1. Microencapsulation Eric Lee, Yik Ning Wong, Miguel Benson,
John Harrison, Albert Kwansa
Client: Dr. Craig Atwood
Advisor: Professor William Murphy

2. Background - HPG Axis Testosterone Target Cells GnRH LH & FSH - LH FSH
Male Gonads
Anterior Pituitary
Hypothalamus
GnRH + -
LH & FSH LH
Leydig
Cells
Sertoli
Inhibin
FSH
Albert:
First some background on the scope and problem of our project.
The HPG axis is comprised of the hypothalamus, anterior pituitary, and the gonads.
Our focus is on the pathway between the leydig cells found in the male gonads and their function of testosterone production.
During proper functioning of the HPG axis, feedback loops afford auto-regulation, however, when complications arise, HPG dysfunction can occur.
Such complications can occur naturally with aging.

3. Background – Hypogonadism
Reduction or loss of gonad function
Diminished testosterone production by leydig cells
Approach: Replace this steroidogenic function via leydig cell transplantation.
Albert:
An example of such a complication is hypogonadism, which can be generally described as the diminished or lost function of the gonads.
Hypogonadism can result in imbalances to the HPG axis and other systems of the body.
Our project is focused upon a specific type of primary hypogonadism in reduced testosterone production.
The approach is thus to restore testosterone to healthy levels by transplanting new leydig cells into the body.

4. Cell Transplantation Challenges with traditional cell transplantation
Immune Response
Advantages of microencapsulation
Cell entrapment
Immunoisolation
Selective transportation
Sustained release of hormones
Micro-scale size reduces diffusion distance
Polyethylene glycol diacrylate (PEGdA)
Albert:
Traditional cell transplantation involves introducing cells from another human or animal donor.
The main challenge with merely transplanting donor cells is immune rejection, because the cells will likely be recognized as foreign, become targeted, and degraded by immune system cells.
Microencapsulation is a method that utilizes a biomaterial to entrap cells into very small particles.
The material properties of the biomaterial can be developed to afford selective transportation of biomolecules and isolation from immune system elements such as antibodies and cells.
Due to the micro-scale size of this system, the resistance to mass transfer of essential biomolecules is decreased as compared to a macro-encapsulation system.

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

6. Last Semesters Work UV exposure time upper limit = 15 min
Theoretical max gel thickness = 250 μm
John:

7. Current Goal
Determine effect on cell viability and testosterone production due to:
Gel thickness…………………
UV exposure………………….
RGD adhesion………………..
Calculated test range: μm
0-15 min
0-1 mol%
John:

8. UV Exposure Time Previous constraints Further constraints
Must be <15 min for cells to survive
Further constraints
Find minimum time to gel completely
Degree of swelling gives measure of degree of polymerization

9. Swelling Experiment Incomplete polymerization Complete polymerization
Less X-links
More room to swell
Complete polymerization
Max links crossed
Decreased swelling capability
Observed at t ≥ 13 min

10. Capsule Radius Long Term implementation
Theoretical maximum radius = 250 m
Equilibrium Swelling Ratio = 3.80
Maximum initial capsule radius = 160m
160m
250m

11. Hydrogel Thickness Simulation of capsule radius
Post swell thickness = 25m ~ 250m
Pre swell thickness = 25m ~ 175m
Tape spacers
Microscope slides
PEGdA Hydrogel
Preset thickness

12. Ultrasound
Determination post swell thickness and thickness of hydrogel sandwich
Confirm swelling calculation
Transducers D
Time = 2*Distance / Speed of sound in water
PEGdA
Jacqueline:
Since gel thickness affects cell viability and should be accurately defined, we will use ultrasound to determine the post-swell thickness and thickness of the hydrogel sandwich. The thickness measured by ultrasound will also confirm the swelling calculation.

13. Disadvantages of Ultrasound
Similar density of hydrogel and water
Contrast agent
Backscattering
Microscope slide surface treatment (Sigmacote)
Transducer D
Jacqueline:
However, there are some disadvantages in the method.
Since the hydrogel has a similar density to water, a contrast agent is needed to increase the density difference at the boundary and obtain a more accurate signal.
Backscattering of ultrasound can also alter the reading.
In order to minimize backscattering, we will treat the microscope slide with SigmaCote to aid in cleaner removal of the gel
This can provide for a smoother gel surface and more accurate signal.
PEGdA
PEGdA
PEGdA w/ Contrast Agent
PEGdA w/o Contrast Agent

14. RGD Adhesion Molecule Promote cell adhesion within PEGdA capsule
RGD peptide
Cell
PEGdA biomaterial
Jacqueline:
The figure on the left shows a cell in our bioinert PEGdA material.
The figure on the right shows a cell and our PEGdA material incorporating RGD peptides.
RGD is a peptide sequence that promotes cell adhesion and is present as a cell binding domain of many extracellular matrix proteins.
With RGD, the cells can form adhesions, which may maintain or enhance cell survival.
Additionally, these cell-peptide interactions may allow for the influence of cell-specific functions such as hormone production.
R = arginine
G = glycine
D = aspartic acid

15. RGD & Spacer Sequence N-term-CGGGR GDSP-C-term Without Spacer
With Spacer
RGD peptide sequence
Integrin cell receptor
Spacer sequence
Biomaterial
Cell protrusion
KEY
Jacqueline:
Here is our peptide sequence, with the RGD sequence and a spacer sequenc.
The spacer lengthens the peptide and may thus increase availability of the RGD binding sequence to cells.
There is a a photosensitive component, which will allow for the peptide to be incorporated into the gel during photopolymerization.

16. Questions?

17. Mathematical Calculations
Equal swelling in all dimension
T = (Swelling Ratio )1/3 x t T t T* t* t T
Volume = t x t x t
(Swelling Ratio)(Volume) = T x T x T