3D Culture Of Human Pluripotent Stem Cells And Construction Of 3D System For Cryopreservation

Human induced pluripotent stem cells(hiPSCs)have the potential to expand indefinitely and differentiate into a variety of cells and tissues.Compared with human embryonic stem cells,hiPSCs not only face less ethical and moral disputes,but also avoid the risk of immune rejection.3D culture is an effective method for scale expansion,but the 3D efficient scale expansion and long-term preservation of hiPSCs are still facing many issues in future clinical applications.Herein,in this thesis,research on the 3D culture of pluripotent stem cells and their cryopreservation in a 3D system was conducted.The main research contents and results are as follows:(1)3D culture of hiPSCs in nanofibrilliar cellulose hydrogel: hiPSCs were cultured in0.8 wt% nanocellulose hydrogels.Cell viability and pluripotency of hiPSCs at different locations inside 3D hydrogel were investigated by calcein/propidium iodide(Calcein/PI)and stem cell markers.The effective diffusion coefficients of glucose,lactate and transforming growth factor(FGF2)and fibroblast growth factor(TGF-β1)within the hydrogel was determined.Then the distribution of nutrient and metabolic products was analyzed throught the hydrogel.Biological compositions of hiPSC including proteins,lipids and nucleic acids were detected using synchrotron radiation infrared microscopy at the single-cell level.Principal component analysis(PCA)method was applied to evaluate the changes in the biochemical composition of cells at different times and different zones inside hydrogel during culture.The experimental results show that TGF-β1 and FGF in the hydrogel exhibits significant different in the concetration between the deep zone and superficial zone,probably a reason for different proliferation rates of cells at different locations.The lactic acid concentration insid the gel continues to accumulate over time,causing the cells exposed to an acidic growth environment,leading to a change in the electrical charge of nanocellulose and proteins in cell culture medium,resulting in further changes in the cell growth environment.All these together,it causes heterogeneity in protein secondary structure and protein glycosylation in cells at different locations and different incubation times,ultimately leading to changes in cell pluripotency.(2)Emulsion construction based on Taylor vortex reactor: The physical model of the vortex reactor was established by COMSOL software.The influence of the gap between the inner and outer barrels of the reactor,vortex speed on the Taylor number and surface tension and system viscosity on the formation of emulsion were analyzed.The Taylor vortex flow was further determined,and the effect of shear force on the particle size distribution of the emulsion was evaluated.Based on the analysis of the fluid shape,the process parameters for transforming laminar flow to wavy Taylor vortex flow was obtained.Eventually,the relevant parameters of the Taylor vortex reactor for emulsion construction was determined.To explore the effects of different emulsification processes and reagent formulations on the stability of the emulsion,a series of experiments were performed.The results show that the viscosity is inversely proportional to the size of the droplets,increasing the vortex speed lead to an increase in the frequency of droplet generation.It is difficult to form droplets when the surface tension is small.Multiple emulsions with high thermodynamic stability at room temperature were obtained by manipulating the parameters of surfactant ratio,emulsification speed,internal and external aqueous formula.(3)Cryopreservation of hiPSCs: In this study,the cryopreservation of hiPSCs in a 3D system was explored.Firstly,hiPSC cells with GFP green fluorescence were constructed.The Taylor vortex reactor was used to encapsulate the hiPSCs in multiple emulsions.The effects of internal and external water phases on the encapsulation efficiency was exploited.The emulsion rate after cryopreservation was further explored.The results show that sucrose in the inner aqueous phase plays an essential role in the cryopreservation process,while the cross-linked sodium alginate in the outer aqueous phase is able to enhance the stability of the multiple emulsions during cryopreservation.The effects of combination of sucrose,EG,DMSO,FBS,glutamine on the cryopreservation of hiPSCs were elucidated.The results reveal that the combination of DMSO and sucrose is better than the combination of EG,but DMSO is not suitable for the cytotoxicity of DMSO in this system.On the contrary,the combined effect of EG with sucrose and FBS is better,the encapsulation of the emulsion can provide better cryopreservation for hiPSC,and the hydrogel in the inner water phase can promote the recovery rate of cells after cryopreservation.