contains supplementary material, which is available to authorized users

  17. Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

    Science.gov (United States)

    Sakata, Ichiro; Park, Won-Mee; Walker, Angela K.; Piper, Paul K.; Chuang, Jen-Chieh; Osborne-Lawrence, Sherri

    2012-01-01

    The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain. PMID:22414807

  18. Effects of viscoelastic ophthalmic solutions on cell cultures

    Directory of Open Access Journals (Sweden)

    Madhavan Hajib

    1998-01-01

    Full Text Available The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken. We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions. Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles. The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy. Other products did not have any adverse effects on the cell lines nor did they show floating particles. The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity. The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective.

  19. Staurosporine induces different cell death forms in cultured rat astrocytes

    International Nuclear Information System (INIS)

    Simenc, Janez; Lipnik-Stangelj, Metoda

    2012-01-01

    Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10 −7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10 −6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis

  20. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    International Nuclear Information System (INIS)

    Dai, R.X.; Djakiew, D.; Dym, M.

    1987-01-01

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125 I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived

  1. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  2. Somatic embryogenesis in cell cultures of Glycine species.

    Science.gov (United States)

    Gamborg, O L; Davis, B P; Stahlhut, R W

    1983-08-01

    This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

  3. Radiation transformation in differentiated human cells in culture

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.; Moriarty, M.; Malone, J.; Byrne, P.; Hennessy, T.

    1986-01-01

    A tissue culture technique is described for human thyroid tissue as an approach to studying mechanisms of human radiation carcinogenesis. Normal human tissue obtained from surgery is treated in one of two ways, depending upon size of specimen. Large pieces are completely digested in trypsin/ collagenase solution to a single cell suspension. Small pieces of tissue are plated as explants following partial digestion in trypsin/collagenase solution. Following irradiation of the primary differentiated monolayers (normally 10 days after plating), the development of transformed characteristics is monitored in the subsequent subcultures. A very high level of morphological and functional differentiation is apparent in the primary cultures. Over a period of approx. 6 months, the irradiated surviving cells continue to grow in culture, unlike the unirradiated controls which senesce after 2-3 subcultures. (UK)

  4. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  5. The bioconversion process of deoxypodophyllotoxin with Linum flavum cell cultures

    NARCIS (Netherlands)

    Koulman, A; Beekman, A.C; Pras, N.; Quax, Wim

    2003-01-01

    The in vitro cell suspension culture of Linum flavum is able to convert high amounts of the 2,7'-cyclolignan deoxypodophyllotoxin into 6-methoxypodophyllotoxin 7-O-glucoside. We studied this conversion in detail by monitoring the intermediates and side-products after feeding different concentrations

  6. Using Haworthia Cultured Cells as an Aid in Teaching Botany

    Science.gov (United States)

    Majumdar, Shyamal K.; Castellano, John M.

    1977-01-01

    Callus induction from species of Haworthia can be done quickly in the laboratory with minimal equipment to study tissue dedifferentiation and cellular redifferentiation. It is shown that the cultured cell can also be used to study and evaluate the effects of various mutagens, carcinogens, and pesticides in controlled environments. (Author/MA)

  7. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system...

  8. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  9. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of E. angustifolia was ...

  10. Ultrastructure of cells of Ulmus americana cultured in vitro and exposed to the culture filtrate of Ceratocystis ulmi

    Science.gov (United States)

    Paula M. Pijut; R. Daniel Lineberger; Subhash C. Domir; Jann M. Ichida; Charles R. Krause

    1990-01-01

    Calli of American elm susceptible and resistant to Dutch elm disease were exposed to a culture filtrate of a pathogenic isolate of Ceratocystis ulmi. Cells from untreated tissue exhibited typical internal composition associated with healthy, actively growing cells. All cells exposed to culture filtrate showed appreciable ultrastructural changes....

  11. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

    Full Text Available Abstract Background In order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial. Objective By employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells. Methods Cigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors. Results Cigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

  12. Porcine platelet lysate as a supplement for animal cell culture

    Science.gov (United States)

    Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

    2007-01-01

    A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

  13. Maintenance of mesenchymal stem cells culture due to the cells with reduced attachment rate

    Directory of Open Access Journals (Sweden)

    Shuvalova N. S.

    2013-01-01

    Full Text Available Aim. The classic detachment techniques lead to changes in cells properties. We offer a simple method of cultivating the population of cells that avoided an influence on the surface structures. Methods. Mesenchymal stem cells (MSC from human umbilical cord matrix were obtained and cultivated in standard conditions. While substituting the culture media by a fresh portion, the conditioned culture medium, where the cells were maintained for three days, was transferred to other culture flacks with addition of serum and growth factors. Results. In the flacks, one day after medium transfer, we observed attached cells with typical MSC morphology. The cultures originated from these cells had the same rate of surface markers expression and clonogenic potential as those replated by standard methods. Conclusions. MSC culture, derived by preserving the cells with reduced attachment ability, actually has the properties of «parent» passage. Using this method with accepted techniques of cells reseeding would allow maintaining the cells that avoided an impact on the cell surface proteins.

  14. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells.

    Science.gov (United States)

    Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou

    2015-07-01

    Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. © 2015 Wiley Periodicals, Inc.

  15. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  16. Transfer plate radioassay using adsorbed anti-insulin antibody to detect insulin secreted by islet cell cultures

    International Nuclear Information System (INIS)

    Scearce, R.M.; Oie, H.K.; Gazdar, A.F.; Chick, W.L.; Eisenbarth, G.S.

    1981-01-01

    A solid-phase radioimmunoassay for detection of insulin synthesized by islet cell clones is described. This assay employs anti-insulin antibody adsorbed onto fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each transfer plate well permits fluid to enter the wells when transfer plates are lowered into microculture wells containing insulin. With this assay it is possible to rapidly screen hundreds of islet cell cultures for insulin production. The authors have used this assay to facilitate cloning of the RIN rat insulinoma cell line. The assay readily detects insulin synthesis by RIN cells and [ 125 I]insulin is not displaced by culture medium from cells which do not produce insulin. The transfer plate format should be applicable to semiautomate other radioimmunoassays. (Auth.)

