Microencapsulation of leydig cells
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Microencapsulation of Leydig cells. Team: Bryan Baxter Tim Eng Joe Zechlinski April Zehm BME 400 October 14, 2005. Client: Dr. Craig Atwood Dr. Sivan Vadakkadath Meethal Miguel Gallego VA Hospital Advisor: Assistant Professor Kristyn Masters Department of Biomedical Engineering.

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Microencapsulation of Leydig cells

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Microencapsulation of leydig cells

Microencapsulation of Leydig cells

Team:

Bryan Baxter

Tim Eng

Joe Zechlinski

April Zehm

BME 400

October 14, 2005


Microencapsulation of leydig cells

Client:Dr. Craig AtwoodDr. Sivan Vadakkadath MeethalMiguel GallegoVA HospitalAdvisor:Assistant Professor Kristyn MastersDepartment of Biomedical Engineering


Overview

Overview

  • Problem Statement

  • Background

  • Design Specifications

  • Recap of previous work

  • New directions


Problem statement

Problem Statement

  • Develop method of encapsulating cells to allow hormone release while providing a physical barrier to the host’s immune system

    Motivation

  • Potential alternative to less desirable treatments

    • Organ transplant

    • Hormone injections

    • Cellular grafts


Microcapsules

Microcapsules

  • Provide physical barrier to immune system

  • Consist of hydrogels

  • Implanted in vivo

  • Time-released hormone therapy

(Uludag et al., 2000)


Client research

Client Research

  • Microencapsulation applications

    • Anti-aging therapy

    • Reproductive disorders

  • Cells and hormones of interest

    • Leydig and Sertoli cells

    • Testosterone, inhibin, activin, FSH, LH

(Ownby, 1999)

(Adapted from Morohashi, 1997)


Design specifications

Biocompatibility

Material properties

Crosslinking procedure

Repeatability of results

Immunoprotection

Controlled pore size (MWCO)

Degradation

Mechanical

Biological

Design Specifications


Previous work

Previous work

  • Polyethylene glycol (PEG)

    • Diacrylated synthetic polymer

    • UV-crosslinked

    • Water-in-oil emulsification


Previous work1

Previous work

  • Bioprinter

    • Modified Epson R200 inkjet printer

    • Piezoelectric droplet generation

    • Linux platform, software interface


Microsphere production

Microsphere Production

  • Microfluidic devices

    • Increased precision and control on microscale

      • Sample and sheath flow rates determine size and quantity of microcapsules

    • Minimal reagents used

(Jeong et al., 2005)


Cell culture

Cell culture

  • Cell line

    • MA-10 cells (mouse cancer Leydig cells)

    • Produce testosterone

  • Cell passaging

    • Split proliferating cells

    • Hemocytometry

    • Cryofreezing

(Saltzman, 2004.)


Peg hydrogels on the macroscale

PEG hydrogels on the macroscale

  • Current Experiments:

    • Acrylation procedure

    • Hydrogel swelling/PEG crosslinking

  • Planned Experiments:

    • Combine with MA-10 cell suspension

12.5x


Viability hormone studies

(http://respiratory-research.com)

Viability/Hormone studies

  • Live/Dead® assay

    • Metabolism (greenlive)

    • Membrane integrity (reddead)

  • Testosterone assay

    • Competitive sandwich ELISA (ALPCO Diagnostics)


References

References

Jeong, W., et al. 2005. Continuous fabrication of biocatalyst immobilized microparticles using photopolymerization and immiscible liquids in microfluidic systems. Langmuir 21: 3738-3741.

Machluf M, Orsola A, Boorjian S, Kershen R, and Atala A. 2003. Microencapsulation of leydig cells: a system for testosterone supplementation. Endocrinology 144:4975-4979.

Morohashi, K. 1997. The ontogenesis of the steroidogenic tissues. Genes to Cells 2: 95-106.

Ownby. 1999. Histology: male reproductive system. http://www.cvm.okstate.edu/instruction/mm_curr/histology/MR/HiMRp3.htm. Accessed February 12, 2005.

Saltzman, W. 2004. Tissue engineering : engineering principles for the design of replacement organs and tissues. New York : Oxford University Press.

Uludag H, De Vos P, and Tresco PA. 2000. Technology of mammalian cell encapsulation. Advanced Drug Delivery Reviews 42:29-64.

http://chemfinder.cambridgesoft.com

http://respiratory-research.com

http://web.indstate.edu/thcme/mwking/glycans.html

http://www.cybercolloids.net/library/alginate/structure.php


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