Objective To investigate the effect of transition between hypoxia and normoxia on proliferation of rabbit mesenchymal stem cells(rMSCs).Methods rMSCs expanded in hypoxia were divided into two groups:L-L group and L-N group.Moreover,rMSCs expanded in normoxia were also divided into two groups:N-N group and N-L group.The L-L and N-L groups were subsequently subcultured in hypoxia,and L-N and N-N groups in normoxia.Growth curve,colony-forming efficiency,glucose and lactate metabolism,and intracellular ROS generation of the four groups were examined.Results In L-L group cell density and colony-forming efficiency were the highest,and intracellular ROS generation was the lowest,whereas reversed results were obtained in N-N group.Cells cultured in hypoxic conditions exhibited higher glucose consumption,lactate production and yield coefficients of lactate from glucose compared to normoxic ones.Conclusion Hypoxia may enhance the proliferation of rMSCs in continuous subcultures.There is a potential link between intracellular ROS generation and expansion of rMSCs.

BACKGROUND: Effect of hypoxia on osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) has been differently reported. Those differences might cause by varying volume fraction of oxygen and varying source of BMMSCs. OBJECTIVE: To compare the biological differences between placenta and BMMSCs under hypoxia.METHODS: Human placenta amniotic mesenchymal stem cells (hAMSCs) and rabbit BMMSCs were isolated by two step proteinases and whole bone marrow adhesion, respectively. hAMSCs and BMMSCs at the same passage were seeded in 12-well plates at an initial cell density of 2 × 10~4 cells per well with α-MEM containing 10% FBS. Then, the cells were cultured under 5% O_2 or 20% O_2 for 12 days. hAMSCs and BMMSCs at the same passage were seeded in 12-well plates at an initial cell density of 1 × 10~5 cells per well with osteogenic medium. Then, the cells were cultured under 5% O_2 or 20% O_2 for 14 days. Cell growth curve, the specific glucose consumption rates and specific lactate production rates, and osteogenic differentiation were detected. RESULTS AND CONCLUSION: Compared to normal oxygen, hypoxia promoted the proliferation and osteogenic differentiation of MSCs. When compared to BMMSCs, statistically significant enhancement of the growth of hAMSCs by hypoxia was observed. hAMSCs cultured under hypoxia exhibited lower glucose consumption and lactate production in contrast with BMMSCs. Furthermore, comparison between hAMSCs and BMMSCs showed that the alkaline phosphatase expression of BMMSCs was significantly enhanced by hypoxia and was markedly higher compared with hAMSCs. The amount of calcium deposition was also enhanced by hypoxia, but there were no statistically significant differences between hAMSCs and BMMSCs.

BACKGROUND: Polydimethylsiloxane (PDMS) is widely used in the basic research on cell biology because of its good biocompatibility and ability to be processed at the micro/nano level. However, cell culture on PDMS has been generally compromised by its strong hydrophobicity.OBJECTIVE: To perform a comparative investigation on the influences of different surface modifications as well as stiffness of PDMS on cellular behaviors.METHODS: PDMS films with varying stiffnesses were subjected to various surface modifications, including serum incubation, type I collagen deposition and air plasma treatment. Bovine articular chondrocytes were seeded on PDMS films and cell adhesion, proliferation and matrix production were characterized using F-actin staining, cell counting kit-8,microscopic examination, Sirius red/Safranin-O staining and quantitative determination of glycosaminoglycans,respectively.RESULTS AND CONCLUSION: Serum incubation, type I collagen deposition and air plasma treatment were all found to promote adhesion and proliferation of bovine articular chondrocytes from the results of F-actin staining and cell prolifereration curve, with air plasma treatment the best. Total amount of glycosaminoglycans (GAG) secretion was only increased by air plasma treatment and GAG/DNA was decreased by all modifications. Stiffness also played roles in cell adhesion, proliferation and GAG production, which was found to be dependent on surface modifications. This study would provide guidance for applying PDMS in cell culture.

