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OBJECTIVE@#To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into neuron- like cells after induction with B27- supplemented serum- free medium.@*METHODS@#HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mL nerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (group C), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometry and the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillary acidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting.@*RESULTS@#Before induction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) and CD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the best in group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantly different between group A and the other 3 groups ( < 0.001 or 0.05).@*CONCLUSIONS@#B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) strongly promotes the cell differentiation.
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As one of the top 10 breakthrough and emerging technologies in the world in 2018, cultured meat has attracted extensive attention due to its advantages of traceable origin, food safety and green sustainable development. Europe and the United States have invested a lot of resources to focus on research about cultured meat, which will affect our domestic meat and food market in the future. At present, the challenge of cultured meat production is how to efficiently simulate the growth environment of animal muscle tissue and realize large-scale production in bioreactor. Although cell tissue engineering has been deeply studied and achieved varying successful application, it is still difficult to obtain large-scale cultured meat production due to the high cost and technical requirements. Therefore, the development of efficient and safe cell culture technology is an urgent problem for large-scale cultured meat production, which can effectively reduce costs and achieve industrial application. In this review, we summarize the research progress of animal cell tissue culture technology used for cultured meat, and highlighted the current challenges and possible strategies in further applications.
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Animals , Bioreactors , Cell Culture Techniques , Meat , Tissue Engineering , United StatesABSTRACT
BACKGROUND: Stem cell therapy requires a serum-free and/or chemically-defined medium for commercialization, but it is difficult to find one that supports long-term expansion of cells without compromising their stemness, particularly for novel stem cells. METHODS: In this study, we tested the efficiency of StemPro® MSC SFM Xeno Free (SFM-XF), a serum-free medium, for the long-term expansion of human fetal cartilage-derived progenitor cells (hFCPCs) from three donors in comparison to that of the conventional α-Modified Eagle's Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS). RESULTS: We found that SFM-XF supported the expansion of hFCPCs for up to 28–30 passages without significant changes in the doubling time, while α-MEM with 10% FBS showed a rapid increase in doubling time at 10–18 passages depending on the donor. Senescence of hFCPCs was not observed until passage 10 in both media but was induced in approximately 15 and 25% of cells at passage 20 in SFM-XF and α-MEM with 10% FBS, respectively. The colony forming ability of hFCPCs in SFX-XF was also comparable to that in α-MEM with 10% FBS. hFCPCs expressed pluripotency genes like Oct-4, Sox-2, Nanog, SCF, and SSEA4 at passage 20 and 31 in SFM-XF; however, this was not observed when cells were cultured in α-MEM with 10% FBS. The ability of hFCPCs to differentiate into three mesodermal lineages decreased gradually in both media but it was less significant in SFM-XF. Finally we found no chromosomal abnormality after long-term culture of hFCPCs until passage 17 by karyotype analysis. CONCLUSION: These results suggest that SFM-XF supports the long-term expansion of hFCPCs without significant phenotypic and chromosomal changes. This study have also shown that hFCPCs can be mass-produced in vitro, proving their commercial value as a novel source for developing cell therapies.
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Humans , Aging , Cartilage , Cell- and Tissue-Based Therapy , Chromosome Aberrations , In Vitro Techniques , Karyotype , Mesoderm , Stem Cells , Tissue DonorsABSTRACT
Aim To explore the best method of neural stem cellextraction and culture, and provide a technical reference for thebasic research of neural stem cells. Methods Different extractionand culture methods of neural stem cells were compared.The rate of ball of neural stem cells and the stability of neuralstem cells in undifferentiated state were observed by extraction offetal and neonatal rats cortex, using different types of medium,different inoculation density, different culture methods, differentmethods of changing liquid and different passage methods. Neuralstem cells' activities were detected by Varioskan LUX MultimodeMicroplate Reader. Results ① The brain cortex of fetalrats of 14 d had higher proportion of neural stem cells, less othercells and more neurospheres than newborn rats of 24 h. ② Neuralstem cells could be stabilized in undifferentiated state by usingserum-free medium, while most of the neural stem cellswere differentiated into neurons and glial cells by using serummedium. ③ Neural stem cells, seeding at 1. 0 ×109 ·L-1 , hada large number of neurosperes and were in good condition. ④Suspension culture was beneficial to form a stable neurosphereand keep the neural stem cells in an undifferentiated state thanadherent culture. ⑤ The state of neurosphere by changing halfof the medium and adding medium without discarding was betterthan that by replacing all medium. ⑥ The neurospheres couldbe separated into single cells by mechanical blow in primary generationand the second generation of neurospheres cultured in serum-free medium. While the percentage of viable cells in neuralstem cells was the highest digested with stem cell lysates afterthree generations and the neurospheres re-formed were better. ⑦Neural stem cells' activity of 14 d fetal rat in Accutase digestiongroup was significantly higher than that of the other threegroups, and the difference was significant (P <0. 05). ConclusionsNeural stem cells proliferate and divide well, with highrate of ball formation and good neurosphere condition, which canmaintain a stable undifferentiated state by extracting the cerebralcortex of 14 d fetal rats, using serum-free medium, inoculatinginto a 25 cm2 flask at a density of 1. 0 × 109 ·L-1 , and takingthe suspension culture (adding the medium 1 ~ 1. 5 mL every 2~3 d and passage every 6 ~8 d ).
