GP014: Effect of different Cryopreservation Media on Human Nucleus Pulposus Cells' Viability and Trilineage Potential

Croft, Andreas S.; Guerrero, Julien; Oswald, Katharina A. C.; Häckel, Sonja; Albers, Christoph E.; Gantenbein, B. (2021). GP014: Effect of different Cryopreservation Media on Human Nucleus Pulposus Cells' Viability and Trilineage Potential (Unpublished). In: ISSLS Virtual Meeting. Milano, italy. 2-4 June 2021.

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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).
Our study aimed to analyze whether commercially available cryopreservation media are more efficient than “commonly used” media in terms of cell viability and maintaining the cell’s possible differentiation potential.
Methods: 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. Consent, according to the local Human Research Act, was given by all patients. To isolate the cells, the tissue was digested with a mild two-step protocol using pronase and collagenase type 2. After subsequent expansion, hNPCs (passages 2-5) were separated and either differentiated into osteogenic, adipogenic, chondrogenic lineages for 21 days or cryo-preserved for one week at -150°C. 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. The trilineage differentiation potential was assessed by quantitative polymerase chain reaction and histological analysis. Cell viability after freezing as well as the cell’s gene expression were analysed as single replicates by two-way ANOVA followed by a Tukey’s multiple comparison test and the histological stains were analysed in technical duplicates by a Kruskal-Wallis test and a Dunn’s multiple comparisons test.
Results: After one week of cryopreservation, the hNPC’s cell viability was comparable for all five conditions, i.e. independent of the cryopreservation medium used (82.3 ± 0.8% cell viability). Furthermore, hNPCs showed evidence for osteogenic differentiation, i.e. matrix calcification, adipogenic differentiation, i.e. significant upregulation of adiponectin as well the the production of lipid droplets, and chondrogenic differentiation, i.e. up to 750-fold upregulation of collagen type 2. Moreover, cryopreservation did not affect the cell’s differentiation potential in the majority of the donors tested.
Discussion: To conclude, “commonly used DMSO-based” 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. However, extensive donor variations were observed throughout our study.

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