School of Medicine and Public Health, University of Newcastle, Callaghan, NSW, Australia.
Immune Health Program, Hunter Medical Research Institute, University of Newcastle, New Lambton Heights, NSW, Australia.
Sci Rep. 2023 Jul 11;13(1):11200. doi: 10.1038/s41598-023-37828-0.
Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)-(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.
原代气道上皮细胞的空气-液界面（ALI）培养广泛用于模拟气道反应。最近的一项进展是开发了增强增殖能力的条件重编程。几种不同的培养基和方案被使用，但即使是细微的差异也可能影响细胞反应。我们比较了形态和功能反应，包括对条件重编程的原代支气管上皮细胞（pBEC）在两种常用培养基于 ALI 分化后对鼻病毒感染的固有免疫反应。pBEC 从健康供体（n=5）中收集，使用γ射线照射的 3T3 成纤维细胞和 Rho 激酶抑制剂进行 CR。CRpBEC 在 PneumaCult（PN-ALI）或基于支气管上皮生长培养基（BEGM）的分化培养基（BEBM：DMEM，50：50，Lonza）-（AB-ALI）中在 ALI 分化 28 天。跨上皮电阻（TEER）、免疫荧光、组织学、纤毛活性、离子通道功能和细胞标志物表达进行分析。通过 RT-qPCR 评估病毒 RNA，并在 Rhinovirus-A1b 感染后通过 LEGENDplex 定量抗病毒蛋白。与 BEGM 培养基相比，在 PneumaCult 中分化的 CRpBEC 更小，TEER 和纤毛拍打频率更低。PneumaCult 培养基培养物表现出更高的 FOXJ1 表达、更多具有更大活动面积的纤毛细胞、更多的细胞内粘蛋白和增加的钙激活氯离子通道电流。然而，病毒 RNA 或宿主抗病毒反应没有显著变化。在两种常用的 ALI 分化培养基中培养的 pBEC 存在明显的结构和功能差异。在设计特定研究问题的 CRpBECs ALI 实验时，需要考虑这些因素。