Based on the requirement of culture conditions for hematopoietic stem and progenitor cells (HSPCs) ex vive expansion, we developed a new-type bioreactor by combining superiorities of static and stirred culture models. Stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand(Flt-3) were used as the cytokines cocktails. The effects of the static and cyclic culture on the expansion characteristics of CD34+ selected cells were compared in the new-type bioreactor. After 7 d cultures, the expansion of total cells in the static culture was 13.86 +/- 4.26 fold, higher than that in the cyclic culture (7.23 +/- 2.67 fold). The analysis of the fold expansion and the proportion of CD34+ cells showed that there was no marked difference between the static culture and the cyclic culture. However, the fold expansion and the proportion of CD34+CD38- cells were higher in the cyclic culture than those in the static culture (3.90 +/- 0.85 versus 1.82 +/- 0.58), which reflected more primary CD34+CD38- cells were obtained in the cyclic culture. The above results demonstrated that both the static culture and the cyclic culture could be used in ex vive expansion of CD34+ cells with the new-type bioreactor. In static culture hematopoietic stem cells differentiated into progenitor cells, whilst the cyclic culture favored the expansion of primary HSPCs.
Antigens, CD34 ; metabolism ; Bioreactors ; Cell Culture Techniques ; methods ; Cell Differentiation ; physiology ; Cell Proliferation ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Stem Cell Factor ; pharmacology

This study is aimed to design a chemically-defined serum free medium (CDSFM) to support in vitro culture of bone marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were isolated from the femoral bones of one-month-old New Zealand Rabbits with density gradient centrifugation. We compared the proliferation capability, cell cycle, colony-forming efficiency, osteogenic and adipogenic differentiation capabilities of BM-MSCs cultured in CDSFM with those cultured in serum-containing medium (SCM). After 10 days culture, BM-MSCs were expanded by 50 folds in CDSFM, while only 40 folds in SCM. EGF, bFGF and hy-drocortisone were the most important additives and significantly stimulated BM-MSCs proliferation. The percentage of cells at G0-G1 cell cycle was 80.31% +/- 0.58% after CDSFM culture, with no significant difference (P>0.05) compared to 75.24% +/- 4.05% for SCM culture. However, the cloning efficiency of BM-MSCs cultured in CDSFM was significantly lower than that in SCM (P<0.01). The expanded BM-MSCs in CDSFM preserved differentiation potentials into mesenchymal lineages in vitro, including adipocytes and osteoblasts. We have designed a chemically-defined serum free medium that could support in vitro proliferation and maintain the properties of BM-MSCs as stem cells, which could be applied to cell-based therapy and biomedical research.
Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Culture Media, Serum-Free ; Mesenchymal Stromal Cells ; cytology ; Rabbits

In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture, serum-free adherent culture and serum-free single-cell suspension culture. We found that less metabolic by-products were produced in both serum-free cultures. In serum-free single-cell suspension batch culture, the viable cell density was highest. These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.
Animals ; Batch Cell Culture Techniques ; methods ; Culture Media, Serum-Free ; Dogs ; Influenza Vaccines ; biosynthesis ; Madin Darby Canine Kidney Cells ; Virus Cultivation ; methods

Mesenchymal stem cells are of great value in tissue engineering and genetic engineering. To study the in vitro dynamic expansion of mesenchymal stem cells by microcarrier technology, we began the research with the investigation of attachment of rabbit mesenchymal stem cells on cytodex 3 microcarriers after inoculation. The result showed a poor attachment efficiency of 16.7% +/- 1.1% under general conditions, so the attachment efficiency must be increased through the optimization of inoculation conditions. Intermittent stirring, inoculation in 50% of the final culture volume and reduction of the fetal bovine serum concentration at inoculation all led to notable increases in attachment efficiency. Ratio of rMSCs attached in aMEM cultures was 39.8% higher than that of DMEM's on the average. When all these optimal conditions were adopted, the attachment efficiency (65.5%) was significantly higher than that (26.6%) under general conditions; accordingly the final growth extents of rMSCs were 4. 50 and 2. 01, respectively. Therefore, the optimization of attachment conditions promoted the expansion of rMSCs on microcarriers.
Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Dextrans ; Mesenchymal Stromal Cells ; cytology ; Microspheres ; Rabbits

