Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics

After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spher...

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Bibliographic Details
Published in:Applied sciences 2021-04, Vol.11 (8), p.3309
Main Authors: Sako, Kosuke, Sakai, Daisuke, Nakamura, Yoshihiko, Matsushita, Erika, Schol, Jordy, Warita, Takayuki, Horikita, Natsumi, Sato, Masato, Watanabe, Masahiko
Format: Article
Language:English
Subjects:
Back pain
Cell culture
Cell proliferation
Cell viability
Cells (biology)
Colonies
Cryopreservation
Degenerative disc disease
Gene expression
Growth factors
Homeostasis
intervertebral disc
Intervertebral discs
Nuclei (cytology)
Nucleus pulposus
nucleus pulposus progenitor cells
Optimization
Phenotypes
Progenitor cells
R&D
Research & development
spheroid colony
Stem cells
Thawing
Tie2
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Summary:After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.
ISSN:2076-3417
2076-3417
DOI:10.3390/app11083309