Theory-based cryopreservation mode of mesenchymal stromal cell spheroids.

Cryopreservation of spheroids requires development of new improved methods. The plasma membranes permeability coefficients for water and cryoprotectants determine time characteristics of mass transfer through the cell membranes, and therefore the optimal modes of cells cryopreservation. Here we proposed an approach to cryopreservation of multicellular spheroids which considers their generalized characteristics as analogues of the membranes' permeability coefficients of the individual cells. We have determined such integral characteristics of spheroids from mesenchymal stromal cells (MSCs) as osmotically inactive volume; permeability coefficients for water and Me2SO molecules and the activation energy of their penetration. Based on these characteristics, we calculated the osmotic behavior of multicellular spheroids under cooling conditions to select the optimal cooling rate. We also determined the optimal cooling rate of spheroids using the probabilistic model developed based on the two-factor theory of cryodamage. From the calculation it follows that the optimal cooling rate of the MSC-based spheroids is 0.75°Ð¡/min. To verify the obtained theoretical estimates, we conducted experiments on freezing MSC-based spheroids under different modes. The obtained results of primary viability screening indicate that freezing at a constant linear cooling rate of 0.75-1.0°Ð¡/min gives a good result. Theoretical prediction of the spheroid osmotic behavior during cooling provided the basis for experimental verification of varying the temperature to which slow cooling should be carried out before immersion in liquid nitrogen. Slow freezing of spheroids to -40 °C followed by immersion in liquid nitrogen was shown to preserve cells better than slow freezing to -80 °C. Obtained data allow more effective use of MSC-based spheroids in drug screening and regenerative medicine.

Closed Vitrification System as A Platform for Cryopreservation of Tissue Engineered Constructs.

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.