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

AGGREGATION CULTURES

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 15

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

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Aggregate culturesAggregation allows:• rapid formation of small units of tissues• intimate contacts between cells leading to

enhancement of cell functionality and viability

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Principals of aggregate cultures• Presence of cell adhesion molecules (CAMs)

on cellular surfaces• Presence of matrices or arteficial anchorage

molecules that facilitate aggregation for cells that would not aggregate naturally

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Cell adhesion

Cell-cellinteractions

Cell-matrix interactions

Soluble ECM

IntegrinsStatic ECM

Cadherins

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Methods of cell aggregation

Aggregation in gravity culture

Aggregation on low adherence surfaces

Aggregation on scaffolds/modified

surfaces

Aggregation in rotation/suspension

cultureAggregation in bioreactors

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Gravity culturesCells can assemble into spheroids naturally in natural or increased gravity. Types of gravity cultures:• Suspension aggregates in bioreactors• Hanging drop cultures• Centrifuged aggregates

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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

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Aggregation in rotation cultureRotation culturefor adherent cells

Rotation culturefor suspension

SamplingportsFill port

LSMMG

NG

Gravitationforce

Gravitationforce

Rotation

Samplingports

Fill portRotation

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Bioreactors and cell aggregationRotating 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

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Cell type aggregates using bioreactors

Bioreactor type Cell type

Rotating wall vessel (RWV)

HepG2, human stem cells, human dermal fibroblasts, human embryonic kidney cells

Spinner flaskChondrocytes, primary mouse and rat hepatocytes, L6 myoblasts, CHO

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Application of the cell aggregates

Cell aggregates Use

CHOProduction of recombinant protein

Human embryonic stem cells

Embryonic body formation and differentiation

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Microgravity culture (hanging drop) I

Cavity slide

Sample placed oncoverslip with loop

Oil drop

Vaseline

180°

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Microgravity culture (hanging drop) II

180° 180°

180°

Time(days)

Outgrowth of plated EBs and spontaneous differentiation into cell types of all three germ

layers

0

2

5

TÁMOP-4.1.2-08/1/A-2009-0011Microwells for uniform embryoid body culture and control of cell-cell contact

40 mm

150 mm

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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)

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Separation and enrichment ofhigh proliferative hepatocyte

Natural cell aggregation

PVLA has a potentiality as an artificial liver material by

varying a coating concentration onto Pts dish

PVLA (Poly N-p-vinyl venzyl D-lactose lactone amide)

ASGP-R

OthersIntegrinEGF-RHGF-R

Fas

HepatocytesASGP-Rhigh

low proliferative

Spheroid formation

HepatocytesASGP-Rlow

high proliferative

+EGF

Regulation of cell shape

Spheroid

100 mg/ml PVLA-coated dish

Coating concentration onto Pts dish

15-20 ng/ml PVLA-coated dish

Spreading

1 mg/mlPVLA-coated dish

100 mg/mlPVLA-coated dish

Roundshape

E-Cadherin

Bile duct

Hepatocyte

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Synthetic cell aggregation ICreation of a polymer bridge to connect cellsTypes:• Natural adhesion molecule• Segment of an extracellular matrix• Polymer matrix

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Synthetic cell aggregation II

Aggregated cellsCells

Bifunctional polymer

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Biotinylated cell cross-linking

Avidin

Multicellular aggregate

Biotinhydrazide

Periodate tested cells

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Chemical modification of surfaces• Chitosan, natural biodegradable polymer

(810 kDa Mw)• Modified PEG (polyethylene glycol)• Lactone modified eudragit• PLGA nanospheres• Lectins and derivatives

