Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Abstract

Background aims

Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.

Methods

MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.

Results

Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.

Conclusions

A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

Introduction

Multipotent mesenchymal stromal cells (MSC) (1) are somatic stem cells, first described by Friedenstein et al. in the 1970s (2), that are located in bone marrow and many other tissues (3). MSC are being used for tissue repair (4), hematopoiesis support (5) and immunomodulation (6). These cells can differentiate into tissues developing from mesoderm, such as bone, cartilage and fat (7), a characteristic that is also explored to define them and evaluate their function in vitro. Other important defining characteristics of MSC are the expression of specific membrane molecules (CD73, CD90 and CD105) together with a lack of expression of hematopoietic markers (CD14, CD34 and CD45), and their ability to adhere to plastic (8). Besides their potential for differentiation and hematopoiesis support, MSC exert an immunosuppressive effect by a variety of mechanisms (9).

Usually, MSC should be expanded ex vivo and cryopreserved before clinical use. However, the optimal protocol for cryopreservation of these cells has not yet been defined. Another important issue is their sensitivity to dimethyl sulfoxide (DMSO) exposure, especially after thawing and before cell infusion, a time lag that can take from minutes to some hours.

We designed this study to unveil MSC sensitivity to cryopreservation and different periods of exposure to DMSO after thawing. Furthermore, we tested a method for cryopreservation and storage of MSC in plastic bags, which facilitates manipulation and clinical use.