1. Microencapsulation of Leydig cells
Team:
Bryan Baxter
Tim Eng
Joe Zechlinski
April Zehm
BME 402
February 24, 2006

2. Client: Dr. Craig Atwood Dr
Client: Dr. Craig Atwood Dr. Sivan Vadakkadath Meethal Miguel Gallego Department of Medicine (UW-Madison) and VA Hospital Advisor: Assistant Professor Kristyn Masters Department of Biomedical Engineering

3. Problem Statement Motivation
Develop method of encapsulating cells to allow hormone release while providing a physical barrier to the host’s immune system
Motivation
Potential alternative to less desirable treatments
Organ transplant
Hormone injections
Cellular grafts

4. Microcapsules Provide physical barrier to immune system
Consist of hydrogels
Implanted in vivo
Time-released hormone therapy

5. Client Research Microencapsulation applications
(Adapted from Morohashi, 1997)
Client Research
Microencapsulation applications
Anti-aging therapy
Reproductive disorders
Cells and hormones of interest
Leydig and Sertoli cells
Testosterone, inhibin, activin, FSH, LH
Leydig (MA-10) cells

6. Previous work Bioprinter Modified Epson R200 inkjet printer
Piezoelectric droplet generation

7. Previous Work Microfluidic devices
Increased precision and control on microscale
Minimal reagents used
(Jeong et al., 2005)

8. Changes in Current Design
Perform hydrogel experiments on the macroscale (200 µL in 96 well plate)
Allows easier characterization of material interactions with cells and proteins
Use 12 kDa PEG rather than 8 kDa to reach 5.0 nm mesh size
Required to block Ig’s, but provide LH diffusion
Incorporate RGD adhesion peptide
Allow cell adhesion to PEGdA backbone
Black line indicates length between crosslinks, which infers mesh size.
PEGdA polymer backbone.

9. Upcoming Experiments Diffusion study
Create fluorescently-labeled solutions of aprotinin, LH, dextrans, and IgG
Place hydrogel into solution
Remove hydrogel and slice to expose inner section
Measure fluorescence to infer protein diffusion

10. Expected Results

11. Viability Studies Culture Leydig cells in PEGdA hydrogels
Measure viability using Live/Dead® assay at 0, 1, 4, and 16 d after polymerization
Metabolism (greenlive)
Membrane integrity (reddead)
Look at effect of RGD incorporation

12. Expected Results

13. Hormone Assay Culture Leydig cells in PEGdA hydrogels
Stimulate cells with LH and/or FSH
Measure testosterone release using ELISA after two days
Testosterone, a steroid hormone and derivative of cholesterol.

14. Expected Results

15. Summary
Diffusion study – verify that Ig’s are blocked, while allowing LH and testosterone diffusion
Viability study – demonstrate that PEGdA material is appropriate cell environment
Hormone study – show that cells remain functional in biomaterial setting

16. References
Jeong, W., et al Continuous fabrication of biocatalyst immobilized microparticles using photopolymerization and immiscible liquids in microfluidic systems. Langmuir 21:
Morohashi, K The ontogenesis of the steroidogenic tissues. Genes to Cells 2: