Journal articles on the topic 'Human cell culture'
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Boyce, Steven T. "Human Cell Culture Protocols." Journal of Investigative Dermatology 108, no. 2 (February 1997): 235. http://dx.doi.org/10.1111/1523-1747.ep12335350.
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Hernáez-Moya, Raquel, Sheyla González, Arantza Urkaregi, Jose Ignacio Pijoan, Sophie X. Deng, and Noelia Andollo. "Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis." International Journal of Molecular Sciences 21, no. 17 (August 25, 2020): 6132. http://dx.doi.org/10.3390/ijms21176132.
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Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients’ health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium—supplemental hormone epithelial medium (SHEM)—supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.
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Skottman, Heli, and Outi Hovatta. "Culture conditions for human embryonic stem cells." Reproduction 132, no. 5 (November 2006): 691–98. http://dx.doi.org/10.1530/rep.1.01079.
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Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.
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Finoli, Anthony, Eva Schmelzer, Patrick Over, Ian Nettleship, and Joerg C. Gerlach. "Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells." BioMed Research International 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/6040146.
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Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.
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Wang, Jing, Qingchen Qiao, Yaxi Sun, Wenting Yu, Jiran Wang, Minjia Zhu, Kai Yang, Xiaofeng Huang, and Yuxing Bai. "Osteogenic Differentiation Effect of Human Periodontal Ligament Stem-Cell Initial Cell Density on Autologous Cells and Human Bone Marrow Stromal Cells." International Journal of Molecular Sciences 24, no. 8 (April 12, 2023): 7133. http://dx.doi.org/10.3390/ijms24087133.
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Stem cells have differentiation and regulation functions. Here, we discussed the impact of cell culture density on stem cell proliferation, osteoblastogenesis, and regulation. To discuss the effect of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the osteogenic differentiation of autologous cells, we found that the hPDLSC proliferation rate decreased with an increase in the initial plating density (0.5–8 × 104 cells/cm2) for the 48 h culture cycle. After hPDLSCs induced osteogenic differentiation for 14 days with different initial cell culture densities, the expression of osteoprotegerin (OPG) and runt-related transcription factor 2(RUNX2) and the OPG/ Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) ratio were the highest in the hPDLSCs initially plated at a density of 2 × 104 cells/cm2, and the average cell calcium concentration was also the highest. To study hPDLSCs regulating the osteoblastic differentiation of other cells, we used 50 μg/mL of secreted exosomes derived from hPDLSCs cultured using different initial cell densities to induce human bone marrow stromal cell (hBMSC) osteogenesis. After 14 days, the results indicated that the gene expression of OPG, Osteocalcin(OCN,)RUNX2, and osterix and the OPG/RANKL ratio were the highest in the 2 × 104 cells/cm2 initial cell density group, and the average calcium concentration was also the highest. This provides a new idea for the clinical application of stem cell osteogenesis.
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Andrews, R. G., J. W. Singer, and I. D. Bernstein. "Human hematopoietic precursors in long-term culture: single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells." Journal of Experimental Medicine 172, no. 1 (July 1, 1990): 355–58. http://dx.doi.org/10.1084/jem.172.1.355.
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CD34+ human marrow cells not expressing T cell-, B cell-, and myeloid cell-associated antigens (TBM-) were cloned by two-color cell sorting into culture wells containing irradiated marrow stromal cells. After 4 wk of culture, 3.7 +/- 2.1% of these cells generated colony-forming cells (CFC), with each of these cells generating 6.3 +/- 5.3 CFC. This was not due to the 0.5 +/- 0.5% CFC present in the purified CD34+ TBM- cells, as less than 1% of CFC persist in these cultures. This is the first demonstration that single immature precursor cells in human long-term cultures generate multiple CFC progeny. The immature nature of these clonable CD34+ TBM- precursors suggests their candidate status as human hematopoietic stem cells.
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Chattong, Supreecha, Ruttachuk Rungsiwiwut, Wittaya Yindeedej, Amornpun Sereemaspun, Kamthorn Pruksananonda, Pramuan Virutamasen, Anant Setpakdee, and Krissanapong Manotham. "Original article. Human dental pulp stem cells as a potential feeder layer for human embryonic stem cell culture." Asian Biomedicine 8, no. 3 (June 1, 2014): 333–43. http://dx.doi.org/10.5372/1905-7415.0803.297.
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AbstractBackground: Human embryonic stem (hES) cells are pluripotent, and can differentiate into three germ layers. Traditionally, cultures of hES cells are maintained in a system containing mouse embryonic fibroblasts as a feeder layer for support of undifferentiated growth. However, contamination by animal cells limits the use of hES cells.Objective: We evaluated the use of human dental pulp stem cells (hDPSCs) as a feeder layer for hES cell culture. It should be possible to obtain a new source of human mesenchymal stem cells for feeder cells to maintain undifferentiated growth of hES cells.Methods: hDPSCs from removed impacted wisdom teeth (third molars) were extracted, cultured, and characterized for mesenchymal stem cell properties. Furthermore, hDPSCs were used as a feeder layer for culturing Chula2 and Chula5 hES cell lines. Finally, hES cell lines grown on hDPSCs feeders were examined embryonic stem cell properties.Results: We found that hDPSCs, which have mesenchymal properties, can support undifferentiated growth of hES cell lines. After prolonged culture (passage 17), these hES cell lines still maintain ES cell properties including typical morphology seen in hES cells, the expression of pluripotency markers (Oct4, Sox2, Nanog, Rex1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), embryoid body formation and retention of a normal karyotype.Conclusion: hDPSCs, derived from the pulp tissue of impacted third molars, are a potential source of human feeder cells for the culture of undifferentiated hES cells.
