Microencapsulation of Leydig cells

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Microencapsulation of Leydig cells

• Team
• Bryan Baxter
• Tim Eng
• Joe Zechlinski
• April Zehm
• BME 402
• February 24, 2006

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ClientDr. Craig AtwoodDr. Sivan Vadakkadath
MeethalMiguel GallegoDepartment of Medicine
(UW-Madison)and VA HospitalAdvisorAssistant
Professor Kristyn MastersDepartment of
Biomedical Engineering
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Problem Statement

• Develop method of encapsulating cells to allow
  hormone release while providing a physical
  barrier to the hosts immune system
• Motivation
• Potential alternative to less desirable
  treatments
• Organ transplant
• Hormone injections
• Cellular grafts

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Microcapsules

• Provide physical barrier to immune system
• Consist of hydrogels
• Implanted in vivo
• Time-released hormone therapy

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Client Research
(Adapted from Morohashi, 1997)

• Microencapsulation applications
• Anti-aging therapy
• Reproductive disorders
• Cells and hormones of interest
• Leydig and Sertoli cells
• Testosterone, inhibin, activin, FSH, LH

Leydig (MA-10) cells
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Previous work

• Bioprinter
• Modified Epson R200 inkjet printer
• Piezoelectric droplet generation

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Previous Work

• Microfluidic devices
• Increased precision and control on microscale
• Minimal reagents used

(Jeong et al., 2005)
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Changes in Current Design

• Perform hydrogel experiments on the macroscale
  (200 µL in 96 well plate)
• Allows easier characterization of material
  interactions with cells and proteins
• Use 12 kDa PEG rather than 8 kDa to reach 5.0 nm
  mesh size
• Required to block Igs, but provide LH diffusion
• Incorporate RGD adhesion peptide
• Allow cell adhesion to PEGdA backbone

Black line indicates length between crosslinks,
which infers mesh size.
PEGdA polymer backbone.
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Upcoming Experiments

• Diffusion study
• Create fluorescently-labeled solutions of
  aprotinin, LH, dextrans, and IgG
• Place hydrogel into solution
• Remove hydrogel and slice to expose inner section
• Measure fluorescence to infer protein diffusion

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Expected Results
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Viability Studies

• Culture Leydig cells in PEGdA hydrogels
• Measure viability using Live/Dead assay at 0, 1,
  4, and 16 d after polymerization
• Metabolism (green?live)
• Membrane integrity (red?dead)
• Look at effect of RGD incorporation

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Expected Results
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Hormone Assay

• Culture Leydig cells in PEGdA hydrogels
• Stimulate cells with LH and/or FSH
• Measure testosterone release using ELISA after
  two days

Testosterone, a steroid hormone and derivative of
cholesterol.
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Expected Results
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Summary

• Diffusion study verify that Igs are blocked,
  while allowing LH and testosterone diffusion
• Viability study demonstrate that PEGdA material
  is appropriate cell environment
• Hormone study show that cells remain functional
  in biomaterial setting

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References

• Jeong, W., et al. 2005. Continuous fabrication
  of biocatalyst immobilized microparticles using
  photopolymerization and immiscible liquids in
  microfluidic systems. Langmuir 21 3738-3741.
• Morohashi, K. 1997. The ontogenesis of the
  steroidogenic tissues. Genes to Cells 2 95-106.