1. Microencapsulation of Leydig cells Team: Bryan Baxter Tim Eng Joe Zechlinski April Zehm BME 400 October 14, 2005

2. Client: Dr. Craig Atwood Dr. Sivan Vadakkadath Meethal Miguel Gallego VA Hospital Advisor: Assistant Professor Kristyn Masters Department of Biomedical Engineering

3. Overview Problem Statement Problem Statement Background Background Design Specifications Design Specifications Recap of previous work Recap of previous work New directions New directions

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

5. Microcapsules Provide physical barrier to immune system Provide physical barrier to immune system Consist of hydrogels Consist of hydrogels Implanted in vivo Implanted in vivo Time-released hormone therapy Time-released hormone therapy (Uludag et al., 2000)

6. Client Research Microencapsulation applications Microencapsulation applications –Anti-aging therapy –Reproductive disorders Cells and hormones of interest Cells and hormones of interest –Leydig and Sertoli cells –Testosterone, inhibin, activin, FSH, LH (Ownby, 1999)(Adapted from Morohashi, 1997)

7. Design Specifications Biocompatibility Biocompatibility –Material properties –Crosslinking procedure Repeatability of results Repeatability of results Immunoprotection Immunoprotection –Controlled pore size (MWCO) Degradation Degradation –Mechanical –Biological

8. Previous work Polyethylene glycol (PEG) – –Diacrylated synthetic polymer – –UV-crosslinked – –Water-in-oil emulsification

9. Previous work Bioprinter – –Modified Epson R200 inkjet printer – –Piezoelectric droplet generation – –Linux platform, software interface

10. Microsphere Production Microfluidic devices Microfluidic devices –Increased precision and control on microscale  Sample and sheath flow rates determine size and quantity of microcapsules –Minimal reagents used (Jeong et al., 2005)

11. Cell culture Cell line Cell line –MA-10 cells (mouse cancer Leydig cells) –Produce testosterone Cell passaging Cell passaging –Split proliferating cells –Hemocytometry –Cryofreezing (Saltzman, 2004.)

12. PEG hydrogels on the macroscale Current Experiments: Current Experiments: –Acrylation procedure –Hydrogel swelling/PEG crosslinking Planned Experiments: Planned Experiments: –Combine with MA-10 cell suspension 12.5x

13. Viability/Hormone studies Live/Dead® assay Live/Dead® assay –Metabolism (green  live) –Membrane integrity (red  dead) Testosterone assay Testosterone assay –Competitive sandwich ELISA (ALPCO Diagnostics) (http://respiratory-research.com)

14. References Jeong, W., et al. 2005. Continuous fabrication of biocatalyst immobilized microparticles using photopolymerization and immiscible liquids in microfluidic systems. Langmuir 21: 3738-3741. Machluf M, Orsola A, Boorjian S, Kershen R, and Atala A. 2003. Microencapsulation of leydig cells: a system for testosterone supplementation. Endocrinology 144:4975- 4979. Morohashi, K. 1997. The ontogenesis of the steroidogenic tissues. Genes to Cells 2: 95- 106. Ownby. 1999. Histology: male reproductive system. http://www.cvm.okstate.edu/instruction/mm_curr/histology/MR/HiMRp3.htm. Accessed February 12, 2005. Saltzman, W. 2004. Tissue engineering : engineering principles for the design of replacement organs and tissues. New York : Oxford University Press. Uludag H, De Vos P, and Tresco PA. 2000. Technology of mammalian cell encapsulation. Advanced Drug Delivery Reviews 42:29-64. http://chemfinder.cambridgesoft.com http://respiratory-research.com http://web.indstate.edu/thcme/mwking/glycans.html http://www.cybercolloids.net/library/alginate/structure.php