An optimized culture system for notochordal cell expansion with retention of phenotype

Abstract

Background Notochordal (NC) cells display therapeutic potential in treating degeneration of the intervertebral disc. However, research on their phenotype and function is limited by low‐cell yields and a lack of appropriate methodology for cell expansion. Utilizing porcine cells, this study aimed to develop an optimized culture system which allows expansion of NC cell populations with retention of phenotype. Methods Post‐natal porcine and foetal human nucleus pulposus tissue was compared histologically and expression of known NC cell marker genes by porcine NC cells was analyzed. Porcine NC cells were isolated from six‐week post‐natal discs and cultured in vitro under varied conditions: (1) DMEM vs αMEM; (2) laminin‐521, fibronectin, gelatin and uncoated tissue culture‐treated polystyrene (TCP); (3) 2% O 2 vs normoxia; (4) αMEM (300 mOsm/L) vs αMEM (400 mOsm/L); (5) surface stiffness of 0.5 and 4 kPa and standard TCP. Adherence, proliferation, morphology and expression of NC cell markers were assessed over a 14‐day culture period. Results Native porcine nucleus pulposus tissue demonstrated similar morphology to human foetal tissue and porcine NC cells expressed known notochordal markers (CD24, KRT8, KRT18, KRT19, and T). Use of αMEM media and laminin‐521‐coated surfaces showed the greatest cell adherence, proliferation and retention of NC cell morphology and phenotype. Proliferation of NC cell populations was further enhanced in hypoxia (2%) and phenotypic retention was improved on 0.5 kPa culture surfaces. Discussion Our model has demonstrated an optimized system in which NC cell populations may be expanded while retaining a notochordal phenotype. Application of this optimized culture system will enable NC cell expansion for detailed phenotypic and functional study, a major advantage over current culture methods described in the literature. Furthermore, the similarities identified between porcine and human NC cells suggest this system will be applicable in human NC cell culture for investigation of their therapeutic potential.

Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus

“…7,[34][35][36][37][38][39][40][41][42] Much of this work consistently shows that NP cell morphology responds to both ligand and stiffness, where soft, laminin-presenting substrates promote a juvenile, healthy rounded morphology, but stiff and/or non-laminin associated ligands allow a spread, fibroblastic-like shape observed in adult NP. 7,15,37,[42][43][44][45][46][47][48] These reports also support the idea that changes in NP cell shape are associated with concomitant increases in expression of phenotypic markers and matrix biosynthesis. come from the FDA-approved drug verteporfin (VP), which was discovered as a TEAD inhibitor via a drug library screen.…”

New horizons in spine research: Disc biology, tissue engineering, biomechanics, translational, and clinical research

“…12 The potential of notochordal cells for therapy of disc disease has the potential for success. However, the limitation of the success of using notochordal cells is mainly due to the reduced yield of the notochordal cells, and retention of their molecular profile in culture.…”

“…Optimized culture system for notochordal cell expansion with retention of the phenotype : Humprey et al, describes an improved and streamlined technique to culture and expand notochordal cells for their potential use for disc treatment and regeneration . The potential of notochordal cells for therapy of disc disease has the potential for success.…”

“…Humprey et al, describes an improved and streamlined technique to culture and expand notochordal cells for their potential use for disc treatment and regeneration. 12 The potential of notochordal cells for therapy of disc disease has the potential for success. However, the limitation of the success of using notochordal cells is mainly due to the reduced yield of the notochordal cells, and retention of their molecular profile in culture.…”

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro

“…2C) for both treated and non-treated groups was observed with an increase in passage. Although unexpected, it is not uncommon to observe increased ACAN expression with advancing NP cell passage, as demonstrated by the work of Ono et al 29) Additionally, the notochordal cell type derived from rat NP tissue might have differentiated to a more chondrogenic phenotype in vitro monolayer culture conditions, resulting in enhanced ACAN expression 30,31) . As such, future studies might benefit from applying an animal model that lacks notochordal cells in their IVD 32) .…”