Objective:To investigate the growth and metabolism of Vero cells immobilized cultured on various microcarriers.Methods:Cell viability and cell morphology of the Vero cells cultured in 1%(v/v) newborn bovine serum DMEM/F12,the growth of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was evaluated on the viable cell density;The specific consumption rate of glucose(qglc),the specific production rate of lactate(qlac),the specific consumption rate of glutamine(qgln),and the specific consumption rate of glutamate(qglu) were used as the evaluation indexes,the metabolism of the Vero cells cultured on various microcarriers was determined.Results:After 7 d in culture the viable cell density of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was 18.4×105 cells/ml,21.9×105 cells/ml,23.9×105 cells/ml and 16.2×105 cells/ml,respectively.And,the Vero cells growth on Cytodex 1 formed compact cell layers with distinct cell morphology;Metabolic indexes of the Vero cells cultured on various microcarriers were basically the same.Conclusion:Among these microcarriers,Cytodex 1 is comparatively good for the growth of Vero cells,and could be used as the favor choice of microcarrier for the large-scale cultivation of Vero cells for viral vaccine production.

Beta-cell transplantation is considered to be the most effective approach to cure type 1 diabetes (T1D). Unfortunately, the scarce availability of donor tissue limits the applicability of this therapy. Recent stem cell research progress shows stem cell therapy may be a potential means to solve this problem. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewable and multipotent adult stem cells which can differentiate into the three germ layers. Here we aimed to investigate whether MSCs could be reprogrammed into insulin-producing cells (IPCs). We isolated and characterized MSCs obtained from rat bone marrow. Then MSCs were induced to transdifferentiate into IPCs under specific conditions containing high concentrations of glucose, activin A, all-trans retinoic acid, and other maturation factors. The induced cells expressed multiple genes related to pancreatic beta-cell development and function, such as insulin1, glucagon, Pdx1, Pax6, and Glut-2. Insulin1 and C-peptide production were identified by immunocytochemistry. In vitro glucose challenge studies showed the induced cells secreted insulin in a glucose-dependent manner, as do normal pancreatic beta-cells. Transplantation of these MSC-derived insulin-positive cells could reverse the hyperglycemia of streptozotcin (STZ)-induced diabetic rats. These results demonstrated that MSCs could be reprogrammed into IPCs and might be a potential autologous cell source for transplantation therapy of T1D.

Apoptosis plays an important role in limiting viable cell density and production performance in large-scale animal cell culture.Overexpression of anti-apoptotic genes is one of the most common strategies used to improve apoptosis resistance of industrial cells.HEK293 cells with adenoviral anti-apoptotic E1B-19K gene were transfected and picked out several representative clones.These clones were further investigated in the apoptotic percentages under various inducing culture conditions and cell growth and metabolism under normal culture condition.The results showed that the overexpression of E1B-19K in HEK293 cells,endowed cells anti-apoptotic ability by decreasing cell death by 60%~80% under inducing culture conditions with reduced glucose,low serum or without glutamine,delayed cells' entry into decay phase by 2 d under normal culture conditions;and had no significant affect on cell metabolisms of glucose,lactate and glutamine.The above results indicate that the overexpression of E1B-19K is a promising strategy to reduce cell death in mammalian cell culture.

Objective: To investigate the effect of p27 inducible-expression on the growth and metabolism of HEK293 cells.Methods: The vector pTet-on and Dox-responsive p27-expressing vector were co-transfected into HEK293 cells and individual clones were isolated randomly.Cell growth and metabolism were assessed by cell-cycle distribution and viable cell density,Qglc,Qlac and Qgln as indexes,respectively.Results: The expression of p27 increased percentage of G1 cells,reduced consumption of glucose and production of lactate of HEK293 cells.Conclusions: The inducible-expression of p27 is an effective strategy for G1-phase arrest of HEK293 cells.

Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.

Objective: To evaluate the influences of constitutively overexpressing human telomerase reverse transcriptase(hTERT) on the growth and metabolism of Vero cells cultured in vitro.Methods: Using the viable cell density,cell viability as the major evaluation indexes,in combination with cell morphology and stretching dynamics,the growth of both the hTERT-overexpressing Vero cell line T1 and wild-type Vero cells cultured in static attachment and suspended microcarriers was evaluated;Using the specific consumption rate of glucose(qglc),specific consumption rate of glutamine(qgln),specific production rate of lactate(qlac) and yield of lactate to glucose(Ylac/glc) as the evaluation indexes,the metabolism of both T1 cells and wild-type Vero cells cultured in static attachment and suspended microcarriers was determined.Results: Constitutively hTERT overexpression in T1 cells resulted in poorer capability of adhesion and extension,and reduced serum dependence,followed by the ability of adhesion independent growth in vitro to some extent.And no significant differences in metabolism between T1 cells and wild-type Vero cells cultured in static attachment and suspended microcarriers were observed.Conclusion: Constitutive overexpression of hTERT could reduce the dependency on serum and adhesion of Vero cells cultured in vitro,which indicated that constitutive overexpression of hTERT is a potential technical approach to optimize the characteristics of animal cells cultured in vitro.

