Abstract
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.
Highlights
Low back pain is the primary cause of disability worldwide and often leads to poor quality of life for patients [1,2]
The ability of EZSPHERE dishes to support nucleus pulposus (NP) cell colony formation was evaluated by determining the colony formation and cell proliferation rates (Figure 1)
Type II collagen is thought to play an important role in the formation of the intervertebral disc (IVD), which suggests that NP cells that can be collected by this culture method should have excellent functionality
Summary
Low back pain is the primary cause of disability worldwide and often leads to poor quality of life for patients [1,2]. Low back pain is thought to relate to intervertebral disc (IVD) degeneration, the mechanisms have not been clearly identified [3]. The IVD is composed of three distinct tissues: the nucleus pulposus (NP), annulus fibrosus (AF), and cartilage endplate. The NP comprises the gelatinous tissue located in the center of the IVD. The AF comprises layered collagen structures that surround the NP [4]. Previous reports have indicated that IVD homeostasis is determined by the NP [5,6] and that IVD degeneration is thought to be caused by dysregulation of extracellular matrix (ECM) homeostasis within the NP tissue [7,8]
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