  17. Imprint lithography provides topographical nanocues to guide cell growth in primary cortical cell culture

    NARCIS (Netherlands)

    Xie, S.; Luttge, R.

    2014-01-01

    In this paper, we describe a technology platform to study the effect of nanocues on the cell growth direction in primary cortical cell culture. Topographical cues to cells are provided using nanoscale features created by Jet and Flash Imprint Lithography, coated with polyethylenimine. We

  18. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.

    Science.gov (United States)

    Kaji, Hirokazu; Camci-Unal, Gulden; Langer, Robert; Khademhosseini, Ali

    2011-03-01

    Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell-cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell-cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. 2010 Elsevier B.V. All rights reserved.

  19. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

    NARCIS (Netherlands)

    Lammers, A.; Vorstenbosch, van C.J.; Erkens, J.H.F.; Smith, H.E.

    2001-01-01

    We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover. we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other

  20. MEMS-based dynamic cell-to-cell culture platforms using electrochemical surface modifications

    International Nuclear Information System (INIS)

    Chang, Jiyoung; Lin, Liwei; Yoon, Sang-Hee; Mofrad, Mohammad R K

    2011-01-01

    MEMS-based biological platforms with the capability of both spatial placements and time releases of living cells for cell-to-cell culture experiments have been designed and demonstrated utilizing electrochemical surface modification effects. The spatial placement is accomplished by electrochemical surface modification of substrate surfaces to be either adhesive or non-adhesive for living cells. The time control is achieved by the electrical activation of the selective indium tin oxide co-culture electrode to allow the migration of living cells onto the electrode to start the cell-to-cell culture studies. Prototype devices have a three-electrode design with an electrode size of 50 × 50 µm 2 and the separation gaps of 2 µm between them. An electrical voltage of −1.5 V has been used to activate the electrodes independently and sequentially to demonstrate the dynamic cell-to-cell culture experiments of NIH 3T3 fibroblast and Madin Darby canine kidney cells. As such, this MEMS platform could be a basic yet versatile tool to characterize transient cell-to-cell interactions

  1. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  2. Mass and Momentum Transport in Microcavities for Diffusion-Dominant Cell Culture Applications

    Science.gov (United States)

    Yew, Alvin G.; Pinero, Daniel; Hsieh, Adam H.; Atencia, Javier

    2012-01-01

    For the informed design of microfluidic devices, it is important to understand transport phenomena at the microscale. This letter outlines an analytically-driven approach to the design of rectangular microcavities extending perpendicular to a perfusion microchannel for microfluidic cell culture devices. We present equations to estimate the spatial transition from advection- to diffusion-dominant transport inside cavities as a function of the geometry and flow conditions. We also estimate the time required for molecules, such as nutrients or drugs to travel from the microchannel to a given depth into the cavity. These analytical predictions can facilitate the rational design of microfluidic devices to optimize and maintain long-term, physiologically-based culture conditions with low fluid shear stress.

  3. A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    2018-01-01

    Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

  4. Cell culture density affects the proliferation activity of human adipose tissue stem cells.

    Science.gov (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2016-01-01

    In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  6. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

    An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

  7. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10–6M) or NAA (10−5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10−7M) and 5.1 days on supraoptimal levels of 2,4-D (10−5M). Cultures grew on NH4+-N alone (from ammonium...... malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...

  8. Trophic Effects of Mesenchymal Stem Cells in Chondrocyte Co-Cultures are Independent of Culture Conditions and Cell Sources

    NARCIS (Netherlands)

    Wu, Ling; Prins, H.J.; Helder, M.; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

  9. Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources

    NARCIS (Netherlands)

    Wu, L.; Prins, H.J.; Helder, M.N.; van Blitterswijk, C.A.; Karperien, M.

    2012-01-01

    Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

  10. A 3D human neural cell culture system for modeling Alzheimer’s disease

    Science.gov (United States)

    Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

  11. The Effect of Spaceflight on Bone Cell Cultures

    Science.gov (United States)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural

  12. Frazzled/DCC facilitates cardiac cell outgrowth and attachment during Drosophila dorsal vessel formation.

    Science.gov (United States)

    Macabenta, Frank D; Jensen, Amber G; Cheng, Yi-Shan; Kramer, Joseph J; Kramer, Sunita G

    2013-08-15

    Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC (Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB) results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance mechanisms contribute to lumen formation by preventing luminal domains from coming together, site-specific Netrin/Frazzled signaling mediates CB attachment. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Monitoring Dynamic Interactions between Breast Cancer Cells and Human Bone Tissue in a Co-Culture Model

    Science.gov (United States)

    Contag, Christopher H.; Lie, Wen-Rong; Bammer, Marie C.; Hardy, Jonathan W.; Schmidt, Tobi L.; Maloney, William J.; King, Bonnie L.

    2015-01-01

    Purpose Bone is a preferential site of breast cancer metastasis and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. Procedures Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell division and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX® immunoassays. Results BLI demonstrated increased MDA-MB-231-fLuc proliferation (pbones, and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc colonization of bone, and MILLIPLEX® profiles of culture supernatants suggested breast/bone crosstalk. Conclusions Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays. PMID:24008275

  14. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  15. Cell chirality: emergence of asymmetry from cell culture.

    Science.gov (United States)

    Wan, Leo Q; Chin, Amanda S; Worley, Kathryn E; Ray, Poulomi

    2016-12-19

    Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

  16. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    International Nuclear Information System (INIS)

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-01-01

    Highlights: ► Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). ► Presence of SCs dramatically increased proliferation and migration of UCMSCs. ► Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of “nurse” cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  17. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People' s Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  18. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    John J Vincent

    Full Text Available The cell intrinsic programming that regulates mammalian primordial germ cell (PGC development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs from mouse embryonic stem cells (ESCs. Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

  19. Lewis Lung Cancer Cells Promote SIGNR1(CD209b)-Mediated Macrophages Polarization Induced by IL-4 to Facilitate Immune Evasion.

