Expansion of human allogeneic liver-derived progenitor cells for liver regenerative therapy in serum-free culture conditions.

Human allogeneic liver-derived progenitor cells (HALPCs) display advanced ability to differentiate into hepatocyte-like cells and exhibit potent immunomodulatory, anti-inflammatory, and anti-fibrotic properties. HALPCs have been successfully manufactured under good manufacturing practice (GMP) and are currently in clinical development. A previous phase 2a trial demonstrated the safety of peripheral intravenous infusions of HALPCs and preliminary evidence of the cells' properties to restore liver function in patients with acute-on-chronic liver failure (ACLF), thus potentially improving their survival. A phase 2b trial is currently ongoing across multiple centers (NCT04229901) to obtain proof-of-concept on efficacy and additional safety. HALPCs are currently manufactured using fetal bovine serum (FBS), which can reveal qualitative and quantitative variations between batches. The use of serum-free medium (SFM) represents an alternative means to overcome this variability while also complying fully with regulations. The aim of this study was to compare current FBS-containing culture conditions with two industry-available GMP-compliant SFMs: StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and PRIME-XV (FUJIFILM Irvine Scientific, Santa Ana, California, USA). The proliferation of HALPCs was significantly stimulated by both SFMs, which shortened both their emergence period and population doubling time. This effect was correlated with a significant improvement in their genetic stability as analyzed by conventional karyotyping. The expression profile (identity and purity) and functionality of HALPCs cultured in SFM were maintained, as demonstrated by flow cytometry and enzyme-linked immunoassay (ELISA), respectively. Their potency, evaluated via prostaglandin E2 (PGE2) secretion, showed a similar effect on CD4+ T-cell proliferation in FBS and SFM conditions. Furthermore, a greater proportion of HALPCs cultured in SFM showed enhanced expression of tissue factor (CD142) compared with the FBS condition. Altogether, SFM conditions enabled consistent HALPC quality to be achieved without altering their expression and functional profiles.

A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

Development of a chemically disclosed serum-free medium for mouse pluripotent stem cells.

Mouse embryonic stem cells (mESCs) have been widely used as a model system to study the basic biology of pluripotency and to develop cell-based therapies. Traditionally, mESCs have been cultured in a medium supplemented with fetal bovine serum (FBS). However, serum with its inconsistent chemical composition has been problematic for reproducibility and for studying the role of specific components. While some serum-free media have been reported, these media contain commercial additives whose detailed components have not been disclosed. Recently, we developed a serum-free medium, DA-X medium, which can maintain a wide variety of adherent cancer lines. In this study, we modified the DA-X medium and established a novel serum-free condition for both naïve mESCs in which all components are chemically defined and disclosed (DA-X-modified medium for robust growth of pluripotent stem cells: DARP medium). The DARP medium fully supports the normal transcriptome and differentiation potential in teratoma and the establishment of mESCs from blastocysts that retain the developmental potential in all three germ layers, including germ cells in chimeric embryos. Utility of chemically defined DA-X medium for primed mouse epiblast stem cells (mEpiSCs) revealed that an optimal amount of cholesterol is required for the robust growth of naïve-state mESCs, but is dispensable for the maintenance of primed-state mEpiSCs. Thus, this study provides reliable and reproducible culture methods to investigate the role of specific components regulating self-renewal and pluripotency in a wide range of pluripotent states.

Development of a serum-free medium for myoblasts long-term expansion and 3D culture for cell-based meat.

Cell-based meat technology provides an effective method to meet the demand for meat, while also posing a huge challenge to the expansion of myoblasts. It is difficult to develop serum-free medium suitable for long-term culture and large-scale expansion of myoblasts, which causes limited understanding of myoblasts expansion. Therefore, this study used C2C12 myoblasts as model cells and developed a serum-free medium for large-scale expansion of myoblasts in vitro using the Plackett-Burman design. The serum-free medium can support short-term proliferation and long-term passage of C2C12 myoblasts, while maintaining myogenic differentiation potential well, which is comparable to those of growth medium containing 10% fetal bovine serum. Based on the C2C12 myoblasts microcarriers serum-free culture system established in this study, the actual expansion folds of myoblasts can reach 43.55 folds after 7 days. Moreover, cell-based meat chunks were preliminarily prepared using glutamine transaminase and edible pigments. The research results provide reference for serum-free culture and large-scale expansion of myoblasts in vitro, laying the foundation for cell-based meat production. PRACTICAL APPLICATION: This study developed a serum-free medium suitable for long-term passage of myoblasts and established a microcarrier serum-free culture system for myoblasts, which is expected to solve the problem of serum-free culture and large-scale expansion of myoblasts in cell culture meat production.

