Low temperature storage of primate embryonic stem cell derived neural precursor cells

Abstract

Objective: Development of an efficient method for low temperature storage of embryonic stem cell derived neural progenitor cells (ESC-NPC). ESC-NPCs carry potential in the study of neuronal cell differentiation and in the cell based treatment of neurodegenerative diseases. The ability to store ESC-NPCs would facilitate research efforts and ultimately will be required in therapeutic applications. Progenitor cell cultures derived from differentiated embryonic stem cells can be produced in large numbers within a few days in contrast to starting from embryonic stem cells which requires 4+ weeks. Design: Prospective study of ESC-NPC survival and differentiation after randomized allocation to vitrification or conventional slow freezing procedures as evaluated by the expression of specific protein markers for NPCs and neurons. Materials/Methods: Rhesus macaque embryonic stem cells derived from in vitro produced blastocysts were cultured to embryoid bodies via cell aggregation in hanging drops and differentiated to ESC-NPC as neurospheres (Kuo et al, Biol Reprod 78, Suppl 1, 2002). The spheres were 300–600 um dia. The vitrification procedure involved sequential exposures to 1) 10% glycerol, 2) 10% glycerol + 20% ethylene glycol and 3) 25% glycerol + 25% ethylene glycol. The spheres were then dropped directly into liquid nitrogen and sealed in vials for storage. Drops were warmed in 0.5 M sucrose and passed through 0.25 M and 0.125 M sucrose prior to rinsing and further culture. Controlled rate slow freezing involved sequential exposure to 5% and 9% glycerol then cooling in vials at -3C/min to -8C for seeding and further cooling at -0.3C/min to -30C prior to storing in liquid nitrogen. Thawing involved incubation in 0.5 M, then 0.2 M sucrose prior to rinsing and culture. Results: In replicate experiments a total of 41 spheres were cryopreserved, thawed and cultured on N2-FGF2 media. Twenty of 25 vitrified spheres survived and attached. Twelve of 16 slowly frozen spheres also survived and attached. After 4 days the surviving cells were dispersed and plated on ornithine-laminin coated cover slips. Immunocytochemistry with the neural progenitor markers nestin and Musashi1 identified 87% and 90% of 900 vitrified cells positive while 72% and 92% of 822 slow freeze cells were positive, respectively. Further culture for 9 days in N3 differentiating media then characterization with antibodies specific for mature neurons, Neu N and MAP2c, identified 93% and 86% of 785 vitrified cells positive while 81% and 95% of 982 slow freeze cells, respectively, were also positive. Less than 10% of 1131 differentiated cells exhibited the marker for glial fibrillary acidic protein. Conclusions: Primate neural progenitor cells can be efficiently stored at low temperatures by vitrification or traditional slow freezing procedures. Both protocols preserve the precursor nature and allow further differentiation to committed cells, thus enhancing the practical availability of progenitor cells for investigation or therapy. Supported by: NIH RR 15199.