    Science.gov (United States)

    Yan, Xiaolong; Li, Wenhai; Pan, Lei; Fu, Enqing; Xie, Yonghong; Chen, Min; Mu, Deguang

    2016-05-01

    Tumor-associated macrophages are a prominent component of lung cancer and contribute to tumor progression by facilitating the immune evasion of cancer cells. DC-SIGN (CD209) assists in the immune evasion of a broad spectrum of pathogens and neoplasms by inhibiting the maturation of DCs and subsequent cytokines production. However, the expression of DC-SIGN in macrophages and its role in mediating immune evasion in lung cancer and the underlying mechanism remain unclear. Our study aimed to identify the immunosuppressive role of SIGNR1 in murine macrophage differentiation and lung cancer progression. We found that SIGNR1-positive RAW264.7 macrophages were enriched in mixed cultures with Lewis lung cancer cells (LLC) (ratio of RAW 264.7 to LLC being 1:1) after stimulation with IL-4. Moreover, LLC-educated macrophages exhibited significantly higher levels of IL-10 but lower IL-12 in response to IL-4 treatment as determined by RT-PCR and ELISA. However, inhibition of SIGNR1 markedly hampered the production of IL-10, indicating that SIGNR1 was indispensable for IL-4+LLC induced macrophage polarization towards the M2 subtype. Furthermore, polarized M2 cells immersed in a tumor microenvironment promoted the migration of LLCs, as measured by transwell assays, but migration was suppressed after blockade of SIGNR1 using CD209b antibody. In addition, IL-4+LLC-educated macrophages reduced the proliferation of the activated T cells and reduced IFN-γ-mediated Th1 response in T cells, while SIGNR1 inhibition rescued Th1 cell functions. In conclusion, murine SIGNR1 expressed in LLC-educated macrophages appears to mediate IL-4-induced RAW264.7 macrophage polarization and thus facilitate lung cancer evasion. © 2015 Wiley Periodicals, Inc.

  20. Aging and senescence of skin cells in culture

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2015-01-01

    Studying age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. The three main cell types that have been studied extensively with respect to cellular...... aging in vitro are dermal fibroblasts, epidermal keratinocytes, and melanocytes. Serial subcultivation of normal diploid skin cells can be performed only a limited number of times, and the emerging senescent phenotype can be categorized into structural, physiological, biochemical, and molecular...... phenotypes, which can be used as biomarkers of cellular aging in vitro. The rate and phenotype of aging are different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. A progressive accumulation...

  1. Propagation and isolation of ranaviruses in cell culture

    DEFF Research Database (Denmark)

    Ariel, Ellen; Nicolajsen, Nicole; Christophersen, Maj-Britt

    2009-01-01

    The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV......), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2......), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28 °C for two weeks. BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells...

  2. Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

    Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters

  3. A microfluidic cell culture array with various oxygen tensions.

    Science.gov (United States)

    Peng, Chien-Chung; Liao, Wei-Hao; Chen, Ying-Hua; Wu, Chueh-Yu; Tung, Yi-Chung

    2013-08-21

    Oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. Therefore, drug testing using conventional in vitro cell models under normoxia often possesses limited prediction capability. A traditional method of setting an oxygen tension in a liquid medium is by saturating it with a gas mixture at the desired level of oxygen, which requires bulky gas cylinders, sophisticated control, and tedious interconnections. Moreover, only a single oxygen tension can be tested at the same time. In this paper, we develop a microfluidic cell culture array platform capable of performing cell culture and drug testing under various oxygen tensions simultaneously. The device is fabricated using an elastomeric material, polydimethylsiloxane (PDMS) and the well-developed multi-layer soft lithography (MSL) technique. The prototype device has 4 × 4 wells, arranged in the same dimensions as a conventional 96-well plate, for cell culture. The oxygen tensions are controlled by spatially confined oxygen scavenging chemical reactions underneath the wells using microfluidics. The platform takes advantage of microfluidic phenomena while exhibiting the combinatorial diversities achieved by microarrays. Importantly, the platform is compatible with existing cell incubators and high-throughput instruments (liquid handling systems and plate readers) for cost-effective setup and straightforward operation. Utilizing the developed platform, we successfully perform drug testing using an anti-cancer drug, triapazamine (TPZ), on adenocarcinomic human alveolar basal epithelial cell line (A549) under three oxygen tensions ranging from 1.4% to normoxia. The developed platform is promising to provide a more meaningful in vitro cell model for various biomedical applications while maintaining desired high throughput capabilities.

  4. Laminin-411 Is a Vascular Ligand for MCAM and Facilitates TH17 Cell Entry into the CNS

    Science.gov (United States)

    Flanagan, Ken; Fitzgerald, Kent; Baker, Jeanne; Regnstrom, Karin; Gardai, Shyra; Bard, Frederique; Mocci, Simonetta; Seto, Pui; You, Monica; Larochelle, Catherine; Prat, Alexandre; Chow, Samuel; Li, Lauri; Vandevert, Chris; Zago, Wagner; Lorenzana, Carlos; Nishioka, Christopher; Hoffman, Jennifer; Botelho, Raquel; Willits, Christopher; Tanaka, Kevin; Johnston, Jennifer; Yednock, Ted

    2012-01-01

    TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation. PMID:22792325

  5. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

    Science.gov (United States)

    Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

    2017-09-01

    Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

  6. A Dialogue with Carl Rogers: Cross-Cultural Challenges of Facilitating Person-Centered Groups in Africa.

    Science.gov (United States)

    Hain-Hill, Alicia; Rogers, Carl R.

    1988-01-01

    Presents brainstorming dialogue with Carl Rogers which was held in January of 1987, shortly before Rogers's death. Explores basic challenges involved in a large-scale, cross-cultural application of person-centered group work in South Africa. (Author)

  7. HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

    Directory of Open Access Journals (Sweden)

    Irna Sufiawati

    Full Text Available Herpes simplex virus (HSV types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD. Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

  8. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Ruth Olmer

    2018-05-01

    Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture

  9. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Calcium exchange, structure, and function in cultured adult myocardial cells

    International Nuclear Information System (INIS)

    Langer, G.A.; Frank, J.S.; Rich, T.L.; Orner, F.B.

