Optimization Of Serum-free Culture Of Umbilical Cord Mesenchymal Stem Cells

Objective:As a kind of stem cells,mesenchymal stem cells(MSCs)have the ability of self-replication and multi-differentiation.Serum is used as an additive to maintain cell growth in vitro.However,there are too many uncontrollable factors,which bring risks to cell culture in vitro.Serum-free culture has become an inevitable trend.The purpose of this study is to compare different types of serum substitutes commonly used in the market on the basis of avoiding serum,to evaluate their advantages and disadvantages and the possibility of using them in large-scale cell culture in vitro,to select a suitable serum substitutes through various identification,to judge the possibility of completely replacing serum by a series of identification,and to explore this.Whether the substitutes can satisfy the continuous passage and expansion of cells at a lower concentration can reduce the cost of culture.It provides data reference for the improvement of cell culture technology in vitro.Method:1.Select the umbilical cord of newborns that meet the criteria,isolate and culture,pass on,part of the cells are frozen for reserve,and the other part is detected and identified according to the traditional serum culture technology and the international criteria for identification of mesenchymal stem cells.2.In contrast with the conventional animal serum culture system,two kinds of commercialized serum substitutes for stem cell culture,i.e.chemical const ituent-limited serum substitutes(K)and platelet lysates(G),were selected.Mesenchymal stem cells isolated from umbilical cord tissue were subcultured continuously and their growth curves were recorded.The effect was evaluate d,and the dryness of the cells was evaluated by morphological observation,flow cytometry,surface markers and induction of trilineage differentiation.3.Ideal serum substitutes(G)were selected through the above experiments.In addition to passage,the serum was replaced by adding components of primary culture medium and cryoprotectant after isolation of umbilical cord tissue.Serum culture medium was used as control to observe the growth status of primary cells.The cells after cryopreservation and resuscitation were subcultured continuously,and the growth curve was counted and drawn to describe the growth status of cells.Flow cytometry and three-line differentiation induction were used to identify the stem of P6 cells.4.Final to find a better serum substitute(G)for stem cell culture in vitro.Four groups of experimental groups were set up according to the concentration of the substitute.The P2 cells were cultured continuously after resuscitation,and the effect of serum substitute concentration on cell culture in vitro was evaluated.5.Cells were cultured in four groups according to different inoculation concentration,using the best additive concentration culture system screened by the above experiments.The cells were cultured in routine serum culture as control.After continuous passage,growth curve was drawn according to the results of counting.Flow cytometry and three-line induction differentiation were performed on P6 cells to evaluate the stem cells.6.Resuscitation of cells frozen with substance G as cryoprotectant,10%FB Sas control group and 5%G as experimental group.Resuscitation cells were subcultured continuously and passed to P15 generation.Growth curve was drawnby each generation counting to reflect cell proliferation.Phenotypic detection and three-line differentiation induction of P15 generation cells were carried out to determine whether the cells were in line with international standards.Mesenchymal stem cell identification criteria.Result:1.Mesenchymal stem cells obtained by traditional serum culture in vitro conform to international mesenchymal stem cells.Identification criteria.2.Mesenchymal stem cells cultured in substance K and G were compared with serum-cultured mesenchymal stem cells.Compared with the P3 generation,the size of mesenchymal stem cells cultured in K medium became larger and the degree of fusion was less than 60%.By P6 generation,the degree of cell fusion was less than 30%,and the number of cells was too small to carry out follow-up experiments.In the whole passage culture process,the fusion degree of G cells in each generation can reach more than 80%,and the cell morphology is good,the number of G cells has no significant difference with the control group.3.Microscopic observation after 2-3 days of primary cell culture showed that a small number of adherent cells crawled around the tissue mass.Out,the cells were short and thick spindle-shaped or hill-shaped,and the cell gap was large and the growth was slow.There was no significant difference between the experimental group and the control group.After 10-14 days of culture,the fusion degree of cells reached more than 50%and the morphology was uniform.There was no significant difference between the two groups.Five days after passage of P1,the fusion degree of cells in the two groups reache more than 80%,and the number of recovered cells in the two groups remained unchanged.After cryopreservation and resuscitation,cells were subcultured to P6 generation.The fusion degree of cells in the experimental group was slightly better than that in the control group,and the number of cells harvested was slightly higher than that in the control group.There was no significant difference between the results of flow cytometry and induction of three-line differentiation in the experimental group and the control group.4.The medium containing 10%FBS was used as the control.Through the passage comparison of different G concentration,it was found that the medium containing 10%FBS contained 10%FBS.Cells culturedin medium conta ining 5%,4%and 3%G had higher amplification efficiency.Cells cultured in medium containing 2%G were similar to the control group,while cells cultured in medium containing 1%G had lower amplification efficiency than the control group.5.The G substance can meet the requirement of low density cell culture.In this experiment,the medium containing 10%FBS was used.10000 cells/cm~2were inoculated as control group.The experimental group used medium containing 5%G.The inoculation density was 2000 cells/cm~2,4000 cells/cm~2,6000 cells/cm~2,8000 cells/cm~2 and 10000 cells/cm~2 respectively.The results showed that under the condition of inoculation density of 2 000 cells/cm~2,the cell disposal and expansion were faster,but with the continuous passage of low density,the cell emerged.Aging,increasing volume and slowing down amplification rate.