Three-Dimensional Lab-On-A-Chip Models Of Ductular Organs On Polymeric Scaffolds

Abstract

The majority of in vitro tissue models in health and disease have been based on two-dimensional (2D) culture systems, which are an inaccurate representation of the three dimensional (3D) complex in vivo. Three dimensional culture systems are more biomimetic in that the micro-environment can be emulated and cell-cell/cell-matrix interactions are more natural, allowing more realistic analysis of drug response and effectiveness. While research into 3D in vitro models has been ongoing for a couple of decades, some models still lack the exact architecture of the in vivo tissue. Ductular tissues are circular and porous in vivo to enable interaction with the surrounding tissue, yet they are modeled using non-porous rectangular channels. To address this technological gap, our lab previously created a lab-on-a-chip (LOC) with a completely circular ductular channel fabricated from a PMMA (poly-methyl methacrylate) body with a sandwiched porous PET (polyethylene terephthalate) membrane to study 3D tissue and organ models. We aimed to develop 3D models of ductular tissues, namely the breast cancer tissue (BCT) and the blood-brain barrier (BBB) using MDA-MB-231 cells, HMT-3522-S1 and T4 cells and ECV-304 cells respectively. We first utilized the LOC previously engineered in our lab and optimized cell attachment using type I collagen and poly-L-lysine, and various seeding densities. In addition, we established a protocol to enable full circumferential cell coverage in the LOC channels by flipping the LOC. Type-I collagen was determined to be the optimal attachment protein with an increase of over 70% relative to controls and p values of less than 0.05. Higher seeding densities resulted in better coverage, and flipping the chip was essential to achieve full coverage of the top and bottom hemichannels, with coverage reaching up to 86% when seeding only the bottom channel, and coverages up to 80% and 70% respectively for upper and bottom channels upon flipping the chip. Second, to address limitations associated with the available LOC design, including meniscus formation in the channel, challenge of imaging the entire channel, the delicate skill required for cell seeding and evaluation of cell’s functionality, we worked towards developing electrospun scaffolds as an alternative model. Electrospinning was used to fabricate ductular scaffolds from natural and synthetic polymers, and their potential as a platform to develop 3D ductular models was evaluated. Cellulose extracted from natural products, and synthetic polymers such as polyethylene oxide (PEO) and polycaprolactone (PCL) were mechanically characterized, tested for biocompatibility and then assessed to produce electrospun scaffolds. The electrospinning parameters of PCL were optimized to get aligned fibers. Cell coverage and viability studies were conducted on the electrospun scaffolds using MDA-MB-231 and ECV-304 cells. Trans-epithelial electrical resistance (TEER) measurements were conducted on PCL scaffolds seeded with ECV-304 cells to evaluate biomimicry of the developed models. The electrospun PCL scaffolds are biocompatible, and the cells achieve full coverage along the duct after 14 days of cell seeding at 20 million cells/mL using the ECV-304 cells, and approximately 70% coverage with the MDA-MB-231 cells. TEER values across the electrospun scaffold increased from 5Ω to 115Ω after 14 days of seeding ECV-304 cells, a significant increase with a p value less than 0.001. The engineered models are expected to enable better prediction of drug efficacy in vivo, since the grown cells and tissues would be interacting and responding to drugs in a more biomimetic manner. The models have a lower cost when compared to animal testing, can reduce the bench to bedside timeframe, as well as provide a platform for modelling various ductular tissues, including glandular ducts and vascular tissue.