    1987-01-01

    Cells digested from adult rat heart and cultured for 14 days demonstrate all the structural elements, in mature form, associated with the process of excitation-contraction (EC) coupling. The transverse tubular (TT) system is well developed with an extensive junctional sarcoplasmic reticulum (JSR). In nonphosphate-containing buffer contraction of the cells is lost as rapidly as zero extracellular Ca concentration ([Ca] 0 ) solution is applied and a negative contraction staircase is produced on increase of stimulation frequency. Structurally and functionally the cells have the characteristics of adult cells in situ. 45 Ca exchange and total 45 Ca measurement in N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES)-buffered perfusate define three components of cellular Ca: 1) a rapidly exchangeable component accounting for 36% of total Ca, 2) a slowly exchangeable component (t/sub 1/2/ 53 min) accounting for 7% total Ca, and 3) the remaining 57% cellular Ca is inexchangeable (demonstrates no significant exchange within 60 min). The slowly exchangeable component can be increased 10-fold within 60 min by addition of phosphate to the perfusate. The Ca distribution and exchange characteristics are little different from those of 3-day cultures of neonatal rat heart previously studied. The results suggest that the cells are representative of adult cells in situ and that both sarcolemmal-bound and sarcoplasmic reticular Ca contribute to the component of Ca that is rapidly exchangeable

  11. Diffusion chamber culture of mouse bone marrow cells, (1)

    International Nuclear Information System (INIS)

    Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

    1980-01-01

    Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

  12. Clonal variation in proliferation rate of cultures of GPK cells.

    Science.gov (United States)

    Riley, P A; Hola, M

    1981-09-01

    Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. The mean interdivision time (IDT) for the culture was 1143 +/- 215 (SD) min. The mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93--10.2 x 10(-4)/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. The data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. The mean generation rate of quadrats was 8.73 x 10(-4)/min (SD = 4.9%). The spatial distribution showed some clustering of cells. The mean local density of the clones (2.25 +/- 0.62 cells/10(-4) cm2) was significantly higher than the quadrat density (1.76 +/- 0.8 cells/10(-4) cm2). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P = 2.7%). The results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.

  13. Vulnerability of cultured canine lung tumor cells to NK cell-mediated cytolysis

    International Nuclear Information System (INIS)

    Haley, P.J.; Kohr, J.M.; Kelly, G.; Muggenburg, B.A.; Guilmette, B.A.

    1988-01-01

    Five cell lines, designated as canine lung epithelial cell (CLEP), derived from radiation induced canine lung tumors and canine thyroid adeno-carcinoma (CTAC) cells were compared for their susceptibility to NK cell-mediated cytolysis using peripheral blood lymphocytes from normal, healthy Beagle dogs as effector cells. Effector cells and chromium 51 radiolabeled target cells were incubated for 16 h at ratios of 12.5:1, 25:1, 50:1, and 100:1. Increasing cytolysis was observed for all cell lines as the effector-to-target-cell ratios increased from 12.5:1 to 100:1. The percent cytotoxicity was significantly less for all lung tumor cell lines as compared to CTAC at the 100:1 ratio. One lung tumor cell line, CLEP-9, had 85% of the lytic vulnerability of the CTAC cell line and significantly greater susceptibility to NK cell-mediated lysis than all of the other lung tumor cell lines. Susceptibility to NK cell cytolysis did not correlate with in vivo malignant behavior of the original tumor. These data suggest that cultured canine lung tumor cells are susceptible to NK cell cytolytic activity in vitro and that at least one of these cell lines (CLEP-9) is a candidate for substitution of the standard canine NK cell target, CTAC, in NK cell assays. The use of lung tumor cells in NK cell assays may provide greater insight into the control of lung tumors by immune mechanisms. (author)

  14. Cultural Differences in Parents' Facilitation of Mathematics Learning: A Comparison of Euro-American and Chinese-American Families.

    Science.gov (United States)

    Huntsinger, Carol S.; Jose, Paul E.

    A longitudinal study examined differences in Chinese-American and Euro-American parents' facilitation of their young children's mathematics learning. Participating in the Time 2 data collection were 36 second-generation Chinese-American and 40 Euro-American first and second graders from well-educated suburban Chicago families. Children were given…

  15. Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

    International Nuclear Information System (INIS)

    Voralia, M.; Semeluk, A.; Wegmann, T.G.

    1987-01-01

    We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation

  16. Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

    Science.gov (United States)

    Sun, Xiuzhi S.; Nguyen, Thu A.

    2013-01-01

    Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing. PMID:23527204

  17. Ultra-thin Polyethylene glycol Coatings for Stem Cell Culture

    Science.gov (United States)

    Schmitt, Samantha K.

    Human mesenchymal stem cells (hMSCs) are a widely accessible and a clinically relevant cell type that are having a transformative impact on regenerative medicine. However, current clinical expansion methods can lead to selective changes in hMSC phenotype resulting from relatively undefined cell culture surfaces. Chemically defined synthetic surfaces can aid in understanding stem cell behavior. In particular we have developed chemically defined ultra-thin coatings that are stable over timeframes relevant to differentiation of hMSCs (several weeks). The approach employs synthesis of a copolymer with distinct chemistry in solution before application to a substrate. This provides wide compositional flexibility and allows for characterization of the orthogonal crosslinking and peptide binding groups. Characterization is done in solution by proton NMR and after crosslinking by X-ray photoelectron spectroscopy (XPS). The solubility of the copolymer in ethanol and low temperature crosslinking, expands its applicability to plastic substrates, in addition to silicon, glass, and gold. Cell adhesive peptides, namely Arg-Gly-Asp (RGD) fragments, are coupled to coating via different chemistries resulting in the urethane, amide or the thioester polymer-peptide bonds. Development of azlactone-based chemistry allowed for coupling in water at low peptide concentrations and resulted in either an amide or thioester bonds, depending on reactants. Characterization of the peptide functionalized coating by XPS, infrared spectroscopy and cell culture assays, showed that the amide linkages can present peptides for multiple weeks, while shorter-term presentation of a few days is possible using the more labile thioester bond. Regardless, coatings promoted initial adhesion and spreading of hMSCs in a peptide density dependent manner. These coatings address the following challenges in chemically defined cell culture simultaneously: (i) substrate adaptability, (ii) scalability over large areas

  18. Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Liu, Xiaozhen; Zhou, Long; Chen, Xi; Liu, Tao; Pan, Guoqing; Cui, Wenguo; Li, Mao; Luo, Zong-Ping; Pei, Ming; Yang, Huilin; Gong, Yihong; He, Fan

    2016-01-01

    Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H_2O_2) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100 μM H_2O_2 dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs. - Highlights: • Decellularization preserved the architecture and components of cell-deposited ECM.

