Team:UCL/Design

Gut-on-Chip

Introduction

To demonstrate a functional prototype of our project, we decided to show our system working under real-world conditions simulated in the lab using a Gut-on-a-Chip design similar to the one described in:
http://pubs.rsc.org/en/Content/ArticleLanding/2012/LC/c2lc40074j#!divAbstract

The idea is to model the rate at which our genetically engineered bacterial culture (E. Coli Nissle) grows and colonizes the gut, and to characterize its expression of 5-HTP, a serotonin precursor that acts as an anti-depressant. With the assistance of Dr. Chiang, from UCL’s Microfluidics Lab, we designed unsing SolidWorks a 3D version of the chip model described in the attachment.

Dr. Paul Sharp, who works with human intestinal epithelial cells at Kings College London, kindly agreed to collaborate with us on this project. He gave us the Caco-2 cells we needed, and adviced us on the best ways to culture them. After attending a lecture by Dr. Marques (UCL Biochemical Engineering), we decided to improve the original Gut-on-a-Chip designed at Harvard University by making it a more realistic mimic of reality and more financially feasible.The new design doesn't require a porous membrane, and is inspired by Dr. Marco's bulging bioreactor. It has been designed under the guidance of Dr. Paul Sharp. In addition to replicating the peristaltic motion of the longitudinal muscles in the intestines like Harvard's design, this model will also replicate the motions created by circular muscles.

GoC Design1 GoC Design2

The microfluidics device will be initially tested using Monkey Kidney Epithelial Cells, which are very similar to Intestinal Epithelial Cells (Caco-2). Experiments were carried to determine the optimum seeding cell density of the cells, and the time they need to adhere.

Monkey Kidney Fibroblast Cell Culture:

Protocol for Determining Optimum Seeding Cell Density:

MATERIALS

Cells at 70% confluency (take cells only when ready)
Medium: DMEM (1x) + GlutaMAXTM-I (Life Technologies, 61965-026) + 10% FBS
10 mL PBS without Ca and Mg (Lonza, BE17-516F)
Trypsin/EDTA (TE) solution
25 mL cell flasks x 2
25 mL centrifuge tube (yellow cap) x 1
5 mL pipettes x 5
10 mL pipettes x 5
1 mL micropipette
Tips for 1 mL micropipette
Pipette dispenser
Tissue paper roll
Ethanol 70%
Detergent
Markers
Rack
USBs
Waste disposal flask
Cell culture in well plates or coverslips
4% paraformaldehyde
PBS without Ca and Mg (Lonza, BE17-516F)
PBS + 0.1% Triton X-100
PBS + 3% BSA
:3000 DAPI or Hoechst in PBS
Aluminum foil
Markers
Rack
USBs
Confocal microscope
Safety glasses

METHODS

Preparation

Warm medium (DMEM (1x) + GlutaMAXTM-I + 10% FBS), PBS and TE solution in a water bath @ 37ºC
Spray tubes, bottles, racks and everything that goes inside the hood with 70% ethanol

Taking off the medium

Take off almost all the medium inside the flask

Washing the cells

Wash gently the cells with PBS

Trypsinization

Cover the flask with 3-5 mL with Trypsin/EDTA solution
Incubate for 3 - 5 minutes @ 37ºC
Disperse the cells by hitting the flask (once)
Watch in the microscope to confirm trypsinization step
Add 5 mL medium to inactivate trypsin
Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)

Adding new medium

Remove medium + TE without touching the pellet
Add 5 mL of medium to the centrifuge tube with cells
Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)

Cell fixation

Fix the cells in 4% paraformaldehyde for 15 min @ RT
Wash with PBS three times (3 times, 5 minutes each)

Cell permeabilization for intracellular biomarkers

Incubate with PBS + 0.1% Triton X-100 for 10 minutes @ RT
Wash with PBS three times (5 minutes each)

Blocking

Block with PBS + 3% BSA for 1 hour @ RT
Wash with PBS (3 times, 5 minutes each)

DAPI/Hoechst addition

Incubate the cells on 1:3000 DAPI for 5 minutes @ 4 ºC
Wash with PBS (3 times, 5 minutes each)

Imaging

Cells were visualized by confocal microscopy. Images were aquired at fluorescence peak excitation and emission wavelengths of 360 nm and 460 nm, respectively.