Difference between revisions of "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.

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

• 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

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
Taking off the medium
3. Take off almost all the medium inside the flask
Washing the cells
4. Wash gently the cells with PBS
Trypsinization
5. 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)
Adding new medium
11. 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)
Cell fixation

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

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)

Blocking

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

DAPI/Hoechst addition

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

Imaging

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

Column: Cell Count (Cells per ul)

50000	25000	12500	6250	3125	1563
781	391	195	98	49	Negative Control

Protocol for Determining Adherence Time:

1. Repeat steps 1 to 12 as described in the protocol above.
2. Seed 6000 cells into 3 wells respectively of 4 96 well plates.
3. Repeat steps 14 to 24 for one plate at intervals of 1 hour.

Adherence Results

1 hour

2 hours

3 hours

4 hours