Cenhrang Khamhbawihum, Leasor Cody W, Thotamune Waruna, Karunarathne Ajith, Baker Lane A, Martin R Scott
Department of Chemistry, Saint Louis University, Saint Louis, Missouri 63103, United States.
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
ACS Meas Sci Au. 2025 Jun 3;5(4):547-558. doi: 10.1021/acsmeasuresciau.5c00045. eCollection 2025 Aug 20.
In this paper, we describe how 3D printing can be used to fabricate a microfluidic-based transwell cell culture system with robust fluidic connections for long-term cell culture and recirculating flow. This approach consists of an electrospun collagen scaffold sandwiched between two laser-cut Teflon membranes that match the fluidic design. Madin-Darby canine kidney (MDCK) cells were cultured on the collagen scaffold to create an epithelial cell monolayer. Introduction of cells into the device was facilitated by a printed reservoir that could be closed after proper cell seeding with minimal effect of the flow profile over the cells. The resulting MDCK cell monolayer was exposed to continuous flow and transport through the cell layer and could be monitored by sampling from the basolateral channel network. COMSOL simulations and flow injection analysis were used to determine the effect of the reservoir geometry on the shear stress that cells experience. A variety of analytical tools were used to assess the effect of flow over the cells in this model. This includes confocal microscopy and potentiometric scanning ion conductance microscopy (to determine morphology and conductance), as well as transendothelial/epithelial electrical resistance (TEER) measurements and reverse transcription-quantitative polymerase chain reaction studies (for gene expression analysis). Finally, a drug transport study with the cell model was carried out using two drugs (caffeine and digoxin) to determine the apparent permeability of high and low permeability drugs, with results being similar to findings from in vivo studies as well as studies where MDCKs have been transfected to form more resistive barriers. This approach holds great promise for the creation of more in vivo-like, flow-based barrier models for transport studies.
在本文中，我们描述了如何使用3D打印制造一种基于微流控的Transwell细胞培养系统，该系统具有坚固的流体连接，可用于长期细胞培养和循环流动。这种方法包括一个夹在两个与流体设计相匹配的激光切割聚四氟乙烯膜之间的电纺胶原蛋白支架。将Madin-Darby犬肾（MDCK）细胞培养在胶原蛋白支架上以形成上皮细胞单层。通过一个打印的储液器便于将细胞引入该装置，在适当接种细胞后可以关闭该储液器，且对细胞上的流动分布影响最小。所得的MDCK细胞单层暴露于连续流动并通过细胞层进行运输，并且可以通过从基底外侧通道网络取样进行监测。使用COMSOL模拟和流动注射分析来确定储液器几何形状对细胞所经历的剪切应力的影响。在该模型中使用了各种分析工具来评估流动对细胞的影响。这包括共聚焦显微镜和电位扫描离子电导显微镜（用于确定形态和电导），以及跨内皮/上皮电阻（TEER）测量和逆转录定量聚合酶链反应研究（用于基因表达分析）。最后，使用两种药物（咖啡因和地高辛）对该细胞模型进行药物转运研究，以确定高渗透性和低渗透性药物的表观渗透率，结果与体内研究以及将MDCK细胞转染以形成更具抗性屏障的研究结果相似。这种方法对于创建更多类似体内的、基于流动的屏障模型用于转运研究具有很大的前景。