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Objective To explore the proliferation-promoting effect of bovine mammary gland epithelial cells (BMECs) co-cultured with umbilical cord mesenchymal stem cells (UC-MSCs) in serum-free culture mediuum.Methods Bovine UC-MSCs and BMECs were selected for co-culturing in direct or indirect contact.In the direct contact culture groups, UC-MSCs and BMECs were co-cultured at concentration ratios of 2∶1, 1∶1, 1∶2, 1∶3, 1∶4, 1∶5, and 1:10, respectively.In the indirect contact culture group, the supernatant of UC-MSCs was used as the conditioned medium to re-suspend BMECs.In the control groups, UC-MSCs and BMECs were cultured alone.The cell growth status in each group was observed at 0, 4, 8, 12, 24, 36, 48, 60, 72 h after culture, and cell proliferation was detected by cell counting kit-8 (CCK-8) assay.Results At 48 h, the optical density of the conditioned medium-BMECs group was significantly higher compared with the control groups (P<0.05).Meanwhile, the optical density in the direct contact group at a concentration ratio of 1∶2 reached the peak, which was extremely significantly higher compared with the control groups (P<0.01) and significantly higher compared with the other direct contact culture groups and the conditioned medium-BMECs group (P<0.05).Conclusions Co-culture of UC-MSCs and BMECs in serum-free culture medium is capable to promote the proliferation of BMECs, and the co-culture by cell-to-cell contact has a better effect.The optimal concentration ratio of UC-MSCs to BMECs is 1∶2, and the optimal culture time is 48 h.
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Objective At present, the methods of separating and identifying nasopharyngeal cancer stem cells are not yet mature.This study was to explore the methods of culturing nasopharyngeal cancer stem cell microspheres and identify the cancerous stem cell biological features of CNE-2 cell microspheres. Methods We conducted suspension culture of human nasopharyngeal cancer CNE2 cells and C666-1 cells in serum-free medium ( SFM) containing growth factors.Then we measured the proportion of CD133 +cells in CNE2 monolayer ( CNE2-MN) and CNE2 microsphere cells ( CNE2-SC) by flow cytometry, determined their in vitro invasiveness through Transwell chamber experiments, and detected their in vivo tumorigenicity via nude mouse experiments.We ob-served the differentiation potency of the CNE2-SCs in the adherent-cultured serum-containing medium and detected the expressions of the cancer stem cell-related genes Bmi-1, Oct4, and Twist1 in CNE2-MN and CNE2-SCs by flow cytometry and RT-PCR analysis. Results In the special blend of SFM, both of the cell lines can form microspheres that can be stably transferred.Fresh SFM prepara-tion, substituting cell separation agent Accutase for pancreatic enzyme for transfer, and maintaining the state of cell suspension contrib-uted to the formation and proliferation of microspheres.Adherent culture with serum-containing medium induced the differentiation of CNE2-MN cells, which exhibited no significant difference from the CNE2 microsphere cells.The CD133 +cells accounted for 98.79%in the CNE2 microspheres, significantly higher than 0.98%in the CNE2 cells (P<0.01).Compared with the CNE2-MN cells, the CNE2-SCs showed highly increased expressions of Bmi-1, Oct4, and Twist1 (P<0.01).The numbers of membrane-penetrating cells in the CNE2-SCs and CNE2-MN cells were 122 ±6 and 36 ±7 per visual field, the former with a stronger invasive ability than the latter genesis of 1 ×106 CNE2-SCs vs that of the same number of CNE2 -MNcells was (2.332 ±0.549) cm 3 sv (0.669 ±0 .278) cm3 ( P<0.01). Conclusion Using suspension culture with a specially prepared SFM, nasopharyngeal cancer CNE2 microsphere cells can be obtained, in which there are large numbers of cancer stem cells.This culture method may provide a base for further studies of naso-pharyngeal cancer stem cells.
