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Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares.
Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.
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Humans , Connective Tissue Cells , Serum Albumin, Bovine , Fibroblasts , Cell- and Tissue-Based TherapyABSTRACT
RESUMEN Objetivo. Describir la influencia del Suero Fetal Bovino (SFB) en la supervivencia, crecimiento y expresión de organelas celulares en las células epiteliales dentales de rata. Materiales y métodos. Cultivos de células epiteliales dentales de rata fueron llevados a cabo a 37°C en una atmosfera húmeda, en ausencia y a una concentración de 10% de SFB. Una evaluación morfológica fue realizada durante la proliferación y confluencia de las células en cultivo. Dobles marcajes por inmunofluorencia fueron efectuados haciendo uso de anticuerpos anti-actina, anti-TOMM20 y anti-LAMP1. Resultados. Se evidenciaron células epiteliales dentales circulares u ovoides con núcleos voluminosos durante la proliferación y confluencias de manera similar en las células cultivas en presencia y ausencia de SFB. La carencia de SFB impactó negativamente la proliferación de las células epiteliales. No fueron observadas alteraciones en la localización de los inmunomarcajes anti-actina, anti-TOMM20 y anti-LAMP1 en las dos condiciones de cultivos experimentales. Conclusiones. La supresión del SFB en el cultivo de células epiteliales dentales de rata disminuyó la supervivencia, proliferación y sugiere no tener un impacto sobre las organelas evaluadas.
ABSTRACT Objective. Describe the influence of Fetal bovine serum (FBS) on the survival, growth and expression of cellular organelles in rat dental epithelial cells. Material and methods. Cell cultures of rat dental epithelial cells were carried out at 37°C in a humid atmosphere, in the absence and at a concentration of 10% FBS. Morphological evaluation was performed during the proliferation and confluence of cell in culture. Double immunofluorescence labels were made using anti-Actin, anti-TOMM20A, and anti-LAMP1 antibodies. Results. Circular or ovoid dental epithelial cells with bulky nuclei were evidenced during proliferation and confluences in a similar manner in culturing cells in the presence and absence of FBS. The lack of FBS negatively impacts the proliferation of epithelial cells. No alterations were observed in the localization of the anti-actin, anti-TOMM20 and anti-LAMP1 immunomarkers in both conditions of experimental cultures. Conclusion. FBS suppression in rat dental epithelial cells decreased survival, proliferation and suggests not having an impact on the organelles evaluated.
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Animals , Cattle , Serum Albumin, Bovine , Cattle , Dental Enamel , Epithelial CellsABSTRACT
Resumen El esmalte dental, tejido más duro del cuerpo humano, es formado por medio de la diferenciación de células epiteliales conocidas como ameloblastos. Recientemente, las células epiteliales dentales de incisivo de rata han sido utilizadas como modelo celular de eventos fisiopatológicos durante la formación del esmalte dental. Sin embargo, en función de los eventos estudiados es meritorio comprender su comportamiento, particularmente cuando la concentración del Suero Fetal Bovino (SFB) varia. El propósito de este trabajo es evaluar el impacto de la concentración del SFB sobre el crecimiento, proliferación y supervivencia de las células epiteliales dentales. Células epiteliales dentales fueron cultivadas en DMEM/F12, en presencia y ausencia de SFB. Observaciones morfológicas e inmunohistoquímicos de la actina, vimentina y fibronectina fueron realizados. Los resultados permitieron constatar que la ausencia de SFB afectó negativamente la proliferación de las células epiteliales dentales, pero mantuvo una expresión óptima de la actina y vimentina. Se identificó una alteración en la expresión de la fibronectina en las células tratadas en ausencia de SFB. En conclusión, la carencia de SFB disminuyo drásticamente la supervivencia, proliferación y expresión de la fibronectina en las células epiteliales dentales de rata.
