In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: An improved isolation and cryopreservation method
In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: An improved isolation and cryopreservation method
复制标题
DOI：
10.1016/j.bone.2005.04.034
复制
发表时间：
2005-10-01
期刊：
影响因子：
4.1
通讯作者：
Odgren, PR
中科院分区：
文献类型：
--
作者：
Gartland, A;Mechler, J;Odgren, PR
简介：分离和培养原代软骨细胞，使其保持其细胞类型并分化为肥大状态，是许多骨骼生长及其调节研究的核心。储存冷冻软骨细胞的能力具有额外的科学和组织工程兴趣。以前的工作已经产生了不同的产量和复杂性的方法，但不允许冷冻储存的细胞，随后在培养中分化。调查的生长板发育不良继发于有缺陷的破骨细胞在啮齿类动物模型的骨石症导致我们适应和修改的培养方法和冷冻保存新生大鼠costochonyte软骨cardiocytes.Methods：软骨细胞分离从解剖肋骨的3天大的幼鼠胶原酶，透明质酸酶，胰蛋白酶连续digestion。这可以在初始蛋白酶处理后立即进行，或者在分离中断后进行，以使仍在部分消化的肋骨雏形中的软骨细胞冷冻，然后解冻用于培养。将细胞接种在平底威尔斯孔中，并使其在不同条件下粘附和生长。培养基的选择允许细胞维持或诱导分化。监测细胞生长，以及几种相关基因的表达：II型和X型胶原蛋白;骨钙素、Sox 9、脂肪细胞FABP、MyoD、聚集蛋白聚糖等。矿化测定茜素红结合，培养物进行了检查，通过光，荧光，和电子microscopic.Results：细胞保留其软骨细胞表型和分化能力，并矿化胶原丰富的细胞外基质，即使在冻融。RT-PCR显示保留软骨细胞特异性基因表达，包括聚集蛋白聚糖和胶原蛋白II。细胞最初具有扁平的“增殖区”外观，并且在汇合后2周，表现出体内观察到的肥大细胞的肿胀和其他显著特征。胶原纤维丰富的细胞外基质，沿着与基质囊泡。切换到胶原X型作为标记肥大没有严格的时间调节，发生在体内，但其表达增加在肥大differentiation.Conclusions：这种方法应证明有价值的研究cbondrocell调节的一种手段，并具有简单的初始解剖的优点，产量的一个纯粹的软骨细胞群体，并能够储存冷冻的原材料用于后续研究。(c)2005年爱思唯尔公司All rights reserved.
Introduction: Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes.Methods: Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy.Results: Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation.Conclusions: This method should prove valuable as a means of studying cbondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies. (c) 2005 Elsevier Inc. All rights reserved.