Difference between revisions of "Team:UCL/Design"

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METHODS
 
METHODS
 
<ol>
 
<ol>
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<li>Pre-warm all reagents to 37C in water bath.  </li>
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Preparation
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<li>After reagents are warmed, spray bottles down with ethanol and prepare the hood as for routine feeding.</li>
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<li> Warm medium (DMEM (1x) + GlutaMAXTM-I + 10% FBS), PBS and TE solution in a water bath @ 37ºC</li>
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<li>Aspirate spent culture media from the cell culture vessel.</li>
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<li> Spray tubes, bottles, racks and everything that goes inside the hood with 70% ethanol</li>
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<li>Wash the cells once with PBS.  Add 5 ml of PBS for every 25 cm2 of culture area.</li>
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<li><b>Taking off the medium</b>
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<li>Aspirate the PBS.</li>
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<li> Take off almost all the medium inside the flask<li>
−
<li>Add 1-2 ml per 25 cm2 of trypsin-EDTA into the culture flask (i.e., 5ml of trypsin-EDTA for a T-75 culture flask), and return the sealed flask to the incubator for 5minutes.</li>
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<li>
−
<li>After incubation, examine the cells under a microscope.  Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask/dish.</li>
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<p><b>Washing the cells</b></p>
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<li>If the cells are not fully detached, place the flask back into the incubator.  Some cells may require some mechanical agitation (including “rapping” the flask or “scraping” the culture surface), BUT THIS IS NOT PREFERRED.</li>
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Wash gently the cells with PBS</li>
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<li>Once the cells have detached, add serum-containing medium to the flask in an amount approximately 2-3X that of the trypsin (i.e., 10-15ml of medium for a T-75 culture flask).  Trypsin will start to act on the excess serum proteins instead of harming the cells.
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<li><p><b>Trypsinization</b></p>
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Note: The medium MUST contain serum in order to act to inhibit the trypsin.  Serum-free media can only be used IF a trypsin inhibitor is used.</li>
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Cover the flask with 3-5 mL with Trypsin/EDTA solution</li>
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<li>Collect the harvested cells and pipet into an appropriately sized centrifuge tube.</li>
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<li> Incubate for 3 - 5 minutes @ 37ºC</li>
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<li>Centrifuge cells for approximately 5 minutes at 200xg (800-1100 rpm, depending on the centrifuge).</li>
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<li>Disperse the cells by hitting the flask (once)</li>
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<li>During centrifugation, label new culture flasks.  Label flasks with cell type, your initials, the new passage number (passage number increases with every split), and today’s date.</li>
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<li> Watch in the microscope to confirm trypsinization step</li>
−
<li>NOTE: Certain cell types are only viable up to a certain passage number, such as primary cells.  Make sure to check this before splitting beyond the appropriate passage number!</li>
+
<li> Add 5 mL medium to inactivate trypsin</li>
−
<li>Following centrifugation, aspirate the media above the cell pellet and resuspend the cells in a logical volume (5-10 ml).</li>
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<li>Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)</li>
−
<li>If necessary, count cells via hemacytometer or coulter counter.</li>
+
<li><p><b>Adding new medium</b></p>
 +
<li> Remove medium + TE without touching the pellet</li>
 +
<li> Add 5 mL of medium to the centrifuge tube with cells</li>
  
  
 
<li>Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)</li>
 
<li>Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)</li>
−
<li>After 3 days, the cell confluency was checked under a microscope to determine the optimum level.</li>
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<!--<li>After 3 days, the cell confluency was checked under a microscope to determine the optimum level.</li>-->
  
 
</ol>
 
</ol>

Revision as of 13:49, 3 September 2015

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
  4. Take off almost all the medium inside the flask

  6. Washing the cells

    Wash gently the cells with PBS

  7. Trypsinization

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

  13. Adding new medium

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


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.