Modular Cell Culture Platform with Passive Fluid Controls for GI Tract - Liver Tissue Co-Culture

Modular Cell Culture Platform with Passive Fluid Controls for GI tract - Liver Tissue Co-Culture              

Mandy B. Esch,¹ Michael L. Shuler²

(1) Dept. of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY; (2) Dept. of Biomedical Engineering, Cornell University, Ithaca, NY

We have developed a microfluidic cell culture platform and used it to culture GI tract epithelial cells and primary liver cells together for 14 days. In the body, these tissues are responsible for the first pass meatbolism of nutrients and drugs. Both, GI tract epithelium and liver were scaled down from in vivo values by a factor of 110,000. Both tissues were perfused at physiologic fluid flow rates that were controlled via passive device elements. Representing the GI tract epithelium, Caco-2 cells maintained a transepithelial resistance (TEER) of 200 to 380 ½cm². Representing human liver tissue, a mixture of human primary nonparenchymal cells (fibrolbasts, stellate and Kupffer cells) and parenchymal cells (hepatocytes), maintained urea and albumin synthesis for the entire culture period of 14 days. The modular design enabled us to culture both tissues separate from each other in order to reach maturity before combining them. The device presents a low cost approach to culturing multiple tissue with ratios of tissue volumes and fluid flow rates that are of physiologic relevance.

Introduction: Patients who suffer from diabetes regularly inject insulin subcutaneously. If they took insulin orally, their stomach and intestine would break it down to such a high degree that even though any remaining insulin would be delivered directly to the liver, its concentration would be too low to be effective. In the case of insulin, the direct delivery to the liver would be an advantage, because that's where it's needed. Drugs that are needed at other organs, such as painkillers, are often additionally broken down in the liver. Considerable efforts go into developing drug formulations that can overcome these obstacles.[1] Animal models do not predict the first pass effects well,[2] and in vitro models of the human GI tract – liver unit would be of significant use when assessing new formulations of drug that seek to increase bioavailability.

Materials and Methods: We designed a modular device using Solidworks and printed it with a 3D Object printer from Stratasys (Israel). We calculated channel resistances so that medium flow rates reach physiologic values when tilted the device at an angle of 18¼. On separate tissue chips we cultured Caco-2 cells (for 16 days), and 150,000 primary human nonparenchymal cells (a mixture of fibrolbasts, stellate and Kupffer cells) and 250,000 hepatocytes (for 7 days). The two tissue chips contained chambers that were scaled down from human tissue volumes 110,000 fold. After maturation, we inserted the GI tract and liver chips into the multi-organ platform and operated the device for 14 days. We replaced 50% of medium with fresh medium dayly. We measured TEER, activity of CYP enzymes (CYP 3A4 and 1A1), and urea and albumin synthesis for 14 days.

Results and Discussion: The devices operated for 14 days without loss of function of the tissues. The GI tract epithelium maintained TEER values between 200 and 380 ½cm² for the entire culture period (Fig.1 A). The liver tissue produced urea (Fig. 1B) and albumin (not shown) at rates we and others have previously observed under fluidic flow.[3] The cells also responded with CYP enzyme activity when stimulated with rifampicin or 3-Methylmethaxocine (results not shown).

Figure 1. A) TEER of Caco-2 cells for 14 days of co-culture. B) Urea synthesis of the liver tissue consisting of primary human liver cells [non-parenchymal cells (fibroblasts, stellate and Kupffer cells) and parenchymal cells (hepatocytes)].

Conclusions: We have developed a modular microfluidic device that operated without loss of tissue function for 21 days. We believe this device design could be useful when utilizing primary cells or stem cells to generate tissues for the purpose of combining them in order to simulate the first pass metabolism in vitro. The device design is expandable, meaning additional tissue modules could be designed and integrated in the future. Ref.: [1] S. Kalra, Diabetol Metab Syndr., 2010, 2, 66. [2] X.Gao, Pharmaceutical Research, 2006, 23(8), 1675. [3] Ebrahimkhani, ADDR, 2014, 69/70, 132.