lecture -the cell system- primary cells€¦ · lecture -the cell system-primary cells - prof. dr....

Martin Luther University Halle-Wittenberg

Lecture -The Cell System-

Primary Cells -

Prof. Dr. Thomas Groth

Biomedical Materials



Content

• Isolation and cultivation of primary cells

• Problem: senescence

• Problem: dedifferentiation in vitro

• Immortalization of cells

• Conditions for in vitro culture

• Options for cultivation of adherent cells


Primary Cells

• Cells that are cultured directly from a subject in vitro Primary cells

• With exception of some tumor cells, primary cell have limited lifespan in vitro

• Require special media and surfaces to survive

• Different tissue types from same species may have quite different media requirements


Utilization of Primary Cells

• Pros:

Immunologic harmless if autologous

Differentiated function for replacement of target tissue (e.g. chondrocytes for cartilage)

• Cons:

Limited amount of cells available

Morbidity at extraction site

Limited division capacity (tissue type, age of patient)

Infected (damaged) cells in diseases

Dedifferentiation during in vitro culture


Extraction of Primary Cells

• Biopsy of relevant tissue

• Mechanical or enzymatic dissociation tissue single cells

• Enzymes trypsin, dispase, collagenase, hyaluronase, et al.

• Explant culture outgrowing of primary cells from biopsy into tissue culture


Isolation of Cells From Tissues

Enzyme Enzyme

Biopsy enzymatic digestion separation, isolation

Biopsy, Explant Migration, Proliferation


Change of Cell Behaviour Due to Isolation

---

Gene expression

Secretion

Organization of cytoskeleton

Phagocytosis procedures

Surface charge

Adhesion behaviour

Permeability, transport processes

Surface antigens (receptors)

Intercellular communication


Cultivation of Primary Cells

• Propagation of cells in vitro preparation of adequate cell numbers for TE

• Division capability depends on tissue type and donor age

• Accessible cell count of biopsies is limited (ca. 60 doublings in vitro) Senescence

• Dedifferentiation in vitro


Cell Cycle

S-Phase

G2-Phase

Mitosis

G1-Phase

G0-Phase

(no cell division)

G1-Phase – Synthesis RNA, proteins, lipids

S-Phase – Replication DNA

G2-Phase – Preparation cell division


Duration of Interphase

MitosisInterphase

Duration of interphase:

blood stem cells, tumor cells days

liver, kidney, gut glands years

neurons, heart muscle lifelong


Examples for Regeneration Capability of Tissues

Tissue New cells/d [%] Life time [d]

Neural cells 0

Liver parenchyma 0.2 - 0.7

Kidney parenchyma 0.3 – 0.4

Thyroid parenchyma 0.3

Bladder epithelium 2 64

Trachea epithelium 2.1 48

Epidermis 5.2 19

Stomach epithelium 56.4 1.8

Small intestine epithelium 79 1.3


Growth Curves of Cells in Vitro

Plateau-PhaseLog-PhaseLag-PhaseX – Population

doubling time


Limited Number of Cell Divisions

• Different division capabilities (growth rate) depending on donor age and tissue type: Maximum about 60 divisions!

• Generation number n cell number N = N0 x 2n with N ~ initial cell number and n ~ number of divisions (usually <60)

• Generation time tG tG = t/n with t ~ reading time


Example of Calculation

• Required cell number for therapeutic intervention (e.g. liver replacementby biohybrid extracorporal liver support) 1011 – 1012 cells in total

• Biopsie yields 106 cells

• N = N0 x 2n with N = 1012, N0 = 106

• N/N0 = 2n 106 = 2n

• log10 106 = n x log102

• log10 106 /log102 = n 6/0.301 = n

• n ~ 20 Passages (generations) required


Correlation Between Life Time, Species and Division Capability of Cells

• Experiments Leonhard Hayflickembryonic fibroblasts max. 50 celldivisions

• Different species correlationbetween life time and number of max. cell divisions of fibroblasts

• Example chicken: max. life time 12 yearsmax. 25 cell divisions

• Example Galapagos - giant turtle: max. life time 175 yearsmax. 130 celldivisions


Relevance of Telomers for Aging

• Chromosome ends (Telomers) repeating units of 6 bases TTAGGG

• Telomers shield chromosomes from degradation

• During cell division loss of repeating units telomers are reduced

• Beginning of embryonic growth telomers 10.000 base pairs long

• Birth telomers only half that size

• Per cell division loss of 8 repeating units

• Loss of telomers per division higher at short-living than long-living birds and mammals

• Loss of telomers loss of functional DNA per division + signal to tumor suppressor gene p53 Stop of division

• Germ cells, stem cells, immortal cancer cells telomerase active

http://www.ch.ic.ac.uk/local/projects/burgoine/origins.txt.html


Telomerase

• Telomerase as Reverse Transcriptase use of RNA template for repair of telomers

• 85% of all human cancer cells expression of human TElomerase Reverse Transcriptase (hTERT)

• RNA template in most body cells expressed expression of hTERT critical for immortalization

