Large Scale Expansion Of Human Placenta-derived Mesenchymal Stem Cells Ex Vitro In Stirred Bioreactor

ObjectiveMesenchymal stem cells(MSCs)give origin to the marrow stromal environment that supports hematopoiesis.These cells present a wide range of differentiation potentials, they can be induced to differentiate into bone,adipose,cartilage,muscle,and endothelium if these cells are cultured under specific permissive conditions.Moreover, the immunogenicity of MSCs are low,using MSCs from unrelated donors as a cell feeder layer for support of hematopoietic progenitor cells(HPCs)can not induce immunological rejection.Therefore,MSCs may have a wide range of potential applications in clinical therapy and it is necessary to develop ex vivo culture systems to scale up the expansion.Conventional culture systems such as T-flasks and gas-permeable blood bags are the most widely used devices for expanding MSCs. However,such static culture systems have several inherent limitations:lack of mixing results in concentration gradients for dissolved nutrient substances and metabolites, lack of readily monitoring or controlling the environmental condition on-line,limit in productivity by the number of cells.Stirred tank bioreactor can provide balanced nutrition for MSCs expansion,in addition,the environmental conditions can be monitored.As a castoff after parturition,along with the ease of accessibility,lack of ethical concerns,placenta may be an attractive source of mesenchymal stem cells for basic and clinical application.In this work,the feasibility of using stirred tank bioreactors to expand human placenta-derived mesench-ymal stem cells(hPDMSCs) was studied.Parallel experiments using T-flasks were also carried out for a comparative study.During the 6 days of culture,the total cell number,the surface markers were determined and metabolites,pH and osmolality of the medium were monitored.Mehtod1.Isolation and culture of hPDMSCs:The harvested pieces of term placentas were obtained and washed several times in PBS,and then the explant method was used to isolate and cultured MSCs.2.Pretreatment the Cyterdex-3 microcarriers and stirred tank bioreactors: Sufficient Cytodex-3 microcarriers were weighed per 10mg/ml and soaked overnight with PBS,autoclaved,coated with gelatin,and then maintaining the microcarriers at 4℃for spare.Stirred tank bioreactors were soaked in ethanol overnight and then used distilled water to wash them.Autoclaving the stirred tank bioreactors for spare.3.Expansion of hPDMSCs ex vitro in stirred tank bioreactors:Inoculating the microcarriers with 1×10⁷ cells respectively to prepare cell suspension.The cell suspension was incubated at 37℃with a water-saturated atmosphere 24 h and then injected it into the stirred tank bioreactors.The stirring speed of the reactor was step increased from 40rpm for 6h,and then reached 60 rpm.Parallel experiments using T-flasks were also carried out for a comparative study.4.Measurement the growing and metabolic changes of hPDMSCs:The cell suspension was sampled every day to measure the expansion rates of hPDMSCs and PH,osmolality,glucose(GL)and lactic acid(LA)of the medium.Stopping the experiement when cells reached to growth inhibiting.5.Immunophenotyping and cell cycle of hPDMSCs by flow cytometry: Unexpanded cells and expanded cells derived from the reactor were detected using a flow cytometer to analyze the expression status of CD13,CD14,CD29,CD31,CD44, CD45,CD73,CD90,CD105,CD166,HLA-DR,HLA-ABC and the change of cell cycle.Result1.Establishment of primary culture:The isolated cells demonstrated a spindle-shape or fibroblast-like morphology and can be passaged more than 20 times. 2.Characterization of by flow cytometry:hPDMSCs were positive for CD13,CD29,CD44,CD73,CD90,CD105,CD166,HLA-ABC and were negative for CD14,CD31,CD45,HLA-DR.3.Growing and metabolic changes of hPDMSCs(1)Growth characteristics of on microcarriers:In control groups,hPDMCs were obviously adherent to on microcarriers,but there were a few of microcarriers to put up bridges.In experimental groups,there were a lot of microcarriers to put up bridges through cell matrix.(2)Growth curves of hPDMCs:In control groups,hPDMCs proliferated leniter. After 4 d,a plateau in the growth was reached,and then cell density decreased gradually.In stirred bioreactors,cells proliferated less slowly than cells that in control groups at the beginning 48h,then cells proliferated fleetly and reached plateau at the 5^thd.(3)Expansion folds of hPDMCs:The average expansion fold in stirred tank bioreactors was 10.55±1.62 per passage,significantly higher than that was 6.10±0.11 in control group(P＜0.05).(4)Metabolic changes of hPDMCs:The average concentration of GL in stirred tank bioreactors was always higher than that in control groups,while the average concentration of LA in stirred tank bioreactor was always lower than that in control groups.(5)The changes of medium PH:pH was mostly kept between 6.8 and 7.4 in the stirred tank bioreactors but 6.5 and 7.4 in control group.(6)The changes of medium osmolality:The osmolality of the culture medium in the stirred tank bioreactor and T-flasks was both nearly kept constant and well controlled in 280 mm Osmol/kg and 320 mm Osmol/kg during the culture period.4.Immunophenotypes and cell cycle of hPDMCs:The immunophenotypes of hPDMCs were not change after being cultured in stirred bioreactor.Cell surface makers were also expressed as:CD13,CD31,CD44,CD45,CD73,CD90,CD166,HLA-ABC, HLA-DR,and cell cycle had not become the distinguishing feature whether cells were cultured in stirred bioreactors(P＞0.05).All these showed that hPDMCs could keep their bionomics when they were cultured in stirred bioreactor.Conclusion1.As along with the ease of accessibility,lack of ethical and law concerns and with abundant MSCs number in,placenta may be a new attractive source of mesenchymal stem cells.2.It was safe and effective to use stirred bioreactor to expand hPDMCs.It not only solved the problem that it is difficult to obtain considerable stem cell seeds,but also offered a new biological evidence for the manufacture of bioreactors.