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Elliott, G., D. van de Meent, J. van Dijk, and M. Mol. "Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time." Mediators of Inflammation 1, no. 6 (1992): 385–90. http://dx.doi.org/10.1155/s0962935192000589.
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Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG) E than confluent ones. As interleukin-1 (IL-1) stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1α (100 U/ml) stimulated PGE2synthesis by proliferating (7 days in culture) but not differentiating (14 days in culture) keratinocytes. Similar effects were observed using tumour necrosis factor-α. Both arachidonic acid (AA) and the calcium ionophore A23187 stimulated PGE2synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s) by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.
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Saito, Hirohisa, Duraisamy Kempuraj, Morimitsu Tomikawa, Hisashi Tomita, Kangmo Ahn, and Yoji Iikura. "Human Mast Cell Colony-Forming Cells in Culture." International Archives of Allergy and Immunology 124, no. 1-3 (2001): 301–3. http://dx.doi.org/10.1159/000053739.
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Rosenzwajg, M., B. Canque, and JC Gluckman. "Human dendritic cell differentiation pathway from CD34+ hematopoietic precursor cells." Blood 87, no. 2 (January 15, 1996): 535–44. http://dx.doi.org/10.1182/blood.v87.2.535.bloodjournal872535.
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The most effective antigen-presenting cells for T lymphocytes are dendritic cells (DCs), the differentiation pathway of which, however, is incompletely characterized. We examined here how DCs differentiated from human cord blood CD34+ progenitor cells cultured with granulocyte- macrophage colony-stimulating factor, tumor necrosis factor-alpha, and stem cell factor. After 5 days, 2 of 3 nonadherent cells were CD13hiHLA- DRhiCD4+, half of them were also CD14+, and < or = 10% were CD1a+. When day-5 sorted CD13hiCD1a- and CD13lo cells were further cultured, CD1a+ cells appeared in the already CD13hi population, whereas CD13hi cells, a minority of which rapidly became CD1a+, emerged from the CD13lo population. By day 12, still 66% of bulk cells in suspension were CD13hi, most of which displayed high forward and side scatters of large granular cells. Half of CD13hi cells were CD1a+. All CD13hi cells expressed to the same extent DR, CD4, costimulatory and adhesion molecules, and various amounts of CD14. CD1a+ cells stimulated allogeneic lymphocytes more than CD13hiCD1a- cells and, although they were CD14+, both cell types were nonspecific esterase-negative nonphagocytic cells and were stronger mixed leukocyte reaction stimulators than were their macrophage counterparts. Eventually, the percentage of CD1a+ cells decreased. However, typical CD1a+ DCs still emerged in culture of sorted day-12 CD13hiCD1a- cells, and adding interleukin-4 to bulk cultures at that time led to the persistence of the CD1a+ population while diminishing CD14 expression. Thus, this system results first in the differentiation of CD13hi precursors that strongly express DR and CD4, from which more mature CD1a+ DCs continuously differentiate all along the culture period.
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Todd, Kyle V., and Ralph A. Tripp. "Vero Cells as a Mammalian Cell Substrate for Human Norovirus." Viruses 12, no. 4 (April 14, 2020): 439. http://dx.doi.org/10.3390/v12040439.
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Human norovirus (HuNoV) is a principal cause of acute gastroenteritis worldwide, particularly in developing countries. Its global prevalence is underscored by more serious morbidity and some mortality in the young (<5 years) and the elderly. To date, there are no licensed vaccines or approved therapeutics for HuNoV, mostly because there are limited cell culture systems and small animal models available. Recently described cell culture systems are not ideal substrates for HuNoV vaccine development because they are not clonal or only support a single strain. In this study, we show Vero cell-based replication of two pandemic GII.4 HuNoV strains and one GII.3 strain and confirm exosome-mediated HuNoV infection in Vero cells. Lastly, we show that trypsin addition to virus cultures or disruption of Vero cell host genes can modestly increase HuNoV replication. These data provide support for Vero cells as a cell culture model for HuNoV.
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Chato-Astrain, Jesús, David Sánchez-Porras, Óscar Darío García-García, Claudia Vairo, María Villar-Vidal, Silvia Villullas, Indalecio Sánchez-Montesinos, Fernando Campos, Ingrid Garzón, and Miguel Alaminos. "Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers." Biomedicines 9, no. 11 (November 6, 2021): 1634. http://dx.doi.org/10.3390/biomedicines9111634.
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Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.
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Ichii, Michiko, Kenji Oritani, Takafumi Yokota, Isao Takahashi, Takahiro Shirogane, Norimitsu Saitoh, Rie Tanigawa, Satomi Kasai, and Yuzuru Kanakura. "Establishment of Stroma-Free Cultures for Human B Lymphopoiesis: Roles of High Cell Density Condition and Mesenchymal Stem Cell-Secreted Factors." Blood 110, no. 11 (November 16, 2007): 1420. http://dx.doi.org/10.1182/blood.v110.11.1420.1420.
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Abstract Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells. Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7. Results: Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential. For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them. When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis. Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week. CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character. Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.
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CAUX, FRÉDÉRIC, JOZSEF TIMAR, ELEMER MOCZAR, KÀROLY LAPIS, and MADELEINE MOCZAR. "Cell-associated glycosaminoglycans in human melanoma cell culture." Biochemical Society Transactions 18, no. 2 (April 1, 1990): 294–95. http://dx.doi.org/10.1042/bst0180294.
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Koh, Benson, Nadiah Sulaiman, Mh Busra Fauzi, Jia Xian Law, Min Hwei Ng, Too Lih Yuan, Abdul Ghani Nur Azurah, Mohd Heikal Mohd Yunus, Ruszymah Bt Hj Idrus, and Muhammad Dain Yazid. "A Three-Dimensional Xeno-Free Culture Condition for Wharton’s Jelly-Mesenchymal Stem Cells: The Pros and Cons." International Journal of Molecular Sciences 24, no. 4 (February 13, 2023): 3745. http://dx.doi.org/10.3390/ijms24043745.