  19. Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaozhen [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); Zhou, Long; Chen, Xi [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Liu, Tao [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pan, Guoqing; Cui, Wenguo; Li, Mao; Luo, Zong-Ping [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pei, Ming [Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV 26506 (United States); Yang, Huilin [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Gong, Yihong, E-mail: gongyih@mail.sysu.edu.cn [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); He, Fan, E-mail: fanhe@suda.edu.cn [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China)

    2016-04-01

    Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H{sub 2}O{sub 2}) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100 μM H{sub 2}O{sub 2} dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs. - Highlights: • Decellularization preserved the architecture and components of cell

  20. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

    International Nuclear Information System (INIS)

    Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M.; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

    2014-01-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM

  1. The release of elements from dental casting alloy into cell-culture medium and artificial saliva.

    Science.gov (United States)

    Can, Gülşen; Akpınar, Gül; Aydın, Ahmet

    2007-04-01

    The biocompatibility of dental casting alloys is a critical issue because these alloys are in long-term intimate contact with oral tissues. Since the biocompatibility of alloys is not completely known; the release of elements from the alloys has been studied. The aim of this study was to compare the elemental release from dental casting alloy during exposure to artificial saliva and cell-culture medium. Twenty specimens made from Ni-Cr alloy were provided in the form of 5 mm diameter discs, 2 mm in thickness with a 7 mm stem attached to one face to facilitate handling. Ten of twenty samples were polished separately using a conventional technique. The remaining ten samples were left sandblasted with 50 mum Al(2)0(3). Ten samples (5 polished, 5 sandblasted) were separately placed into cell-culture wells with Dulbecco's Modified Eagle's Medium. The other ten samples were placed separately into cell-culture wells with artificial saliva. The samples were subjected in contact with these medium for 30 days. These medium were collected every 7 days. The cell-culture medium and artificial saliva without alloy samples were subjected to elemental analyses as a control. At the end of the exposure time, Atomic Absorption Spectrometry (AAS) was used to determine the release of elements from the alloys into all collected medium. Statistical analyses were assessed with two-way ANOVA. In general, the elemental release occurred with in all medium. The elemental releases of sandblasted alloys were higher than polished alloys. Artificial saliva was found to cause more release from the samples. In both media, Ni released from polished and sandblasted alloys were higher than Cr and Mo. The results suggest that the release of elements from the alloys might have correlated with the environments and the surface of dental alloy.

  2. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Kazumi; Kinoshita, Takaaki [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Uemura, Takeshi [Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Motohashi, Hozumi [Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Watanabe, Yohei; Ebihara, Tatsuhiko [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Nishiyama, Hidetoshi [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Sato, Mari [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Suga, Mitsuo [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Maruyama, Yuusuke; Tsuji, Noriko M. [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Sato, Chikara, E-mail: ti-sato@aist.go.jp [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan)

    2014-08-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM.

  3. Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

    International Nuclear Information System (INIS)

    Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

    1986-01-01

    Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with 3 H-thymidine. Cachetin treatment (10 -6 to 10 -10 M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and 3 H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (∼ 50%) at 10 -10 M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation

  4. Radiation-induced bystander effects in cultured human stem cells.

    Directory of Open Access Journals (Sweden)

    Mykyta V Sokolov

    2010-12-01

    Full Text Available The radiation-induced "bystander effect" (RIBE was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR. RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.Human bone-marrow mesenchymal stem cells (hMSC and embryonic stem cells (hESC were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05. A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05.These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies.

  5. Handbook of plant cell culture. Volume 2. Crop species

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, W.R.; Evans, D.A.; Ammirato, P.V.; Yamada, Y. (eds.)

    1984-01-01

    In this volume the state-of-the-art plant cell culture techniques described in the first volume are applied to several agricultural and horticultural crops. In 21 chapters, they include maize, oats, wheat, beans, red clover and other forage legumes, asparagus, celery, cassava, sweet potato, banana, pawpaw, apple, grapes, conifers, date palm, rubber, sugarcane and tobacco. Each chapter contains (1) detailed protocols to serve as the foundation for current research, (2) a critical review of the literature, and (3) in-depth evaluations of the potential shown by plant cell culture for crop improvement. The history and economic importance of each crop are discussed. This volume also includes an essay, ''Oil from plants'', by M. Calvin.

  6. An introduction to plant cell culture: the future ahead.

    Science.gov (United States)

    Loyola-Vargas, Víctor M; Ochoa-Alejo, Neftalí

    2012-01-01

    Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an experimental necessity for solving important fundamental questions in plant biology, but they currently represent very useful biotechnological tools for a series of important applications such as commercial micropropagation of different plant species, generation of disease-free plant materials, production of haploid and doublehaploid plants, induction of epigenetic or genetic variation for the isolation of variant plants, obtention of novel hybrid plants through the rescue of hybrid embryos or somatic cell fusion from intra- or intergeneric sources, conservation of valuable plant germplasm, and is the keystone for genetic engineering of plants to produce disease and pest resistant varieties, to engineer metabolic pathways with the aim of producing specific secondary metabolites or as an alternative for biopharming. Some other miscellaneous applications involve the utilization of in vitro cultures to test toxic compounds and the possibilities of removing them (bioremediation), interaction of root cultures with nematodes or mycorrhiza, or the use of shoot cultures to maintain plant viruses. With the increased worldwide demand for biofuels, it seems that PTC will certainly be fundamental for engineering different plants species in order to increase the diversity of biofuel options, lower the price marketing, and enhance the production efficiency. Several aspects and applications of PTC such as those mentioned above are the focus of this edition.

