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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
Aggregate Unioncultures

  • Aggregationallows:

  • rapid formation of smallunitsoftissues

  • intimatecontactsbetweencellsleadingtoenhancement of cellfunctionality and viability


Principals of aggregate cultures
Principals Union of aggregatecultures

  • Presence of celladhesionmolecules (CAMs) oncellularsurfaces

  • Presence of matricesorarteficialanchoragemoleculesthatfacilitateaggregationforcellsthatwouldnotaggregatenaturally


Cell adhesion
Cell Unionadhesion

Soluble ECM

Cell-cell

interactions

Cadherins

Cell-matrixinteractions

Integrins

Static ECM


Methods of cell aggregation
Methods Union of cellaggregation

Aggregationonlowadherencesurfaces

Aggregation on scaffolds/modified surfaces

Aggregationinrotation/suspensionculture

Aggregationinbioreactors

Aggregationingravityculture


Gravity cultures
Gravity Unioncultures

  • Cellscanassembleintospheroidsnaturallyinnaturalorincreasedgravity.

  • Types of gravitycultures:

  • Suspensionaggregatesinbioreactors

  • Hanging dropcultures

  • Centrifugedaggregates


Suspension aggregate cultures
Suspension aggregate cultures Union

  • 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 Union

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 Unioncell 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 Union(hanging drop)I

Sample placed on

coverslip with loop

Vaseline

Cavity slide

180°

Oil drop


Microgravity culture hanging drop ii
Microgravity culture Union(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
Microwells Union for uniform embryoid body culture and control of cell-cell contact

40 mm

150 mm


Aggregation on low adherence surfaces
Aggregation on low adherence surfaces Union

  • 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 Unioncell 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 aggregation UnionI

  • 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 Unioncell aggregationII

Bifunctional polymer

Cells

Aggregatedcells


Biotinylated cell cross linking
Biotinylated Union cell cross-linking

Avidin

Biotin

hydrazide

Multicellular aggregate

Periodate tested cells


Chemical modification of surfaces
Chemical modification of surfaces Union

  • Chitosan, natural biodegradable polymer (810kDa Mw)

  • Modified PEG (polyethylene glycol)

  • Lactone modified eudragit

  • PLGA nanospheres

  • Lectins and derivatives


Chitosan
Chitosan Union


Modified peg
Modified Union 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
Lactone Unionmodifiedeudragit

Counter-ions

+

+

+

+

-

-

-

-

-

+

+

+

+

-

-

COO-

Co-ions

COOH

COOH

COO-

HOOC

pH > 6

-OOC

COOH

-OOC

COO-

HOOC

COOH

COO-


Plga nanospheres
PLGA Unionnanospheres

Disperse

phase

Highpressure

water out

Pump

Continuous

phase

Pre-mixing

Pump

Magneticstirrer

Highpressure

waterin


Lectins and derivatives i
Lectins Union and derivativesI

  • Cellsurfacecarbohydrateboundproteinsbindtolectins

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

  • Aslectinsmediatespecific, transient, cell-celladhesionevents, areusefulincellsurfacemodificationtoincreasecellularinteractions


Lectins and derivatives ii
Lectins Union 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 glycans recognised by pha
    Types Union of N-glycansrecognisedby PHA


    Cell aggregation on scaffolds
    Cell Unionaggregationonscaffolds

    • Aggregation of homotypic and heterotypiccells

    • Biotinilation of proteins and usingavidinascross-linker


    Nanostructured scaffolds
    Nanostructured Unionscaffolds

    • Selfassemblingscaffoldmaterial

    • Nanocomposites

    • Nanofibres


    Nanomaterials for aggregate cultures
    Nanomaterials Unionforaggregatecultures


    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 Unionprinciples of tissue printing

    • No scaffold

    • Purifiedcellsformedintoclusters

    • Cellclustersusedas „bio-ink”

    • 3D tissue is printed usingtheability of cellclusterstofuse


    Cell clusters fuse into micro tissues
    Cell Unionclustersfuseintomicro-tissues


    Cell clusters fuse into micro tissue shapes
    Cell Unionclustersfuseintomicro-tissueshapes

    Closelyplacedcellaggregates and embryonicheartmesenchymalfragmentscanfuseto ring ortube-likestructures


    Organ printing
    Organ Union printing

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

    • Types of tissue printing:

    • Laser printing (osteosarcoma, embryoniccarcinoma)

    • Ink-jet printing (hippocampal and corticalneurons)


    The first tissue printer
    The Unionfirsttissue printer


    Mature organ specific primary cells i
    Mature, organ specific primary cells UnionI

    Cell culture

    Biopsy

    Purification

    Cells for

    engineering


    Mature organ specific primary cells ii
    Mature, organ specific primary cells UnionII

    Purification

    Cells for

    engineering

    Tissue specific resident stem cell

    Biopsy

    Cell cultures

    Differentiated tissue cells


    Mature tissue specific cells in tissue engineering
    Mature Uniontissuespecificcellsintissueengineering

    • Biopsyorresection

    • Purification

    • Regainingproliferationcapacityincellculture

    • Redifferentiation


    Generation of blood vessels
    Generation Union of bloodvessels

    Importanttohold pressure


    Application of blood vessels
    Application Union of bloodvessels

    • Coronaryheartdisease, bypass

    • Treatment of trombosis

    • Accidentalbloodvesseldamage

    • Generation of complextissues


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