Abstract Dental enamel, the hardest tissue in the human body is formed by the differentiation of epithelial cells known as ameloblasts. Recently rat incisor dental epithelial cells have been used as a cellular model of pathophysiological events during tooth enamel formation. However depending on the events studied, it is necessary to understand the behavior of these cells, particularly when the concentration of Bovine Fetal Serum (FBS) varies. The purpose of this work is to evaluate the impact of FBS concentration on the growth, proliferation, and survival of dental epithelial cells. Dental epithelial cells were cultured in DMEM/F12 culture medium in the absence and presence of 10% FBS. Morphological and immunohistochemical observations of actin, vimentin, and fibronectin were performed. The results confirm that the absence of FBS negatively affected the proliferation of dental epithelial cells but maintained an optimal expression of actin and vimentin. An alteration in the expression of fibronectin in the cells treated in the absence of FBS was identified. In conclusion, the lack of FBS dramatically decreased the survival, proliferation, and expression of fibronectin in rat dental epithelial cells.
RESUMO O esmalte dentário, o tecido mais duro do corpo humano, é formado pela diferenciação de células epiteliais conhecidas como ameloblastos. Recentemente, células epiteliais dentárias de incisivo de rato têm sido utilizadas como modelo celular de eventos fisiopatológicos durante a formação do esmalte dentário. No entanto, dependendo dos eventos estudados, é meritório entender seu comportamento, particularmente quando a concentração de soro fetal bovino (FBS) varia. O objetivo deste trabalho é avaliar o impacto da concentração de SFB no crescimento, proliferação e sobrevivência de células epiteliais dentárias. Células epiteliais dentárias foram cultivadas em DMEM / F12, na presença e ausência de SFB. Observações morfológicas e imunohistoquímicas de actina, vimentina e fibronectina foram realizadas. Os resultados permitiram confirmar que a ausência de SFB afetou negativamente a proliferação de células epiteliais dentárias, mas manteve uma ótima expressão de actina e vimentina. Uma alteração na expressão de fibronectina nas células tratadas na ausência de SFB foi identificada. Em conclusão, a falta de SFB diminuiu drasticamente a sobrevivência, proliferação e expressão de fibronectina em células epiteliais dentárias de ratos
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Objective To establish a culture method for primary human nail matrix cells in serumfree media.Methods Nail matrix tissues were collected from 9 patients,who received nail or toe amputation and nail bed repair in Peking University Shenzhen Hospital between January 2016 and December 2016,and cultured in the serum-free DEME/F-12 media at a 37℃ incubator with an atmosphere of 5％ CO2 in air for 2-3 days.Then,primary human nail matrix cells were cultured in keratinocyte serumfree media (CnT-07),and the morphology of human nail matrix cells was observed by microscopy during the culture process.Immunofluorescence cytochemistry with anti-keratin 5 (K5) and K10 was performed to identify the acquired cells,and flow cytometry to analyze the cell purity.Results After 2 or 3 days of the culture,some cells began to crawl out from the tissue.On day 10,large cell masses were formed,some cells were morphologically similar to epithelioid cells arranged in a paving stone-like pattern,and some were flat giving a spindle-shaped or star-shaped appearance.Immunofluorescence cytochemistry showed that some cells could express both K5 and K10,which proved the existence of nail matrix cells,and 37.6％ of the cells expressed K10.Conclusion Human primary nail matrix cells could be successfully cultured by using the tissue culture method with serum-free culture media,and the nail matrix cells cultured in vitro can express both K5 and K10.
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Objective@#To explore the biological characteristics of synovial fluid-derived mesenchymal stem cells (SF-MSCs) cultured in serum-free medium and the ability of in vitro reconstruction of three-dimensional cartilage combined with scaffold material.@*Methods@#Human SF-MSCs were cultured in serum medium and mesenchymal stem cells medium-serum free (MSCM-sf) respectively, then the proliferative ability and morphology of SF-MSCs were compared; The third passage SF-MSCs cultured in MSCM-sf were identified by flow cytometry, three-way(chondrogenic, osteogenic, adipogenic)differentiation assay and induced for chondrogenic differentiation when combined with polyglycolic acid/polylactic acid (PGA/PLA).@*Results@#SF-MSCs cultured in MSCM-sf had better morphology and proliferative ability than that cultured in serum medium. The expression levels of positive markers of the third passage SF-MSCs cultured in MSCM-sf, such as CD73 (99.5%), CD90 (98.9%) and CD105 (96.5%), were more than 95%. However, the overall negative markers (CD34, HLA-DR and CD11b) expressed less than 2%. Three-way differentiation staining was positive. The combination of SF-MSCs and PGA / PLA can be induced into cartilage in vitro.@*Conclusions@#SF-MSCs cultured in MSCM-sf can be amplified under the condition of maintaining the stem cell characteristics, and can be combined with PGA/PLA scaffold to construct three-dimensional cartilage in vitro.
