Improvement Of The Stability Of Calcium Alginate Microbeads In RPMI 1640 Culture Medium By Polyvinylpyrrolidone Modification

Objective:RPMI 1640 medium is a commonly used medium for in vitro culture of various cell types.Calcium alginate microbeads can provide a three-dimensional growth microenvironment for cells,which is closer to in vivo.And cell function maintenance is also better than the conventional two-dimensional culture.However,a large number of the conventional calcium alginate microbeads disrupt quickly in RPMI 1640 medium,which results in cell leakage.In contrast,the microbeads soaked in DMEM medium can remain intact.This indicates that the stability of the conventional calcium alginate microbeads is very poor in RPMI 1640 medium.Therefore,cells cultured in RPMI 1640 medium cannot grow in the conventional calcium alginate microbeads for three-dimensional culture.In order to improve this key problem,this study investigated how to improve the culture stability of microbeads in RPMI 1640 medium.Because calcium ions in microbeads maintain the stability of the three-dimensional network,the poor stability may be related to the loss of calcium ions.In the first part of this study,we systematically evaluated the differences in the stability of the conventional calcium alginate microbeads between in RPMI 1640 and DMEM medium.By analyzing the differences of calcium ion concentration in these two medium,we investigated the improvement of the microbead stability by adding proper amount of calcium ions in RPMI 1640 medium.In the second part of this study,composite microbeads modified with polyvinylpyrrolidone（PVP）were further designed and prepared to improve the stability of microbeads instead of adding calcium ions.The effect of PVP concentration on the culture stability of composite microbeads in RPMI 1640 medium was systematically studied,and the optimal PVP concentration that was helpful to cell growth and good culture stability of microbeads was identified through three-dimensional cell culture in vitro.Method:In the first part of this study,DMEM+10%（v/v）FBS was used as the control group,and the morphology,particle size,swelling,sphericity and breakage rate of the conventional calcium alginate microbeads soaked in RPMI 1640+10%（v/v）FBS culture medium were tested under the conditions of simulated cell culture.The stability of the conventional microbeads cultured in RPMI 1640 medium was evaluated by qualitative and quantitative analysis.In addition,calcium ions were added to RPMI 1640 medium,which was equal to the calcium ion concentration in DMEM medium.The effect of calcium ions on reducing the breakage rate of microbeads was evaluated.In the second part of this study,in order to solve the limitation of calcium ion supplement,PVP modified calcium alginate composite microbeads were further designed and prepared,and the effects of PVP concentration on the morphology,particle size,swelling,sphericity and breakage rate of composite microbeads in RPMI 1640+10%（v/v）FBS culture medium was studied.The specific coordination molecular configuration and bond energy formed between PVP,sodium alginate and calcium ions were analyzed by quantum chemistry calculation,and the mechanism of the regulation of calcium ions in mediating 3D network was clarified.The surface and internal structure of the composite hydrogels were analyzed by 3D measurement laser microscope.Moreover,L929 cells were embedded in composite microbeads for three-dimensional culture in vitro using RPMI 1640+10%（v/v）FBS.During the culture time,the changes of morphology,particle size,swelling,sphericity and breakage rate of composite microbeads were investigated.According to cell growth and microbead stability,the optimal PVP concentration was finally obtained.Results:In the first part of this study,we found that the stability of the conventional alginate microbeads cultured in RPMI1640+10%（v/v）FBS medium was very poor,and the breakage occurred after 24h culture,and the breakage rate reached 100%on Day 10,while no breakage was found in DMEM+10%（v/v）FBS medium.In addition,the adding of calcium ions into the RPMI 1640 culture medium（equal to the concentration in DMEM）had the limited inhibitory on the breakage rate,and the microbeads still had significant breakage,which indicated that the effect of adding exogenous calcium ions was very limited.In the second part of this study,PVP modified alginate composite microbeads were successfully prepared,and it was found that with the increase of the final concentration of PVP,the breakage rate of composite microbeads cultured in RPMI 1640+10%（v/v）FBS culture medium gradually decreased,which indicated that the addition of PVP could improve the culture stability of composite microbeads.In addition,through the quantum chemical calculation,we found that PVP and sodium alginate could form a new molecular configuration,namely,GG-Ca²⁺-PVP,through the coordination of calcium ions.The bonding energy of GG-Ca²⁺-PVP was 524.6 kcal/mol,which was very close to the bonding energy of GG-Ca²⁺-GG（555.4 kcal/mol）as the gel skeleton.This indicated that PVP formed a relatively stable three-dimensional interpenetrating network with sodium alginate through the coordination with calcium ions,which improved the culture stability of composite microbeads in RPMI 1640 medium.Moreover,through3D measurement laser microscope,we found that the addition of PVP made the surface of the composite hydrogel more flat and the internal structure more compact.In addition,L929 cells could survive,proliferate and form cell clusters in the microbeads containing 4%（w/v）PVP,and these microbeads had good stability,which indicated that 4%（w/v）was the optimal PVP concentration.Conclusions:1.Compared with DMEM+10%（v/v）FBS culture medium,the conventional alginate microbeads soaked in RPMI 1640+10%（v/v）FBS culture medium had very poor stability,and the inhibition effect of adding proper amount of calcium ions on the breakage rate was still limited.2.PVP formed a relatively stable three-dimensional interpenetrating network with sodium alginate through the coordination with calcium ions,which made the structure of the composite hydrogel more compact,which could effectively improve the culture stability of composite microbeads in RPMI 1640+10%（v/v）FBS medium.4%（w/v）PVP was the optimal final concentration.Under this condition,L929 cells in composite microbeads could survive,proliferate,and form cell clusters,and the breakage rate of composite microbeads in RPMI 1640 medium could be significantly inhibited,and the stability of composite microbeads could be significantly improved.Therefore,this study provides a new method for improving the cell culture stability of calcium alginate microbeads.