Dominique Chabot1, Antoine Lewin2, Lionel Loubaki1, RenÉe Bazin3. 1. Medical Affairs and Innovation, Héma-Québec, Québec (Qc), Canada; Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec (Qc), Canada. 2. Medical Affairs and Innovation, Héma-Québec, Québec (Qc), Canada; Faculty of Medicine and Health Science, University of Sherbrooke, Sherbrooke (Qc) Canada. 3. Medical Affairs and Innovation, Héma-Québec, Québec (Qc), Canada; Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec (Qc), Canada. Electronic address: renee.bazin@hema-quebec.qc.ca.
Abstract
BACKGROUND: We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events. METHODS: MSC prepared from six umbilical cords were frozen to -130°C and immediately transferred in a dry ice container or exposed to room temperature for 2 to 10 min (warming events) prior to storage in liquid nitrogen. Viability, functionality (inhibition of T-cell proliferation), adhesion and expression of various integrins were evaluated. RESULTS: The monotonic loss of functional activity with time was proportional to the length of warming events to which MSC were subjected and correlated with the monotonic loss of adhesion capacity. In contrast, post-thaw viability assessment did not predict functional impairment. Interestingly, flow cytometry analyses revealed the emergence of a FSClow population present in the viable cell fraction of freshly thawed MSC, which displayed poor adhesion capacity and expressed low levels of integrin β5. The prevalence of this FSClow population increased with the length of warming events and correlated with impaired functional and adhesion properties. DISCUSSION: Our results reveal that loss of functional activity (4-day test) induced by transient warming events could be predicted by evaluating adhesion (2-hr test) or FSC profile (10-min test) of MSC immediately post-thaw. These observations could lead to the development of surrogate tests for rapidly assessing the functional quality of cryopreserved MSC.
BACKGROUND: We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events. METHODS: MSC prepared from six umbilical cords were frozen to -130°C and immediately transferred in a dry ice container or exposed to room temperature for 2 to 10 min (warming events) prior to storage in liquid nitrogen. Viability, functionality (inhibition of T-cell proliferation), adhesion and expression of various integrins were evaluated. RESULTS: The monotonic loss of functional activity with time was proportional to the length of warming events to which MSC were subjected and correlated with the monotonic loss of adhesion capacity. In contrast, post-thaw viability assessment did not predict functional impairment. Interestingly, flow cytometry analyses revealed the emergence of a FSClow population present in the viable cell fraction of freshly thawed MSC, which displayed poor adhesion capacity and expressed low levels of integrin β5. The prevalence of this FSClow population increased with the length of warming events and correlated with impaired functional and adhesion properties. DISCUSSION: Our results reveal that loss of functional activity (4-day test) induced by transient warming events could be predicted by evaluating adhesion (2-hr test) or FSC profile (10-min test) of MSC immediately post-thaw. These observations could lead to the development of surrogate tests for rapidly assessing the functional quality of cryopreserved MSC.