Development of Serum-Free Media for Cryopreservation of Hydrogel Encapsulated Cell-Based Therapeutics

• Published in: Cellular and molecular bioengineering 2022-10, Vol.15 (5), p.425-437
• Main Authors: Cui, Yufei, Nash, Amanda M., Castillo, Bertha, Sanchez Solis, Leonardo D., Aghlara-Fotovat, Samira, Levitan, Maya, Kim, Boram, Diehl, Michael, Veiseh, Omid
• Format: Article
• Language: English
• Subjects: Alginates; Alginic acid; Biological and Medical Physics; Biomaterials; Biomedical Engineering and Bioengineering; Biomedical Engineering/Biotechnology; Biophysics; Cell Biology; Cell viability; Composite functions; Cryopreservation; Dimethyl sulfoxide; Encapsulation; Engineering; Freeze-thaw; Freezing; Hydrogels; Mesenchymal stem cells; Permeability; S.I. : 2022 CMBE Young Innovators; Secretion; Structural integrity; Thawing
• ISSN: 1865-5025; 1865-5033
• DOI: 10.1007/s12195-022-00739-7

Introduction While hydrogel encapsulation of cells has been developed to treat multiple diseases, methods to cryopreserve and maintain the composite function of therapeutic encapsulated cell products are still needed to facilitate their storage and distribution. While methods to preserve encapsulated cells, and post-synthesis have received recent attention, effective preservation mediums have not been fully defined. Methods We employed a two-tiered screen of an initial library of 32 different cryopreservation agent (CPA) formulations composed of different cell-permeable and impermeable agents. Formulations were assayed using dark field microscopy to evaluate alginate hydrogel matrix integrity, followed by cell viability analyses and measurements of functional secretion activity. Results The structural integrity of large > 1 mm alginate capsules were highly sensitive to freezing and thawing in media alone but could be recovered by a number of CPA formulations containing different cell-permeable and impermeable agents. Subsequent viability screens identified two top-performing CPA formulations that maximized capsule integrity and cell viability after storage at − 80 °C. The top formulation (10% Dimethyl sulfoxide (DMSO) and 0.3 M glucose) was demonstrated to preserve hydrogel integrity and retain cell viability beyond a critical USA FDA set 70% viability threshold while maintaining protein secretion and resultant cell potency. Conclusions This prioritized screen identified a cryopreservation solution that maintains the integrity of large alginate capsules and yields high viabilities and potency. Importantly, this formulation is serum-free, non-toxic, and can support the development of clinically translatable encapsulated cell-based therapeutics.