Construction And Study Of Microencapsulated Dynamic Co-culture System Of Hematopoietic Stem/Progenitor Cells

At present,the expansion of hematopoietic stem/progenitor cells(HS/PCs)in vitro is one of the effective ways to solve the shortage of HS/PCs for clinical transplantation.However,the in vitro culture of HS/PCs is usually accompanied by the loss of their long-term proliferation activity.Therefore,how to effectively maintain the stem cell activity of HS/PCs and achieve their large-scale expansion is a key issue that needs to be solved urgently.With the research and discovery of the physiological structure and function regulation of the hematopoietic system,in vitro biomimetic construction of the hematopoietic microenvironment to achieve its effective expansion has become one of the current research hotspots.Among the different methods of constructing HS/PCs microenvironment based on biomaterials,the data on the comprehensive effect of multi parameters of solid media,stromal cells and dynamic environment are very limited.This study systematically constructed a microencapsulated dynamic co-culture mode of HS/PCs in vitro,coupled with stromal cells,hydrogel microencapsulated HS/PCs,and three-dimensional dynamic culture in order to achieve effective expansion of HS/PCs.The mouse parietal pre-osteoblasts(MC3T3-E1)were used as stromal cells in the dynamic co-culture system of HS/PCs.The microspheres suitable for the adhesion culture of the MC3T3-E1 cells were constructed and evaluated in vitro.Using polylactic acid(PLLA)as the substrate and gelatin solution as the dispersant,three kinds of polylactic acid-based microspheres were prepared by emulsification solvent volatilization method,and the biophysical properties of the three polylactic acid-based microspheres were comprehensively investigated.It was found that polylactic acid/nano-hydroxyapatite(PLLA/nHA)microspheres have significant advantages in promoting cell adhesion,proliferation and osteogenic differentiation.Therefore,the PLLA/nHA microspheres were selected as the adhesion carriers of MC3T3-E1 cells in dynamic co-culture system.Calcium alginate/gelatin beads were constructed and optimized for HS/PCs embedding culture in vitro.The influence of calcium chloride,sodium alginate and gelatin on the physical properties of alginate beads,such as particle size,pore distribution,viscoelasticity,swelling,permeability,shrinkage and dissolution time was revealed.And,it was found that the cells embedded in the gel beads prepared with calcium chloride,sodium alginate and gelatin at concentrations of 2.5%,2.0%and 0.5%(W/V%)had the best proliferation activity.Thus,this proportion of calcium alginate/gelatin beads would be used to embed HS/PCs.Fluent software was used to numerically simulate the flow field environment of two dynamic co-culture models(the rotary cell culture system(RCCS)and the spinner flask(SF))at different speeds,as well as the force analysis of PLLA/nHA microspheres and calcium alginate/gelatin beads randomly arranged in them,so as to assist in selecting the appropriate operating conditions.RCCS could maintain a relatively stable flow field environment.The overall range of fluid shear stress on the two spheres at 40 rpm was 0.001 Pa-1.108 Pa,which met the needs of the two kinds of cells for the fluid shear stress environment.And this rotational speed was set as the operating condition of RCCS.In SF,two fluid cycles were generated in the upper and lower regions of the blade,and a relatively stationary fluid circulation region was formed at the bottom.The velocity and pressure in the center of these regions was relatively low,which was not conducive to material exchange.The overall range of the fluid shear stress on the two spheres at 45 rpm was 0.000 Pa-2.072 Pa,which was taken as the operating condition of SF after comparative analysis.The separation and purification of umbilical cord blood mononuclear cells(MNCs)and CD34+cells were completed,and the growth characteristics of the two kinds of cells in single parameter static suspension culture were investigated.The survival status of the two kinds of cells gradually deteriorated with the extension of the culture time.Under short-term culture(<7 days),the expansion fold of total nuclear cell number was 1.00±0.11 times and the content of CD45+CD34+and CD34+CD38-cells subpopulations in MNCs were 1.17%±0.15%and 0.96%±0.04%,respectively.The total nucleated cells in CD34+cells were expanded by 5.63±0.54 times,corresponding to the contents of the two subgroups were 47.67%±9.29%and 2.84%±0.23%,respectively.Serum-free single parameter static suspension culture could not promote the expansion of HS/PCs and maintain its original stem cell activity over a long period of time(14 days).The effect of serum-free multi-parameter integration culture mode coupling with the hydrogel encapsulation,stromal cells,and three-dimensional dynamic flow field environment on the expansion activity of HS/PCs was systematically explored.After 14 days of microencapsulated culture of MNCs,the contents of CD45+CD34+and CD34+CD38-cell subpopulations in the RCCS-Co group were 1.76%±0.16%and 2.61%±0.14%,respectively,and the total nucleated cell expansion ratio was 6.56±0.32 times,which was higher than other groups(P<0.001),the average expansion folds of the corresponding two subpopulations were 7.85 times and 7.53 times,respectively.After 14 days of microencapsulated culture of CD34+cells,the total nucleated cells in the RCCS-Co group were 11.87±0.74 times,and the contents of the two cell subpopulations were 67.57%±3.34%and 12.57%±1.27%,respectively,which were also higher than other groups(P<0.05),and the average amplification multiples of the two cell subpopulations were 9.39 times and 15.72 times,respectively.The encapsulated CD34+cells in RCCS-Co group had stronger proliferation and colony forming ability.The co-culture of MC3T3-E1 cells and HS/PCs stimulated MC3T3-E1 cells to accelerate the secretion of related factors to a certain extent.The HS/PCs microencapsulated dynamic co-culture model constructed in this study was more conducive to the expansion of CD34+cells and the maintenance of the activity of primary stem cells than the single parameter static suspension culture.In summary,this study constructed a new type of HS/PCs microencapsulated dynamic co-culture mode.This serum-free multi-parameter integration culture mode,while satisfying the dynamic isolation and co-culture of two kinds of cells,effectively maintained the stem cell activity of HS/PCs and promoted their expansion.It was expected to provide high-quality HS/PCs for related basic research,and also provide experimental basis for hydrogel microencapsulation culture of HS/PCs.