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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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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

aggregation cultures
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
aggregate cultures
Aggregatecultures
  • Aggregationallows:
  • rapid formation of smallunitsoftissues
  • intimatecontactsbetweencellsleadingtoenhancement of cellfunctionality and viability
principals of aggregate cultures
Principals of aggregatecultures
  • Presence of celladhesionmolecules (CAMs) oncellularsurfaces
  • Presence of matricesorarteficialanchoragemoleculesthatfacilitateaggregationforcellsthatwouldnotaggregatenaturally
cell adhesion
Celladhesion

Soluble ECM

Cell-cell

interactions

Cadherins

Cell-matrixinteractions

Integrins

Static ECM

methods of cell aggregation
Methods of cellaggregation

Aggregationonlowadherencesurfaces

Aggregation on scaffolds/modified surfaces

Aggregationinrotation/suspensionculture

Aggregationinbioreactors

Aggregationingravityculture

gravity cultures
Gravitycultures
  • Cellscanassembleintospheroidsnaturallyinnaturalorincreasedgravity.
  • Types of gravitycultures:
  • Suspensionaggregatesinbioreactors
  • Hanging dropcultures
  • Centrifugedaggregates
suspension aggregate cultures
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
aggregation in rotation culture
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

bioreactors and c ell a ggregation
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
microgravity culture hanging drop i
Microgravity culture (hanging drop)I

Sample placed on

coverslip with loop

Vaseline

Cavity slide

180°

Oil drop

microgravity culture hanging drop ii
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

aggregation on low adherence surfaces
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)
natural c ell a ggregation
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

synthetic cell aggregation i
Synthetic cell aggregationI
  • Creation of a polymer bridge to connect cells
  • Types:
  • Natural adhesion molecule
  • Segment of an extracellular matrix
  • Polymer matrix
synthetic c ell a ggregation ii
Synthetic cell aggregationII

Bifunctional polymer

Cells

Aggregatedcells

biotinylated cell cross linking
Biotinylated cell cross-linking

Avidin

Biotin

hydrazide

Multicellular aggregate

Periodate tested cells

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

lactone modified eudragit
Lactonemodifiedeudragit

Counter-ions

+

+

+

+

-

-

-

-

-

+

+

+

+

-

-

COO-

Co-ions

COOH

COOH

COO-

HOOC

pH > 6

-OOC

COOH

-OOC

COO-

HOOC

COOH

COO-

plga nanospheres
PLGA nanospheres

Disperse

phase

Highpressure

water out

Pump

Continuous

phase

Pre-mixing

Pump

Magneticstirrer

Highpressure

waterin

lectins and derivatives i
Lectins and derivativesI
  • Cellsurfacecarbohydrateboundproteinsbindtolectins
  • Lectins, orphytohemagglutinins (PHA),areproteins of nonenzymatic, nonimmuneoriginthatbindcarbohydratesreversiblywithoutinducinganychangeinthecarbohydratebinding
  • Aslectinsmediatespecific, transient, cell-celladhesionevents, areusefulincellsurfacemodificationtoincreasecellularinteractions
lectins and derivatives ii
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.
cell aggregation on scaffolds
Cellaggregationonscaffolds
  • Aggregation of homotypic and heterotypiccells
  • Biotinilation of proteins and usingavidinascross-linker
nanostructured scaffolds
Nanostructuredscaffolds
  • Selfassemblingscaffoldmaterial
  • Nanocomposites
  • Nanofibres
tissue printing
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
main principles of tissue printing
Main principles of tissue printing
  • No scaffold
  • Purifiedcellsformedintoclusters
  • Cellclustersusedas „bio-ink”
  • 3D tissue is printed usingtheability of cellclusterstofuse
cell clusters fuse into micro tissue shapes
Cellclustersfuseintomicro-tissueshapes

Closelyplacedcellaggregates and embryonicheartmesenchymalfragmentscanfuseto ring ortube-likestructures

organ printing
Organ printing
  • 3D printing: depositingcellsonbiomaterialsin a rapid layer-by-layerfashion
  • Types of tissue printing:
  • Laser printing (osteosarcoma, embryoniccarcinoma)
  • Ink-jet printing (hippocampal and corticalneurons)
mature organ specific primary cells i
Mature, organ specific primary cellsI

Cell culture

Biopsy

Purification

Cells for

engineering

mature organ specific primary cells ii
Mature, organ specific primary cellsII

Purification

Cells for

engineering

Tissue specific resident stem cell

Biopsy

Cell cultures

Differentiated tissue cells

mature tissue specific cells in tissue engineering
Maturetissuespecificcellsintissueengineering
  • Biopsyorresection
  • Purification
  • Regainingproliferationcapacityincellculture
  • Redifferentiation
generation of blood vessels
Generation of bloodvessels

Importanttohold pressure

application of blood vessels
Application of bloodvessels
  • Coronaryheartdisease, bypass
  • Treatment of trombosis
  • Accidentalbloodvesseldamage
  • Generation of complextissues
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