  7. [Biological characteristics of mesenchymal stem cell and hematopoietic stem cell in the co-culture system].

    Science.gov (United States)

    Wei, Wei; Xu, Chao; Ye, Zhi-Yong; Huang, Xiao-Jun; Yuan, Jia-En; Ma, Tian-Bao; Lin, Han-Biao; Chen, Xiu-Qiong

    2016-10-25

    The aim of the present study was to obtain the qualified hematopoietic stem/progenitor cells (HSC/HPC) and human umbilical cord-mesenchymal stem cells (MSC) in vitro in the co-culture system. Cord blood mononuclear cells were separated from umbilical cord blood by Ficoll lymphocyte separation medium, and then CD34 + HSC was collected by MACS immunomagnetic beads. The selected CD34 + HSC/HPC and MSC were transferred into culture flask. IMDM culture medium with 15% AB-type cord plasma supplemented with interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt-3L) factors were used as the co-culture system for the amplification of HSC/HPC and MSC. The cellular growth status and proliferation on day 6 and 10 after co-culture were observed by using inverted microscope. The percentage of positive expression of CD34 in HSC/HPC, as well as the percentages of positive expressions of CD105, CD90, CD73, CD45, CD34 and HLA-DR in the 4 th generation MSC, was tested by flow cytometry. Semisolid colony culture was used to test the HSC/HPC colony forming ability. The osteogenic, chondrogenesis and adipogenic ability of the 4 th generation MSC were assessed. The karyotype analysis of MSC was conducted by colchicines. The results demonstrated that the HSC/HPC of co-culture group showed higher ability of amplification, CFU-GM and higher CD34 + percentage compared with the control group. The co-cultured MSC maintained the ability to differentiate into bone cells, fat cells and chondrocytes. And the karyotype stability of MSC remained normal. These results reveal that the appropriate co-culture system for MSC and HSC is developed, and via this co-culture system we could gain both two kinds of these cells. The MSCs under the co-culture system maintain the biological characteristics. The CFU-GM ability, cell counting and the flow cytometry results of HSC/HPC under the co-culture system are conform to the criterion, showing that

  8. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    Science.gov (United States)

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  9. Enhanced chondrocyte culture and growth on biologically inspired nanofibrous cell culture dishes.

    Science.gov (United States)

    Bhardwaj, Garima; Webster, Thomas J

    2016-01-01

    Chondral and osteochondral defects affect a large number of people in which treatment options are currently limited. Due to its ability to mimic the natural nanofibrous structure of cartilage, this current in vitro study aimed at introducing a new scaffold, called XanoMatrix™, for cartilage regeneration. In addition, this same scaffold is introduced here as a new substrate onto which to study chondrocyte functions. Current studies on chondrocyte functions are limited due to nonbiologically inspired cell culture substrates. With its polyethylene terephthalate and cellulose acetate composition, good mechanical properties and nanofibrous structure resembling an extracellular matrix, XanoMatrix offers an ideal surface for chondrocyte growth and proliferation. This current study demonstrated that the XanoMatrix scaffolds promote chondrocyte growth and proliferation as compared with the Corning and Falcon surfaces normally used for chondrocyte cell culture. The XanoMatrix scaffolds also have greater hydrophobicity, three-dimensional surface area, and greater tensile strength, making them ideal candidates for alternative treatment options for chondral and osteochondral defects as well as cell culture substrates to study chondrocyte functions.

  10. Cell division requirement for activation of murine leukemia virus in cell culture by irradiation

    International Nuclear Information System (INIS)

    Otten, J.A.; Quarles, J.M.; Tennant, R.W.

    1976-01-01

    Actively dividing cultures of AKR mouse cells were exposed to relatively low dose-rates of γ radiation and tested for activation of endogenous leukemia viruses. Efficient and reproducible induction of virus was obtained with actively dividing cells, but cultures deprived of serum to inhibit cell division before and during γ irradiation were not activated, even when medium with serum was added immediately after irradiation. These results show that cell division was required for virus induction but that a stable intermediate similar to the state induced by halogenated pyrimidines was not formed. In actively dividing AKR cell cultures, virus activation appeared to be proportional to the dose of γ radiation; the estimated frequency of activation was 1-8 x 10 - 5 per exposed cell and the efficiency of activation was approximately 0.012 inductions per cell per rad. Other normal primary and established mouse cell cultures tested were not activated by γ radiation. The requirement of cell division for radiation and chemical activation may reflect some common mechanism for initiation of virus expression

  11. Mesenchymal stem cells cultured on magnetic nanowire substrates

    Science.gov (United States)

    Perez, Jose E.; Ravasi, Timothy; Kosel, Jürgen

    2017-02-01

    Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing h

  12. Mesenchymal stem cells cultured on magnetic nanowire substrates

    KAUST Repository

    Perez, Jose E.

    2016-12-28

    Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing h

  13. New 2-arylbenzofuran metabolite from cell cultures of Morus alba.

    Science.gov (United States)

    Zhang, De-Wu; Tao, Xiao-Yu; Yu, Li-Yan; Dai, Jun-Gui

    2015-01-01

    A new 2-arylbenzofuran compound, 5-dehydroxy-moracin U (1), along with 10 known compounds (2-11), were isolated from cell cultures of Morus alba. Their structures were elucidated on the basis of extensive spectroscopic analyses. The anti-inflammatory activity assay of 1-8 showed that 2 and 8 exhibited significant inhibitory effect on LPS-induced NO production with the values of 76.4% and 98.7% at 10(- 5) M, respectively.

