N A Trufanova1, V S Zaikov2, A V Zinchenko2, A Yu Petrenko2, Y A Petrenko3. 1. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkov, Ukraine. n.a.trufan@gmail.com. 2. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkov, Ukraine. 3. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkov, Ukraine. Department of Neuroscience, Institute of Experimental Medicine, Prague, Czech Republic.
Abstract
BACKGROUND: Cryopreservation of mesenchymal stromal cells (MSCs) and MSCs-based tissue engineered constructs (TECs) is a promising strategy for regenerative medicine. OBJECTIVE: To examine vitrification system consisting of multicomponent vitreous solution, closed type container, human adult MSCs and two-step exposure procedure as a platform for cryopreservation of MSCs-based TECs. MATERIALS AND METHODS: Vitrification properties of solutions were studied by visual analysis and calorimetry. Viability (trypan blue, MTT-test), metabolic activity (Alamar Blue assay) and adhesion of cells were assessed both after exposure with vitreous solutions and following rapid cooling-thawing in standard cryovials. RESULTS: The feasibility of the vitrification system was tested on MSCs suspensions (S-MSCs) and alginate encapsulated MSCs (AE-MSCs). The minimal concentrations of cryoprotectants, which allowed avoiding ice formation during rapid cooling and rewarming comprised 10 % for dimethylsulfoxide, 20 % for ethylene glycol, 20 % for 1.2-propanediol and 0.5 M sucrose. To achieve viability and metabolic activity rates of AE-MSCs comparable to S-MSCs after vitrification the extension of the exposure time within the same vitreous solution was sufficient. After vitrification both S-MSCs and AE-MSCs retained the capacity to osteogenic and adipogenic differentiation. CONCLUSION: Data demonstrate that this vitrification system can be used as a platform for development of effective protocols for cryopreservation of MSCs-based TECs.
BACKGROUND: Cryopreservation of mesenchymal stromal cells (MSCs) and MSCs-based tissue engineered constructs (TECs) is a promising strategy for regenerative medicine. OBJECTIVE: To examine vitrification system consisting of multicomponent vitreous solution, closed type container, human adult MSCs and two-step exposure procedure as a platform for cryopreservation of MSCs-based TECs. MATERIALS AND METHODS: Vitrification properties of solutions were studied by visual analysis and calorimetry. Viability (trypan blue, MTT-test), metabolic activity (Alamar Blue assay) and adhesion of cells were assessed both after exposure with vitreous solutions and following rapid cooling-thawing in standard cryovials. RESULTS: The feasibility of the vitrification system was tested on MSCs suspensions (S-MSCs) and alginate encapsulated MSCs (AE-MSCs). The minimal concentrations of cryoprotectants, which allowed avoiding ice formation during rapid cooling and rewarming comprised 10 % for dimethylsulfoxide, 20 % for ethylene glycol, 20 % for 1.2-propanediol and 0.5 M sucrose. To achieve viability and metabolic activity rates of AE-MSCs comparable to S-MSCs after vitrification the extension of the exposure time within the same vitreous solution was sufficient. After vitrification both S-MSCs and AE-MSCs retained the capacity to osteogenic and adipogenic differentiation. CONCLUSION: Data demonstrate that this vitrification system can be used as a platform for development of effective protocols for cryopreservation of MSCs-based TECs.