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Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton’s Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.
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Prewitz, Marina, Friedrich Philipp Seib, Martin Bornhaeuser, and Carsten Werner. "Engineering Biomimetic Culture Systems: Impact On Human Bone Marrow-Derived Stem Cells." Blood 114, no. 22 (November 20, 2009): 3628. http://dx.doi.org/10.1182/blood.v114.22.3628.3628.
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Abstract Abstract 3628 Poster Board III-564 The bone marrow (BM) harbours haematopoietic stem/progenitor cells (HSCs) in anatomically distinct sites (niches) where HSCs are subjected to regulatory cues such as cytokines, cell-cell contacts and extra-cellular matrix (ECM) all of which control stem cell fate. In particular mesenchymal stromal cells (MSCs) are an integral part of the bone marrow and are known to be key regulators of the HSC niche. We have previously shown that bio-artificial scaffolds can have a significant impact on the in vitro behaviour of MSCs. Here, we are therefore focussing on the role of (native) ECM within the MSC-HSC microenvironment by building on our previous findings and published data (Seib et al.,Tissue Eng Part A., 2009 in press). Thus the aim of the current study is (a) to identify niche-specific ECM components and (b) the use of such ECMs for in vitro culture of BM-derived stem cells. To mimic the natural ECM composition of the BM, different ECM types were generated from BM-derived cells using (a) Dexter cultures, (b) standard MSC cultures, (c) MSCs subjected to osteogenic differentiation. After 10 days of culture those MSC-derived ECMs were decellularised using 0.5% Triton-X and 20mM NH4OH leaving only the ECM behind (verified by scanning electron microscopy). Those ECMs were used as a substrate for a second culture of MSCs, which were analysed for their proliferation and differentiation potential. Cell-free ECM from standard MSC cultures improved MSC proliferation compared to cells grown on regular tissue culture plastic (TCP) over the period of 8 days. Most notably, all cell-free ECM preparations lead to a significant difference in the cytoskeletal arrangement of MSCs during the first 2 days of culture compared to TCP controls. Cultivation of MSCs on native ECM provided a guiding structure for those cells to grow into, and helped to maintain an elongated cell shape compared to substantial cell spreading on TCP (roundness 0.2 versus 0.5 and cell area of 2.2 versus 8.2mm2, respectively, p<0.001, n=60. A factor of 1 was set to equate to a perfect circle). Next, we investigate if native ECM could either directly improve HSC cultures or maximise MSC feeder characteristics. For the latter set of studies MSCs were initially cultured for 7 days on cell-free ECM (from standard MSC cultures) and subsequently co-cultured with human peripheral blood CD34+ HSCs in serum free medium supplemented with cytokines (Tpo, Flt3, and SCF at 10ng/ml). Following a 14 day culture period up to 3.5-fold more CD34+ cells were present in ECM co-cultures compared to TCP co-cultures that was accompanied with an overall expansion of CD45+ cells of 109-fold versus 35-fold, respectively. Our data suggest that ECM preparations derived from MSCs might be useful to accomplish better expansion of HSCs under defined culture conditions. In addition, this system permits the identification of bimolecular key components that can be utilized in the future design of simple and robust carrier systems for improved HSC maintenance in vitro. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Disclosures: No relevant conflicts of interest to declare.
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Eglen, Richard M., and Terry Reisine. "Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 2: Spheroids, Organoids, and Disease Modeling." SLAS TECHNOLOGY: Translating Life Sciences Innovation 24, no. 1 (January 22, 2019): 18–27. http://dx.doi.org/10.1177/2472630318803275.
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Human induced pluripotent stem cells (HiPSCs) provide several advantages for drug discovery, but principally they provide a source of clinically relevant tissue. Furthermore, the use of HiPSCs cultured in three-dimensional (3D) systems, as opposed to traditional two-dimensional (2D) culture approaches, better represents the complex tissue architecture in vivo. The use of HiPSCs in 3D spheroid and organoid culture is now growing, but particularly when using myocardial, intestinal enteric nervous system, and retinal cell lines. However, organoid cell culture is perhaps making the most notable impact in research and drug discovery, in which 3D neuronal cell cultures allow direct modeling of cortical cell layering and neuronal circuit activity. Given the specific degeneration seen in discrete neuronal circuitry in Alzheimer’s disease (AD) and Parkinson’s disease (PD), HiPSC culture systems are proving to be a major advance. In the present review, the second part of a two-part review, we discuss novel methods in which 3D cell culture systems (principally organoids) are now being used to provide insights into disease mechanisms. (The use of HiPSCs in target identification was reviewed in detail in Part 1.)
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Liu, Haijun, Xiaoniu Dai, Yusi Cheng, Shencun Fang, Yingming Zhang, Xingang Wang, Wei Zhang, Hong Liao, Honghong Yao, and Jie Chao. "MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 2 (January 15, 2016): L121—L132. http://dx.doi.org/10.1152/ajplung.00278.2015.
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Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.
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Chan, Martin C. W., Y. P. Wong, and Wai K. Leung. "Cell Culture Assay for Human Noroviruses." Emerging Infectious Diseases 13, no. 7 (July 2007): 1117–18. http://dx.doi.org/10.3201/eid1307.070131.
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Straub, Timothy M., Kerstin Höner zu Bentrup, Patricia Orosz-Coghlan, Alice Dohnalkova, Brooke K. Mayer, Rachel A. Bartholomew, Catherine O. Valdez, et al. "Cell Culture Assay for Human Noroviruses." Emerging Infectious Diseases 13, no. 7 (July 2007): 1117–18. http://dx.doi.org/10.3201/eid1307.070566.