The National Institutes of Health resource for stem cell research


Causes for Limited Division Capability• Replicative senescence of cells

• Regulators tumor suppressor genes pRB(retinoblastoma protein) and p53 (src kinase), as well as Telomers

• Telomer length reduced due to cell division somatic cells telomerase inactive

• (85-90% of all cancer cells telomerase active)

• p53 - late G1 Phase cell division blocking the access to S-Phase

• RB1 - slows down the transition from G0/G1 into S-Phase

• P53 + pRB inactive in cancer cells

• (Viral oncoproteins SV 40 LT and HPV E6 disable tumorsuppressor genes pRB and p53)


Example for Senescence of Cells

Embryonic murine fibroblasts: top normal cell culture, bottom senescent cells, right MDCK-cells


Barriers to Cell Immortalization

M0 – Stop cell divisions of epithelial cells

M1 – ageing of cell (fibroblasts division stop, metabolism active)

M2 – Either apoptosis of cells or immortalization

TMM – Telomerase maintenance mechanism

RAS – Signal transducer (see lecture signal transduction)


Immortalization of Cells I

• Telomerase hTERC RNA subunit + hTERT catalytic + reverse transcriptase-subunit

• Ectopic expression of hTERT immortalization of pre-senescent fibroblasts and endothelial cells in vitro (normal karyotype, normal behaviour at reduction of serum)

• Epithelial cells despite hTERT – senescence!!!


Immortalization of Cells II

• Ectopic expression of SV 40 T-Antigen disables pRB + p53 tumor supressor genes Problem: no growth control + dedifferentiation

• HPV-16 E6/E7 – transfection of chondrocytes with vector preservation of differentiation without neoplastic potential possible

Oncogene (2005) 24, 7729–7745


Problem II – Dedifferentiation

In vivo:

Specific ECM + neighbouring cells mix of fixed and soluble signals around

typical cell morphology

Expression of cell-typical proteins

Transport functions, etc

Isolation of cells:

Mechanical, enzymatic treatment

Change of cell shape

Loss of surface structures

Cultivation in vitro:

Adhesion substrate change of cell shape

Loss of neighbour cells (e.g. epithelium-mesenchymal-interaction)

Change of medium composition


Dedifferentiation in Vitro

American Journal of Pathology. 2005;166:1077-1088.)© 2005 American Society for Investigative Pathology

Transdifferentiation of Mature Rat Hepatocytes into Bile Duct-Like Cells in VitroYuji Nishikawa*, Yuko Doi, Hitoshi Watanabe, Takuo Tokairin*, Yasufumi Omori*, Mu Su*, Toshiaki Yoshioka* and Katsuhiko Enomoto

Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitroReischl, J. ; Prelle, K.5; Schöll, H. ; Neumüller, C.2; Einspanier, R.; Sinowatz, F. and Wolf, E.5: Cell Tissue Res. 296 (1999) S. 371-383

http://ajp.amjpathol.org/misc/terms.shtml


Examples for Dedifferentiation

• Cartilage tissue cells in vitro - production of unspecific collagenes (collagen I instead of collagen II)

• Liver cells in vitro - loss of cytochrome P450 isoenzymes, urea synthesis, etc.

• Endothelium cells - pro-coagulant, pro-inflammatory behaviour

• Epithelium cells - change of transport properties


Dedifferentiation of Primary Chondrocytes in Vitro


Dedifferentiation of Primary Chondrocytes in Vitro

Immunofluorescence staining of primary chondrocytes after 1 (A & B) and 4 (D &E) weeks against collagen II (A&D) and collagen i (D&E)


Comparison of Media Compositions

human serum

Medium 199

Basal Medium Eagle

Williams Medium E

DulbeccosModified Eagle Me

pH-value 7.4 7.4 7.4 7.4 7.4

Na+

[mmol]142 139 146 144 158

Cl- 103 125 111 117 116

K+ 4 5.1 4.8 4.8 4.8

Ca++ 2.5 1.5 1.4 1.4 1.3

Glucose (mg/ml)

100 99 94 186 382

mOsm 290 270 286 288 323


Additional Medium Supplements

• Essential amino acids for protein metabolism

• Vitamines like biotin, cholinchloride, D-Ca-panthotenate, folin acid, nicotinamide, et al.

• Components for DNA and RNA-synthesis and energy metabolism (e.g. Adenine, Thymidine, Glucose, Sodium pyruvate, etc.)

• Buffer substances for pH-value stability e.g. NaHCO3 or HEPES

• Color-indicators for pH-value of the medium like phenol red


Serum

• Serum: heterogeneous mixture of proteins - growth and adhesion supporting properties!!!

• Production by blood coagulation !

• Mixture of proteins (transport proteins, growth factors), hormones, electrolytes and other components

• Origin other species like cow (fetal, new born), horse, goat, sheep, rabbit, chicken, etc.


