Animal Cell Cultures

8/3/2019 Animal Cell Cultures

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Animal Cell Biotechnology


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ANIMAL CELL BIOTECHNOLOGY

Human health and well-being is threatened bynumerous diseases. These may be infectious diseasesor diseases due to some malfunction in the body.Finding remedies for and, if possible, preventing human

disease is a major area of scientific research.Biotechnology research focuses on optimizing theproduction process for bacterial and virus vaccines andpharmaceutical proteins. For this we make use of

detailed metabolic models in mammals, which in turnare also used to study the effect of food components onmammalian cell physiology.


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Pharmaceutical Proteins

Animal cell culture increasingly attracts interest dueto the ability of animal cells to produce qualitativeexcellent pharmaceutical proteins. Examples of such

proteins are Erythropoietin (Epo) and Follicle-stimulating hormone (FSH). However, compared tomicrobial production systems, animal cell culturesare characterized by low biomass concentrations,

low productivities, slow growth rates, and a highshear sensitivity. Our research focuses on themedium, process and reactor design addressing theabove mentioned problems.


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Erythropoietin (Epo)

the principal factor responsible for the regulationof red blood cell production during steady-stateconditions and for accelerating recovery of red bloodcell mass following hemorrhage

Follicle-stimulating hormone (FSH)

produced by the Pituitary gland,causes the follicle to develop in females,

then stimulates the follicle to produce estrogen,and helps to stimulate sperm production in males ---When FSH is elevated, in males, it generally reflects adefect in sperm production, or castration.


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Stem cells&tissue engineering Skin, Liver

Bone and Cartilage Pharmaceuticals MKZ (ID-Lelystad)

Classical swine fever (Bayer) Small pox

Remicade (Centocor)

-Glucosidase (Pharming)

Animal cell culture


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History

Animal cell culture

Why, media, applications, comparison

microbial cell culture

Regulations


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History

1880 Roux Embryonic chicken in saline

1900 Harrison

Anchorage dependent Nutrients Relative slow growth rate

Doubling 1 day vs 20 minutes bacteria Contamination

frog embryo


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characteristics for in vitrocell growth:

Cells require an anchor like the lymph clots and thecover slip.Cells require nutrients provided by the lymph.

Cells grow very slow; 20 hours doubling timecompared to 20 minutes for bacteria This means cellcultures are vulnerable to contamination

1900 Harrison


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History

Carrel (surgeon, 1923)

Aseptic techniques

Carrel Flask

1912-1946 Culture Chicken Embryo Fibroblast

Plasma+tissue homogenate


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Polio

1940/1950s Major Polio epidemics


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Polio

Antibiotics: Penicillin and streptomycin

Polio vaccine first product

primary monkey kidney cellshuman diploid lung fibroblast


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1950s

Hela cell line: human carcinoma

Chemically defined media (Eagle, Earle)ConsistencySterilizationReduced chance of contamination

Insect cell lines (Grace)


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Recombinant DNA 1970

All proteins in E-coli

No post-translational modifications

Glycosylation Folding

Inclusionexcretion

Large complex proteins requireanimal cells


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MicrocarriersVan Wezel 1960s RIVM

Monkey Kidney cells

Polio

http://www.rivm.nl/en/
http://www.rivm.nl/en/http://www.rivm.nl/en/


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Hybridoma

1975 Kohler and Milstein

B-cellantibody

Mortal

Myelomasingle chain

immortal

Hybridoma

monoclonal antibodyimmortal


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Serum-free media

Genetic modification cells

Bioreactors Large-scale (20 m3)

Nowadays


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Physiological studies Understand function of cells (Toxicology)

Tissue engineering (Skin, Cartilage, Liver)

Biologicals (pharmaceutical proteins)

Monoclonal Antibodies

Hormones (EPO, FSH) Enzymes (-glucosidase)

Vaccines (Polio)


