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/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.


SolidWorks1 SolidWorks2

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.

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:


Monkey kidney cells1 Monkey kidney cells2 Monkey kidney cells2 Monkey kidney cells2

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

  4. Washing the cells

    Wash gently the cells with PBS

  5. Trypsinization

    Cover the flask with 3-5 mL with Trypsin/EDTA solution
  6. Incubate for 3 - 5 minutes @ 37ºC
  7. Disperse the cells by hitting the flask (once)
  8. Watch in the microscope to confirm trypsinization step
  9. Add 5 mL medium to inactivate trypsin
  10. Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)
  11. Adding new medium
    Remove medium + TE without touching the pellet
  12. Add 5 mL of medium to the centrifuge tube with cells
  13. Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)
    14. Cell fixation
  14. Fix the cells in 4% paraformaldehyde for 15 min @ RT
  15. Wash with PBS three times (3 times, 5 minutes each)

    16. Cell permeabilization for intracellular biomarkers

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

    18. Blocking

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

    20. DAPI/Hoechst addition

  20. Incubate the cells with secondary antibody (1:200) in PBS + 1% BSA for 1 hour @ RT
  21. Wash with PBS (3 times, 5 minutes each)
  22. Incubate the cells on 1:3000 DAPI or Hoechst for 5 minutes @ 4 ºC
  23. Wash with PBS (3 times, 5 minutes each)

    24. Imaging

  24. Take pictures in a confocal microscope according to the desired channel (bright field, DAPI, Texas Red, GFP) at 4x, 10x, 20x



Column: Cell Count

  1. 50000
  2. 25000
  3. 12500
  4. 6250
  5. 3125
  6. 1563
  7. 781
  8. 391
  9. 195
  10. 98
  11. 49
  12. 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.