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

2. Hypogonadism 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

3. 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
Reduced diffusion distance

4. Microcapsule Parameters
Degradation
Size exclusion via mesh size
Testosterone, Wastes
LH, FSH, O2, Nutrients
Antibodies
Biocompatibility
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
Microcapsule Size

5. Polyethylene glycol (PEG)
Synthetic polymer
Systematically variable mesh size
Non-biodegradable
Sustained cell protection
Bio-inert
Difficult for cells & proteins to adhere
Pre-cursor Solution:
10% PEGdA MW12000
0.05% I2959
PBS diluent
± cell suspension
PEGdA macromers
Photopolymerization
(365nm UV light)
Polymerization & cross-linking via free-radical mechanism O n
PEGdA
Swelling
PEGdA hydrogel
H2O

6. Problem Design Statement
To investigate the effects of hydrogel thickness on the viability of human prostate cancer cells embedded within a polyethylene glycol diacrylate hydrogel. Additionally, to assess the polymerization and cross-linking phenomena of PEGdA macromers and the diffusive behavior of progesterone through a PEGdA hydrogel matrix.
The overall goal of this project is to design an encapsulation system that offers efficient immunoprotection and effective diffusion of oxygen, nutrients, hormones, and metabolic wastes.
This system, along with embedded human prostate cancer cells, will enable the restoration of un-regulated hormone levels commonly observed in elders, and retard the symptoms of aging.

7. Previous Work Used capsule size of 100µm diameter
Observed cell viability out to 7 days and detected negligible testosterone release
15 min of UV exposure = threshold for sustained cell viability
Current approach for improvements
Microcapsule size
UV exposure time

8. UV Exposure Time vs. Degree of Hydrogel cross-linking
14.5 minutes of UV exposure is sufficient for cross linking
3D Swelling Ratio = 3.8

9. Capsule Diameter Post-Swell Testing Range = 25µm ~ 250µm
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 (µm)
Percent Change in Oxygen
Concentration

10. Hydrogel sandwich Simulation of capsule radius
Sigmacote surface treatment to aid PEGdA removal
Post-swell thickness = 25m ~ 250m
Pre-swell thickness = 25m ~ 175m
PEGdA Hydrogel
Microscope slides
Preset thickness
Tape spacers
Sigmacote

11. Ultrasound Confirmation of swelling calculation
Determine pre/post swell thickness of hydrogel sandwich
Transducer
Water D
PEGdA
Microscope Slide
Distance (D) = (1/2) x [Time x Speed of Sound]

12. Ultrasound Result Linear swollen ratio is 1.54 Swelling =1.54
And swelling time =3.54-3d
Because 3-D and 1-D

13. Progesterone Diffusion

14. Progesterone Diffusion
Observed Progesterone release over time
High progesterone levels after 5 hours
Progesterone level exceeded linear range of calibrated curve
Data variability
Sex hormone capable of diffusing out of PEGdA network

15. Cell Viability Results
Statistical Analysis:
2-sample t-test α = 0.05 * * * * * * *
Cell Titer-BlueTM Cell Viability Assay
* Denotes significant drop from day 2 to day 3
* Denotes significant drop from day 3 to day 4

16. Cell Viability Discussion
Further data is needed to establish a meaningful trend and interpretation
Fluorescence readings close to that of the negative control (cell culture medium)
Increase number of cells per well and/or increase incubation time to 3 or 4 hours

17. Overall Conclusions PEGdA suitable material for cell encapsulation
Sub-lethal UV time 14.5 min
The mesh size achieved allows for the diffusion of progesterone
Need to extend cell viability studies for more concrete interpretation

18. Future Work
Continue to assess hydrogel thickness effect on cell viability (extended studies)
Evaluate effects of gel thickness on hormone release
2-D & 3-D studies of the effects of RGD cell adhesion peptides on cell function
Fabrication of micro-spheres of specified diameter
In vivo analysis of encapsulation system

19. References
ALPCO Diagnostics (2004). Progesterone EIA: For the direct quantitative determination of Progesterone by enzyme Immunoassay in human serum. 11-PROGH-305 Version 4.0
Cruise, G. M., Hegre, O. D., Scharp, D. S., & Hubbell, J. A. (1998). A sensitivity study of the key parameters in the interfacial photopolymerization of poly(ethylene glycol) diacrylate upon porcine islets. Biotechnology and bioengineering, 57(6),
Cruise, G. M., Scharp, D. S., & Hubbell, J. A. (1998). Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels. Biomaterials, 19(14),
Diramio, J. A., Kisaalita, W. S., Majetich, G. F., & Shimkus, J. M. (2005). Poly(ethylene glycol) methacrylate/dimethacrylate hydrogels for controlled release of hydrophobic drugs. Biotechnology progress, 21(4),
Kizilel, S., Perez-Luna, V. H., & Teymour, F. (2004). Photopolymerization of poly(ethylene glycol) diacrylate on eosin- functionalized surfaces. Langmuir : the ACS journal of surfaces and colloids, 20(20),
Kizilel, S., Sawardecker, E., Teymour, F., & Perez-Luna, V. H. (2006). Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization. Biomaterials, 27(8),
Martens, P. J., Bryant, S. J., & Anseth, K. S. (2003). Tailoring the degradation of hydrogels formed from multivinyl poly(ethylene glycol) and poly(vinyl alcohol) macromers for cartilage tissue engineering. Biomacromolecules, 4(2),
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:
Nuttelman, C. R., Tripodi, M. C., & Anseth, K. S. (2005). Synthetic hydrogel niches that promote hMSC viability. Matrix biology : journal of the International Society for Matrix Biology, 24(3),
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):

20. Special Thanks Chemistry Department
Dr. Daesung Lee
Yi-Jin Kim
VA hospital
Dr. Craig Atwood
Miguel Gallego
Andrea Wilson
Ryan Haasl
Promega Corporation
Lydia Hwang for her vital donation of project resources
CS Hyde Company
School of Medicine and Public Health, Medical Physics
Dr. Tim Stiles for his help in ultrasound measurements
Pharmacy Department
Dr. John Kao
Graduate student Amy Chung for her endless generosity
Biomedical Engineering Department
Dr. Kristyn Masters and lab
Dr. William Murphy and lab
Dr. Brenda Ogle and lab

21. Micro
Albert Kwansa Eric Lee

22. encaps John Harrison Miguel Benson Client: Dr. Craig Atwood
Advisor: Professor William Murphy

23. ulation
Yik Ning Wong