Gargus Matthew, Niu Chao, Shaker Anisa
Department of Medicine, Keck School of Medicine, University of Southern California.
Department of Medicine, Keck School of Medicine, University of Southern California;
J Vis Exp. 2015 Jan 18(95):52215. doi: 10.3791/52215.
Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express α-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.
小鼠和人食管肌成纤维细胞通过酶消化法获得。取新生（8 - 12日龄）小鼠食管，切碎、洗涤，用胶原酶和分散酶进行酶消化25分钟。人食管切除标本去除固有肌层和外膜，将剩余的黏膜切碎，用胶原酶和分散酶进行酶消化长达6小时。培养的细胞表达α - SMA和波形蛋白，结蛋白表达微弱或完全不表达。培养条件不利于上皮细胞、造血细胞或内皮细胞生长。通过对潜在污染的造血细胞和内皮细胞的细胞表面标志物表达进行流式细胞术评估，进一步确认培养纯度。所描述的技术操作简单，能持续产生非造血、非内皮的基质细胞。该技术的局限性是分子生物学研究中使用原代培养所固有的，即在不同小鼠或人类建立的培养物之间不可避免地存在变异性。然而，与细胞系相比，原代培养更能代表体内状态。这些方法还使研究人员能够从不同的临床和实验条件中分离和培养基质细胞，从而进行组间比较。特征明确的食管基质细胞也可用于研究食管疾病中上皮 - 基质相互作用的功能研究。
