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
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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
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