Nishizawa Satoru, Kanazawa Sanshiro, Fujihara Yuko, Asawa Yukiyo, Nagata Satoru, Harai Motohiro, Hikita Atsuhiko, Takato Tsuyoshi, Hoshi Kazuto
Translational Research Center, The University of Tokyo Hospital, Tokyo, Japan.
Department of Cell and Tissue Engineering (Fujisoft) and Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Biores Open Access. 2020 Mar 3;9(1):51-63. doi: 10.1089/biores.2019.0058. eCollection 2020.
Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the and production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.
以往用于修复和重建因事故和疾病而缺失的耳廓和鼻子时使用的是硅胶植入物，而如今一种使用组织工程软骨的新治疗方法备受关注。在这种方法中，培养细胞的质量很重要，因为它会影响治疗效果。然而，软骨细胞的标志物，尤其是耳廓软骨细胞的标志物尚未确立。本研究的目的是建立一种最佳标志物，以评估作为再生软骨组织细胞来源的培养耳廓软骨细胞的质量。使用微阵列方法全面比较人未分化和去分化耳廓软骨细胞之间的基因表达水平，以研究一种候选质量控制指标，其在分化细胞中表达水平高，但在去分化细胞中显著降低。我们确定胶质纤维酸性蛋白（GFAP）是一种在耳廓软骨细胞中随传代次数减少的标志物。在关节软骨细胞、肋软骨细胞或成纤维细胞中未检测到GFAP，而在细胞培养中需要将这些细胞与耳廓软骨细胞区分开来。在培养的耳廓软骨细胞中观察到GFAP mRNA表达，并且还在这些细胞的细胞裂解物和培养上清液中测量了GFAP蛋白水平。然而，随着孵育时间的增加，从细胞裂解物中的mRNA和蛋白检测到的GFAP水平显著降低。相比之下，细胞上清液中的蛋白量不受孵育时间的影响。此外，培养细胞上清液中GFAP的蛋白水平与这些细胞的软骨基质的 和 产生相关。通过测量培养的耳廓软骨细胞培养上清液中的GFAP蛋白水平，可以预测这些细胞中软骨基质的生产力。因此，GFAP被视为培养耳廓软骨细胞纯度和特性的标志物。
