Manifestation of Novel Social Challenges of the European Union
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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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Aggregation cultures

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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


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


Aggregatecultures

  • Aggregationallows:

  • rapid formation of smallunitsoftissues

  • intimatecontactsbetweencellsleadingtoenhancement of cellfunctionality and viability


Principals of aggregatecultures

  • Presence of celladhesionmolecules (CAMs) oncellularsurfaces

  • Presence of matricesorarteficialanchoragemoleculesthatfacilitateaggregationforcellsthatwouldnotaggregatenaturally


Celladhesion

Soluble ECM

Cell-cell

interactions

Cadherins

Cell-matrixinteractions

Integrins

Static ECM


Methods of cellaggregation

Aggregationonlowadherencesurfaces

Aggregation on scaffolds/modified surfaces

Aggregationinrotation/suspensionculture

Aggregationinbioreactors

Aggregationingravityculture


Gravitycultures

  • Cellscanassembleintospheroidsnaturallyinnaturalorincreasedgravity.

  • Types of gravitycultures:

  • Suspensionaggregatesinbioreactors

  • Hanging dropcultures

  • Centrifugedaggregates


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

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 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


Cell type aggregates using bioreactors


Application of the cell aggregates


Microgravity culture (hanging drop)I

Sample placed on

coverslip with loop

Vaseline

Cavity slide

180°

Oil drop


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


Microwells for uniform embryoid body culture and control of cell-cell contact

40 mm

150 mm


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 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 aggregationI

  • Creation of a polymer bridge to connect cells

  • Types:

  • Natural adhesion molecule

  • Segment of an extracellular matrix

  • Polymer matrix


Synthetic cell aggregationII

Bifunctional polymer

Cells

Aggregatedcells


Biotinylated cell cross-linking

Avidin

Biotin

hydrazide

Multicellular aggregate

Periodate tested cells


Chemical modification of surfaces

  • Chitosan, natural biodegradable polymer (810kDa Mw)

  • Modified PEG (polyethylene glycol)

  • Lactone modified eudragit

  • PLGA nanospheres

  • Lectins and derivatives


Chitosan


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


Lactonemodifiedeudragit

Counter-ions

+

+

+

+

-

-

-

-

-

+

+

+

+

-

-

COO-

Co-ions

COOH

COOH

COO-

HOOC

pH > 6

-OOC

COOH

-OOC

COO-

HOOC

COOH

COO-


PLGA nanospheres

Disperse

phase

Highpressure

water out

Pump

Continuous

phase

Pre-mixing

Pump

Magneticstirrer

Highpressure

waterin


Lectins and derivativesI

  • Cellsurfacecarbohydrateboundproteinsbindtolectins

  • Lectins, orphytohemagglutinins (PHA),areproteins of nonenzymatic, nonimmuneoriginthatbindcarbohydratesreversiblywithoutinducinganychangeinthecarbohydratebinding

  • Aslectinsmediatespecific, transient, cell-celladhesionevents, areusefulincellsurfacemodificationtoincreasecellularinteractions


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.


  • Types of N-glycansrecognisedby PHA


    Cellaggregationonscaffolds

    • Aggregation of homotypic and heterotypiccells

    • Biotinilation of proteins and usingavidinascross-linker


    Nanostructuredscaffolds

    • Selfassemblingscaffoldmaterial

    • Nanocomposites

    • Nanofibres


    Nanomaterialsforaggregatecultures


    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

    • No scaffold

    • Purifiedcellsformedintoclusters

    • Cellclustersusedas „bio-ink”

    • 3D tissue is printed usingtheability of cellclusterstofuse


    Cellclustersfuseintomicro-tissues


    Cellclustersfuseintomicro-tissueshapes

    Closelyplacedcellaggregates and embryonicheartmesenchymalfragmentscanfuseto ring ortube-likestructures


    Organ printing

    • 3D printing: depositingcellsonbiomaterialsin a rapid layer-by-layerfashion

    • Types of tissue printing:

    • Laser printing (osteosarcoma, embryoniccarcinoma)

    • Ink-jet printing (hippocampal and corticalneurons)


    The firsttissue printer


    Mature, organ specific primary cellsI

    Cell culture

    Biopsy

    Purification

    Cells for

    engineering


    Mature, organ specific primary cellsII

    Purification

    Cells for

    engineering

    Tissue specific resident stem cell

    Biopsy

    Cell cultures

    Differentiated tissue cells


    Maturetissuespecificcellsintissueengineering

    • Biopsyorresection

    • Purification

    • Regainingproliferationcapacityincellculture

    • Redifferentiation


    Generation of bloodvessels

    Importanttohold pressure


    Application of bloodvessels

    • Coronaryheartdisease, bypass

    • Treatment of trombosis

    • Accidentalbloodvesseldamage

    • Generation of complextissues


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