1. Background - HPG Axis Target Cells GnRH + - Testosterone LH & FSH LH
Male Gonads
Anterior Pituitary
Hypothalamus
GnRH + -
Testosterone
LH & FSH LH
Leydig
Cells
Sertoli
Inhibin
HPG axis
Regulates secretion of hormones namely testosterone, which is released by leydig cells.
Testosterone
FSH

2. Micro
Albert Kwansa

3. Client: Dr. Craig Atwood Advisor: Professor Murphy
encaps
Eric Lee John Harrison
Client: Dr. Craig Atwood
Advisor: Professor Murphy

4. ulation
Yik Ning Wong

5. Background – Hypogonadism
General: Reduction or loss of gonad function
Target function: Testosterone production by leydig cells found in male gonads
Approach: Restore steroidogenic function of leydig cells

6. Cell Transplantation Challenges with traditional cell transplantation
Immune Response
Foreign Body Reaction
Advantages of microencapsulation
Cell entrapment
Immunoisolation
Selective transportation
Sustained release of hormones from entrapped cells
Micro-scale capsule size

7. Microcapsule Parameters
Microcapsule Size
Size exclusion via mesh size
LH, FSH, O2, Nutrients
Antibodies
Testosterone,
Wastes
Biocompatibility
Degradation
Mesh size
Allow diffusion of nutrients, gases, wastes, and hormones
Prevent large immune molecules (antibodies) from penetrating capsule
Microcapsule diameter
Sufficient diffusion of gases (oxygen) and nutrients regardless of distance from exterior capsule surface
Degradation
Remain intact long enough to sustain a critical cell mass and provide adequate hormone release
Biocompatibility
Avoid host response
Non-toxic degradation products
Minimize protein adsorption and exterior cell adhesion

8. Polyethylene glycol (PEG)
Synthetic polymer
Systematically variable mesh size
Non-biodegradable
Sustained cell protection
Bio-inert
Difficult for cells & proteins to adhere
PEG O
PEGdA O O HO H O n n O

9. Previous Work Used capsule size of 100µm diameter
Observed cell viability out to 8 days and detected negligible testosterone release
Current approach for improvements
Microcapsule size
UV exposure time
Adhesion peptide incorporation

10. Project Design Statement
Design PEGdA hydrogels for the encapsulation of Murine Leydig Tumor Cells in an effort to increase cell viability and testosterone secretion.
PEGDA hydrogels must provide immunoprotection and allow effective diffusion of oxygen, nutrients, hormones, and metabolic wastes.

11. Thickness Parameter Testing Range = 25µm ~ 250µm
Tissue Implant size = 40µm ~ 200µm
Percent Change in Oxygen Concentration at Various Hydrogel
Thicknesses as Compared to the Oxygen Concentration
at the Site of Implantation
-60.0%
-50.0%
-40.0%
-30.0%
-20.0%
-10.0%
0.0% 50
100
150
200
250
Thickness (Micrometer)
Percent Change in Oxygen
Concentration

12. Thickness Methodology
Tape Spacers
Liquid PEGdA
Microscope Slide (Base)
Microscope Slide (Top)
Ready for UV Exposure

13. UV Exposure on Mechanical Properties of PEGdA
Cross Linking (Swelling Ratio)
Mesh Size
Stiffness of the PEGdA Network

14. UV & PEGdA Hydrogel Swelling
Fabricate thin gels under different UV times & take digital snapshot
Perfuse w/DI H2O, wait until equilibrium swelling is attained, and take second digital snapshot
Compute change in volume via imaging software

15. Swelling Ratio Methodology
Reduced Hydrogel
Swollen Hydrogel

16. UV radiation on Cell Viability
PC3 cell culture in 10% serum media
Manual counting via hemacytometer
UV Time: 0 to min intervals
Incubation for 18 hours at 37oC
Cell Titer-Blue Cell Viability Assay  Fluorescence
Normalization of fluorescence to cell number
A Cell Titer-Blue Assay was performed to assess cell viability 18 hrs after UV exposure.

17. Cell viability 18 hrs after UV exposure
Dramatic decrease in cell viability was observed at time equal and greater than 10 minutes.

18. Expected RGD Results RGD effects on secretion
RGD of different concentration and cells are injected into PEGdA
0 – 2.5 mM RGD
RGD effects on secretion
RGD can promote cell adhesion but is not selective.
RGD would enhance adhesion of both encapsulated cells and macrophages exterior to the capsule.
Incorporation of RGD presents a trade-off between potential influence on cell viability and function and the attack by macrophages.

19. Expected RGD Results Macrophage Density adherence on RGD-PEG
Adherent Macrophage Density

20. Pilot Study Conclusion
Mesh size: 4-5 nm
UV exposure time: <10mins
RGD concentration: <2.5mM

21. Future Work
Perform cell viability experiments up to 10 min at smaller increments
Cell counting via PicoGreen DNA Assay
The PicoGreen DNA Assay would allow for a more accurate representation of cell quantity and would eliminate some of the complications that arise from a non-homogeneous cell suspension.

22. References
Mellott. M, Searcy. K, Pishko. M (2001). Release of protein from highly cross-linked hydrogels of poly(ethylene glycol) diacrylate fabricated by UV polymerization. Biomaterials 22(9):
Muschler. G, Nakamoto C, Griffth L (2004). Engineering Principles of Clinical Cell-Based Tissue Engineering, The Journal of Bone and Joint Surgery (American) 86:
Yang. F, Williams. C, Wang. D, Lee. H (2004) The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials Oct;26(30):

23. Acknowledgements Dr. Craig Atwood, VA Hospital
Professor William Murphy
Professor Kristyn Masters
Professor John Kao
Dr. Daesung Lee
Amy Chung
Yi Jin Kim
Eun Jin Cho