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Rihs, F., J. Kesselring, and C. Meier. "Human oligodendrocytes in dissociated cell culture." Acta Neuropathologica 78, no. 3 (1989): 317–24. http://dx.doi.org/10.1007/bf00687762.
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Miyamoto, Yoshitaka, Masashi Ikeuchi, Hirofumi Noguchi, Tohru Yagi, and Shuji Hayashi. "Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an in vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture." Cell Medicine 9, no. 1-2 (January 2017): 35–44. http://dx.doi.org/10.3727/215517916x693096.
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The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.
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Su, Kuei-Ying, Takuya Nojima, Masayuki Kuraoka, M. Anthony Moody, and Garnett Kelsoe. "An in vitro culture system for studying the human B cell repertoire (P1459)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 174.12. http://dx.doi.org/10.4049/jimmunol.190.supp.174.12.
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Abstract Defining the human B-cell repertoire is important for understanding human B-cell development and autoimmunity. Currently, re-expression of immunoglobulins from single B cells is the standard method to study human B-cell repertoire; however, this method is labor-intensive and costly as it requires large numbers of recombinant antibodies to be generated by single-cell PCR and expression of recombinant heavy and light chain pairs. We have developed an alternative method to characterize the B-cell repertoire by culture that supports the proliferation and differentiation of human pre-B, immature/transitional, and mature B cells recovered from bone marrow, fetal liver, or peripheral blood. Briefly, single B cells are cultured in IL-2, IL-4, IL-21, and BAFF on CD154+ stromal cells. This culture supports extensive cell proliferation and differentiation into IgG-secreting plasmacytes. The monoclonal IgG antibodies in each cell culture can be readily screened by ELISA, Luminex, or immunofluorescence against endogenous and exogenous antigens. We find that autoantibodies from pre-B cell cultures have higher avidities for self-antigens than do those from immature/transitional and mature B cell cultures, suggesting the loss of high avidity cells at the first tolerance checkpoint. V(D)J rearrangements are easily recovered from selected clones for sequencing and expression. This culture method offers a pathway to determine B-cell repertoires with high throughput and reduced cost.
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Rungsiwiwut, Ruttachuk, Praewphan Ingrungruanglert, Pranee Numchaisrika, Pramuan Virutamasen, Tatsanee Phermthai, and Kamthorn Pruksananonda. "Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines." Stem Cells International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/4626048.
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Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.
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Chute, John P., Abha A. Saini, Dennis J. Chute, Mark R. Wells, William B. Clark, David M. Harlan, Jenny Park, Margaret K. Stull, Curt Civin, and Thomas A. Davis. "Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow." Blood 100, no. 13 (December 15, 2002): 4433–39. http://dx.doi.org/10.1182/blood-2002-04-1238.
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Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cell (SRC). Coculture of human ABM CD34+ cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34+ cells, induced more than 95% of the CD34+CD38− subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34+ cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34+ cells to be 1 in 9.9 × 105 cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 × 105cells. All mice that received transplants of HUBEC-cultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow.
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Чернов, А. Н., Е. П. Баранцевич, Э. С. Галимова, and М. М. Галагудза. "Cell cultures of human malignant tumors in development of new anticancer therapies." Nauchno-prakticheskii zhurnal «Patogenez», no. 4() (January 30, 2018): 13–23. http://dx.doi.org/10.25557/gm.2018.4.9744.
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Современный эффективный скрининг новых противоопухолевых химиопрепаратов и биологических препаратов на доклиническом этапе невозможен без применения моделей культур опухолевых клеток. К таким моделям относят первичные культуры клеток и клеточные линии опухолей человека, культивируемые в двумерной (2D) и трехмерной (3D) системах. В обзоре обсуждаются различные аспекты применения моделей клеточных культур неоплазий человека, их актуальность в исследованиях противоопухолевой эффективности препаратов. Current effective preclinical screening of new anticancer chemotherapies and biological medicines requires cancer cell culture models. Such models include primary cell cultures and human tumor cell lines cultured in two-dimensional (2D) and three-dimensional (3D) systems. This review discussed different aspects of using human tumor cell culture models and their relevance for studying efficacy of antitumor drugs.
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Kirshenbaum, A. S., J. P. Goff, S. C. Dreskin, A. M. Irani, L. B. Schwartz, and D. D. Metcalfe. "IL-3-dependent growth of basophil-like cells and mastlike cells from human bone marrow." Journal of Immunology 142, no. 7 (April 1, 1989): 2424–29. http://dx.doi.org/10.4049/jimmunol.142.7.2424.
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Abstract Human bone marrow cultured in the presence of human rIL-3 has been reported to give rise to basophils. In contrast, mouse bone marrow, cultured in the presence of mouse IL-3, leads to the growth of mast cells. To determine if human rIL-3 might also stimulate the growth of human mast cells, we cultured human bone marrow in the presence of human rIL-3 in suspension cultures, methylcellulose, and in "interphase" cultures where cells are layered over agar. The presence of mast cells was determined using a variety of histochemical techniques. In agreement with previous reports, basophil-like cells were identified in all culture systems. Mastlike cells were identified only in interphase cultures. By 3 wk, such cultures consisted of basophil-like cells (20 to 50%) and mastlike cells (1 to 5%). Cultures supplemented with rIL-4 showed no additional increase in basophil-like and mastlike cells. Both basophil-like and mastlike cells fluoresced with o-phthaldialdehyde and exhibited IgE receptors. Unlike basophil-like cells, mastlike cells were chloroacetate esterase, amidase, and human mast cell tryptase positive. We conclude that human rIL-3 can support the growth of human mastlike cells under selected culture conditions.