The hematopoietic repopulating ability of fresh and cultured CD34+ cells and CD34- cells derived from cord blood were compared by nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Fresh CD34+ cells and CD34- cells were isolated from fresh cord blood. Cultured CD34+ cells and CD34- cells were separated from cultured mononuclear cells (MNC). We transplanted these cells into sublethally irradiated NOD/SCID mice via the tail vein and sacrificed surviving mice after 6 weeks. The peripheral blood, spleen and bone marrow from each mouse were harvested for flow cytometry, colony-forming cells and human Alu sequences analyses. The proportions of CD45+ cells and human multilineage hematopoietic cells in NOD/SCID mice received CD34+ cells were close to that in the mice received both CD34+ cells and CD34- cells, while it was significantly higher than that in the mice received CD34- cells. Six weeks after transplantation, all the mice injected with cultured CD34- cells dead. The survival rate of mice injected with cultured CD34+ cells was 66.7%. All of the mice injected with both cultured CD34- and CD34+ cells survived. Moreover, CD45+ cells could be detected in all surviving mice, and human CD34, CD3, CD19, CD33 and CD71 antigen also could be detected on these CD45+ cells. The results showed that both fresh and cultured CD34+ cells had the capability of engraftment and hematopoiesis reconstitution, but CD34- cells hadn't the ability. However, CD34- cells had assistant effect on the hematopoietic repopulating ability of CD34+ cells.
Animals ; Cells, Cultured ; Cord Blood Stem Cell Transplantation ; Embryonic Stem Cells ; cytology ; Fetal Blood ; cytology ; Hematopoiesis ; physiology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

TNFR-Fc is an important fusion protein that has great potential in therapeutic and diagnostic applications. We developed an efficient fed-batch process for GS-CHO cells to produce TNFR-Fc. The rationale of this fed-batch process relies on the supply of sufficient nutrients to meet the requirements of cell metabolism. The optimal feed medium was designed through ration design. A metabolically responsive feeding strategy was designed and dynamically adjusted based on the residual glucose concentration determined off-line. In this process, the maximal viable cell density and antibody concentration reached above 9.4x10(6) cells/mL and 207 mg/L, respectively. Compared with the batch process, the newly developed fed-batch process increased the cell yield by 3.4 fold and the final antibody concentration by 3 fold. This fed-batch process would therefore facilitate the production of therapeutic antibody by GS-CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Etanercept ; Glucose ; analysis ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Agitation plays an important role in the hyaluronic acid (HA) fermentation process. However, views about the effect of agitation on HA production remain controversial. We investigated the effect of agitation on cell growth and HA synthesis during HA fermentation process by using Computational Fluid Dynamics (CFD) technology. The results showed that the biomass and HA yield changed a little with the increase of impeller speed, but the HA molecular weight firstly increased and then decreased. The results of phase agitation control strategy demonstrated that the influence of agitation on the HA molecular weight mainly exhibited at the stage of HA synthesis. Moreover, the CFD simulation results indicated that when impeller speed increased, the mixing time reduced while the shear rate increased significantly. The removal of anchor could moderate the contradiction between the mixing time and shear rate, and finally the HA molecular weight increased by 23.9%. The results of this work could provide guidelines for optimizing the HA fermentation, as well as the bioreactor design and scaling up.
Bioreactors ; Fermentation ; Hyaluronic Acid ; biosynthesis ; chemistry ; Hydrodynamics ; Numerical Analysis, Computer-Assisted ; Shear Strength ; Streptococcus equi ; growth & development ; metabolism