Vitrification And 3D Culture Of Stem Cells Based On Hydrogel Encapsulation

Stem cells has been widely applied in biomedical engineering,cryopreservation of stem cells is particularly important to meet the ever increasing demand of stem cells.Conventional slow-freezing of stem cells may cause inevitable cell damage,vitrification as a novel cryopreservation method has recently been used to preserve stem cells.Conventional vitrified cells often require high concentrations of cryoprotectants,so that the sample is completely vitrified during the freezing process,there is no ice formation during the rewarming process,so as to ensure that the cells have a higher survival rate after freezing and rewarming.To address these problem,two strategies were employed,using high concentration of penetrating cryoprotectants(CPA)or reducing sample volume(to achieve sufficient cooling/rewarming rate).In the one hand,high concentrations of penetrating CPA(≈8M)is toxic cells,in the other hand,reducing the sample volume tends to complicate the freezing process and increase the workload,meanwhile,the loss rate of cell during the freezing process increased.In view of the existing problems of vitrification,we first studied the potential mechanism of freezing damage,we studied the water transport properties and the probability of intracellular ice formation(PIF)of pig adipose-derived stem cells(pADSCs)during cryopreservation by cryomicroscope.Three cooling rates(5℃/min,10℃/min and 15℃/min)were performed to observe the volume changes of pADSCs during freezing.A mathematical model was used to fit the experimental data to obtain two transmembrane water transport parameters of pADSCs,they were permeability coefficient of water(Lpg)and the activation energy(ELP),which would be helpful for the optimization of freezing protocol and better understand the osmotic damages.As for the PIF of the pADSCs during the freezing process,we tested three different cooling rates(20℃/min,30℃/min and 60℃/min)and founded the most suitable cooling rate for cryopreservation.In this paper,the hydrogel was used to inhibit the intracellular ice formation during the vitrification and rewarming.In the past researches,small volume(Diameter<250μm)of hydrogels and high concentration(≈ 8M)of CPAs were employed to achieve vitrifivation.In this paper,pADSCs were encapsulated in alginate microcapsules(large volume with diameter>500μm)with core-shell structure for partial vitrification(there exist ice crystals outside the microcapsules while the hydrogel inhibited the ice formation within the capsules,which protect the cell from damages of ice.),which great improved the encapsulation efficiency.In our research,we achieved rapid encapsulation of large volumes(more than a few hundred liters of sample,frequently used in cell therapy and transplantation)of cell suspensions.In addition,ultra-low concentration of CPAs(≈2 M penetrating CPA and 0.5M nonpenetrating CPA)were added to the sample.Cell-laden microcapsules were directly plugged into the liquid nitrogen for freezing,we obtained high survival rate of cryopreserved cells.Encapsulation can greatly improve the survival rate of cells with 4 groups of CPAs(with different formulation),from 24%to 73%,25%to 71%,25%to 63%and 21%to 56%respectively.The results demonstrated that hydrogel microcapsules can effectively inhibit the formation and propagation of ice crystals during the freezing and rewarming process.Microcapsules with large volume has great potential in the stem cells-based therapies.In order to improve the generation of microcapsules,we discard the traditional oil-based method,three biocompatible water-soluble solvents were used to produce sodium alginate microcapsules which can better protect the cell viabilities.Comparing with conventional water-soluble solvents-based microcapsule generation method,our method can easily adjust the diameter of the microcapsules and the relative thickness of the shell and nucleus.The peripheral solution is a water-soluble solution with excellent biocompatibility so that the viabilities of cells or other biological samples could be saved during the cross-link process.pADSCs were encapsulated in the capsules for 3D cell culture,since the cells were in a biocompatible solution during the entire encapsulation and cross-linking process,the cells experienced minor damages so that the cells grow well and gathered into cluster on the seventh day,no obvious cell death were observed.In order to further improve the efficiency of encapsulation,we replaced the oil phase with with calcium chloride solution to produce microfibers with core-shell structure.The shape of the microfibers is more similar to blood vessels,tendons and nerves,so the 3D culture of the cell-laden micorfibers has more significance in bionics.Microfiber-like hydrogels were used to encapsulate pADSCs suspension,the microfiber were wrapped with gauze and then plugged into the liquid nitrogen for vitrification,survival rate of more than 70%was obtained with the 1M penetrating CPAs(0.5M PG and 0.5 EG),It is worth noting that we used CaCl2 solution instead of oil phase,avoid the separation of microfiber from oil phase.In this study,microfibers were used to encapsulate cells which is more efficient than microcapsules.What’s more,microfibers is easier to hold and operate,there needn’t any special container.Microfiber can be directly plugged into liquid nitrogen to achieve a higher cooling/rewarming rate than microcapsules loaded in cryovials or plastic straws.