Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

(1)

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

AGGREGATION CULTURES

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue engineering – Lecture 15

(3)

Aggregate cultures

Aggregation allows:

• rapid formation of small units of tissues

• intimate contacts between cells leading to

enhancement of cell functionality and viability

(4)

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

(5)

Cell adhesion

Cell-cell interactions

Cell-matrix interactions

Soluble ECM

Integrins

Static ECM Cadherins

(6)

Methods of cell aggregation

Aggregation in gravity culture Aggregation on low

adherence surfaces

Aggregation on

scaffolds/modified surfaces

Aggregation in

rotation/suspension culture Aggregation in bioreactors

(7)

Gravity cultures

Cells can assemble into spheroids naturally in natural or increased gravity.

Types of gravity cultures:

• Suspension aggregates in bioreactors

• Hanging drop cultures

• Centrifuged aggregates

(8)

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

(9)

Aggregation in rotation culture

Rotation culture

for adherent cells

Rotation culture

for suspension

Sampling ports Fill port

LSMMG

NG Gravitation

force

Gravitation force

Rotation Sampling

ports

Fill port

Rotation

(10)

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

(11)

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 flask Chondrocytes, primary mouse and rat hepatocytes, L6 myoblasts, CHO

(12)

Application of the cell aggregates

Cell aggregates Use

CHO Production of recombinant

protein Human embryonic

stem cells

Embryonic body formation and differentiation

(13)

Microgravity culture (hanging drop) I

Cavity slide

Sample placed on coverslip with loop

Oil drop Vaseline

180°

(14)

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

(15)

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

40 mm

150 mm

(16)

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)

(17)

Separation and enrichment of high 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

Others Integrin

EGF-R HGF-R

Fas

Hepatocytes ASGP-R^high low proliferative

Spheroid formation

Hepatocytes ASGP-R^low 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

Roundshape

E-Cadherin Bile duct

Hepatocyte

(18)

Synthetic cell aggregation I

Creation of a polymer bridge to connect cells Types:

• Natural adhesion molecule

• Segment of an extracellular matrix

• Polymer matrix

(19)

Synthetic cell aggregation II

Aggregated cells Cells

Bifunctional polymer

(20)

Biotinylated cell cross-linking

Avidin

Multicellular aggregate Biotin

hydrazide

Periodate tested cells

(21)

Chemical modification of surfaces

• Chitosan, natural biodegradable polymer (810 kDa Mw)

• Modified PEG (polyethylene glycol)

• Lactone modified eudragit

• PLGA nanospheres

• Lectins and derivatives

(22)

Chitosan

(23)

Modified PEG

MA(PEG)_n

Methyl-PEG_n-Amine

Methyl-(#ethyleneglycol) amine H₂N

CH₃ O

O

MA(PEG)₈ M.W. 383.48 Spacer arm 29.7 Å

[ ]8

CH₃ H₂N

O

MA(PEG)₁₂ M.W. 559.69 Spacer arm 43.9 Å

H₂N [ ]12

CH₃ O

MA(PEG)₂₄ M.W. 1088.32 Spacer arm 86.1 Å

[ ]24

CH₃ H₂N

O

(24)

Lactone modified eudragit

pH > 6

HOOC

COOH

COOH HOOC

COOH

Counter-ions Co-ions

COO- -OOC

-OOC

COO-

+ -

+

+ - - +

(25)

PLGA nanospheres

Continuous phase

Disperse phase

Pump

Pre-mixing

Magnetic stirrer

High pressure water in High pressure

water out

(26)

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

(27)

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.

(28)

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)

(29)

Cell aggregation on scaffolds

• Aggregation of homotypic and heterotypic cells

• Biotinilation of proteins and using avidin as cross- linker

(30)

Nanostructured scaffolds

• Self assembling scaffold material

• Nanocomposites

• Nanofibres

(31)

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

(32)

TISSUE PRINTING

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue engineering – Lecture 16

(33)

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

(34)

Cell clusters fuse into micro-tissues

(35)

Cell clusters fuse into micro-tissue shapes

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

(36)

Organ printing

3D printing: depositing cells on biomaterials in a rapid layer-by-layer fashion

Types of tissue printing:

• Laser printing (osteosarcoma, embryonic carcinoma)

• Ink-jet printing (hippocampal and cortical neurons)

(37)

The first tissue printer

(38)

Mature, organ specific primary cells I

Biopsy Purification

Cell culture

Cells for engineering

(39)

Mature, organ specific primary cells II

Biopsy

Purification

Cells for engineering

Differentiated tissue cells

Tissue specific resident

stem cell Cell cultures

(40)

engineering

• Biopsy or resection

• Purification

• Regaining proliferation capacity in cell culture

• Redifferentiation

(41)

Generation of blood vessels

Important to hold pressure

(42)

Application of blood vessels

• Coronary heart disease, bypass

• Treatment of trombosis

• Accidental blood vessel damage

• Generation of complex tissues