Multiplication of human Chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity
Multiplication of human Chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity
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DOI：
10.1089/107632704322791754
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发表时间：
2004-01-01
影响因子：
--
通讯作者：
Van Osch, GJVM
中科院分区：
文献类型：
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作者：
Mandl, EW;Van der Veen, SW;Van Osch, GJVM
为了用组织工程技术治疗软骨缺损，供体细胞的增殖是必不可少的。然而，在单层扩增培养软骨细胞的增殖过程中，软骨细胞将失去其表型并产生劣质基质（去分化）。低接种密度和传代更广泛地发生去分化。为了获得高质量的软骨，增殖的细胞恢复其软骨表型（再分化能力）是重要的。在接种密度和传代方面，缺乏单层软骨细胞增殖的“金标准”。在许多现有的研究中，使用了不同的细胞密度，使得这些研究的结果难以比较。因此，我们进行了比较研究，以了解接种密度和传代对细胞再分化能力的影响。从得到的数据，我们推导出接种密度在单层培养的细胞扩增是足够的和快速的，而细胞保持再分化的能力。作为指导原则，我们计算出，至少需要20倍的增殖才能用我们获得的供体软骨细胞填充平均4 cm（2）的软骨缺损。在这项研究中，我们使用了5名儿童的耳软骨细胞。在单层培养中使用了四种不同的接种密度，范围为3500至30，000个细胞/cm（2）。将细胞培养4代。在藻酸盐珠中额外培养3周后，研究了扩张的软骨细胞再分化的能力（再分化能力），并通过糖胺聚糖产生和I型胶原、II型胶原、弹性蛋白和成纤维细胞的免疫组织化学染色进行评估。标记物（11-fibrau）。一般来说，我们发现传代和降低接种密度都能增加扩增的软骨细胞，但同时降低了去分化能力。在进一步分析我们的数据，根据提出的指导方针，我们发现，与较低的接种密度，足够的增殖（20倍），达到在更短的时间和更少的传代比在较高的接种密度。重要的是，这些软骨细胞的再分化能力得以保留。在较高接种密度下，其增殖能力相当于甚至超过20倍的软骨细胞，而后者在单层培养中需要更多的时间和更多的传代次数。因此，对于软骨组织工程的目的，我们建议，扩大培养低接种密度是优选的。
To treat a cartilage defect with tissue-engineering techniques, multiplication of donor cells is essential. However, during this multiplication in monolayer expansion culture chondrocytes will lose their phenotype and produce matrix of inferior quality (dedifferentiation). Dedifferentiation occurs more extensively with low seeding densities and passaging. To obtain cartilage of good quality it is important that the multiplicated cells regain their cartilaginous phenotype (redifferentiation capacity). A "gold standard" for the multiplication of chondrocytes in monolayer, with respect to seeding density and passaging, is lacking. In numerous available studies, various cell densities have been used, making comparison of the results of these studies difficult. Therefore, we performed a comparative study to gain insight concerning the effect of seeding density and passaging on the capacity of cells to redifferentiate. From the resulting data we deduced the seeding density in monolayer culture for which cell expansion is both sufficient and fast, while the cells retain a capacity to redifferentiate. As a guideline we calculated that, at minimum, 20-fold multiplication is needed to fill an average cartilage defect of 4 cm(2) with the amount of donor chondrocytes we obtained. For this study we used isolated ear chondrocytes from five children. Four different seeding densities in monolayer culture were used, ranging from 3500 to 30,000 cells/cm(2). The cells were cultured for four passages. The capacity of the expanded chondrocytes to redifferentiate (redifferentiation capacity) was studied after an additional 3-week culture in alginate beads and was assessed by glycosaminoglycan production and immunohistochemical stainings for collagen type I, collagen type II, elastin, and a fibroblast marker (11-fibrau). In general, we found that both passaging and decreasing seeding density yielded an increase in expanded chondrocytes, but at the same time decreased the dedifferentiation capacity. In further analyzing our data according to the proposed guidelines we found that with lower seeding densities sufficient multiplication (20 times) was reached in less time and with less passaging than at higher seeding densities. Importantly, the redifferentiation capacity of these chondrocytes was preserved. It was equal to or even surpassed that of chondrocytes multiplied 20 times at higher seeding densities, which required more time and more passages in monolayer culture. Thus, for cartilage tissue-engineering purposes we propose that expansion culture with low seeding densities is preferable.