Cancer Institute, Department of Haematology, University College London, London, UK.
Department of Biochemical Engineering, University College London, London, UK.
Sci Rep. 2021 Jun 11;11(1):12392. doi: 10.1038/s41598-021-91939-0.
Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.
用于临床替代非功能性上呼吸道黏膜的工程化上皮细胞片被视为药品，应按照良好生产规范（GMP）进行生产。目前，用于研究的上皮细胞生长的金标准是使用生长停滞的鼠 3T3 J2 饲养层，但这些细胞不能作为符合 GMP 要求的原材料使用。我们使用猪黏膜组织，展示了一种从单个活检中获得和培养非角化鳞状上皮细胞和成纤维细胞的新方法，用生长停滞的原代成纤维细胞饲养层替代 3T3 J2，并使用混合人血小板裂解物（HPL）作为培养基血清补充物替代胎牛血清（FBS）。最初使用 Octodissociator 对细胞进行半自动分离，并将得到的细胞悬液冷冻保存以备将来使用。与金标准的 3T3 J2 和含 FBS 的培养基相比，细胞的生长、活力、干细胞群体或分化为成熟上皮细胞的能力没有下降。此外，该方法在人颊组织中得到了复制，提供了足够数量和质量的细胞来创建组织工程化片。