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BACKGROUND:Until now, there is yet no complete recovery from spinal cord injury in terms of structure and functional recoveries. Neurotrophic factors have limited effects on nerve regeneration. Currently, stem cel transplantation may be an effective way to repair spinal cord injury. OBJECTIVE:To separate, cultivate and purify mouse spinal cord-derived neural stem cels using serum-free suspension method folowed by morphological observation, immunofluorescence technology and multi-lineage differentiation experiments. METHODS:By using the suspension culture method, mouse spinal cord-derived neural stem cels at embryonic day 13.5 were cultured and purified. Cel morphology changes were observed under inverted microscope. Cel proliferation ability was detected using cel counting kit-8. Nestin and Sox2 expression was detected by immunofluorescence technology. Multilineage differentiation of spinal cord-derived neural stem cels at passage 4 was detected by natural differentiation method in order to prove the differentiation ability. RESULTS AND CONCLUSION: Serum-free medium suspension culture method was successfuly applied to separate spinal cord-derived neural stem cels. Cultured cels had good proliferative ability and highly expressed Nestin and Sox2 that was in accordance with the results of DAPI nucleus staining, suggesting the high purity of cels. After induction, the cels could express both Tuj1 and GFAP, indicating the cels had good differentiation potential. This experiment has successfuly established the isolation, culture, identification system of spinal cord-derived neural stem cels, providing experimental basis for subsequent studies of neural stem cels.
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BACKGROUND:Human embryonic stem cels are able to self-renew indefinitely and have the capacity to differentiate into al three germ layers (ectoderm, endoderm and mesoderm). These properties imply great potential in the basic research and clinical application, including regenerative medicine, drug screening and toxins, early human embryo, cel transplantation, gene therapy,etc. However, it is a substantial chalenge to develop efficient techniques for their large-scale culture under defined conditions, and for controling and directing their differentiation. For therapeutic purposes, many scholars are trying to establish methods for maintaining pluripotency in defined xeno-free conditions and scalable culture systems. OBJECTIVE:To discuss the progress of serum-free culture systems in human embryonic stem cel research reported in recent years and to highlight the chalenges and advances being made towards the development of serum-free and xeno-free culture systems suitable for therapeutic applications. METHODS:A computer-based search of CNKI and PubMed academic database was performed for articles addressing serum-free culture systems of human embryonic stem cels published from 2008 to 2015. Repetitive and old articles were excluded. Finaly, 58 articles were summarized. RESULTS AND CONCLUSION:Several groups have attempted to exclude individual animal components by using feeder-free matrices, feeder cels of human origin, or defined xeno-free media, aiming to select a suitable matrix and medium that can minimize or not use heterologous components, in order to obtain cel lines at clinical level. However, the current cel products are far from clinical application. There are stil many problems to be solved, such as standardization, normalization and individualization of cel products. With the normative development of stem cel research and industry, human embryonic stem cel products are expected to be widely used in clinic.
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BACKGROUND:Lung cancer stem cel s are tightly related to the treatment and prognosis of lung cancer. We can provide more references for clinical diagnosis and treatment of lung cancer through the study on the tumorigenicity and surface markers of lung cancer stem cel s. OBJECTIVE:To explore the enrichment methods for lung cancer stem cel s and cel ular tumorigenicity. METHODS:Lung cancer stem cel s were induced in serum-free culture medium containing epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. Then, the expressions of related surface markers were detected using immunofluorescence method. After that, mice were implanted subcutaneous with lung cancer stem cel spheres to understand the tumorigenicity of lung cancer stem cel s. RESULTS AND CONCLUSION:Lung cancer stem cel s under serum-free induction and culture were changed to sphere-forming cel s, and the immunofluorescence detection showed that over 80%of sphere-forming cel s were positive for CCSP, SP-C and OCT4. After transplantation of sphere-forming cel s, the mice showed a high tumorigenicity. These findings indicate that sphere-forming cel s are formed after serum-free suspension culture of lung cancer stem cel s, which have a higher tumorigenicity.
