Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
Vanderbilt Institute for Integrative Biosystems Research and Education, Vanderbilt University, Nashville, TN, USA.
Transl Vis Sci Technol. 2023 Nov 1;12(11):28. doi: 10.1167/tvst.12.11.28.
Cells grown in milliliter volume devices have difficulty measuring low-abundance secreted factors due to low resulting concentrations. Using microfluidic devices increases concentration; however, the constrained geometry makes phenotypic characterization with transepithelial electrical resistance more difficult and less reliable. Our device resolves this problem.
We designed and built a novel microfluidic "Puck" assembly using laser-cut pieces from preformed sheets of silicone and commercial off-the-shelf parts. Transwell membranes containing polarized retinal pigment epithelial (RPE) cells were reversibly sealed within the Puck and used to study polarized protein secretion. Protein secretion from the apical and basal surfaces in response to hypoxic conditions was quantified using an immunoassay method. Computational fluid modeling was performed on the chamber design.
Under hypoxic culture conditions (7% O2), basal vascular endothelial growth factor (VEGF) secretion by polarized RPE cells increased significantly from 1.40 to 1.68 ng/mL over the first 2 hours (P < 0.0013) and remained stably elevated through 4 hours. Conversely, VEGF secretion from the apical side remained constant under the same hypoxic conditions.
The Puck can be used to measure spatiotemporal protein secretion by polarized cells into apical and basal microniches in response to environmental conditions. Computational model results support the absence of biologically significant shear stress to the cells caused by the device.
The Puck can be used validate the mature phenotypic health of autologous induced pluripotent stem cells (iPSC)-derived RPE cells prior to transplantation.
由于最终浓度较低，在毫升体积设备中生长的细胞难以测量低丰度分泌因子。使用微流控设备会增加浓度；然而，受约束的几何形状使得跨上皮电阻的表型特征变得更加困难和不可靠。我们的设备解决了这个问题。
我们使用预先成型的硅酮片和商业现货零件通过激光切割制作了新颖的微流控“ puck”组件，并对其进行了设计和构建。含有极化视网膜色素上皮（RPE）细胞的 Transwell 膜可在 puck 内可逆密封，并用于研究极化蛋白分泌。使用免疫测定法定量测定缺氧条件下从顶侧和底侧表面分泌的蛋白质。对腔室设计进行了计算流体建模。
在缺氧培养条件（7%O2）下，极化 RPE 细胞的基底血管内皮生长因子（VEGF）分泌在最初 2 小时内从 1.40 增加到 1.68ng/ml（P<0.0013），并在 4 小时内保持稳定升高。相反，在相同的缺氧条件下，从顶侧分泌的 VEGF 保持不变。
puck 可用于测量极化细胞在响应环境条件时向顶侧和底侧微区时空分泌的蛋白质。计算模型结果支持设备不会对细胞产生具有生物学意义的剪切力。
曹毅
