Propagating large amounts of human corneal stromal cells (hCSCs) in vitro while maintaining the physiological quality of their phenotypes is necessary for their application in cell therapy. Here, a novel medium to propagate hCSCs obtained from small incision lenticule extraction (SMILE)‐derived lenticules was investigated and the feasibility of intrastromal injection of these hCSCs was assessed. Primary hCSCs were cultured in porcine corneal stroma extract (pCSE) with RIFA medium including ROCK inhibitor Y27632, insulin‐transferrin‐selenium, fibroblast growth factor 2, L‐ascorbate 2‐phosphate and 0.5% FBS (RIFA medium + pCSE). Protein profiling of the pCSE was identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC‐MS/MS). After subculturing in RIFA medium + pCSE or 10% FBS normal medium (NM), hCSCs at P4 were transplanted into mouse corneal stroma. Compared with NM, ALDH3A1, keratocan and lumican were significantly more expressed in the RIFA medium + pCSE. ALDH3A1 was also more expressed in the RIFA medium + pCSE than in the RIFA medium. Fibronectin and α‐SMA were less expressed in the RIFA medium + pCSE than in the NM. Using Metascape analysis, the pCSE with its anti‐fibrosis, pro‐proliferation and anti‐apoptosis activities, was beneficial for hCSC cultivation. The intrastromally implanted hCSCs in the RIFA medium + pCSE had positive CD34 expression but negative CD45 expression 35 days after injection. We provide a valuable new medium that is advantageous for the proliferation of hCSCs with the properties of physiological keratocytes. Intrastromal injection of hCSCs in RIFA medium + pCSE has the potential for clinical cell therapy.

中文翻译：

---

猪角膜基质提取物对 SMILE 衍生微透镜角膜细胞的影响


在体外繁殖大量人角膜基质细胞（hCSC），同时保持其表型的生理质量，对于其在细胞治疗中的应用是必要的。在这里，研究了一种从小切口微透镜提取 (SMILE) 衍生的微透镜获得的 hCSC 增殖新介质，并评估了这些 hCSC 基质内注射的可行性。原代 hCSC 在猪角膜基质提取物 (pCSE) 和 RIFA 培养基中培养，其中包含 ROCK 抑制剂 Y27632、胰岛素-转铁蛋白-硒、成纤维细胞生长因子 2、L-抗坏血酸 2-磷酸和 0.5% FBS（RIFA 培养基 + pCSE）。 pCSE 的蛋白质谱通过纳米级液相色谱与串联质谱联用（纳米 LC-MS/MS）进行鉴定。在 RIFA 培养基 + pCSE 或 10% FBS 正常培养基 (NM) 中传代培养后，将 P4 时的 hCSC 移植到小鼠角膜基质中。与 NM 相比，ALDH3A1、keratocan 和 lumican 在 RIFA 培养基 + pCSE 中的表达显着更高。 ALDH3A1 在 RIFA 培养基 + pCSE 中的表达量也高于 RIFA 培养基中的表达量。纤连蛋白和 α-SMA 在 RIFA 培养基 + pCSE 中的表达低于 NM。使用 Metascape 分析，pCSE 具有抗纤维化、促增殖和抗凋亡活性，有利于 hCSC 的培养。注射后35天，基质内植入的hCSC在RIFA培养基+pCSE中具有阳性CD34表达，但阴性CD45表达。我们提供了一种有价值的新培养基，有利于 hCSC 的增殖，并具有生理性角膜细胞的特性。基质内注射 RIFA 培养基 + pCSE 中的 hCSC 具有临床细胞治疗的潜力。