Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.
Department of Thoracic Medicine, University College Hospital, London, UK.
J Tissue Eng Regen Med. 2018 Jan;12(1):e313-e317. doi: 10.1002/term.2466. Epub 2017 Aug 22.
Autologous airway epithelial cells have been used in clinical tissue-engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures, but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compared endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co-culture of samples with 3T3-J2 feeder cells in culture medium containing a Rho-associated protein kinase inhibitor, Y-27632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures.
自体气道上皮细胞已用于临床组织工程气道移植程序，以协助黏膜再生和恢复黏液纤毛清除功能。然而，由于这些干预措施的紧迫性，上皮细胞的扩增时间有限，并且患者的上皮再生速度较慢。人呼吸道上皮细胞可以从小活检或支气管镜检查过程中的刷取物中扩增，但尚未研究从患者中获取组织的最佳方式。在这里，我们比较了支气管内刷检和支气管内活检样本在细胞数量及其启动基底上皮干细胞培养方面的能力。我们发现，与传统技术相比，直接将样本与 3T3-J2 饲养细胞共培养，并在含有 Rho 相关蛋白激酶抑制剂 Y-27632 的培养基中培养，可在培养的早期关键阶段选择性地扩增更多数量的基底上皮干细胞。此外，我们通过细胞刷检样本或通过活检的酶消化来建立起始细胞培养的细胞悬液，优于外植体培养。从冷冻保存的支气管活检样本开始建立原代上皮细胞培养物，提示冷冻保存可以消除活检样本采集的临床机构与上皮细胞培养的专业实验室之间的紧密接近要求。总体而言，我们的研究结果为未来气道细胞治疗或生物工程气道移植程序中加快上皮细胞准备提供了思路。
