Microencapsulation of murine leydig tumor cells for cell transplantation

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
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
• 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

8
Pore Capsule Size

• 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
Teflon film (spacer)
Coverslip

• Diffusion distance
• UV exposure
• Cell adhesion peptides (RGD)

hydrogel
PEGdA
Petri dish bottom
12
Experimental Protocol
Experimental 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) Cell viability Testosterone production Positive control Cells not exposed to long wavelength UV light
Long wavelength UV lamp exposure time (cells in smallest possible gel thickness) Cell viability Testosterone production Positive control Cells in gels not exposed to long wavelength UV light Negative control Unseeded gels
Addition of RGD adhesion peptide into hydrogel scaffold Cell viability Testosterone production Negative control Cells embedded in gels with no adhesion peptides
13
Materials List
Reagent Supplier Quantity/Cost
PEG SIGMA 1kg/40.00 (mw12000)
Triethylamine SIGMA 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 Donated
14

• Questions?