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Objectives To isolate cancer stem cells (CSCs) in pancreatic cancer cell lines PANC1 and ASPC-1 with serum-free medium( SFM ), and to detect the expression of miR-590-3p in CSCs. Methods PANC1 and ASPC-1 cells was cultured in serum-free medium. The monoclonal formation, differentiation and cell cycle, half inhibitory concentration ( IC50 ), and the expression of the surface markers CD24 + , CD44 + were detected. qRT-PCR was used to detect the expression of miR-590-3p. Results After SFM culture, (0.94 ±0.53 ) ％ of ASPC-1 and (0.57 + 0. 12 ) ％ PANC1 survived, and they formed spheres, and could continuously passage in vitro. Cell spheres differentiation recurred when serum was supplemented in SFM. The G0/G1 stage proportion, CD24+ , CD44 + , CD24+ CD44+ cells proportion, IC50 in ASPC-1 cell were (75.3 ± 5.4)％,0.96％ ～ 2.01％, 27.52％ ～ 34.47％, 0.35％ ～ 0.44％ and (224.37 ± 5.71 ) μg/ml, which were significantly higher than that those in parent cell [ (43.7 ± 3.8 ) ％, 0. 38％ ～ 0.42％, 17.65％ ～ 18.25％,0.05％ ～0.08％, (11.43 ±2.10)μg/ml, P＜0.05]. The G0/G1 stage proportion, CD24+ ,CD44+ ,CD24 +CD44 + cells proportion, IC50 in PANC 1 cell were ( 80. 1 ± 4.7) ％, 5.31％ ～ 9.84％, 72.05％ ～ 93.06％,4.91％ ～5.21％, (296.58±4.27) μg/ml, which were significantly higher than that those in parent cell [ (46.1 ±5.3)％, 4.09％ ～4.97％, 47.71％ ～55.66％, 1.48％ ～2.63％, (26.17 ±3.81)μg/ml, P＜0.05]. The expression of miR-590-3p in ASPC-1, PANC1 spheres was 4.67 and 4.52 times higher than the expression in parent cell lines. Conclusions Pancreatic cancer cell spheres can be isolated from ASPC-1, PANC1 by culture with SFM. miR-590-3p is up-regulated and may play an important role in regulating biological characteristics of pancreatic cancer stem cells.
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Objective To obtain colon cancer spheroid cells from human colon cancer cell lines cultured in serum-free medium (SFM),and investigate the proliferative and migratory properties of colon cancer spheroid cells.Methods Human colon cancer cell lines HCT116 and HT29 were cultured in SFM,and then the generation of spheroid cells was observed.The expression of stem cell surface marker CD133 was detected by flow cytometry,and the proliferative and migratory properties of colon cancer spheroid cells were detected by cell counting kit-8 and Transwell migration assay,respectively.All data were analyzed by using the t test.Results Spheroid cells were obtained from colon cancer cell lines HCT116 and HT29 in SFM.The ratios of spheroid cells with positive expression of CD133 generated by HCT116 and HT29 were 75.44％ ± 11.41％ and 76.22％ ± 14.23％,respectively.Compared with original colon cancer cells cultured in serum supplemented medium,the number of HCT116 and HT29 spheroid cells with positive expression of CD133 was significantly greater (t =11.43,9.17,P ＜ 0.05 ),and the proliferative and migratory abilities were much stronger also.Conclusion Colon cancer spheroid cells cultured in SFM have higher positive expression of CD133 and stronger proliferative and migratory abilities,and it can be utilized as a feasible model for further studies of colonic stem cells.
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Objective To establish a method of primary culture of human preadipocyte in a serum-free medium. Methods Collangenase digestion was used to dispart preadipocyte.The cells were identified by microscopy stained by oil red O and determination of the activity of Glycerol-3-phosphate dehydronase(G-3-PDH). Results The primary cultured human preadipocyte proliferated in the aserum-free medium successfully.In the differential serum-free medium the cells turned to be round on the 4 th day.The adipose drops began to cumulate in the cells,and to the most quantity until the 21 st day.Conclusion The human preadipocyte can be primarily cultured and induced to differentiate in serum-free medium,which is the base for researching the effects of hormones and factors to the proliferation and differentiation of preadipocyte.
