Effect of different cryopreservation media on human nucleus pulposus cells' viability and trilineage potential

Abstract

Introduction: Low back pain (LBP) is a significant cause of disability in many countries, affecting more than half a billion people worldwide. In the past, progenitor cells have been found within the nucleus pulposus (NP) of the human intervertebral disc (IVD). However, in the context of cell therapy, little is known about the effect of cryopreservation and expansion on here called "heterogenic" human NP cells (hNPCs), and whether commercially available cryopreservation media are more efficient than "commonly used" media in terms of cell viability. Materials: In this study, hNPCs from four trauma patients (age 40.5 ± 14.3 years) and two patients with degenerated IVDs (age 24 and 46 years), undergoing spinal surgery, were collected. To isolate hNPCs, the tissue was digested with a mild two-step protocol. After subsequent expansion, hNPCs at passages 2-5 were separated and either cryo-preserved for 1 week at −150 C or differentiated into osteogenic, adipogenic, or chondrogenic lineages for 21 days. Cryopreservation was performed with five different media to compare their effect on the cell's viability and differentiation potential. Cell viability was determined with flow cytometry using propidium iodide and the trilineage differentiation potential was assessed by quantitative polymerase chain reaction and histological analysis. Results: After 1 week of cryopreservation, the hNPC's cell viability was comparable for all conditions, that is, independent of the cryopreservation medium used (82.3 ± 0.8% of cell viability). Furthermore, hNPCs from trauma patients showed some evidence for adipogenic and chondrogenic differentiation and at lower levels, this and evidence of osteogenic differentiation could be confirmed with hNPCs from degenerated discs. Moreover, cryopreservation did not affect the cell's differentiation potential in the majority of the cases tested. Conclusion: "Commonly used" cryopreservation media seem to perform just as well as commercially available media in terms of cell viability and the overall maintenance of the hNPCs trilineage differentiation potential.

Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs

“…[158][159][160] Interestingly, a recent study reported that NP cells derived from trauma patients showed higher adipogenic and chondrogenic potential than those derived from degenerated IVDs. 161 Thus, we are more inclined to hypothesize that ECs, pericytes, and NPPCs are rare in the IVD, and acute trauma may induce local regeneration, which accounts for the unwanted distribution variability across donors. Due to the scarcity of desirable samples of healthy disks from young patients with vertebral fractures, this needs to be explored in future studies.…”

Screening for Growth-Factor Combinations Enabling Synergistic Differentiation of Human MSC to Nucleus Pulposus Cell-Like Cells

“…Overall, a need exists for novel regenerative strategies to treat intervertebral disc diseases. Currently, cell therapy [26] and growth factor injection [17] is gaining momentum, however, further validation and optimization of different regenerative products will likely be needed [64]. One potential approach for enhancing cellular products is priming the cells before transplantation [65], to promote an optimal anabolic state, such that the transplanted cells have the highest potential to contribute to the reorganization of the disc matrix or otherwise direct regional cells to do so [30].…”

Investigation of the Mitigation of DMSO-Induced Cytotoxicity by Hyaluronic Acid following Cryopreservation of Human Nucleus Pulposus Cells

“…Additionally, we developed a wholetissue culture method to enable the expansion and maintenance of these Tie2-positive NPC [39]. However, several challenges remain for the adaptation of these progenitor cells as a clinical cell transplantation product, such as methods to produce large amounts of highly potent and safe cells that are ready for transplantation at the time of intervention [40]. The process of freezing and thawing cells is known to have a negative impact on their viability and potential, which may decrease the effectiveness of cell therapy [41,42].…”