Publication: DEVELOPMENT OF DIMETHYL SULFOXIDE-FREE FORMULATIONS FOR CRYOPRESERVATION AND LYOPHILIZATION OF HUMAN CELLS
Date
2023
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Publisher
International Medical University
Abstract
Introduction: The current standard for storing human cells involves using DMSO and liquid nitrogen, but this method requires specialized equipment and has associated toxicity. This study aims to develop a DMSO-free preservation protocol potentially through lyophilization.
Methods: Trehalose and sucrose at 20-800 mM, hydroxyethyl starch (HES) at 25-350 mg/mL, poly(vinyl alcohol) (PVA) at 1-34 mg/mL and glycerol at 1-10%v/v were evaluated for its toxicity on fibroblasts (MRC5), kidney (HEK293) or mesenchymal stem cells (MSCs). Next, tri-excipients formulations were compared with 10% v/v DMSO during freezing and dehydration. The effect of freezing rate, primary drying duration or choice of reconstitution medium on the cell viability were assessed as well. Cell viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay or trypan blue staining. The optimized protocol was evaluated on other human cells and storage at -20 oC and -80 oC for ≤30 days. Molecular dynamics (MD) simulation was used to elucidate the molecular mechanisms of the sugar and polymer combination under freezing conditions.
Results and Discussion: The formulations failed to provide adequate protection during lyophilization. However, (i) 100 mM trehalose, 150 mg/mL HES and 2% v/v glycerol, (ii) 100 mM sucrose, 150 mg/mL HES and 2% v/v glycerol and (iii) 50 mM trehalose, 50 mM sucrose, 150 mg/mL HES and 2% v/v glycerol showed similar or better cryoprotective capabilities than 10% v/v DMSO during freezing and storage. MD simulation showed the sugar-polymers exert cryoprotective effect through hygroscopicity and water redirection. Conclusion: The optimized formulations demonstrated great potential in replacing DMSO as a cryoprotective agent. However, further investigation is warranted in lyophilization. We hypothesized that intracellular damage was the cause of cell death.
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Keywords
Cell Death, Cryoprotective Agents, Trehalose, Sucrose, Freeze Drying