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

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.

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

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

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

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

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

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)

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?

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

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

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

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

Questions?