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.

Objective: To establish a mammalian inducible expression system with the non-toxic biotin as an inducer.Methods: This system comprised three key components: E.coli biotin ligase(BirA),streptavidin-tetracyclin repressor(SA-TetR) and biotinylation signal-VP16 transactivation domain(AT-VP16).The three genes were introduced into a tricistronic expression vector,and co-transfected 293f cells together with the response vector.The EGFP taken for a reporter gene,EGFP fluorescent intensities were detected under different biotin concentrations.Results: With the increasing of biotin concentrations,EGFP displayed sequential OFF-ON-OFF expression profiles,the fluorescence intensity of EGFP in the induced state was about 3 times that in the inhibited state.Conclusion: The expression of the target gene could be reversibly regulated by adjusting the concentration of biotin.This is a promising inducible expression system.

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.

By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established.

Objective:Through suspension adaptation,to endow the Chinese hamster ovary cells(CHO cells) with suspension growth characteristics and rapid growth ability. Methods:Suspension culture was carried out in 100 mL shaker flasks with 35 mL culture volume and shaking at 90 r / min at an initial inoculation density of 3×105 cells / mL. The culture medium was supplemented with 1% serum,1 g / L Pluronic F-68 and 25 μg / mL dextran sulfate. When the total cell density exceeded 2×106 cells / mL,the cells were subcultured to 3×105 cells / mL. Results:Compared with the original cells,the average specific growth rate of the cells adapted to suspension culture increased from 0.27 / d to 0.48 / d,the highest density of total cells increased from 2.5×106 cells / mL to 6.3×106 cells / mL,and the activity of interest protein increased from the 2 781 U / mL to 8 878 U / mL. The average specific consumption rate of glucose of cells adapted to suspension culture was 1.42 μmol / (106 cells·d),which was lower than that of the original cells [2.16 μmol / (106 cells·d)]. Conclusion:The adherent CHO cells lines adapted to suspension culture can not only grow rapidly under suspension culture conditions,but also improve utilization efficiency to glucose of the cells.

Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 microm and a viable cell density greater than 5 x 10(6) cells x ml(-1) were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1 x 10(7) cells x ml(-1) resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7 x 10(11) GTU ml(-1) at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect.

Fed-batch culture is the predominant mode of the current animal cell culture process for many recombinant protein production. Fed-batch culture operation is mainly based on the nutrient continuous consumption and demand of cells to design continuous or semi-continuous concentration feed medium to maintain or support high-density cell growth and improve volumetric productivity of target protein in the reactor. The main methods to improve production efficiency of fed-batch cells culture include the optimization of medium design, selection and optimization of feed strategy and regulation of cell metabolism.

背景:近来发现羊水中含有具有多向分化潜能的干细胞,具有向内胚层、中胚层和外胚层分化的能力.目的:建立羊水多潜能干细胞的体外培养方法,并探讨其生物学特性.设计、时间及地点:单一样本实验,于2007-04/2008-01在解放军军事医学科学院生物工程研究所完成.材料:5份羊水标本来自孕16～22周流产孕妇自愿捐献.方法:从人孕中期羊水中,采用贴壁筛选法分离羊水多潜能干细胞.主要观察指标:采用免疫荧光、流式细胞术和反转录聚合酶链反应等方法分析细胞的生物学特性和鉴定细胞表型.结果:体外培养的羊水多潜能干细胞呈梭形或类上皮细胞样,细胞增殖迅速,细胞倍增时间约为24h;细胞周期中G1、S、G2期的细胞所占百分比分别为72.88%、5.77%、21.35%;细胞表达Oct4、Nanog、SSEA-4、CD44和CD105,不表达CD34和CD45.结论:采用贴壁筛选法获得的羊水多潜能干细胞增殖能力强,同时表达胚胎和成体干细胞的部分标志,可能为介于胚胎干细胞与成体干细胞之间的一种过渡阶段的干细胞.

