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Aggregation cultures

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Aggregation cultures

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 15 Aggregationcultures

  3. Aggregatecultures • Aggregationallows: • rapid formation of smallunitsoftissues • intimatecontactsbetweencellsleadingtoenhancement of cellfunctionality and viability

  4. Principals of aggregatecultures • Presence of celladhesionmolecules (CAMs) oncellularsurfaces • Presence of matricesorarteficialanchoragemoleculesthatfacilitateaggregationforcellsthatwouldnotaggregatenaturally

  5. Celladhesion Soluble ECM Cell-cell interactions Cadherins Cell-matrixinteractions Integrins Static ECM

  6. Methods of cellaggregation Aggregationonlowadherencesurfaces Aggregation on scaffolds/modified surfaces Aggregationinrotation/suspensionculture Aggregationinbioreactors Aggregationingravityculture

  7. Gravitycultures • Cellscanassembleintospheroidsnaturallyinnaturalorincreasedgravity. • Types of gravitycultures: • Suspensionaggregatesinbioreactors • Hanging dropcultures • Centrifugedaggregates

  8. Suspension aggregate cultures • Cells suspended at very high densities • Placed into rotation conditions to increase probability of cell collision and consequent aggregation • Rotation conditions can be produced by placing suspension cultures in Petri dishes or plates on shakers, or cell suspensions into bioreactors

  9. Aggregation in rotation culture Rotation culture for suspension Rotation culture for adherent cells Gravitation force Gravitation force Sampling ports Fill port Sampling ports Rotation Rotation Fill port NG LSMMG

  10. Bioreactors and cell aggregation • Rotating wall vessel: bioreactor to stimulate microgravity and maintains aggregates in a suspended state. Sheer forces are minimal. • High aspect rotation vessel (HARV) • Slow turning lateral vessel (STLV) • Spinner flasks (stirred tank bioreactors): exist in different sizes possible scaling up for aggregates

  11. Cell type aggregates using bioreactors

  12. Application of the cell aggregates

  13. Microgravity culture (hanging drop)I Sample placed on coverslip with loop Vaseline Cavity slide 180° Oil drop

  14. Microgravity culture (hanging drop)II Time (days) 0 180° 180° 180° 2 5 Outgrowth of plated EBs and spontaneous differentiationinto cell types of all three germ layers

  15. Microwells for uniform embryoid body culture and control of cell-cell contact 40 mm 150 mm

  16. Aggregation on low adherence surfaces • Low adherence surfaces promote suspension cultures • Increase cell to cell adherence • Some extracellular matrix coated surfaces increase cell locomotion and cell to cell aggregation (e.g. Matrigel)

  17. Natural cell aggregation Hepatocyte HGF-R EGF-R Integrin Others Fas Bile duct PVLA has a potentiality as an artificialliver material by varying a coating concentration onto Ptsdish E-Cadherin ASGP-R PVLA (Poly N-p-vinyl venzyl D-lactose lactone amide) Regulation of cell shape Spheroid formation Separation and enrichment of high proliferative hepatocyte +EGF Spreading Roundshape Spheroid Hepatocytes ASGP-Rlow high proliferative Hepatocytes ASGP-Rhigh low proliferative 1mg/ml PVLA-coated dish 100 mg/ml PVLA-coated dish 100 mg/ml PVLA-coated dish 15-20 ng/ml PVLA-coated dish Coating concentration onto Pts dish

  18. Synthetic cell aggregationI • Creation of a polymer bridge to connect cells • Types: • Natural adhesion molecule • Segment of an extracellular matrix • Polymer matrix

  19. Synthetic cell aggregationII Bifunctional polymer Cells Aggregatedcells

  20. Biotinylated cell cross-linking Avidin Biotin hydrazide Multicellular aggregate Periodate tested cells

  21. Chemical modification of surfaces • Chitosan, natural biodegradable polymer (810kDa Mw) • Modified PEG (polyethylene glycol) • Lactone modified eudragit • PLGA nanospheres • Lectins and derivatives

