Microencapsulation of Microencapsulation of Leydig cellsLeydig cells

Team:Team:Bryan BaxterBryan Baxter

Tim Eng Tim Eng Joe Zechlinski Joe Zechlinski

April ZehmApril Zehm

BME 400BME 400October 14, 2005October 14, 2005

Client:Client:Dr. Craig AtwoodDr. Craig Atwood

Dr. Sivan Vadakkadath MeethalDr. Sivan Vadakkadath MeethalMiguel GallegoMiguel Gallego

VA HospitalVA Hospital

Advisor:Advisor:Assistant Professor Kristyn MastersAssistant Professor Kristyn Masters

Department of Biomedical EngineeringDepartment of Biomedical Engineering

OverviewOverview

Problem StatementProblem Statement BackgroundBackground Design SpecificationsDesign Specifications Recap of previous Recap of previous

workwork New directionsNew directions

Problem StatementProblem Statement

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

MotivationMotivation Potential alternative to less desirable Potential alternative to less desirable

treatmentstreatments– Organ transplantOrgan transplant– Hormone injectionsHormone injections– Cellular graftsCellular grafts

MicrocapsulesMicrocapsules Provide physical Provide physical

barrier to immune barrier to immune systemsystem

Consist of Consist of hydrogelshydrogels

Implanted Implanted in vivoin vivo Time-released Time-released

hormone therapyhormone therapy(Uludag et al., 2000)

Client ResearchClient Research

Microencapsulation Microencapsulation applicationsapplications– Anti-aging therapyAnti-aging therapy– Reproductive disordersReproductive disorders

Cells and hormones Cells and hormones of interestof interest– Leydig and Sertoli cellsLeydig and Sertoli cells– Testosterone, inhibin, Testosterone, inhibin,

activin, FSH, LHactivin, FSH, LH

(Ownby, 1999) (Adapted from Morohashi, 1997)

Design SpecificationsDesign Specifications

BiocompatibilityBiocompatibility– Material propertiesMaterial properties– Crosslinking Crosslinking

procedureprocedure Repeatability of Repeatability of

resultsresults

ImmunoprotectionImmunoprotection– Controlled pore size Controlled pore size

(MWCO) (MWCO) DegradationDegradation

– MechanicalMechanical– BiologicalBiological

Previous workPrevious work Polyethylene glycol

(PEG)– Diacrylated synthetic

polymer– UV-crosslinked– Water-in-oil

emulsification

Previous workPrevious work Bioprinter

– Modified Epson R200 inkjet printer

– Piezoelectric droplet generation

– Linux platform, software interface

Microsphere ProductionMicrosphere Production Microfluidic devicesMicrofluidic devices

– Increased precision and control on microscaleIncreased precision and control on microscale Sample and sheath flow rates determine size and Sample and sheath flow rates determine size and

quantity of microcapsulesquantity of microcapsules

– Minimal reagents usedMinimal reagents used

(Jeong et al., 2005)

Cell cultureCell culture

Cell lineCell line– MA-10 cells (mouse MA-10 cells (mouse

cancer Leydig cells)cancer Leydig cells)– Produce testosteroneProduce testosterone

Cell passagingCell passaging– Split proliferating cellsSplit proliferating cells– HemocytometryHemocytometry– Cryofreezing Cryofreezing

(Saltzman, 2004.)

PEG hydrogels on the PEG hydrogels on the macroscalemacroscale Current Experiments:Current Experiments:

– Acrylation procedureAcrylation procedure– Hydrogel swelling/PEG Hydrogel swelling/PEG

crosslinkingcrosslinking Planned Experiments:Planned Experiments:

– Combine with MA-10 Combine with MA-10 cell suspensioncell suspension

12.5x

Viability/Hormone studiesViability/Hormone studies

Live/Dead® assayLive/Dead® assay– Metabolism Metabolism

(green(greenlive)live)– Membrane integrity Membrane integrity

(red(reddead)dead) Testosterone assayTestosterone assay

– Competitive sandwich Competitive sandwich ELISA (ALPCO ELISA (ALPCO Diagnostics)Diagnostics)

(http://respiratory-research.com)

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

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

http://chemfinder.cambridgesoft.com

http://respiratory-research.com

http://web.indstate.edu/thcme/mwking/glycans.htmlhttp://www.cybercolloids.net/library/alginate/structure.phphttp://www.cybercolloids.net/library/alginate/structure.php