microencapsulation of leydig cells team: bryan baxter tim eng joe zechlinski april zehm bme 400...
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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