Cryopreservation Does Not Affect Proliferation and Multipotency of Murine Neural Precursor Cells

Abstract

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70ºC or in liquid nitrogen in a ratecontrolled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)-and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70°C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells. Stem Cells

Effect of Dimethyl Sulfoxide (DMSO) on Cryopreservation of Porcine Mesenchymal Stem Cells (pMSCs)

“…Further, the expression levels of genes among different groups were inversely proporProliferation capacity of pMSCs in ex vivo culture is an essential characteristic of stem cells and it is often tional to the concentration of DMSO. However, the gene expression levels were similar to that of fresh pMSCs in tested by CFU-F assay (23,27). The numbers and size of the colonies in CFU-F assay are influenced by the the postthaw culturing of pMSCs up to 90% confluence (Fig.…”

“…Similar observations were also made in the earlier repression of Bcl-2 and Bak genes before and after cryopreservation of pMSCs at different intervals of time. The ports on adult stem cells, such as HSCs and neural precursor cells in humans (39) and mice (23). Similarities low level of expression of Bcl-2 and Bak genes in pMSCs immediately after thawing indicates the detriin survivability of pMSCs cryopreserved at different concentrations of 5% and 10% DMSO suggest that 5% mental effect of DMSO on cryopreserved cells.…”

“…Several authors have reported the successful cryopreservation of HSCs, MSCs, and neuronal stem propagation of cryopreserved pMSCs. However, no difference in the propagation of pMSCs cryopreserved in cells using 10% DMSO (14,23,25). DMSO is not only responsible for exerting toxic side effects on transfusion 5% and 10% DMSO was observed in this study.…”

The Microenvironment of Embryoid Bodies Modulated the Commitment to Neural Lineage Postcryopreservation

“…[8][9][10] For all these applications, development of an efficient cryopreservation process amenable for the distribution and storage of PSCderived NPCs with desired three-dimensional (3D) structure is a critical step to ensure the cell quality and to accelerate the derivation of different neural cell types. 4,[11][12][13] NPCs are usually derived from PSCs through the formation of embryoid bodies (EBs), the aggregate structure mimicking embryonic development. 9,14 NPC derivation from PSCs has a lengthy procedure that could last up to 6-14 weeks.…”

“…18,19 For PSC-derived NPCs, cryopreservation of the dissociated single cells caused significant apoptosis and required treatment with Rho-associated protein kinase (ROCK) inhibitors or caspase inhibitors to maintain cell viability. 11,20 Although cryopreservation of adult neurospheres is feasible, cryopreservation of EBs for neural differentiation has not been well studied. To date, there are only a few studies for cryopreservation of spontaneously differentiated EBs.…”