DMSO-free Cryopreservation of Bone Marrow-derived Stem Cells

ABSTRACT Stem cell transplantation in the form of bone marrowperipheral blood or cord blood derived cells is commonly performed for reconstitution of the hematopoietic system of patients suffering from cancers and non malignant diseasesCurrent practice for cryopreservation of hematopoietic and mesenchymal stem cells is to employ the cell permeating cryoprotectant dimethylsulfoxideoften with an additional non permeating cryoprotectantand long term storage in vapor phase nitrogenCThe ultimate objective of this proposal is to develop a cryopreservation method that avoids concerns associated with dimethylsulfoxide and allows cells to be stored long term at or belowC for yearsWe have developed prototypic freezing protocols based upon a nanoparticle delivery strategy to load stem cells with a disaccharide trehaloseThis technology avoids use of the commonly employed cell permeating cryoprotectantdimethylsulfoxidewhich has patient cytotoxicity concernsby replacing it with trehaloseTrehalose accumulation is a mechanism used in nature by organisms to survive freezing conditionsIn this proposal we plan to evaluate nanoparticle delivered trehalosenTreusing methods developed by our collaboratorProfHeHe has demonstrated preservation of human adiposederived stem cells using nTre without compromising either the stemness or multilineage differentiation potential of the cellsThis proposal is to further test the nTre DMSO free cryopreservation of bone marrow derived stem cells with the following two specific aimsIn Aimwe will optimize bone marrow stem cell cryopreservation with nTreBone marrow derived hematopoietic and mesenchymal stem cells will be evaluated with appropriate controlsAssays for stemness and differentiation to evaluate the post thawing outcomes will be performed on the cryopreserved cells versus fresh controlsIn Aimwe will validate the optimized cryopreservation method for long term storage for up tomonths at bothC andC for both stem cell typesThe proposed studies will be considered successful if the nTre preserves viability and functiondifferentiation potentialof andgtof fresh control bone marrow stem cellsIn a future Phase II SBIR proposal we will verify the hematopoietic reconstitution potential of human disaccharide preserved stem cells by transplantation in a humanized mouse modelThe reconstitution potential of the cryopreserved cells will also be evaluated versus controls by analysis of human cell markers and secondary transplantation PROJECT NARRATIVE Cheaper and safer storage technologies are necessary for hematopoietic stem cell preservation and other stem cell based therapies in developmentThe technology in this proposal avoids use of cryoprotectants with patient cytotoxicity concerns and should permit long term storage atC in mechanical storage freezers as well as colder mechanical or nitrogen cooled storage systemsThe US and worldwide markets for hematopoietic stem cell services are estimated to be andgtand andgtproceduresrespectivelyvalued at several billion dollarsThere are also overclinical trials registered utilizing mesenchymal stem cells for various cellular therapeutic approachesThe ability to store and manage stem cell cold chain at supracryogenic temperaturesCusing a DMSO free system would greatly facilitate this emerging fieldOur technology may provide preservation methods for stem cell derived cell and tissue therapies that impact thousands of US patients annually