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Westphal, Manfred, Hildergard Nausch, and Hans-Dietrich Herrmann. "Cyst Fluids of Malignant Human Brain Tumors Contain Substances That Stimulate the Growth of Cultured Human Gliomas of Various Histological Type." Neurosurgery 25, no. 2 (August 1, 1989): 196–201. http://dx.doi.org/10.1227/00006123-198908000-00007.
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Abstract The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.
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SHARMA, REKHA, HIMANI SHARMA, SONIKA AHLAWAT, and M. S. TANTIA. "An efficient method of generating skin fibroblast cells for cell banking." Indian Journal of Animal Sciences 88, no. 8 (September 6, 2018): 905–9. http://dx.doi.org/10.56093/ijans.v88i8.82937.
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An optimum cell culture medium is used to generate fibroblast cells for cell banking. Here, we describe a method for obtaining higher number of pure fibroblast cells in much shorter duration from skin tissue explants. Two pronged strategy was used, first two different culture media were selected, one for preferential generation of fibroblast cells in primary culture (Human fibroblast specific media- HifibroXL) and another for their faster multiplication in secondary culture (DMEM + Ham's F12). Secondly, tissue explants were cultured not only once but up to six times increasing the generation of primary cells per se. This method initially standardized with buffalo (Bubalus bubalis) skin tissue explants works efficiently with camel (Camelus bactrianus) and horse (Equs caballus). Modified cell culture method increases the efficiency of establishing fibroblast cell banks by reducing the cost both in terms of consumables and human effort. It is well suited for today's fast-paced conservation laboratories.
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Edvardsson, Louise, Josefina Dykes, and Tor Olofsson. "Gene Expression Profiles, Differentiation and Lineage Commitment of Human Myeloid Progenitors." Blood 104, no. 11 (November 16, 2004): 4120. http://dx.doi.org/10.1182/blood.v104.11.4120.4120.
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Abstract With the objective to further elucidate the mechanism behind commitment to erythroid and neutrophil lineages, we isolated by cell sorting common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), based on the surface expression of CD123 (IL3R-α) and CD45RA on human CD34+ bone marrow cells (first described by Manz et al. PNAS, 2002;99:11872). Methylcellulose cultures supporting the growth of myeloid and erythroid progenitors, and real-time RT-PCR mapping the gene expression of Flt3, c-kit, TpoR, GATA-2, GATA-1, SCL, NF-E2, EpoR, ABO, β-globin, GPA, PU.1, C/EBPα, C/EBPε, G-CSFR, proteinase 3 (PR3) and lactoferrin, were used to validate and characterize the progenitors and their progeny. Cell sorted progenitors were labeled with CFDA, SE to track cell division and cultured in suspension to induce neutrophil or erythroid differentiation (SCF+G-CSF for neutrophil and Epo+IL–3+GM–CSF for erythroid culture). After 3–5 days, cells that had gone through 1–8 divisions were sorted and changes in clonogenicity and gene expression were studied. The CMP-population retained some clonogenicity after as many divisions as were tested (at the most six divisions in erythroid and eight in neutrophil culture) and the CMPs differentiated along the lineage defined by the culture system, as evidenced both by the methylcellulose cultures and an increasing expression of GATA-1 and EpoR in erythroid and PU.1, G-CSFR and PR3 in neutrophil cultures, respectively. On the other hand, the GMP-population displayed granulocyte/monocyte (G/M)-differentiation irrespective of the culture system used, although it divided fewer times and lost its clonogenic capacity faster in erythroid culture. Little or no clonogenicity remained after 4–5 divisions in erythroid culture, while some colony-forming capacity remained even after seven divisions in neutrophil culture (maximum number tested). The increased expression of the granulopoiesis-associated genes was also less pronounced in the erythroid culture. The MEP-population dominated by erythroid differentiation capacity retained colony-forming capacity for at least 6–7 divisions in both erythroid and neutrophil cultures, although with a higher overall clonogenicity in erythroid culture. Unexpectedly, however, MEPs were restricted to G/M-differentiation when cultured in neutrophil culture. In cells from erythroid culture the expression of GATA-1, EpoR and β-globin increased, while a corresponding pattern was seen for PU.1, G-CSFR and PR3 in neutrophil culture. Overall, our data support the progenitor classification, based on the surface expression of CD123 and CD45RA, with regard to CMP and GMP populations but question it with regard to the MEP-population. The change in differentiation course for the MEPs in neutrophil culture could be a result of an initially present G/M-potential or a less strict commitment susceptible to cytokine-induced redifferentiation.
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Okamoto, M., Y. Ishida, A. Keogh, and A. Strain. "Evaluation of the Function of Primary Human Hepatocytes Co-Cultured with the Human Hepatic Stellate Cell (HSC) Line LI90." International Journal of Artificial Organs 21, no. 6 (June 1998): 353–59. http://dx.doi.org/10.1177/039139889802100607.
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Most bioartificial liver devices utilise primary hepatocytes alone although some have considered the use of non parenchymal cells in addition. However the effects of co-culture of human hepatocytes with different sinusoidal cell types has not been fully investigated. In this study we have examined the influence of co-culturing primary human hepatocytes with the human hepatic stellate cell (HSC) line, LI90. Cultures were monitored by light microscopy and on days 4, 8 and 14 urea synthesis and cytochrome P450 activity were measured. Morphologically LI90 cells proliferated to fill spaces between and into adjacent islands of hepatocytes. On day 14 cytochrome P450 activity in co-culture was significantly improved compared to hepatocytes cultured alone. By contrast, urea synthesis in hepatocytes was unaffected by single or co-culture. Therefore it can be concluded that a combination of primary human hepatocytes with LI90 cells is beneficial for growth and some stability of hepatocytes and may therefore be appropriate for seeding bioartificial liver devices.
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De Simone, Uliana, Francesca Caloni, Laura Gribaldo, and Teresa Coccini. "Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds." International Journal of Toxicology 36, no. 6 (November 2017): 463–77. http://dx.doi.org/10.1177/1091581817739428.
