Cryopreservation Of Mesenchymal Stem Cell-Laden Hydrogel Constructs

Since mesenchymal stem cell-laden hydrogel constructs has extremely important application value in tissue engineering and regenerative medicine,their preservation is quite important.On the one hand,stem cell-laden magnetic nanocomposite hydrogels(NCHs)have short shelf life at ambient temperature,and continuous cultivation of them is expensive and time-consuming.Therefore,banking stem cell-laden magnetic NCHs at cryogenic temperatures is essential to provide the "off-the-shelf" availability for cell-based clinical applications.Vitrification method has attracted people’s attention because of its unique features.Vitrification can convert a liquid to a glassy state without ice formation at vapor-phase nitrogen temperatures and thus does not cause cell damage.In addition,vitrification process is fast and simple,and do not need expensive equipment and complex programmable cooling process.On the other hand,conventional cryogenic preservation largely relies on laboratory conditions and complicated containers,such as liquid nitrogen tanks,which is difficult for delivery and transportation of stem cells and their hydrogel constructs between different locations and cities to meet clinical needs.Hypothermic storage can be a simple and effective method for short-term storage of stem cells and their hydrogel constructs.In this work,first the mechanism of NCHs on cryogenic recovery of stem cells by cryomicroscope experiment was explored.Then,the vitrification of stem cell-Fe3O4 magnetic NCH microcapsules was studied,and the electromagnetic rewarming effects of nanocomposite hydrogel composed of Fe3O4 nanoparticles and alginate hydrogels on mesenchymal stem cells were explored.The results show that,the NCHs have good biocompatibility.Then,the magnetothermal effects of NCHs in magnetic field were evaluated.Uniformly dispersed MNPs inside hydrogel constructs effectively suppressed ice nucleation and ice crystal growth via enhancement of warming.Moreover,binding of alginate hydrogel with water molecules decreased freezable water during cooling and warming.Patterning heating could be further achieved using patterned magnetic NCH structures.A quick,controllable thermal response was achieved via coordinated regulation of MNP concentration and distribution and external magnetic fields.Stem cells and NCH microcapsules were preserved with high quality(intact structures and full function)according to evaluation of cell viability,attachment efficiency,cell proliferation and cell differentiation post-vitrification.Recovered magnetic NCH microcapsules encapsulated with stem cells could be further utilized for 3D magnetic levitation culture and subsequent magnetic self-assembly.This work provides a new approach for cryogenic storage and recovery of NCH microcapsules encapsulated with stem cells.In addition,the hypothermic storage of stem cells and their hydrogel microcapsules were explored.The cell viability and cell attachment of unencapsulated stem cells,cells encapsulated with alginate hydrogel and cells encapsulated with alginate hydrogel and polyvinyl alcohol at 4℃ and 25℃ for 7 days were compared.The results show that ambient temperature was more appropriate for hypothermic storage of stem cells and their hydrogel microcapsules.Mesenchymal stem cells could be preserved for 7 days and still had good attachment ability.This research enables short-term storage of stem cells and their hydrogel microcapsules,which facilitates cell delivery and cell-based therapy.The exploration of cryopreservation of mesenchymal stem cell-laden hydrogel constructs is beneficial to provide great support for their wide application in the field of biomedicine.Among various shapes of stem cell-laden alginate hydrogel constructs,microfibers can be folded or woven to form complex three-dimensional structures due to their long,thin,and flexible characteristics,so that they can be more flexibly applied for tissue engineering.However,the research on cryopreservation of cell-laden microfiber constructs is still limited at present.In the following work,mesenchymal stem cell-laden hydrogel microfibers were prepared,and they were preserved by vitrification and hypothermic storage methods.Firstly,appropriate concentration of cryoprotective agent for vitrification of mesenchymal stem cell-laden hydrogel microfibers was explored,and the concentration and current intensity of nanoparticles used for electromagnetic rewarming were optimized.Secondly,the cell survival of mesenchymal stem cell-laden hydrogel microfibers stored at 25℃ for 7 days at hypothermic temperature was studied.In addition,in order to further apply the cryopreserved mesenchymal stem cell-laden hydrogel microfibers for wound repair,the drug-loaded Fe3O4@SiO2-IPN microfibers were prepared,and their magnetothermal heating effects,controlled drug release abilities and antibacterial effect were evaluated.After that,these two kinds of microfibers were assembled and woven,and were preliminary applied for wound repair.This work provides approaches and strategies for the cryopreservation of mesenchymal stem cell-laden hydrogel microfibers,and combines cryopreservation,stem cell therapy and controlled drug release to provide wide prospect for application of multifunctional dressings in tissue engineering and regenerative medicine.