High Cell Density Culture System Research Articles

Gelatin microspheres releasing transforming growth factor drive in vitro chondrogenesis of human periosteum derived cells in micromass culture

For cartilage tissue engineering, several in vitro culture methodologies have displayed potential for the chondrogenic differentiation of mesenchymal stem cells (MSCs). Micromasses, cell aggregates or pellets, and cell sheets are all structures with high cell density that provides for abundant cell-cell interactions, which have been demonstrated to be important for chondrogenesis. Recently, these culture systems have been improved via the incorporation of growth factor releasing components such as degradable microspheres within the structures, further enhancing chondrogenesis. Herein, we incorporated different amounts of gelatin microspheres releasing transforming growth factor β1 (TGF-β1) into micromasses composed of human periosteum derived cells (hPDCs), an MSC-like cell population. The aim of this research was to investigate chondrogenic stimulation by TGF-β1 delivery from these degradable microspheres in comparison to exogenous supplementation with TGF-β1 in the culture medium. Microscopy showed that the gelatin microspheres could be successfully incorporated within hPDC micromasses without interfering with the formation of the structure, while biochemical analysis and histology demonstrated increasing DNA content at week 2 and accumulation of glycosaminoglycan and collagen at weeks 2 and 4. Importantly, similar chondrogenesis was achieved when TGF-β1 was delivered from the microspheres compared to controls with TGF-β1 in the medium. Increasing the amount of growth factor within the micromasses by increasing the amount of microspheres added did not further improve chondrogenesis of the hPDCs. These findings demonstrate the potential of using cytokine releasing, gelatin microspheres to enhance the chondrogenic capabilities of hPDC micromasses as an alternative to supplementation of the culture medium with growth factors. Statement of significanceGelatin microspheres are utilized for growth factor delivery to enhance chondrogenesis of mesenchymal stem cells (MSCs) in high cell density culture systems. Herein, we employ a new combination of these microspheres with micromasses of human periosteum-derived cells, which possess ease of isolation, excellent expansion potential, and MSC-like differentiation capabilities. The resulting localized delivery of transforming growth factor β1 increases glycosaminoglycan and collagen production within the micromasses without exogenous stimulation in the medium. This unique combination is able to drive chondrogenesis up to similar levels as seen in micromasses that do receive exogenous stimulation. The addition of growth factor releasing microspheres to high cell density micromasses has the potential to reduce costs associated with this strategy for cartilage tissue engineering.

High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.

New Approach for Differentiation of Bone Marrow Mesenchymal Stem Cells Toward Chondrocyte Cells With Overexpression of MicroRNA-140

Mesenchymal stem cells are widely stimulated by transforming growth factor beta-3 (TGFβ3) for chondrocyte differentiation. The objective of our study was to establish a new method for differentiation of human mesenchymal stem cells toward chondrocyte by overexpression of MicroRNA-140 (miR-140), and also this method was compared with method of induction with TGFβ3 in high-cell density culture systems. Mesenchymal stem cells were harvested from bone marrow of human. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were cultured in monolayer culture system and were harvested after days 7, 14, and 21. Real time polymerase chain reaction (RT-PCR) was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry technique. Bone marrow mesenchymal stem cells (BMSCs) were transducted with recombinant Lenti virus, and miR-140 was expressed. Immunocytochemical method confirmed the differentiation of BMSC toward chondrocyte with handling cartilage matrix genes. Also real-time PCR showed that after expression of miR-140 in transducted BMSCs significantly increased gene expression of collagen type II and aggrecan and downregulated expression of collagen type I when compared with the mRNA levels measured in nontransducted BMSCs. These results were compatible compared with TGFβ3 induction method as control positive. In this study, we described a new approach and technique that may be applied for differentiation of BMSCs to chondrocyte instead of stimulation with TGFβ3. Our data implies that miR-140 is a potent chondrogenic differentiation inducer for BMSCs, and we have shown increasing chondrogenic differentiation by using miR-140 overexpression.

In vitro evidences of epithelial to mesenchymal transition in low cell-density cultured human fetal hepatocytes

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells.To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches.

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.

Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

Continuous beer production was investigated in a high cell-density culture system which consisted of two stages for the fermentation and sedimentation of yeast cells. The continuous culture was carried out for a fermentation time of 5,500 h without contamination, at varying dilution rates and fermentation temperatures in the ranges of 0.017-0.033 h −1 and 6.5–8.5°C, respectively. This process was found to be suitable for continuous and stable beer brewing. Under these conditions, the cell concentration in the first stage was about 80 times as high as that in the exit of the second stage. Concentrations of viable cells, sugar and ethanol were maintained at 1.3 × 10 9 cells/ml, 25 and 36 g/ l, respectively, and were hardly affected by fermentation temperature. Concentrations of ethyl acetate, isoamyl alcohol and isoamyl acetate were similar in the fermentation temperature ranges of 6.5–8.5°C, and the amounts at a fermentation temperature of 7°C were comparable to those of lager-type beer. Diacetyl flavor, which is known to be an effluent component that causes deterioration in the second stag e (young beer), was maintained at 1.2 ppm at a dilution rate and fermentation temperature of 0.022 h −1 and 7°C, respectively. The diacetyl flavor was due to the accumulation of vicinal diketone, the precursor of which is acetohydroxy acid. The acetohydroxy acid was converted to vicinal diketone by pretreatment at 60°C for 30 min. The vicinal diketone was then consumed by the yeast during after-fermentation at a fermentation temperature of 3°C. Using this method, total vicinal diketone decreased below 0.3 ppm for an after-fermentation time of 6.8 h, which was 225 times as fast as that of after-fermentation without the pretreatment. This process may make it possible to achieve continuous beer fermentation from the fermentation stage to after-fermentation for diacetyl removal.

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.