Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Abstract

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods:The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated.

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization

“…Cryoprotective agents prevent the formation of these ice crystals, 84 nonetheless the effectiveness therefore may depend on the cell type. Mature NPCs have been shown to maintain high viability after cryopreservation with the use of dimethylsulfoxide (DMSO) and fetal calf serum (FCS) as cryoprotective agents, 85,86 and is the most utilized methodology in the spine field 87 . Yet, there is little information on the cryopreservation of NCs.…”

“…Anne Camus received funding from Region Pays de la Loire, RFI BIOREGATE (CAVEODISC), and the French Society of Rheumatology (Spherodisc). The authors would like to thank the other members of the spine community whom contributed to early discussions of the article planning whom are authors from the prior NP methods paper 87 …”

“…The greatest levels of cell adhesion and proliferation, morphology, and expression of NC phenotypic markers (CD24, KRT8, KRT18, KRT19, and TBXT) were observed with the use of laminin-521-coated surfaces with 0.5 kPa stiffness and αMEM media, under 2% pO 2 , 400 mOsm/kg culture conditions, where highest number of vacuolated NC cells (around 70%) were retained.137 This indicates that the vacuolated NC phenotype can also be maintained under specific 2D culture conditions, where at least some population doubling is possible. 137 However, culture within such conditions can be difficult to obtain across laboratories and thus where proliferation of NCs is not required we recommend that clusters of isolated NCs are maintained as clusters and cultured within alginate beads in αMEM media, under physiological O 2 concentration (1%-5%) and osmolarity (400 mOsm/L), in FCS free media such as that described for alginate culture of NPCs with the inclusion of insulin-transferrin-selenium, Albumax and L-Proline,87 as this enables the retention of their in vivo morphologyF I G U R E 1 1 Micromass culture of mouse notochordal cell (NC) Immunofluorescence co-localization: Immunofluorescence images showing Ki-67+/Brachyury+ proliferative NCs, coexpressing SOX9+/Brachyury+, AQUAPORIN3+/Brachyury+, and Caveolin-1+/Brachyury+ NCspecific markers, respectively and showing vacuoles within the cytoplasm where the calcein-AM fluorophore is excluded (yellow arrowhead).…”

Monocytic fibrocyte-like cell enrichment and myofibroblastic adaptation causes nucleus pulposus fibrosis and associates with disc degeneration severity

“…Pronase digestion was commonly deployed for releasing single cells from the NP in scRNA-seq and flow cytometry assays 28 . However, the digestion can also results in extensively membrane proteins degradation, and thereby affect the antibody recognition of surface molecules 29 .…”

“…In severely degenerated IVDs (grade IV-V), NP tissues were more fibrotic and hardly distinguishable from inner AF, so we harvested the tissue presumably at the central region of the NP. Primary NP cells were extracted by sequential enzymes digestion of pronase (0.25%, Roche Diagnostics) and collagenase II (600U/ml, Worthington Biochemical) 28 , and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Biosera) and 1% penicillin/streptavidin (P/S, Invitrogen) at 37 °C and 5% CO 2 .…”