| Cryopreservation is an indispensable method for long-term storage of cells,cellbiomaterial constructs,tissues and even organs,and is widely used in biomedical fields such as regenerative medicine,tissue engineering,and cell therapy.Vitrification is an emerging cryopreservation method that can avoid the formation of ice crystals during cooling process.However,traditional vitrification methods usually require high concentrations(approximately 6-8 M)of permeable cryoprotectants(pCPAs),which can cause osmotic damage and biochemical toxicity,and may lead to uncontrolled differentiation of stem cells.Therefore,it is of great significance to optimize and screen cryoprotectants and to explore a novel and efficient vitrification preservation method with low concentration of pCPAs.In low concentration of pCPAs vitrification,recrystallization/devitrification during the rewarming process can cause fatal damage to biosamples,therefore,the ice recrystallization inhibition has become the focus of research.In this paper,a multifunctional nanocomposite hydrogel system is formed by combining materials with physical field response and recrystallization inhibition function to explore its application in vitrification of biosamples.In this paper,the nucleation temperature,melting temperature,melting enthalpy and supercooling degree of cryoprotectants with different concentrations and components and nano-cryoprotectants during cooling and rewarming were systematically studied by differential scanning calorimetry(DSC).The results show that the higher the concentration of cryoprotectants is,the better the performance of icecrystal inhibition is when the type of cryoprotectants is unchanged,and the combination of non-permeable cryoprotectant sugars and pCPAs can effectively improve the effect of protection at low temperature.The study of nano-cryoprotectants shows that GO-Fe3O4 nanocomposites(NCs)can increase the nucleation temperature through heterogeneous nucleation,and have almost no effect on melting temperature.Therefore,the addition of NCs can reduce the supercooling of the system,and avoid the formation of sharp ice crystals damaging biosamples,which is conducive to cryopreservation.Further,DSC was used to carry out thermal analysis of nanocomposite hydrogels(NCHs)containing GO-Fe3O4.The results show that the NCHs under interacting with cryoprotectants are more conducive to inhibiting ice crystals,and the addition of GO-Fe3O4 NCs can reduce the supercooling degree of hydrogels to a certain extent.In summary,thermal analysis of cryoprotectants and functional materials in cryopreservation is carried out to explore the factors affecting the growth of ice crystals,and provide theoretical guidance for the optimization and selection of cryoprotectants and the application of functional materials in cryopreservation.The use of functional materials provides a new way for the cryopreservation of biosamples.We separately investigated the effects of responsive nanomaterials and hydrogels on vitrification of mesenchymal stem cells(MSCs).We combined GO-Fe3O4NCs with cryoprotectants as nano-cryoprotectants for vitrification of MSCs.The effects of different laser intensities and current intensities on the activity of MSCs after preservation were explored by using the thermal effect of GO-Fe3O4 NCs under laser and alternating magnetic field.In this study,two groups of multi-component combined cryoprotectants CPA#1(1 M EG+1 M PG+10%Dex+1.2 M Tre)and CPA#2(2 M EG+2 M PG+5%Dex+0.5 M Tre)were used for cryopreservation.The results shows that the cell activity of laser-assisted rewarming is significantly higher than that without laser when the nano-cryoprotectants(CPA#1+NCs and CPA#2+NCs)are used for preservation,and the cell survival increases with the increase of laser intensity.The electromagnetic effect can also significantly improve the survival rate of stem cells in cryopreservation mediated by nano-cryoprotectants,and as the current intensity increases,the cell viability also improves.The results of laser and electromagnetic heating show that the cell viability is further improved by dual-mode heating.Besides,the effect of alginate hydrogels on the vitrification of MSCs was explored.The results show that encapsulation of MSCs with alginate hydrogels significantly improves the cell viability after cryopreservation(CPA#1 and CPA#2),and the size of microcapsules has no significant effect on the survival rate of MSCs after cryopreservation.In summary,the GO-Fe3O4 nanomaterials and alginate hydrogels were used to act on stem cells respectively,and the results show that the two materials can improve the preservation effect,but the cell survival rate is still not ideal(both are less than 70%).The result of cryopreservation with CPA#2 is better than that of CPA#1,but considering that CPA#1 is a kind of more ideal cryoprotectants(with lower pCPAs concentration),it is of great significance to further explore new strategies to achieve high-quality vitrification preservation with low concentration of pCPAs(such as CPA#1).In order to achieve the vitrification preservation of stem cells at low concentration of pCPAs,GO-Fe3O4 NCs with dual laser and electromagnetic responses were introduced into the alginate hydrogels to form a GO-Fe3O4/alginate nanocomposite hydrogel system for vitrification under the condition of using optimized nanocryoprotectants(CPA#1+NCs).The results show that the cell viability of suspended stem cells is only about 10%using vitrification method with low concentration of pCPAs,while the use of GO-Fe3O4/alginate nanocomposite hydrogel system,the viability of cells in constructs increases to 80.1%under the condition of simultaneous laser and electromagnetic heating(4 W/cm2,15 A).The 7-day 3D culture result of the vitrified stem cell constructs shows that the stem cell constructs still have intact structure after a long period of culture,and the cell survival rate is similar to that of the fresh group.In addition,the induced differentiation experiment proves that the MSCs still have the ability of adipogenic,osteogenic and chondrogenic differentiation post vitrification.The study of ice inhibition mechanism of GO-Fe3O4/alginate nanocomposite hydrogels discovers that GO-Fe3O4 nanomaterials and alginate hydrogels can effectively inhibit ice recrystallization,and GO-Fe3O4 nanomaterials can realize laser and electromagnetic dual-mode heating,which shorten the recrystallization time during rewarming.In conclusion,GO-Fe3O4 nanomaterials and alginate hydrogels can be used as ice recrystallization inhibitors,while GO-Fe3O4 nanomaterials in the microcapsules and nano-cryoprotectants can act as local heat sources to increase the rewarming rate and inhibit ice recrystallization under the laser and alternating magnetic field.Therefore,the multifunctional nanocomposite hydrogel system is a kind of novel and high-efficiency of cryopreservation strategy,which can effectively inhibit the ice recrystallization by multiple functions,and achieves a low concentration of pCPAs(2 M)vitrifcation of stem cell constructs to meet the clinical needs of "ready to use",thus greatly promoting the development of stem cell-based treatment and regenerative medicine. |
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