  14. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  15. Photodamage of the cells in culture sensitized with bilirubin

    Science.gov (United States)

    Kozlenkova, O. A.; Plavskaya, L. G.; Mikulich, A. V.; Leusenko, I. A.; Tretyakova, A. I.; Plavskii, V. Yu

    2016-08-01

    It has been shown that exposure to radiation of LED sources of light with an emission band maximum at about 465 and 520 nm having substantially identical damaging effects on animal cells in culture, that are in a logarithmic growth phase and preincubated with pigment. Photobiological effect is caused by photodynamic processes involving singlet oxygen generated by triplet excited sensitizer. Mono-exponential type dependence of cell survival on the energy dose indicates that it is bilirubin that acts as a sensitizer but not its photoproducts. The inclusion of bilirubin in the cells, where it is primarily localized in the mitochondria cells, it is accompanied by multiple amplification photochemical stability compared to pigment molecules bound with albumin

  16. Neural differentiation of adipose-derived stem cells by indirect co-culture with Schwann cells

    Directory of Open Access Journals (Sweden)

    Li Xiaojie

    2009-01-01

    Full Text Available To investigate whether adipose-derived stem cells (ADSCs could be subject to neural differentiation induced only by Schwann cell (SC factors, we co-cultured ADSCs and SCs in transwell culture dishes. Immunoassaying, Western blot analysis, and RT-PCR were performed (1, 3, 7, 14 d and the co-cultured ADSCs showed gene and protein expression of S-100, Nestin, and GFAP. Further, qRT-PCR disclosed relative quantitative differences in the above three gene expressions. We think ADSCs can undergo induced neural differentiation by being co-cultured with SCs, and such differentia­tions begin 1 day after co-culture, become apparent after 7 days, and thereafter remain stable till the 14th day.

  17. Computerized microfluidic cell culture using elastomeric channels and Braille displays.

    Science.gov (United States)

    Gu, Wei; Zhu, Xiaoyue; Futai, Nobuyuki; Cho, Brenda S; Takayama, Shuichi

    2004-11-09

    Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use.

  18. Cell cultures for schistosomes - Chances of success or wishful thinking?

    Science.gov (United States)

    Quack, T; Wippersteg, V; Grevelding, C G

    2010-08-01

    Due to their worldwide importance for human and animal health, schistosomes are in the focus of national and international research activities. Their aims are to elucidate the genome, the transcriptome, the proteome and the glycome of schistosomes with the expectation to understand the biology of these blood flukes and to identify new candidate antigens for the development of a vaccine, or target molecules for the design of novel pharmaceutical compounds. All of these efforts have delivered a vast amount of information about the genetic equipment of schistosomes. In the emerging era of post-genomic research, however, methods and tools are necessary to interpret all available data and to characterise molecules of interest in more detail. In addition to transgenesis, it is generally accepted that cell lines for schistosomes are among the requirements to overcome present research limitations. In our commentary the prospect of establishing cell cultures for schistosomes is discussed. To this end we summarise the comprehensive endeavours made in the past regarding the establishment of invertebrate cell lines pointing to critical parameters that should be considered when making new attempts towards schistosome cell culturing. Furthermore, based on preliminary data with pilot-character, we discuss recent advances indicating the possibility of overcoming existing restrictions with respect to the 'immortalisation' of cells by oncogenes. Copyright 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  19. Agent-Based Computational Modeling of Cell Culture ...

    Science.gov (United States)

    Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assumed a “fried egg shape” but became increasingly cuboidal with increasing confluency. The surface area presented by each cell to the overlying medium varies from cell-to-cell and is a determinant of diffusional flux of toxicant from the medium into the cell. Thus, dose varies among cells for a given concentration of toxicant in the medium. Computer code describing diffusion of H2O2 from medium into each cell and clearance of H2O2 was calibrated against H2O2 time-course data (25, 50, or 75 uM H2O2 for 60 min) obtained with the Amplex Red assay for the medium and the H2O2-sensitive fluorescent reporter, HyPer, for cytosol. Cellular H2O2 concentrations peaked at about 5 min and were near baseline by 10 min. The model predicted a skewed distribution of surface areas, with between cell variation usually 2 fold or less. Predicted variability in cellular dose was in rough agreement with the variation in the HyPer data. These results are preliminary, as the model was not calibrated to the morphology of a specific cell type. Future work will involve morphology model calibration against human bronchial epithelial (BEAS-2B) cells. Our results show, however, the potential of agent-based modeling

  20. CDB-4124 does not cause apoptosis in cultured fibroid cells.

    Science.gov (United States)

    Roeder, Hilary; Jayes, Friederike; Feng, Liping; Leppert, Phyllis C

    2011-09-01

    Selective progesterone receptor modulators (SPRMs), such as asoprisnil (J867) and ulipristal (CDB-2914), have been shown to reduce fibroid volume in vivo and to induce apoptosis in vitro. CDB-4124 (telapristone), a SPRM with different side groups, also reduced fibroid volume in vivo, and we hypothesized that this SPRM would also cause apoptosis in cultured fibroid cells. Immortalized, progesterone receptor-positive fibroid cells, known to be capable of apoptosis, were grown to 80% confluence in serum-containing media. Cells were then treated for 48 hours in serum-free media with 0, 10, 100, or 1000 nmol/L CDB-4124. Actinomycin-D and staurosporine were used as positive controls to induce apoptosis. Apoptosis was quantified using a TUNEL-fluorescein kit. Images were captured with a widefield-fluorescence microscope and analyzed using MetaMorph image analysis software. To validate results, Western blots of total cell lysates were probed for cleaved caspase-3 (c-CASP3). Experiments were repeated 3 times using independent cell batches. Analysis of 19 712 nuclei indicated 14.8% ± 10.9% (mean ± SEM), 8.4% ± 4.6%, 8.2% ± 4.7%, and 9.3% ± 6.3% apoptosis in 0, 10, 100, and 1000 nmol/L CDB-4124-treated cells, respectively. There was no evidence of elevated c-CASP3 over vehicle control after treatment with CDB-4124. CDB-4124 did not significantly induce apoptosis in cultured fibroid cells under the conditions described suggesting apoptosis may not be the main pathway responsible for CDB-4124-induced fibroid shrinkage. Variations in SPRM biological effects may be due to differences in fibroid source cells, binding kinetics, or extracellular matrix characteristics, and can be exploited in further investigations of the mechanisms of action of SPRMs in fibroid biology.

  1. Flow field measurements in the cell culture unit

    Science.gov (United States)

    Walker, Stephen; Wilder, Mike; Dimanlig, Arsenio; Jagger, Justin; Searby, Nancy

    2002-01-01

    The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the

  2. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

    Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

  3. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

    Science.gov (United States)

    Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

    2016-11-01

    To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

  4. Regulation of adipose-tissue-derived stromal cell orientation and motility in 2D- and 3D-cultures by direct-current electrical field.

