| Objective:The ex vivo adipogenic differentiation of human bone marrow mesenchymal stem cells is mainly carried out in two-dimensional(2D)culture system so far,however the adipogenic differentiation efficiency in which is still far from optimal.This study was aimed to investigate the effects of zinc and sodium alginate hydrogel culture on the proliferation and adipogenic differentiation of human bone marrow mesenchymal stem cells and develop an optimized culture system.Methods:(1)Human bone marrow mesenchymal stem cells were isolated with density gradient centrifugation and cultured in low glucose DMEM 10%FBS.Third-generation hBM-MSCs(2×10~4/cm~2)were seeded in 96-well plates after trypsinization and optical density value at 450nm was measured to determine the time point at which hBM-MSCs was at the proliferation peak.(2)Third-generation hBM-MSCs(2×10~4/cm~2)were seeded in 96-well plates after trypsinization.hBM-MSCs were cultured in low glucose DMEM 10%FBS containing zinc of 0.001,0.004,0.016,0.064 and 0.256 mmol/L,respectively as experiment groups and in low glucose DMEM 10%FBS without zinc as control until the time of proliferation peak.Optical density value at 450nm was measured to determine the suitable zinc concentrations.(3)Third-generation hBM-MSCs(2×10~4/cm~2)were seeded in 24-well plates after trypsinization.hBM-MSCs were cultured in low glucose DMEM 10%FBS and the medium was changed every 3 days.Adipogenic differentiation was induced 3 days after the hBM-MSCs were confluent to contact inhibition with induction medium with different zinc concentrations for 21 days.Adipocytes were stained with oil red O and optical density value at 492nm was measured to investigate the effects of different zinc concentrations on the adipogenic differentiation of hBM-MSCs.(4)mRNA expression of adipogenic genes PPAR?,C/EBP?and FABP4 were measured with quantitative real-time polymerase chain reaction(qRT-PCR)to investigate the effects of different zinc concentrations on the adipogenic genes expression of hBM-MSCs.(5)Third-generation hBM-MSCs(5×10~6/ml)were resuspended in 1.5%sodium alginate solution and the cell suspension was dropped into 0.1 mol/L calcium chloride to form microspheres.Microspheres were cultured in 24-well plates with low glucose DMEM 10%FBS and the medium was changed every 3 days.The same hBM-MSCs(2×10~4/cm~2)were seeded in 24-well plates as 2D control.The morphology of hBM-MSCs in 2D and 3D culture systems was observed by optical microscope and the proliferation was measured by CCK-8.(6)Third-generation hBM-MSCs(5×10~6/ml)were resuspended in 1.5%sodium alginate solution and the cell suspension was dropped into 0.1 mol/L calcium chloride to form microspheres.Microspheres were cultured in 24-well plates with low glucose DMEM 10%FBS and the medium was changed every 3 days.The same hBM-MSCs(2×10~4/cm~2)were seeded in 24-well plates as 2D control.Adipogenic differentiation in 2D and 3D culture was induced 3 days after the hBM-MSCs in 2D culture were confluent to contact inhibition with induction medium for 21 days.Adipocytes were stained with oil red O.The morphology of adipocytes was observed by optical microscope and optical density value at 492nm was measured to compare the effects of 2D and 3D culture on the adipogenic differentiation of hBM-MSCs.Results:(1)hBM-MSCs were adhering to the culture plates and in growth stagnancy on the first day after seeding.Cell growth increased slowly since the second day and hBM-MSCs were in the latent phase.Since the fifth day,Cell growth significantly increased and cell doubling time notably reduced until proliferation reaching its peak on the sixth day,during which it's logarithmic growth phase.Cell growth slowed down since the seventh day,gradually reaching the stagnate phase.(2)There was no significant difference in cell viability between groups with either0.001 or 0.064 mmo/L zinc and the control(P>0.05).Cell viability significantly increased in groups with 0.004(P<0.05)and 0.016 mmo/L(P<0.001)zinc,indicating 0.004 and 0.016 mmo/L zinc promoted the proliferation of hBM-MSCs.Cell viability notably reduced in the group with 0.256 mmo/L(P<0.01)zinc,indicating 0.256 mmo/L zinc inhibited the proliferation of hBM-MSCs.These data suggested zinc promoted cell proliferation in certain concentrations with a dose-dependent manner.(3)The adipogenic differentiation of hBM-MSCs was significantly enchanced in groups with 0.001,0.004,0.016 and 0.064 mmo/L zinc compared to control(P<0.001).These data suggested zinc promoted the adipogenic differentiation of hBM-MSCs in certain concentrations with a dose-dependent manner and the optimal concentration was 0.064 mmol/L.(4)The expression of adipogenic genes PPAR?,C/EBP?and FABP4 significantly increased in groups with 0.001,0.004,0.016 and 0.064 mmo/L zinc compared to control(P<0.001).These data suggested zinc promoted the expression of adipogenic genes of hBM-MSCs in certain concentrations with a dose-dependent manner and the optimal concentration was 0.064 mmol/L.(5)The hBM-MSCs encapsuled in sodium alginate hydrogel microspheres were evenly distributed in round-shape and able to grow well.The microspheres were suspended in culture medium and maintained three-dimensional structure.The hBM-MSCs in 2D culture adhered to the plates 1 day after seeding in spindle-or polygonal-shape and started to spread and grow in a radial or circinate pattern.(6)Cell growth was significantly faster in 2D culture than 3D culture in every single point-in-time examined(day1,3,5,7)(P<0.001)and it's 3.8 times faster than that of3D culture on day 7.These data suggested the proliferation of hBM-MSCs in 2D culture was more active than that in 3D culture.(7)The hBM-MSCs encapsuled in sodium alginate hydrogel microspheres were successfully induced into adipocytes with round-shape and multiple small round lipid droplets in the cytoplasm similar to the adipocytes in vivo,while the adipocytes induced from hBM-MSCs in 2D culture remained adhering to the plates in elongated spindle-or polygonal-shape with multiple small round lipid droplets in the cytoplasm.Only 40-50%of hBM-MSCs were eventually induced into adipocytes in 2D culture after 21-days adipogenic induction,while 80-90%of hBM-MSCs in sodium alginate hydrogel microspheres differentiated into adipocytes.Optical density value at 492nm also demonstrated that the adipogenic differentiation efficiency in 3D culture was 2times of that in 2D culture(P<0.001).These data suggested sodium alginate hydrogel microspheres 3D culture significantly increased the adipogenic differentiation efficiency of hBM-MSCs.Conclusions:(1)Zinc promoted cell viability and proliferation in certain concentrations with a dose-dependent manner.(2)As an ingredient of adipogenic induction medium,zinc promoted the proliferation and adipogenic differentiation of hBM-MSCs.(3)The hBM-MSCs encapsuled in sodium alginate hydrogel microspheres were evenly distributed in round-shape and able to grow well,however the proliferation of hBM-MSCs in 3D culture was slower than that in 2D culture in the early culture.(4)Sodium alginate hydrogel was a favorable 3D culture system for hBM-MSCs as being able to increase the adipogenic differentiation efficiency of hBM-MSCs. |
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