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Chitosan

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Modified PEG

MA(PEG)nMethyl-PEGn-Amine

Methyl-(#ethyleneglycol) amineH2N

CH3O

OO

O

MA(PEG)8M.W. 383.48

Spacer arm 29.7 Å

[ ]8CH3

H2NO

MA(PEG)12M.W. 559.69

Spacer arm 43.9 Å

H2N[ ]12

CH3O

MA(PEG)24M.W. 1088.32

Spacer arm 86.1 Å

[ ]24CH3

H2NO

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Lactone modified eudragit

pH > 6

HOOC

COOH

COOH

HOOC

COOH

COOH

Counter-ions

Co-ions

COO-

-OOC

-OOC

COO-

COO-

COO-

+

-

+ -

+ -

+

-

+-

+

-+

-+

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PLGA nanospheres

Continuousphase

Disperse phase

Pump

Pump

Pre-mixing

Magnetic stirrer

High pressurewater in

High pressurewater out

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Lectins and derivatives I• Cell surface carbohydrate bound proteins

bind to lectins• Lectins, or phytohemagglutinins (PHA), are

proteins of nonenzymatic, nonimmune origin that bind carbohydrates reversibly without inducing any change in the carbohydrate binding

• As lectins mediate specific, transient, cell-cell adhesion events, are useful in cell surface modification to increase cellular interactions

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Lectins and derivatives II• Six lectin families are recognized: 

– legume lectins, – cereal lectins, – P-, C-, and S-type lectins, and – pentraxis,

with the latter four occurring in animals.  • Lectins bind a variety of cells having cell-surface glycoproteins or

glycolipids such as erythrocytes, leukemia cells, yeasts, and several types of bacteria.  

• Several specificity groups have been identified, such as mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, L-fuctose, and N-acetylneraminic acid.

• The presence of two or more binding sites for each lectin molecule allows the agglutination of many cell types.  

• Lectin binding, however, is saccharide-specific.

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Types of N-glycans recognised by PHA

Type PHA

Bisected di-, tridiantennary complex-type N-glycan

Phaseolus vulgaris Erythroagglutinin (E-PHA)Inhibitor: GalNAc

Tri- and tetraantennary complex-type N-glycan

Phaseolus vulgaris Leukoagglutinin (L-PHA)Inhibitor: GalNAc

Tri- and tetraantennary complex-type N-glycan

Datura stramonium Agglutinin(DSA)Inhibitor: Chitotriose (GlcNAc3)

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Cell aggregation on scaffolds• Aggregation of homotypic and heterotypic

cells• Biotinilation of proteins and using avidin as

cross-linker

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Nanostructured scaffolds• Self assembling scaffold material• Nanocomposites• Nanofibres

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Nanomaterials for aggregate cultures

Material Description Examples of Application

Fullerenes Hexagonal and pentagonal carbon atoms

Encapsulation of therapeutics, imaging

Quantum dots Semiconductor nanocrystals Imaging and biosensing

Liposomes Phospholipids Drug and gene delivery

Dedrimers Polymer structures Drug and gene delivery

Gold nanoparticles Colloid gold Cellular imaging, biosensing

Super-paramagnetic iron oxide

Iron oxide MRI contrast agent

TISSUE PRINTING

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 16

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

TÁMOP-4.1.2-08/1/A-2009-0011

Main principles of tissue printing• No scaffold• Purified cells formed into clusters• Cell clusters used as „bio-ink”• 3D tissue is printed using the ability of cell

clusters to fuse

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Cell clusters fuse into micro-tissues

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Cell clusters fuse into micro-tissue shapes

Closely placed cell aggregates and embryonic heart mesenchymal fragments can fuse to ring or tube-like structures

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Organ printing3D printing: depositing cells on biomaterials in a rapid layer-by-layer fashionTypes of tissue printing:• Laser printing (osteosarcoma, embryonic

carcinoma)• Ink-jet printing (hippocampal and cortical

neurons)

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The first tissue printer

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Mature, organ specific primary cells I

Biopsy PurificationCell culture

Cells forengineering

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Mature, organ specific primary cells II

Biopsy

Purification

Cells forengineering

Differentiated tissue cells

Tissue specific resident stem cell Cell cultures

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Mature tissue specific cells in tissue engineering• Biopsy or resection• Purification• Regaining proliferation capacity in cell culture• Redifferentiation

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Generation of blood vessels

Important to hold pressure

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Application of blood vessels• Coronary heart disease, bypass• Treatment of trombosis• Accidental blood vessel damage• Generation of complex tissues