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Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell–cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 μM), (2) Fe3O4 nanoparticles (1-100 μg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron–astrocyte metabolic interactions.
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Richter-Dahlfors, Agneta, Ursula Heczko, R. Mark Meloche, B. Brett Finlay, and Alison M. J. Buchan. "Helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function." American Journal of Physiology-Gastrointestinal and Liver Physiology 275, no. 3 (September 1, 1998): G393—G401. http://dx.doi.org/10.1152/ajpgi.1998.275.3.g393.
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Although Helicobacter pylori infection increases gastrin secretion, it is unknown whether this is a direct effect or requires activation of the immune system. We developed an H. pylori-infected human primary antral epithelial cell culture model to address this question. This culture protocol favors growth of H. pylori, and infected cultures could be maintained for up to 48 h. These cultures were enriched for gastrin (10–40%), somatostatin (2–5%), and gastric mucin (60–80%) cells but did not contain immunocytes. Bacterial attachment occurred in a random manner within 2 h of infection, although bacterial density was lower than in sections from infected patients. After 24 or 48 h, the bacterial microcolonies were similar in size to those seen in vivo, and at 24 h ultrastructural studies demonstrated well-developed pedestal formation underlying the bacteria. Coculture with H. pylori increased basal but not stimulated gastrin secretion at all time points >2 h. In conclusion, a newly developed cell culture model has been used to characterize the interactions between H. pylori and normal human antral epithelial cells.
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Bayley, Jean-Pierre, Heggert G. Rebel, Kimberly Scheurwater, Dominique Duesman, Juan Zhang, Francesca Schiavi, Esther Korpershoek, Jeroen C. Jansen, Abbey Schepers, and Peter Devilee. "Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas." PLOS ONE 17, no. 9 (September 30, 2022): e0274478. http://dx.doi.org/10.1371/journal.pone.0274478.
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The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4–8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures.
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Klein, Christian, Christian Meller, and Edgar Schäfer. "Human Primary Odontoblast-like Cell Cultures—A Focused Review Regarding Cell Characterization." Journal of Clinical Medicine 11, no. 18 (September 8, 2022): 5296. http://dx.doi.org/10.3390/jcm11185296.
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Cell cultures can provide useful in vitro models. Since odontoblasts are postmitotic cells, they cannot be expanded in cell cultures. Due to their extension into the dentin, injuries are inevitable during isolation. Therefore, “odontoblast-like” cell culture models have been established. Nowadays, there is no accepted definition of odontoblast-like cell cultures, i.e., isolation, induction, and characterization of cells are not standardized. Furthermore, no quality-control procedures are defined yet. Thus, the aim of this review was to evaluate both the methods used for establishment of cell cultures and the validity of molecular methods used for their characterization. An electronic search was performed in February 2022 using the Medline, Scopus, and Web of Science database identifying publications that used human primary odontoblast-like cell cultures as models and were published between 2016 and 2022. Data related to (I) cell culture conditions, (II) stem cell screening, (III) induction media, (IV) mineralization, and (V) cell characterization were analyzed. The included publications were not able to confirm an odontoblast-like nature of their cell cultures. For their characterization, not only a similarity to dentin but also a distinction from bone must be demonstrated. This is challenging, due to the developmental and evolutionary proximity of these two tissue types.
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Frias, Ana, Christopher D. Porada, Kirsten B. Crapnell, Joaquim M. S. Cabral, Esmail D. Zanjani, and Graca Almeida-Porada. "Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System." Blood 104, no. 11 (November 16, 2004): 2888. http://dx.doi.org/10.1182/blood.v104.11.2888.2888.
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Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.
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Su, Kuei-Ying, Masayuki Kuraoka, Akiko Watanabe, and Garnett Kelsoe. "Efficient culture of human naïve and memory B cells (LYM5P.711)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 134.16. http://dx.doi.org/10.4049/jimmunol.194.supp.134.16.
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Abstract The ability to culture and expand B cell numbers in vitro has become a useful tool for studying human immunity. A limitation of current methods in human B cell cultures is the capacity to support continued proliferation by mature B cells. We have adapted the culture methods established by Luo et al. (Blood, 2009) to efficiently (60% cloning efficiency) activate and expand both naïve and memory human B cells. Briefly, naïve or memory B cells recovered from blood are cultured with cytokines on CD154+ feeder cells; this culture supports extensive B cell proliferation, with approximately 103 fold increases in a week; by splitting cultures onto new feeder layers, culture-derived (CD) B cells continuously proliferate, achieving 106 fold increases by day 16. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells upregulate AID expression, undergo isotype switching without V(D)J hypermutation, and terminally differentiate into plasmacytes. CD B cells are readily cryopreserved and retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. We have examined the APC function of CD B cells and find that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.
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Rühle, Alexander, Marie Lies, Maren Strack, Ramon Lopez Perez, Birgit Bieber, Andreas R. Thomsen, Peter Bronsert, et al. "Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma." International Journal of Molecular Sciences 23, no. 14 (July 12, 2022): 7689. http://dx.doi.org/10.3390/ijms23147689.
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Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.
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Yaghoubi, Yoda, Majid Zamani, Adel Naimi, Ali Hassanzadeh, Nastaran Gharibeh, Javad Madani, Roza Motevali, et al. "Human CD34+ hematopoietic stem cells culture in humanized culture medium for cell therapy." Gene Reports 20 (September 2020): 100718. http://dx.doi.org/10.1016/j.genrep.2020.100718.
