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A method of microencapsulating Leydig cells for the long-term time release of male reproductive hormones in vivo is desired. Challenges such as biocompatibility, patient immune response, and perfusion of encapsulated cells must be addressed. The current design uses a polyethylene glycol diacrylate (PEGDA) polymer mixed with a cell suspension. Microcapsule production may be achieved with a microfluidics approach, followed by UV light exposure to cause polymerization, or cross-linking, of the polymer-cell mixture into a stable hydrogel.
PEGdA Biomaterial
Figure 1. Structure of (top) poly(ethylene glycol) and (bottom) poly(ethylene glycol) diacrylate.
Microsphere Production
Figure 2. Schematic of microfluidic device used to produce microspheres.
Figure 3. Microfluidic device showing (right) flow controllers and (center) PDMS microchannel component. (Left) UV light is projected upon the exit tubing to cause polymerization.
Figure 4. Microfluidic device in operation. PEGdA droplets are visible surrounded by mineral oil sheath flow.
Leydig Cell Culture
Figure 6. MA-10 cells: (left) Bright field image at 200x. (Middle) Cells labeled with fluorescent Live/Dead reagents 10 minutes after plating (green=live, red=dead), 100x. (Right) Cells labeled with Live/Dead reagents 1 day after plating, 200x.
Figure 7. LIVE/DEAD assay performed on MA-10 cells encapsulated in 25 uL PEGdA hydrogels, taken at various time points. (Top row) Calcien fluoresence at 517 nm (shown in grayscale) indicating live cells. (Bottom row) Ethidium homodimer fluorescence at 617 nm (shown in grayscale) indicating dead cells. Images are un-altered. Experiment was performed in triplicate, with a representative gel shown for demonstrative purposes.
Current Experiments
Currently, we are characterizing the PEGdA biomaterial. This involves a protein diffusion study, viability study, and hormone study. These are conducted on the macroscale (ie. by polymerizing 20-200 uL PEGdA hydrogels in 384- or 96-well plates) so that preliminary data can be obtained. Refer to the ’BME 402 Midsemester Presentation’ file for more details on experimental design and predicted results.
We have conducted a diffusion study using BSA, 70 kDa dextran, and 500 kDa dextran. However, it has been difficult to assess protein diffusion into the hydrogel versus protein coating the external surface. Rinsing with TBST and PBS did not remove protein/dextran adsorbed to the outer surface of the gels. Sectioning via cryostat into 200 um slices did not improve results.
A viability study was conducted over 8 d using non-modified PEGdA. Excellent cell viability was observed up to 8 d, though high cell proliferation may also be an issue. Cells appeared to be highly aggregated.
A testosterone ELISA was also performed on encapsulated MA-10 cells, 7 d after seeding them in the PEGdA material. After stimulating the cells with LH or LH/FSH, increased testosterone secretion was observed, but the results were not statistically significant.
| Week | Reporting Period Beginning | Activities |
|---|---|---|
| 1 | January 20 | Met with advisor. Discussed experiments. |
| 2 | January 27 | Began writing protocols for diffusion experiments. |
| 3 | February 3 | Constructed materials and reagents list. |
| 4 | February 10 | Re-evaulating diffusion study protocols and ordering supplies. |
| 5 | February 17 | Receiving supplies and reordering. |
| 6 | February 24 | Midsemester presentation and planning outreach activity. |
| 7 | March 3 | Began PEG acrylation and protein labeling with fluorescein. Final prep for outreach activity. |
| 8 | March 10 | Started first diffusion study. |
| 9 | March 17 | Repeated first diffusion study, and started viability assay. |
| 10 | March 24 | Measuring data from diffusion study using cryostat, and repeating viability assay. |
| 11 | March 31 | Start hormone assay using samples from second viability assay trial. |
| 12 | April 7 | Report results of diffusion study. |
| 13 | April 14 | Report results of viability and hormone studies. |
| 14 | April 21 | Final presentation (actually at 12:20pm May 5, ECB Lobby) |
| 15 | April 28 | Closing meetings with client and advisor. Graduation! |
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BME 400 (Fall 2005) Final Report (Feb 1 2006, 4250 kb) |
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BME 400 (Fall 2005) Final Poster (Feb 1 2006, 718 kb) |
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Product Design Specifications (2/1/06) (Feb 1 2006, 28 kb) |
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BME 402 Midsemester Presentation (Mar 1 2006, 2209 kb) |
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Outreach Presentation: Engineers in Today's Society, for middle school students (Apr 11 2006, 3104 kb) |
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Outreach Summary: Engineers in Today's Society, for middle school students (Apr 26 2006, 23 kb) |
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BME 402 (Spring 2006) Final Report (Apr 28 2006, 232 kb) |
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BME 402 (Spring 2006) Final Poster (May 3 2006, 5252 kb) |