Problems with Sera

• Serum for majority of cells no physiological liquid

• Variation of quality from batch to batch (dependent on animal source)

• Sera can contain inhibitors for cells

• In heterogeneous cultures (e.g. explants) growth undesirable cells (e.g. fibroblasts)

• Uptake of serum proteins by human cells acquire immunogenicity

• Risk of viral infections e.g. Retroviruses and BSE (Bovine Spongiform Encephalopathy)

• High costs

• attempts of “serum-free” cultivation


Supplements for Serum-Free Cultures

Additive Cell types Application

Transferrin All 1 - 20 µg/ml

Trace elements All Depends on cell type

DL-a-Tocopherol All 0.01 - 1 µg/ml

Sodium selenite All 20 nm

Progesterone Epithelium cells 1 - 10 nm

Poly-D-Lysine Fibroblasts 0.1mg/ml

Ascorbic acid All 10 - 100 µg/ml

Hydrocortisone Epithelium cells 1 - 10 nm

Insulin All 1 - 10 µg/ml


Supplements for Preservation of the Phenotype (Examples)

• EGF (1 - 10 ng/ml) fibroblast, endothelium cells, glial cells

• aFGF (3 - 100 ng/ml) mesoderm, 3T3, amnion fibroblasts

• bFGF (0.3 - 10 ng/ml) mesoderm, 3T3, amnion fibroblasts

• G-CSF (0.05 - 5 ng/ml) bone marrow cells

• GM-CSF (0.003 - 1 ng/ml) bone marrow cells

• IGF II (20 - 200 ng/ml) chondrocytes, 3T3


Anchorage Factors

• Adherent-dependent cells growth on tissue culture plates not optimal

• Serum-free culture no “attachment”-factors

• surface coating of culture flasks with:

Collagen, gelatine

Fibronectin, superfibronectin, fibronectin-fragments, Laminin, thrombospondin, vitronectin

Matrigel - ECM gel from mouse tumor cell line

Glycosaminoglycans like heparin, chitosan

Poly-L-Lysine


Application of Anchorage Factors I

• Collagen I: epithelium cells, muscle cells, neurons

• Collagen II: chondrocytes

• Collagen IV: epithelium cells, endothelium cells, muscle cells, neurons

• Gelatine: many different cell types

• Chondroitin sulfate: chondrocytes, neurons


Application of Anchorage Actors II

• Fibronectin: epithelium cells, endothelium cells, fibroblasts, mesenchymal cells, neurons

• Laminin: epithelium cells, endothelium cells, hepatocytes, muscle cells, Schwann cells

• Thrombospondin: osteoblasts, endothelium cells, neurons

• Vitronectin: endothelium cells, osteosarcoma cells


Possibilities of in Vitro Culture I

Adherend cultures in cell culture flasks

Materials: polystyrene, plasma-treated

Pro: high cell numbers

Adherend cultures on membrane inserts (epithelium cells)

Materials: cellulose acetate, polycarbonate

Pro: polarization of cells possible (basal, apical)


Possibilities of in Vitro Culture II

Perfusion culturesDifferent geometries:

fixed bed, flat bed, hollow fibre design

Materials: cellulose acetate, polycarbonate, polysulfone

Pro: high cell numbers, continuous replacement of media

delivery of nutrients, oxygen (convective)

removal (toxic) metabolitesMinucell-Bioreactor


Possibilities of in Vitro Culture III

Culture on microcarriers in suspension (spinner flasks)

Good supply of the cells

High cell numbers accessible

Matrix material degradable?

Application as suspension for Tissue Engineering

Example “Skin out of a tube”

Example: Kidney epithelium cells from a pork


Mass Cultivation of Adherend Cells


Tissue Spheroids

• Exploitation of aggregation potential of cells scaffold-free production of tissue equivalents

• Fabrication by cultivation:

in agitated flasks, roller bottles, spinner flasks

on non-adhesive surfaces

compression based on centrifugation

on cell culture inserts

within hanging droplets


Making of Tissue Spheroids


Tissue Spheroids

• Fabrication from single or mixed cell populations

• Size control optimization of supply with oxygen + nutrients

• Cell motility during formation intercellular organization including polarization, formation of extracellular matrix high functionality, no dedifferentiation

• Compatibility with low cell numbers and volumes

• Application possible with many cell types


Formation of MIN6 Aggregates on Agarose Chips

MIN6 aggregates on agarose chip [4x, 10x]. MIN6 aggregates outside agarose chip [10x] – free floating.

Micromoulds made of agarose


Tissue Spheroids

Confocal pictures of spheroids from two cell types

a) hepatocytes / endothelium cells b) chondrocytes

Application of spheroids as transplants, 3D tissue models, pharmaceutical studies, etc.


Literature

• W.W. Minuth, R. Strehl, K. Schumacher, Zukunftstechnologie Tissue Engineering, Wiley VCH 2003

• T. Lindl, Zell- und Gewebkultur, Spektrum/Gustav Fischer Verlag 2000

• JM Kelm & M. Fussenegger, Microscale tissue engineering using gravity-enforced cell assembly, Trends in Biotechnology 22 (2004) 195-202

• WC Hahn, Immmortalization and Transformation of Human Cells. Molecules and Cells 13 (2002) 351-361

• B.Best, Mechanisms of Aging

lecture -the cell system- primary cells€¦ · lecture -the cell system-primary cells - prof. dr....

Documents