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With the expanding scale of animal cell culture and increasing demand of biopharmaceutical,the development of serum-free medium based on cell lines and products has become a major task in the field of cell engineer.The statistical methods and tools are popular in the processes of design medium and can be used to evaluate the multiple factors and their interaction scientifically and effectively.The novel microarray and proteomic analysis can improve the performance of identifying the role of medium.The aim is to provide some idea for serum-free medium study through systematically summarizing the newly and commonly used serum-free medium study and design approaches and their characteristics.
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Human peripheral blood mononuclear cells (PBMCs) activated with Con-A release a soluble factor which augments the expression of class I major histocompatibility complex (MHC) antigens by a variety of tumour cells. Previous attempts to purify this factor called MHC-activating factor (AF) (MHC-AF) made us realize that the presence of large numbers and quantities of irrelevant fetal calf serum proteins in the culture supernatants of the activated human PBMCs, interfered with the purification procedure. It was therefore necessary to standardize the use of a serum free culture medium to generate human MHC-AF. In the present communication we have tried several types of culture media and have identified DCCM-2 as the most suitable culture medium to generate human MHC-AF. MHC-AF generated in DCCM-2 medium appears to be a protein molecule resistant to pH 2 treatment but sensitive to heat treatment (56°C × 45 min) and treatment with proteolytic enzymes trypsin and chymotrypsin.
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Objective: To compare serum-free medium AIMV with standard serum-containing medium in culturing cells activated with IFN-?,IL-2 and anti-CD3. Methods: Peripheral blood mononuclear cells (PBMC) separated from donors were cultured with IFN-?,IL-2 and anti-CD3 in serum-free medium AIMV or in standard serum-containing medium separately. Proliferation, phenotypes and cytokine secretion of cells cultured in the two different media were compared. The inhibitory effects on hepatitis B virus replication in vivo were observed after transfusion of cells cultured in different media. Results: Compared with cells cultured in standard serum-containing medium, cells cultured in serum-free medium AIMV could proliferate well enough to be applied to immunotherapy; More percentage of cells cultured in AIMV expressed CD25; Cells cultured in serum-free medium secreted more IFN-?; After transfusion of cells cultured in AIMV, hepatitis B replication could be inhibited more evidently. Conclusion: Serum-free medium AIMV was better than serum-containing medium in culturing cells for immunotherapy.
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Microvascular endothelial cells from rat lungs were cultured in serum-free medium supplemented with an endothelial growth substance, insulin, hydrocortisone and so on. Five to seven days after plating, cultured cells formed a monolayer. They were identified as endothelial cells by morphology and by positive immunohistochemistry for factor VIII-related antigen, a marker for endothelia cells. Differences between gelatin coated culture plates and plastic culture plates in endothelial cell proliferation were evaluated. Cells plated on uncoated plastic plates had a spindle-shaped morphology and did not express factor VIII-related antigen. Two types of medium, serum-free medium containing endothelial growth substance and basal medium supplemented with 20% fetal calf serum, were also compared in primary culture. In contrast with the serum-free medium, cells cultured in the serum-containing medium showed fibroblast-like morphology and did not express factor VIII-related antigen. These results suggest that a gelatin substratum and serum-free medium containing endothelial growth supplement are necessary for in vitro proliferation of microvascular endothelial cells isolated from rat lungs. The culture method and conditions outlined here allow the proliferation of pure microvascular endothelial cells from rat lungs. It may be useful in studying hematogenous metastasis to the lung and the role of microvascular endothelium in other pulmonary disease.
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Male , Rats , Animals , Blood Physiological Phenomena , Cell Separation/methods , Cells, Cultured , Endothelium, Vascular/cytology , Extracellular Matrix/physiology , Gelatin/pharmacology , Immunohistochemistry , Lung/blood supply , Rats, Inbred StrainsABSTRACT
Objective:To compare expansion and kill activation of A-NK cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also detest the assist effect of EL-12 on A-NK/IL-2 treatment and the morphology character of killed cells through electroscope.Methods:Proliferation ability of the cells cultured in the two different media was compared by MTT method in vitro. The morphology of the killed tumor target cells by adherent natural killer cells (A-NK) was observed through electroscope. Results:All of A-NK cells cultured in deferent condition could rapidly proliferate(P