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BACKGROUND:Ischemia microenvironment contributes mostly to the low survival rate of rat bone marrow mesenchymal stem cells after transplantation. Hydrogen sulfide (H2S) can protect various cells and tissue models against apoptosis and injury. OBJECTIVE:To detect the cellapoptosis and viability, content of H2S in supernatant, and the expression of H2S synthetase after different time of hypoxia and serum deprivation cultivation of rat bone marrow mesenchymal stem cells. METHODS:The passage 3 rat bone marrow mesenchymal stem cells were divided into five different cultivation time groups:0-, 3-, 6-, 12-and 24-hour groups. After enough hypoxia and serum deprivation cultivated time, the cellapoptosis was detected by SubG1, the cellviability was determined by cellcounting kit-8, the content of H 2S in supernatant was measured by N,N-dimethyl-p-phenylenediamin and the expression of H2S synthetase by RT-PCR and western blot. RESULTS AND CONCLUSION:Compared to the normal cultivation group, after different hypoxia and serum deprivation cultivated time, the cellapoptosis increased and cellviability decreased significantly. The longer hypoxia and serum deprivation cultivated time caused the more cellapoptosis and the lower cellviability. The contents of H2S and its synthetase were also suppressed by hypoxia and serum deprivation cultivation. The difference was statistical y significant. These findings suggest that hypoxia and serum deprivation cultivation can inhibit the generation of H 2 expression of its synthetase.
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Células dendríticas (DCs) são as principais células apresentadoras de antígeno do sistema imune, capazes de estimular o linfócito T a iniciar resposta imune especifica. Vacinas de DCs vêm sendo utilizadas como forma de tratamento imunoterápico adjuvante para várias neoplasias. Protocolos para geração dessas células têm sido desenvolvidos e o método ideal de produção para uso clínico ainda necessita ser definido. É fundamental a definição de protocolos e reagentes que ofereçam, a partir de células mononucleares do sangue periférico, células dendríticas seguras e funcionais para uso clínico. A suplementação de meios de cultura com soro de origem animal e humano leva á riscos de xenosensibilização e transmissão de doenças. O uso do soro autólogo parece oferecer menos riscos ao paciente, porém a presença de fatores imunossupressores nesse soro poderia interferir na qualidade das DCs produzidas. Vários tipos de meios livres de soro, baseados nas boas práticas de produção - "good manufacture practice" (GMP), têm sido utilizados recentemente e parecem ser uma opção viável. O objetivo desse estudo foi avaliar os resultados da diferenciação, maturação e funcionalidade de DCs de pacientes com LMA, produzidas em meios livres de soro e em meio suplementado com soro autólogo. Concluímos que os meios de cultura livres de soro foram eficientes na produção de DCs para fins imunoterápicos em pacientes com LMA. Em contrapartida, o uso de soro autólogo parece interferir na capacidade funcional das DCs geradas.
Dendritic cells (DCs) are the main antigen-presenting cells of the immune system, capable of stimulating T lymphocytes to initiate specific immune responses. Vaccines based on DCs have been used as a treatment adjuvant immunotherapy for various malignancies. Protocols for generating these cells have been developed and the optimal method of production for clinical use remains to be defined. There is a great interest in the definition of protocols and reagents providing from peripheral blood mononuclear cells, functional and safe dendritic cells for clinical use. Supplementation of culture media with serum from animal and human leads to reactions due the animal proteins and transmission of disease. The use of autologous serum seems to offer less risk to the patient, but the presence of immunosuppressive factors may affect the quality of the DCs produced. Several types of serum-free media, based on "good manufacture practice" (GMP), have been used recently and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation and function of DCs from AML patients, generated in serum-free media and media supplemented with autologous serum. We concluded that the serum-free media were efficient in the production of DCs for immunotherapy in AML patients. However, the use of autologous serum appears to interfere with the functional capacity of generated DCs.
