Humphreys Matthew D, Ward Lizzy, Richardson Stephen M, Hoyland Judith A
Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health University of Manchester Manchester UK.
NIHR Manchester Musculoskeletal Biomedical Research Unit, Central Manchester Foundation Trust, Manchester Academic Health Science Centre Manchester UK.
JOR Spine. 2018 Jul 26;1(3):e1028. doi: 10.1002/jsp2.1028. eCollection 2018 Sep.
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.
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 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.
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.
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.
脊索细胞在治疗椎间盘退变方面显示出治疗潜力。然而，由于细胞产量低以及缺乏合适的细胞扩增方法，对其表型和功能的研究受到限制。本研究利用猪细胞，旨在开发一种优化的培养系统，能够扩增脊索细胞群体并保持其表型。
对出生后的猪和胎儿人类髓核组织进行组织学比较，并分析猪脊索细胞中已知脊索细胞标志物基因的表达。从出生后六周的椎间盘分离猪脊索细胞，并在不同条件下进行体外培养：（1）DMEM与αMEM；（2）层粘连蛋白-521、纤连蛋白、明胶和未包被的组织培养处理聚苯乙烯（TCP）；（3）2%氧气与常氧；（4）αMEM（300 mOsm/L）与αMEM（400 mOsm/L）；（5）表面硬度为0.5和4 kPa以及标准TCP。在14天的培养期内评估细胞的黏附、增殖、形态以及脊索细胞标志物的表达。
天然猪髓核组织显示出与人类胎儿组织相似的形态，并且猪脊索细胞表达已知的脊索标志物（CD24、KRT8、KRT18、KRT19和T）。使用αMEM培养基和层粘连蛋白-521包被的表面显示出最大的细胞黏附、增殖以及脊索细胞形态和表型的保留。在低氧（2%）条件下，脊索细胞群体的增殖进一步增强，并且在0.5 kPa培养表面上的表型保留得到改善。
我们的模型展示了一种优化的系统，在该系统中脊索细胞群体可以扩增同时保持脊索表型。这种优化培养系统的应用将使脊索细胞得以扩增，用于详细的表型和功能研究，这是相对于文献中描述的当前培养方法的一个主要优势。此外，猪和人类脊索细胞之间发现的相似性表明该系统将适用于人类脊索细胞培养，以研究其治疗潜力。