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Toxaphene

Probes for:

pH

Dissolved Oxygen

Temperature

Gas inlet

Nitrogen

Oxygen

CO2

Gas outlet

Heating

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 50 100 150 200

Viable-cell concentration (109 cells.dm-3)

Culture time (hours)


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An example of the use of animal-cell culture in thestudy of toxicology is shown. Toxaphene is a pesticide

used in the US until the eighties in cotton farming andin fish industry to remove all fish out of lakes with theintention to culture one specific type of fish. It is amixture of 180 types of polychlorinated cyclichydrocarbons and very persistent. Here a culture of

hepatocytes on microcarriers is exposed to differenttoxaphene concentrations of 0 and 10 microgram perliter. In the 10 microgram/l culture no growth occurredand no oxygen was consumed, while glucose was

rapidly converted to lactate. Probably toxapheneinhibits processes in the mitochondria thus inhibitingrespiration. The only way to generate ATP is throughglycolysis. Apparently this is only just enough tomaintain cells and not for growth.


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Cartilage

http://www.isotis.com/
http://www.isotis.com/isotis/isotiswebv3.nsf/lookupDefaultHomepages/$first


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EPO http://www.amgen.com/


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Erythropoeitin is a glycoprotein hormoneproduced in the kidneys that stimulates red-blood-cell production in the bone marrow.Patients with kidney failure produce lessEPO and consequently have lower levels ofred blood cells (up to 1/3 of a normalperson) and have severe anemia. Epogen

solves this problem. Because the activeprotein is modified after translation it canonly be produced in animal cells in thecorrect form.

Erythropoeitin (EPO)


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Multicellular environment

Complex nutritional requirements

20 amino acids, minerals, vitamins, glucose,serum/growth factors

Fragile/ shear sensitive

Oxygenation

Limited life-span

Transformed cells, cancerous cells

Bad growth

Slow growth rates, low biomass, cell death


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0.0

0.0

0.0

0.0

0.1

1.0

10.0

0 50 100Time (h)

Cx(g

/l)

YeastHybridoma

Growth curve

Minimum

densityLag phase

exponential

Stationary

decline


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Desinfection step

Tissue isolation

Incubation&growth

Primary cells


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Passage number

70 generations

Ln(total cells)


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Transformation

Characteristics Infinite life span

High growthpotential

Low growth factordependence

Suspension growth

aneuploid

Methods Mutagens

Viruses

Oncogens Spontaneous

Tumors

Not all transformed cell lines can form tumors, butall tumors contain transformed cells


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Normal cells?

Diploid

Anchoragedependent

Finite life span Non-malignant


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Basic Media

Glucose

Amino acids

HCO3

Mammalian Insect4 g/l 2.5 g/l

0.01-0.15 g/l 0.1-1.5 g/l

Glutamine 1 g/l 1 g/l3.5 g/l 0.35 g/l

H2PO4 0.1 g/l 1 g/l

pH 7.2 6.4Osmolarity 300 mOsm 350 mOsm

Salts

Vitamins/Spore4.5 g/l NaCl 1g/l MgSO4,KCl

More Less


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Media

Insect cells

Fumaric & alfa-

keto-glutaric acid,Malic & succinicacid

Maltose & Sucrose&Threhalose

Mammalian cells

Pyruvate

Hypoxanthine,thymidine

Linoleic acid

Hepes buffer Phenol red


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Serum

0-20% Serum Growth factors

Transferrin (Fe) Lipids

Insulin

Shear protection Detoxification

Problems Infectious agents

(viruses, prions)

Variablecomposition

Expensive

High proteincontent problems inDSP


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Serum

Serum/protein free media Transferrine / ferrous citrate

Lipid concentrate Extracts Yeast extract

Insulin

Bovine Serum Albumin (f.a. transport/detoxification) Pluronic (shear protectant)

Completely mammalian origin free (MOF)


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Comparison with Yeast/Bacteria Syste

Mammalian Insect Yeast Bacteria

Size (m)

Dry cell weight(10-10 g/cell)

Productivity (g/g/day)

15 20 5 1

300 600 10 1

1 0.1

Biomass (g/dm3) 0.5 2 2020

Doubling time (h) 12-48 28-48 0.3 0.3


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Cont

Mammalian Insect Yeast Bacteria

secretion

DSP

Scale-up

yes Lytic variable no

difficult simple

simple complex

Post-transl.Mod.

