(542b) 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.