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/2013/IB/c3ib40126j#!divAbstract
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, in the device. Dr. Chiang, from UCL’s very own Microfluidics Lab, has already drawn up the 3d design described in the attachment using SolidWorks.
Dr. Paul Sharp, who works with human intestinal epithelial cell models at Kings College London, has kindly agreed to collaborate with us on this project, give us the Caco-2 cells we need, and advice us on the best ways to culture them. We then decided to improve on 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 (UCL Biochemical Engineering) 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.
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
- Phosphate-buffered saline (PBS), pH 7.2-7.4
- Trypsin-EDTA (0.05% trypsin)
- Serum-containing cell culture medium (e.g., 10% FBS in DMEM)
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 with secondary antibody (1:200) in PBS + 1% BSA for 1 hour @ RT
- Wash with PBS (3 times, 5 minutes each)
- Incubate the cells on 1:3000 DAPI or Hoechst for 5 minutes @ 4 ºC
- Wash with PBS (3 times, 5 minutes each)
Imaging
- Take pictures in a confocal microscope according to the desired channel (bright field, DAPI, Texas Red, GFP) at 4x, 10x, 20x
Column: Cell Count
- 50000
- 25000
- 12500
- 6250
- 3125
- 1563
- 781
- 391
- 195
- 98
- 49
- Negative Control
Protocol for Determining Adherence Time:
Cells were pipetted into a 96 well plate at the optimum seeding density
At intervals of 1 hour, the medium from 1 column was removed
The cells were stained with DAPI, and cell counting was done under the microscope.