    Science.gov (United States)

    Yang, Gang; Long, Haiyan; Ren, Xiaomei; Ma, Kunlong; Xiao, Zhenghua; Wang, Ying; Guo, Yingqiang

    2017-02-01

    Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane-protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose-tissue-derived stromal cells (ADSCs) in 2D-culture on plastic culture dishes and in 3D-culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L-lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological-strength EFs in a homemade EF-bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time-lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D-culture aligned vertically to EF vector and kept a good cell survival rate. In 3D-culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D-culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors. © 2017 Japanese Society of Developmental Biologists.

  5. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    NARCIS (Netherlands)

    C. Buecker (Christa); H.H. Chen; J.M. Polo (Jose); L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)

    2010-01-01

    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human

  6. Is cell culture a risky business? Risk analysis based on scientist survey data.

    Science.gov (United States)

    Shannon, Mark; Capes-Davis, Amanda; Eggington, Elaine; Georghiou, Ronnie; Huschtscha, Lily I; Moy, Elsa; Power, Melinda; Reddel, Roger R; Arthur, Jonathan W

    2016-02-01

    Cell culture is a technique that requires vigilance from the researcher. Common cell culture problems, including contamination with microorganisms or cells from other cultures, can place the reliability and reproducibility of cell culture work at risk. Here we use survey data, contributed by research scientists based in Australia and New Zealand, to assess common cell culture risks and how these risks are managed in practice. Respondents show that sharing of cell lines between laboratories continues to be widespread. Arrangements for mycoplasma and authentication testing are increasingly in place, although scientists are often uncertain how to perform authentication testing. Additional risks are identified for preparation of frozen stocks, storage and shipping. © 2015 UICC.

  7. Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

    Science.gov (United States)

    van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

    2014-09-01

    The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    Science.gov (United States)

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-01-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc. PMID:24037521

  9. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

    1999-01-01

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  10. Isolation and culture of neural crest cells from embryonic murine neural tube.

    Science.gov (United States)

    Pfaltzgraff, Elise R; Mundell, Nathan A; Labosky, Patricia A

    2012-06-02

    The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from

  11. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2011-07-29

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  12. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    Science.gov (United States)

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-07-30

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

  13. Effects of 2,3-iminosqualene in cultured cells

    International Nuclear Information System (INIS)

    Popjak, G.; Meenan, A.; Nes, W.D.

    1987-01-01

    2,3-Iminosqualene added to culture media 10 ug/ml) of rat hepatoma (H4-II-E-C3) or Chinese hamster ovary (CHO) cells irreversibly inactivates the squalene-oxide: lanosterol cyclase, but it does not inhibit general polyprenyl synthesis either from [ 14 C]acetate or [ 14 C]mevalonate. Isq added to lipoprotein-containing media of H4 cells causes in 24 hr an over twofold rise in HMG-CoA reductase and abolishes the repressive effect of mevalonate (MVA) on the reductase. H4 cells synthesize from [2- 14 C]-MVA labelled squalene, squalene-2,3-oxide, squalene-2,3-22,23-dioxide, but very little sterol. The conversion of MVA to these polyprenyls in the presence of Isq is as efficient as its conversion to squalene and cholesterol in control cells. They conclude that the repressor of HMG-CoA reductase derived from MVA is a sterol - whatever might be the nature of that sterol - and not a nonsteroidal derivative of MVA metabolism. H4 cells exposed to Isq in lipid-depleted media die in 48-72 hr, but can be rescued by LDL, but not by free cholesterol or MVA. CHO cells are more resistant than H4 cells and succumb only after 8-9 days' exposure to Isq

  14. Digital microfluidics for automated hanging drop cell spheroid culture.

    Science.gov (United States)

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. © 2014 Society for Laboratory Automation and Screening.

  15. Cell thickness of UV absorption by the cell: relation to UV action spectrum shift in mammalian cells in culture

    International Nuclear Information System (INIS)

    Sakharov, V.H.; Voronkova, L.N.; Blokhin, A.V.

    1985-01-01

    By means of reconstruction of series half - thin transverse sections the three - dimensional morphometry of SPEV cells for a series of their specific states in culture is performed: for exponential growth in a monolayer, in a merged monolayer, in the mitosis phase, for giant cells and suspension cells. In the monolayer the cell thickness in its central part depended mainly on the nucleus thickness and in average changed but slightly despite a wide range of changes in volumes of nuclei and cells and their density in culture. The cell thickness has noticeably increased in mitosis. For the above states of cells UV radiation absorption spectra are determined. It is shown that a certain shift of action spectrus of death of mammalian cells as compared with that for bacterial cell can be a seguence of selfshielding and not differences in the nature of active chromophores

  16. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-05

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.

  17. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  18. Design of a tablet computer app for facilitation of a molecular blood culture test in clinical microbiology and preliminary usability evaluation

    DEFF Research Database (Denmark)

    Samson, Lasse L.; Pape-Haugaard, Louise; Meltzer, Michelle C.

    2016-01-01

    through specialized applications (apps) while supporting the mobility of the users. The use of apps for mobile phones and tablet computers may support workflow of complex tasks, for example, molecular-based diagnostic tests in clinical microbiology. Multiplex Blood Culture Test (MuxBCT) is a molecular......-based diagnostic test used for rapid identification of pathogens in positive blood cultures. To facilitate the workflow of the MuxBCT, a specialized tablet computer app was developed as an accessory to the diagnostic test. The app aims to reduce the complexity of the test by step-by-step guidance of microscopy...... and to assist users in reaching an exact bacterial or fungal diagnosis based on blood specimen observations and controls. Additionally, the app allows for entry of test results, and communication thereof to the laboratory information system (LIS). OBJECTIVE: The objective of the study was to describe the design...

  19. Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

    Science.gov (United States)

    Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

    2016-02-01

    The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

  20. Periodic heat shock accelerated the chondrogenic differentiation of human mesenchymal stem cells in pellet culture.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Osteoarthritis (OA is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼ 6 weeks and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41 °C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.