  22. Chitosan

  23. Modified PEG MA(PEG)n Methyl-PEGn-Amine Methyl-(#ethyleneglycol) amine H2N O O O O CH3 MA(PEG)8 M.W. 383.48 Spacerarm 29.7 Å MA(PEG)12 M.W. 559.69 Spacerarm 43.9 Å MA(PEG)24 M.W. 1088.32 Spacer arm 86.1 Å [ ]8 [ ]12 [ ]24 CH3 CH3 CH3 O O O H2N H2N H2N

  24. Lactonemodifiedeudragit Counter-ions + + + + - - - - - + + + + - - COO- Co-ions COOH COOH COO- HOOC pH > 6 -OOC COOH -OOC COO- HOOC COOH COO-

  25. PLGA nanospheres Disperse phase Highpressure water out Pump Continuous phase Pre-mixing Pump Magneticstirrer Highpressure waterin

  26. Lectins and derivativesI • Cellsurfacecarbohydrateboundproteinsbindtolectins • Lectins, orphytohemagglutinins (PHA),areproteins of nonenzymatic, nonimmuneoriginthatbindcarbohydratesreversiblywithoutinducinganychangeinthecarbohydratebinding • Aslectinsmediatespecific, transient, cell-celladhesionevents, areusefulincellsurfacemodificationtoincreasecellularinteractions

  27. Lectins and derivativesII • Sixlectinfamiliesarerecognized:  • legumelectins, • cereallectins, • P-, C-, and S-typelectins, and • pentraxis, • withthelatterfouroccurringinanimals.   • Lectinsbind a variety of cellshavingcell-surfaceglycoproteinsorglycolipidssuchaserythrocytes, leukemiacells, yeasts, and severaltypes of bacteria.   • Severalspecificitygroupshavebeenidentified, suchasmannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, L-fuctose, and N-acetylneraminicacid. • The presence of twoor more bindingsitesforeachlectinmoleculeallowstheagglutination of manycelltypes.   • Lectinbinding, however, is saccharide-specific.

  28. Types of N-glycansrecognisedby PHA

  29. Cellaggregationonscaffolds • Aggregation of homotypic and heterotypiccells • Biotinilation of proteins and usingavidinascross-linker

  30. Nanostructuredscaffolds • Selfassemblingscaffoldmaterial • Nanocomposites • Nanofibres

  31. Nanomaterialsforaggregatecultures

  32. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 16 Tissue printing

  33. Main principles of tissue printing • No scaffold • Purifiedcellsformedintoclusters • Cellclustersusedas „bio-ink” • 3D tissue is printed usingtheability of cellclusterstofuse

  34. Cellclustersfuseintomicro-tissues

  35. Cellclustersfuseintomicro-tissueshapes Closelyplacedcellaggregates and embryonicheartmesenchymalfragmentscanfuseto ring ortube-likestructures

  36. Organ printing • 3D printing: depositingcellsonbiomaterialsin a rapid layer-by-layerfashion • Types of tissue printing: • Laser printing (osteosarcoma, embryoniccarcinoma) • Ink-jet printing (hippocampal and corticalneurons)

  37. The firsttissue printer

  38. Mature, organ specific primary cellsI Cell culture Biopsy Purification Cells for engineering

  39. Mature, organ specific primary cellsII Purification Cells for engineering Tissue specific resident stem cell Biopsy Cell cultures Differentiated tissue cells

  40. Maturetissuespecificcellsintissueengineering • Biopsyorresection • Purification • Regainingproliferationcapacityincellculture • Redifferentiation

  41. Generation of bloodvessels Importanttohold pressure

  42. Application of bloodvessels • Coronaryheartdisease, bypass • Treatment of trombosis • Accidentalbloodvesseldamage • Generation of complextissues

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