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

Recombinant bovine FGF1 promotes muscle satellite cells mitochondrial fission and proliferation in serum-free conditions.

Cell cultured meat is a novel and promising technology, but developing specific culture medium for muscle cells remains one of the main technical obstacles. FGF1 signaling is reported to promote proliferation and maintain proliferative capacity of satellite cells. However, the effect of FGF1 as a supplement to serum-free medium on satellite cells in vitro culture is still unclear. In this study, an efficient method for the production of soluble and biologically active recombinant bovine FGF1 (rbFGF1) protein in Escherichia coli was established. The soluble expression level of TrxA-rbFGF1 fusion protein was 562 mg/L in shake flasks, resulting in 5.5 mg of pure rbFGF1 from 0.1 L of starting culture. In serum-free culture conditions, rbFGF1 effectively promoted the proliferation and regulated the mitochondrial morphology and function of C2C12 myoblasts.rbFGF1 activated extracellular signal-regulated kinases1/2 (ERK1/2) signaling in C2C12 myoblasts, which further stimulated dynamin related protein 1 (DRP1) Ser616 phosphorylation. These findings highlighted the potential application of rbFGF1 in developing effective serum-free medium for cultured meat production.

Optimization of adaptation parameters from adhesion cell culture in serum-containing media to suspension in chemically defined media by superlative box design.

A new design of experiments-superlative box design (SBD), was adopted to optimize the adaptation of Chinese hamster ovary cells from adhesion culture to serum-free suspension culture. It is a general trend to use a serum-free medium instead of a serum-containing medium. The advantage of serum-free medium (chemically defended) is that it does not contain unknown components and avoids safety issues. SBD requires fewer experiments while ensuring a sufficient number of experiments and uniformity in the distribution of experiments amongst all the factors. Six factors were considered in this experimental design with 43 runs plus three more repeating center runs. The cell line was adapted to serum-free media by gradually reducing serum, and from adherent to suspension by rotating at various speeds in a shake flask. Response surface methodology was applied to find the optimum condition. The optimized cell density reached 7.02 × 105 cells/mL, calculated by the quadratic model. Experiments validated the predicted cell adaptation with the maximum cell density. Three suspension runs were selected randomly to perform in the bioreactor to validate cell stability and production homogeneity. This study provides an efficient method to transfer adherent cells to suspension cells and is the first to successfully use SBD and establish a parameter quadratic optimization model.

Recombinant Protein Production from Stable CHO Cell Pools.

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.

Human umbilical cord mesenchymal stem cells differentiate into neuron-like cells after induction with B27-supplemented serum-free medium / 南方医科大学学报

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.

Application of cell culture techniques in cultured meat-a review / 生物工程学报

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.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Characterization of Human Fetal Cartilage Progenitor Cells During Long-Term Expansion in a Xeno-Free Medium

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.

Comparative study on extraction and culture method of neural stem cells / 中国药理学通报

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 ).

Proliferation-promoting effect of umbilical cord mesenchymal stem cells on co-cultured bovine mammary gland epithelial cells / 中国实验动物学报

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.

Human nasopharyngeal cancer stem cell microspheres:Culture and biological characterization / 医学研究生学报

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.

Biological properties of colon cancer spheroid cells cultured in serum-free medium / 中华消化外科杂志

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.

Expression of miR-590-3p in pancreatic cancer stem cells / 中华胰腺病杂志

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.

Primary culture of human preadipocyte in a serum-free medium / 基础医学与临床

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.

The Study and Design Methods of Serum-Free Medium for Animal Cells / 中国生物工程杂志

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.

Comparison of Serum-Free Medium AIMV with Standard Serum-Containing Medium in Culturing Cells Activated with IFN-?,IL-2 and Anti-CD3 / 中国肿瘤生物治疗杂志

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.