1. Microencapsulation of murine leydig tumor cells for cell transplantation
Albert Kwansa - Leader
John Harrison - Communicator
Yik Ning Wong - BSAC
Eric Lee - BWIG
Advisor: Professor William Murphy
Client: Dr. Craig Atwood

2. Client Introduction Dr. Craig Atwood, Geriatrics Research
~The Laboratory of Endocrinology, Aging and Disease (LEAD)
Project Motivation:
Decrease in testosterone production by leydig cells can disrupt the HPG axis and lead to a variety of disorders.

3. Background - HPG Axis - - GnRH LH & FSH Testosterone + - - LH FSH
Hypothalamus - -
GnRH
LH & FSH
Testosterone +
Anterior Pituitary - -
Target Cells LH
FSH
Testosterone
Inhibin + +
Male Gonads
Testosterone
Leydig
Cells
Sertoli
Cells

4. Background - Hypogonadism
Primary & Secondary sources
Congenital defects
Acquired disorders
Natural occurrence with aging
Possible symptoms
Bone & muscle atrophy
Reduced mental acuity
Infertility

5. Microencapsulation Replace the streroidogenic function of leydig cells
Tiny particles are surrounded by a coating to give small capsules with many useful properties
Immuno-isolation against host response
Criteria for microencapsulation
Sufficient diffusion distance
Provide sustained release of hormone

6. Parameters Mesh size Microcapsule diameter Degradation Thickness
Pore size of hydrogel
Allow diffusion of nutrients, gases, wastes, and hormones
Prevent large immune molecules (antibodies) and cells
Microcapsule diameter
Sufficient diffusion of gases (oxygen) and nutrients
Avoid cell necrosis and hypoxia
Degradation
Remain intact long enough to sustain a critical cell mass and provide adequate hormone release
Thickness
Affect diffusion rate

7. Material Selection Polyethylene glycol (PEG) Synthetic polymer (pure)
Minimize protein adsorption  reduced fibroblast overgrowth
Allows for chemical modification to suit specific purposes
Difficult for cells to adhere O H HO n
PEG O n
PEGdA

8. Pore & Capsule Size Immunoprotection and Hypoxia Mesh size of 4-5nm
Testosterone,
Wastes
LH, FSH, O2, Nutrients
Antibodies
Size exclusion via mesh size
Encapsulated leydig cells
Immunoprotection and Hypoxia
Mesh size of 4-5nm
Human antibodies: 5.4nm (IgG)
FSH(2.2nm), LH(3nm)
Capsule size of 100µm
Rule of thumb
PEGdA (MW 12000)

9. Past work- Assays
Diffusion into/out-of PEGdA network (BSA-Fluorescein)
Observed diffusion
Cell viability (LIVE/DEAD assay [qualitative])
Cells viable up to 8 days
Florescence strongly concentrated at the edges of hydrogels
Hypoxia? Immune response? Effect of UV?
Testosterone production (ELISA)
Inconclusive results
Cell anchorage? Effect of UV?

10. Project Status
Current goal – Extend cell viability and increase testosterone production

11. Experimental Design PEGdA hydrogel Diffusion distance UV exposure
Teflon film
(spacer)
Coverslip
Diffusion distance
UV exposure
Cell adhesion peptides (RGD)
PEGdA
hydrogel
Petri dish
bottom

12. Experimental Protocol
Variable
Quantification
Parameters
Controls
Gel Thickness
[25 – 250 mm]
Cell viability
Testosterone production
Negative control:
Unseeded gels of varying thickness
Long wavelength UV lamp exposure time (cells only; no gels)
Positive control:
Cells not exposed to long wavelength UV light
Long wavelength UV lamp exposure time (cells in smallest possible gel thickness)
Cells in gels not exposed to long wavelength UV light
Unseeded gels
Addition of RGD adhesion peptide into hydrogel scaffold
Cells embedded in gels with no adhesion peptides

13. Materials List Reagent Supplier Quantity/Cost PEG SIGMA
1kg/$40.00 (mw=12000)
Triethylamine
100mL/$10.00
Acryloyl chloride
ALFA SAER
Ethyl ether
UW-Chem Lab
100mL/donated
Irgacure 2959 photoinitatior
CIBA
Free sample
Cell Titer BlueTM
Promega
Donated
Testosterone assay (ELISA)
Client

14. Questions?