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Le, Quy, Tiffany A. Hylkema, Sommer Castro, Jenny L. Smith, Amanda R. Leonti, Thao T. Tang, Cynthia Nourigat-Mckay, et al. "Endothelial Cell Niche Promotes Leukemic Transformation of Human Cord Blood Stem/Progenitor Cells Expressing CBFA2T3-GLIS2 Oncogenic Fusion." Blood 138, Supplement 1 (November 5, 2021): 360. http://dx.doi.org/10.1182/blood-2021-148916.
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Abstract The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo. Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells. The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs. To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs. Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E). To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs. Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. Figure 1 Figure 1. Disclosures Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.
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Harrnand, M. F. "Human cell culture and characterization of cell/biomaterial interface." Clinical Materials 11, no. 1-4 (January 1992): 145–50. http://dx.doi.org/10.1016/0267-6605(92)90040-z.
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Tung, Nguyen Thanh, Thi Pham Thi Diem, Dang Ngoc Sang, Do Thi Thao, and Hoang Tan Quang. "Biomass Accumulation of Gynostemma Pentaphyllum (Thunb.) Makino in Cell Suspension Cultures inhibiting Human Cancer Cell Growth." Research Journal of Biotechnology 17, no. 3 (February 25, 2022): 61–68. http://dx.doi.org/10.25303/1703rjbt6168.
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Gynostemma pentaphyllum (Thunb.) Makino (GpM) is a medicinal plant in traditional medicine throughout Asia for the treatment of several diseases including cancer. GpM plant cell suspension cultures provide a time and cost effective well-controlled means promising a high-yielding biomass production of pharmaceutical compounds. The purpose of the current work is to investigate the effect of GpM cell suspension cultures on human cancer cell lines growth. The biomass was produced by cell suspension culture of GpM callus into 250 mL Erlenmeyer flask containing 50 mL of liquid medium culture. Gypenosides in GpM were confirmed by HPLC. Pharmacological activities of GpM extract were tested on human cancer cell line (HepG2, Huh7, A549 and HL-60) using Sulforhodamine B (SRB) cytotoxicity assay and Tetrazolium (MTT) assay. We successfully produced 5.485 ± 0.223 g GpM fresh biomass per 250 mL Erlenmeyer flask after 18 days culture. Total gypenosides and Rb1 in dry cell suspension were 48.844 ± 3.933 mg/g and 0.041 ± 0.004 mg/g. The crude extract from GpM cell suspension cultures exhibited significant cancerous cell growth inhibition in a dose dependent manner. From the MTT assay and SRB cytotoxicity assay, it is obvious that GpM cell suspension culture extract at 200 μg/mL significantly inhibited the growth of multiple human cancer cells including hepatoma cell lines (HepG2, Huh7), lung carcinoma cell line (A549) and leukemia cell line (HL-60). Anti-cancer cell proliferation properties of GpM cell suspension culture were significantly higher than those of natural plants’ extracts. In this framework, GpM in cell suspension cultures was found to inhibit the proliferation of several human cancer cells. Biomass accumulation of GpM in cell suspension cultures may contribute to the development of efficient strategies for plant-derived anticancer compound production.
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Huhtala, Anne, Sami K. Nurmi, Hanna Tähti, Lotta Salminen, Päivi Alajuuma, Immo Rantala, Heikki Helin, and Hannu Uusitalo. "The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line." Alternatives to Laboratory Animals 31, no. 4 (July 2003): 409–17. http://dx.doi.org/10.1177/026119290303100407.
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Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.
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Quesenberry, P. J., M. Del Tatto, D. Berz, T. Miner, T. Ng, E. S. Winer, J. Aliotta, et al. "Marrow cell genetic phenotype change induced by human lung cancer cells." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 11108. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.11108.
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11108 Background: Murine lung-derived microvesicles are capable of inducing lung-specific mRNA in marrow cells, when co-cultured across from these cells, but separated from them by a cell-impermeable (0.4 micron) membrane. These converted murine marrow cells showed mRNA elevations, lung-specific protein production and enhanced capacity to convert to lung epithelial cells after in vivo transplantation into irradiated mice. We examine here whether fresh tissue from lung cancer patients would have the same capacity to genetically alter co-cultured human marrow cells. Methods: Lung cancer samples were collected from 5 patients undergoing surgery. Minced tumor tissue at 50–100 mg was co-cultured in a semi-permeable culture plate insert opposite 3.0 ×106 human marrow cells. The marrow cells were harvested after 2–7 days of co-culture. Marrow cell RNA was analyzed for lung specific mRNA using real time RT-PCR. Relative levels of gene expression was expressed a fold increase compared to level in controls. Results: Lung cancers studied were adenocarcinoma, endobronchial alveolar carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma and squamous cell carcinoma. mRNAs for aquaporin 1–5, specific for type I pneumocytes and surfactant A-D, specific for type II pneumocytes, were measured. Aquaporin I was elevated in marrow cells from co culture with all lung cancers; elevations ranging from 2.15 to 56.7 fold (mean 23 fold). Similarly surfactant B mRNA was induced in marrow cells by all lung cancers with fold elevations ranging from 7.9 to 2164 (mean fold elevation 668). More variable elevations were also seen with aquaporin 3, 4, and 5, surfactant A, surfactant C, and surfactant D. Ultracentrifugation (28,000 g) of conditioned media from these cancers revealed the presence of microvesicles with diameters of 100–180 nm. Conclusions: These observations indicate that the genetic phenotype of cells in the vicinity of lung cancer cells can be altered and that these alterations might be mediated by microvesicle transfer of genetic information. No significant financial relationships to disclose.
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Muramoto, Garrett G., Rachid Safi, Alice Salter, Heather Himburg, Nelson J. Chao, Donald McDonnell, and John P. Chute. "The Retinoid X Receptor Regulates Human Hematopoietic Stem Cell Fate." Blood 108, no. 11 (November 16, 2006): 1324. http://dx.doi.org/10.1182/blood.v108.11.1324.1324.