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Humans , Male , Female , Adult , Dendritic Cells , Immunotherapy , Leukemia, Myeloid, Acute/therapy , Culture Media, Serum-FreeABSTRACT
Objective To investigate the tissue structure of the human scalp hair follicles,the ability of hair follicle regeneration in vitro and the influence elements of growth and proliferation of follicle organ.Methods The paraffin sections of the 15 cases of human scalps and 150 cases of scalp hair follicle tissues were prepared,and morphological characteristics and organizational structures was observed by HE staining.The experience and growth pattern of the hair follicle that was cultured in Williams E medium in vitro were summarized.Results Human scalp contained a large number of hair follicles and sebaceous glands,sweat glands,and other skin accessories.Epithelial cells were rich in hair follicles and poorly differentiated in structure,and grew in the regular direction in scalp tissue.The Williams E was used to culture successfully human scalp hair follicles.Hair follicles grew with the fastest speed in the first 5 days.Compared with other media,the use of serum-free Williams E medium in the hair follicle in vitro experiments achieved better results.Conclusions Human scalp contained a large number of hair follicles and sebaceous glands,sweat glands,and other skin accessories.Williams E medium without serum medium was more conducive than other medium for hair follicle culture in vitro,so that regeneration of hair follicles in vitro with a better capacity created favorable conditions for the delay in hair transplant surgery.
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BACKGROUND:Restoration of neurological functions after the damaged peripheral nerve is reconstructed is a hot topic in existing research. Within a short term fol owing peripheral nerve injury, nerve and muscle begin to develop irreversible degeneration. Restoration of the damaged nerve requires delayed degeneration and basic microenvironment. OBJECTIVE:To investigate the protective effect of cardiotrophin-1 on PC12 cells and Schwann cells. METHODS:Schwann cells and PC12 cells were obtained and cultured in complete medium, serum-free medium and 50 ℃ medium, respectively. cells in cardiotrophin-1 group were treated with exogenous cardiotrophin-1 solvent, while those in the control group were treated with equivalent Dulbecco’s modified Eagle’s medium, for 24 hours. The survival rate for PC12 cells and Schwann cells was determined using cellCounting Kit-8 colorimetric method. The lactate dehydrogenase activity in supernatant was detected by lactate dehydrogenase kit, and the malondialdehyde content and superoxide dismutase activity were measured by thiobarbituricaicd and xanthine oxidese method respectively. RESULTS AND CONCLUSION:The survival rate of PC12 cells and Schwann cells in cardiotrophin-1 group was obviously increased, lactate dehydrogenase releasing and malondialdehyde content were obviously decreased, superoxide dismutase activity was dramatical y improved compared with control group. Exogenous cardiotrophin-1 reduces the injury caused by ischemia and heat stress stimulation for PC12 cells and Schwann cells. The mechanism of protection may be related to the expression of anti-apoptosis protein activated by the combination of cardiotrophin-1 and its receptors.
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Objective:To isolate breast cancer stem/progenitor cells from human breast cancer and study their proliferation and differentiation biological characteristics over long-term passages in vitro. Methods:Human breast cancer stem/progenitor cells were enriched in suspension cultures as nonadherent mammospheres(MS). Serial sphere formation assay was performed to determine self-renewal ability of mammosphere-derived cells (MSDC). Differentiation was induced by culturing MSDC in DMEM-F12 supplemented with serum but without growth factors. The ratio of CD44~+/CD24~(-/low) cell population was evaluated by flow cytometry(FCM). Results:The mammospheres were formed after inoculation of primary breast cancer cells into culcutre medium with growth factors but without serum. The mammospheres contained undifferentiated cells similar to stem cells, which had self-renewal and extensive proliferation capabilities. With increasing passages, the cells tended to adhere and differentiate. The number of adhering and differentiating cells increased, and the amount and size of mammospheres decreased. The CD44~+/CD24~(-/low) cell population was enriched in the basal-like molecular subtype of human breast tumors. The biological behaviors of mammospheres varied between different specimens.Conclusion:Cancer cells with stem cell properties of self-renewal, indefinite proliferation and multi-lineage differentiation widely existed in human breast cancer tissues. The biological behaviors varied because of different origin of specimens and changed under the effects of environmental factors.