BACKGROUND: Extract of injured pancreatic gland contains specific growth factor, which can promote β cell proliferation and insulin secretion, resulting in insulin regeneration and cell differentiation. OBJECTIVE: To investigate effects of the injured rat pancreatic extract on in vitro trans-differentiation of bone marrow mesenchymal stem cells (BMSCs) into insulin-like cells. DESIGN, TIME AND SETTING: A cytology in vitro experiment was performed at the Institute of Biotechnology, Academy of Military Medical Sciences of Chinese PLA from June 2005 to March 2007. MATERIALS: Six clean male Sprague-Dawley rats were obtained from the Animal Experimental Center of Academy of Military Medical Sciences. Activin A, Retinoic Acid and trypsin inhibitor were bought from Sigma, USA. Streptozotocin was purchased from Sigma company. METHODS: Streptozotocin was intraperitoneally infused into rats. Two days later, pancreatic tissues were obtained when blood glucose exceeded 10 mmol/L. Homogenate was prepared in phosphate buffer saline containing pancreatic protein inhibitor. Supernatant was obtained after twice centrifuge, which was injured pancreatic extract. BMSCs were isolated from rats by density gradient centrifugation. The passage cells were randomly divided into 4 groups when 70%-80% passage cells were confluent. BMSCs in the control group were incubated in low-glucose DMEM containing 2% fetal bovine serum for 11 days. BMSCs in the Activin A and Retinoic Acid (AR) group were separately treated with Activin A and Retinoic Acid each for 24 hours, and with basic fibroblast growth factor, nicotinic amide, nicotinic amide+exendin each for 3 days. BMSCs in the Activin A, Retinoic Acid and injured pancreatic extract (AR+E) group were treated with injured pancreatic extract on the basis of former groups. BMSCs in the injured pancreatic extract (E) group were only incubated with injured pancreatic extract for 11 days. MAIN OUTCOME MEASURES: Phenotypes of cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Insulin secretion levels were determined by enzyme labeled immunosorbent assay (ELISA). RESULTS: More insulin-positive cells existed in AR and AR+E groups, and insulin-like structure was bigger and more in the AR+E group compared with the AR group. There were a few insulin-positive cells and insulin-like structure in the E group. Insulin-1 mRNA expression existed in the experimental groups. Insulin secretion was detected in supernatant. Insulin secretion levels were greater in the AR+E group compared with the AR group, and secretion levels were the lowest in the E group (P < 0.05). Each index was negative in the control group. CONCLUSION: On the basis of previous study of Activin A and Retinoic Acid and other maturation factors, the injured pancreatic extract can increase the efficiency of BMSCs differentiation into insulin-like cells, resulting in elevating differentiation efficiency.

By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose qglc), specific production rate of lactate (qlac), and yield of lactate from glucose (Ylac/glc) as the evaluation indexes, the effects of hydrodynamics on aggregates formation, growth and metabolism of HEK 293 cells in suspension culture were examined in 250 mL spinner flasks by setting the agitation rates at 25, 50, 75, and 100 r/min, respectively. It was found that agitation plays an important role in HEK 293 cell aggregates formation and cell aggregates size distribution. After 7 days of cultivation in spinner flasks operated at 50 r/min and 75 r/min, the average diameter of HEK 293 cell aggregates was 201 μm and 175 μm, respectively, with the fraction of aggregates larger than or equivalent to 225 μm less than 10 %. The cell viability was kept above 90 % with the metabolic indexes, including qglc, qlac, and Ylac/glc kept constant. These results demonstrated that hydrodynamics derived from the proper agitation of the impeller play a decisive role in controlling the formation and size distribution of HEK 293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK 293 cells in suspended aggregates.

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

Recently we have generated a novel anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3×CD20) capable of cross-linking CD20 positive tumor cells and CD3 positive T cells. Here we have analyzed the in vitro efficacy of bscCD3×CD20 in mediating Ramous human B-lymphoma cell lysis in the presence of T-enriched human peripheral blood lymphocytes (PBLs). Obvious apoptosis characters were observed by Annexin V/PI (AV/PI) stained and scanning electron microscopy. As evaluated by a nonradioactive cytotoxicity assay, bscCD3×CD20 showed potent bioactivity of mediating human B-lymphoma cell lysis in the presence of T-enriched human PBLs. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose- and time-dependent effect on the mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5 μg/mL with the ratio of the E:T used being 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBLs. Among the upregulated apoptogenes, ATM and p53 showed an increase of 187 times and 15 times, respectively, which suggested that the ATM-p53 pathway may be the main apoptosis way of Ramous cells being induced by T cells in the presence of the bscCD3×CD20.