未经证实:用组织特异性髓核细胞(NPC)重新填充退变的椎间盘(IVD)已被证明可以促进各种物种的再生。然而,NPC作为基于细胞的疗法的适用性受到可以从供体IVD中提取的低细胞数量以及由于其退化的表型而可能有限的再生能力的阻碍。为了优化膨胀条件,我们在3D培养模型中研究了扩增过程中增加培养基渗透压对犬NPCs表型及其产生健康细胞外基质(ECM)的能力的影响.
UNASSIGNED:将狗NPC在标准渗透压为300mOsm/L的扩增培养基中扩增或在常氧和低氧条件下调节至400或500mOsm/L。扩展后,在具有标准渗透压的软骨形成培养基中,在3D培养模型中培养NPC。读出参数包括细胞增殖率,形态学,表型和健康的ECM生产。
UNASSIGNED:将扩增培养基的摩尔渗透压浓度从300增加到500mOsm/L导致NPC具有更圆形的形态和更低的细胞增殖率,并伴随着几种健康的NPC和祖细胞标记在基因上的表达(KRT18,ACAN,COL2,CD73,CD90)和蛋白质(ACAN,PAX1,CD24,TEK,CD73)水平。以500mOsm/L扩增的NPC能够在3D培养期间保留其大部分表型标记并产生健康的ECM,而与扩增期间使用的氧水平无关。
未经评估:总而言之,我们的发现表明,在扩张过程中增加中等渗透压导致NPC群体具有改善的表型,这可以增强基于细胞的治疗IVD再生的潜力。
UNASSIGNED: Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model.
UNASSIGNED: Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production.
UNASSIGNED: Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion.
UNASSIGNED: Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration.