-
-
Skip navigation
(Access key: S).
Contact information.
Embryonic stem cells (ESCs) have the capacity to differentiate into every cell type in the body, and therefore can theoretically be used to generate cells and tissues to cure a variety of diseases. Our client in the Odorico Lab (Department of Surgery) has derived foregut-committed cell lines from ESCs (which correspond to progenitor cells of the gut region that develops primarily into pancreas) and would like to differentiate these ESCs into insulin-producing pancreatic beta-like cells. These cells could replace or supplement transplanted donor beta cells. The mechanisms required to differentiate ESCs into these pancreatic cells is currently unknown, and this device would aid in researching such mechanisms. Our client would like to test a large number of growth factors for their ability to affect conversion of these precursor cells to mature insulin-secreting cells. In addition, a recapitulation of the 3-dimentional embryonic environment to prompt cells to adopt a pancreatic cell fate, perhaps using a Matrigel substrate, is desirable. A small scale cell culture using microfluidics to set up growth factor gradients is one approach that could be successful.
From left to right: Dhaval Desai, Tim Pearce, Jonathan Baran, Tess Rollmann
Picture showing the cells in the channel before the addition of media in the source and sink.
Picture of the cells after they flowed out after the addition of additional media in the source and the sink.
Picture of the gradient formation with a membrane after four hours.
Picture of gradient formation with a membrane after 7.5 hours.
Picture of gradient formation with the agarose plug.
We are currently introducing cells into our microchannels for the first time. We are initally going to use AR42J cells to test viability within the channel. Also since we are no longer going to suspend the cells in matrigel, we have to change our design by adding a high resistance membrane. Our original channel design has to be tested and verified with this change.
| Week | Reporting Period Beginning | Activities |
|---|---|---|
| 1 | January 25 | We meet with our clients at the end of last semester, and discussed the design changes that need to take place. Also we decided an emphasis should be placed on cell viability within the channels because this could potentially require design modifications. |
| 2 | February 1 | Our clients have decided that rather than using the stem cells, we should use the AR42J cell line which has been shown to differentiante in a concentration gradient. |
| 3 | February 8 | We recieved the membrane which are going to be added to our design. We are going to set up our inital experiements by placing the AR42 cells in the channel and determining how long they can live in different channel dimmensions. |
| 4 | February 15 | Initial test results: Placed cells in the mirochannel and placed the source and sink on top. Due to the lack of a high resistance membrane, flow of the media occured causing the cells to move to the end of the channel. Next a source and sink were not placed on top, this caused the media to evaporate and the cells to die within 20 hours. |
| 5 | February 22 | Further Results: Cell adhesion is still a problem. The cells seem to cling together rather than to the gelatin on the glass slide. Tissue culture treated plastic seems to be a better option. Also inital results with the gradient with a membrane were not successful, dextran did not move through the membrane, |
| 6 | February 29 | Rather than introducing the cells first, then adding the source and sink, the source and the sink were added and the cells were flowed in. However, still the results were similar with the cells being flushed to either end of the channel. Also we performed a test on the extent of cell viability if evaportation is decreased. This was done by filling a channel with cells and media and covering the ports with another layer of PDMS. The experiment is still in progress. |
| 7 | March 7 | The cells have been determined to adhere to the channel best when inserted and left covered with a slab of PDMS for a day. We are now working on gradient formation with the membranes. We have been in contact with a graduate student about how he was able to get the membranes to work. |
| 8 | March 14 | We are continuing to work on the gradient formation with the membranes in place. To get the membranes to adhere to the PDMS plasma bonding is being used. |
| 9 | March 21 | Gradient formation seemed to be witnessed after 7.5 hours. |
| 10 | March 28 | New techniques were tried to try to ensure that air bubbles were not in the gradient. These techniques include: placing the channel in a vacuum and forcing liquid through the membrane with a pipette. Both of these techniques resulted in leakage. |
| 11 | April 4 | Since the membrane does not seem to be working correctly, agarose will be tried. First an agarose "plug" will be placed over the source port to try to act like membrane. Also a redesigned channel will be used with a "T" configuration. The configuration will allow agarose to enter in one channel and media to enter the other. The agarose channel will then block off the media channel causing a high resistance pathway to result. |
| 12 | April 11 | One gradient was established with the agarose plug method. The "T" channel resulted in leakage of agarose, and no results were obtained. |
| 13 | April 18 | After much experimentation, we told our clients that the best method is the 3-D Matrigel method, we recommended that they further pursue this option. |
| 14 | April 25 | |
| 15 | May 2 |
 |
Mid-Semester Presentation (Mar 6 2008, 4056 kb) |
 |
Final Poster (May 1 2008, 2847 kb) |
 |
Final Journal Article (May 12 2008, 200 kb) |