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Objective To isolate and identify prostate cancer stem-like cells from human prostatic carcinoma cell line PC-3,in order to set a foundation for further investigation of human prostate cancer stem cells (hPCSC). Methods Prostatic carcinoma cells line PC-3 was cultivated in serum-free medium (SFM) supplemented with growth factor,with the same cells cultivated in serum-supplemented medium (SSM) supplemented with growth factor to serve as the control. The proportion of prostate cancer stem-like cells was detected by flow cytometry,and prostate cancer stem-alike cells were identified by cell immunofluorescence,then the proliferating potentiality and doubling time with prostatic carcinoma cells PC-3 were compared by MTT. The process of inducing differentiation and the dual immunofluorescence expression after differentiation of prostate cancer stem-alike cells were observed. Results In SFM consisting of human epidermal growth factor (EGF),human basic fibroblast growth factor (bFGF) and human leukaemia inhibitory factor (LIF),a few PC-3 cells could survive and form suspended cell spheres,and the proportion of prostate cancer stem-alike cells (1.43%) was significantly increased compared with that cultured in SSM (0.41%,P
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Objective To establish a purified model of rat retinal ganglion cells (RGCs) cultured by serum-free medium,and provide a good cell model to investigate the damage of RGCs in glaucoma,retinal ischemia,and degenerative retinopathy. Methods Two monoclonal antibodies,anti-rat SIRP (OX-41) against rat macrophage and antibody against rat Thy-1 (OX-7),were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley (SD) rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor (CNTF) meeting the neuronal cell's special requirements.Photomicrographs illustration,immunfluorescence staining of Thy-1,calcein-acetoxymethyl ester (calcein-AM) fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells,91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day,many cells had uniform configuration,large body,and long protuberances. At the 14th day,over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining,which stained the living cells only,showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size,good configuration,and high purity,which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents.
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Objective To explore the feasibility of culturing dermal papillae cells (DPC) of hu- man hair in a serum-flee medium,and to observe the growth characteristics of these cells.Methods Cell culture flasks (plates) were pretreated with fibronectin,and DPC (2nd passage) were incubated with Williams E serum-flee medium supplemented with insulin-transferrin-selenite (ITS).Cells were observed by an inverted phase-contrast microscope.Proliferation of DPC was evaluated with 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and by their growth curve.Results In a serum-free medium,2nd passage DPC adhered to the flask surface within two to four hours of incubation; two to three days later,confluence,of the cells was observed,without noticeable proliferation.Four days later,cell connection was interrupted,isolated cells or cell clusters were seen,and detachment of some cells from the flask surface was observed.One to two weeks later,most cells had died.After incubation with 4% bovine serum for ten hours,cell proliferation was observed surrounding the remaining viable cell colonies. DPC growth curve showed stagnant phase and slow growth phase;however,log growth phase was not ob- served.Conclusion DPC could be successfully cultured in serum-free medium.However,the culture con- dition needs to be further optimized.
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Objective To establish a method of culturing mesenchymal stem cells from umbilical cord blood. Methods The mononuclear cell fraction of umbilical cord blood was isolated with the aid of a Ficoll/Hypaque gradient, then MSC were cultivated in serum containing medium or serum-free medium. The serum containing medium (SC) consisted of LG-DMEM supplemented with 10% fetal calf serum and 10-6M hydrocortisone, with a final cell concentration of 1?106 cells/ml. The serum-free medium (SF) was supplemented with 1?10 -6M hydrocortisone and 40ng/ml fibronectin with a final cell concentration of 1?106 cells/ml. After a week, 50% of the media were renewed. Before reaching the monolayer phase cells were trypsinised and re-cultured under similar conditions for the another 3 weeks. Cells were counted at the beginning and the end of the culturing period. Phenotypes were identifiad by PAS, NAE, ALP staining. Identification of surface markers and cell cycle analysis of MSCs were performed with flow cytometry. Results Adherent cells appeared 24h after plating of mononuclear cells. The cells formed adherent heterogeneous cell populations after 4-7 days in culture, and they consisted of round and spindle-like cells. In the primary passage of culture, the cells proliferated slowly and became confluent in 14-20 days. When subcultured, the heterogeneous cell populations became homogeneous assuming flat and fibroblast-like shape. Though colonies in the medium containing serum formed earlier than those in the serum-free medium, passage times and cell morphology were the same. The total cell numbers in SF group were lower than those of SC group. The percentage of cells at G0-G1 cell cycle was higher in SF group than that of SC group with significant difference. By flow cytometry analysis, cells of two groups were negative for CD34, CD13 and CD45, but strongly positive for specific surface markers such as CD166, CD29, CD105 and CD54. Conclusion Serum-free medium was superior to medium containing serum for culturing MSCs.