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Abstract Our laboratory has recently demonstrated that competitive inhibition of the enzyme, aldehyde dehydrogenase-1 (ALDH-1), in human hematopoietic stem cells (HSCs) facilitates the 3.4-fold expansion of primitive cells capable of repopulating non-obese diabetic/severe combined immune deficient (NOD/SCID) mice. Since ALDH-1 activity is required for the intracellular production of retinoic acids, we hypothesized that retinoid signaling could be fundamentally involved in human HSC fate determinations. In order to demonstrate a link between ALDH-1 activity and retinoid signaling in human HSCs, we cultured human CD34+CD38−lin- HSC-enriched cells with retinaldehyde, which is a specific substrate for ALDH-1, and found that this induced a 10-fold increase in the expression of cEBP-epsilon, which is a Retinoic Acid Receptor (RAR)-dependent transcription factor. Moreover, when the competitive inhibitor of ALDH-1, diethylaminobenzaldehyde (DEAB), was added to HSC cultures supplemented with retinaldehyde, the expression of cEBP-epsilon was completely blocked, confirming a causal relationship between ALDH-1 activity and retinoid signaling in HSCs. We then sought to determine whether direct agonism or antagonism of the RAR or Retinoid X Receptor (RXR) could alter human HSC fate in short-term culture. We cultured primary human bone marrow (BM) and cord blood (CB) CD34+CD38−lin- HSC-enriched cells with hematopoietic cytokines, thrombopoietin (T), stem cell factor (S) and flt-3 ligand (F) in the presence and absence of several agonists and antagonists of RAR and RXR. Seven day culture of BM or CB CD34+CD38−lin- cells with TSF alone resulted in a loss of CD34+CD38− cells in culture, a pronounced increase in colony forming cell (CFC) activity and a complete loss of primitive SCID-repopulating cells (SRCs) compared to input, reflecting HSC differentiation during culture. Interestingly, the addition of either all-trans retinoic acid (ATRA), an agonist of RAR and RXR, or TTNPB, a selective RAR agonist, induced an accelerated differentiation of HSCs in culture compared to cytokines alone. Surprisingly, the addition of LGD815 (courtesy of Ligand Pharmaceuticals, San Diego, CA), a selective RAR antagonist, had no effect on the differentiation of HSCs that otherwise occurred in response to cytokines alone. However, the addition of LGD101506, a partial antagonist of RXR, preferentially maintained phenotypic CD34+CD38− cells in culture and inhibited CFC production in response to cytokines. Moreover, RXR antagonism maintained or induced the expansion of SRC content in culture, whereas SRC content was completely lost following cytokine culture alone. Taken together, these data demonstrate for the first time that RXR signaling contributes to the intrinsic regulation of human HSC fate. Pharmacologic inhibition of RXR signaling impedes the normal differentiation of HSCs that otherwise occurs in response to proliferation-inducing cytokines. These data provide further evidence for the role of retinoid signaling in the differentiation program of human HSCs and indicate that RXR and its heterodimeric partners are novel targets for the purpose of facilitating human HSC expansion.
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Chitturi Suryaprakash, Ravi Teja, Omar Kujan, Kate Shearston, and Camile S. Farah. "Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review." International Journal of Molecular Sciences 21, no. 24 (December 14, 2020): 9520. http://dx.doi.org/10.3390/ijms21249520.
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Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.
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Lucey, Brendan P., Walter A. Nelson-Rees, and Grover M. Hutchins. "Henrietta Lacks, HeLa Cells, and Cell Culture Contamination." Archives of Pathology & Laboratory Medicine 133, no. 9 (September 1, 2009): 1463–67. http://dx.doi.org/10.5858/133.9.1463.
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Abstract Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.
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Grigull, Nele Pascale, Julia Isabelle Redeker, Bärbel Schmitt, Maximilian Michael Saller, Veronika Schönitzer, and Susanne Mayer-Wagner. "Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel." International Journal of Molecular Sciences 21, no. 8 (April 16, 2020): 2785. http://dx.doi.org/10.3390/ijms21082785.
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Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.
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Kanno, Kaho, Tomohisa Sakaue, Mika Hamaguchi, Kenji Namiguchi, Daisuke Nanba, Jun Aono, Mie Kurata, Junya Masumoto, Shigeki Higashiyama, and Hironori Izutani. "Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness." International Journal of Molecular Sciences 22, no. 19 (September 29, 2021): 10534. http://dx.doi.org/10.3390/ijms221910534.
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The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.
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Usui, Satoko, Takeshi Shimizu, Kenichiro Fujita, Chikako Kishioka, and Yasuo Sakakura. "Secretory Cell Differentiation and Mucus Secretion in Cultures of Human Nasal Epithelial Cells: Use of a Monoclonal Antibody to Study Human Nasal Mucin." Annals of Otology, Rhinology & Laryngology 109, no. 3 (March 2000): 271–77. http://dx.doi.org/10.1177/000348940010900307.
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We have developed an air-liquid interface culture system for human nasal epithelial cells that differentiate into mucociliary phenotypes in a defined serum-free medium. Dissociated cells obtained from nasal polyps were cultured on a collagen gel substrate. At confluence, the cells lost characteristics of differentiated cells, and secretory cell and ciliated cell differentiation appeared after 7 days in an air-liquid interface. After 21 days, about half of the epithelial cells were stained with Alcian blue—periodic acid—Schiff stain or monoclonal antibody HCS18, which was directed against human nasal mucin specific for epithelial secretory (goblet) cells. The quantitative examination using the antibody HCS 18 revealed that the antibody-reactive nasal mucin was secreted only on the apical side of the cultures, and interleukin-1 β and tumor necrosis factor α stimulated these mucus secretions. The culture system with an antimucin monoclonal antibody developed in this study should be useful for studying polarized mucus secretion from human nasal epithelial cells.