++ + +/- --

Nutritional Complex simple

Media cost ($/dm3) 20 20


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animal cell Culture?

Tissue engineering/physiology

Obvious

Pharmaceutical protein & vaccine production

Product quality < post-translational modifications

Glycosylation, Sulfonation and folding

Activity

Immunogenity (wanted for vaccines, not wanted forothers)

In-vivo half life

Excretion vs inclusion bodies


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Cells vs whole organs/animals

Physiology studies: Guinea Pigs

Isolating effects on cellular level

Reproducibility

Ethics

Tissue engineering:

Genetically modified organisms as organ donor

Non-ethical

Risks

Patients own material: stem cells


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Cells vs whole animals

Pharmaceutical proteins Economic

Product concentration/down-streamprocessing

Scale-up

Proven technology

Time to market

Reproducibility, robustness

Validation/safety


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Canine KidneyEpithelial

Common cell lines

BHK

CHO

PER-C6

MDCK

Vero

L

3T3

Baby hamster kidneyFibroblast

Chinese Hamster OvaryEpithelial

Mouse fibroblastFibroblast

Mouse tumor fibroblastFibroblast

Monkey KidneyFibroblast

Human KeratonocytEpithelial


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Common cell lines

HeLa

Namalwa

MRC 5

WI-38

Human cervical carcinomaEpithelial

Human lymphoblastoma cellLymphoblast

Human embryonic lungfibroblast

Fibroblast

Human embryonic lungFibroblast

Sf21,Sf9 Insect Spodoptera Frugiperda

T.ni 5 Insect trichopluisa niHigh Five


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Products/Proteins

TPA: Tissue Plasmogenic Activator GenentechFSH: Follicle stimulating Hormone DiosynthEPO: Erythropoin Amgen

Interferon BiogenFactor VIII blood clotting NovoMonoclonal antibodies Centocor

Remicade

ReoproContract production DSM Biologics


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Vaccines

Polio RIVMRabies Pasteur MerieuxFlue Duphar

Foot and mouth disease ID-LelystadClassical swine fever ID-Lelystad/Bayer

Other Intervet


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Dangers

1955 Bad Polio vaccinebatch

250 people ill

11 dead


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Food and Drug Administration

(FDA)

Food and Drug Act

Biological and non-biological

Public Health Act

Biological, pre-market approval


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Over the counter drugs (aspirin)

Prescription (most biologicals)

Pioneer

Generic

Products

New Drug Application

Abbreviated NDA


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Biologicals

Viruses Therapeutic sera

Toxins & antitoxin

Vaccines

Blood products

Cell & gene therapeutics

Therapeutic proteins

Oligonucleotides (antisense)

Peptides/hormones


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Approval Processes

Preclinical

Phase 1

Phase 2

Phase 3

Basic safety: dosagesCells,Guinea pigs

Pharmacological actions,Product safety & side effects

20-50humans

Effectiveness: optimal dosage,

application scheme etc.

50-200

patientsFinal safety and effectivenessHundreds

patients

Investigational new drug application (IND)


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Product License Application

Establishment License Application

Only for biologicals

All changes must be FDA approved

Product must be in phase 3 Good Manufacturing Practice (GMP)

Applications


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Time, finances, scale

Basic research

IND application

Phase 1

Phase 2

Phase 3PLA 1-3 3-100

1-3 0.5-5 1

0.5-2

0.5-35-100 10

1-5 100-1000

10 250

Years Dollars106

Prod. Scaledm3

Total


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Fast track

Clear public health advantage

Orphan drugs

Live threatening diseases Clear therapeutic advances


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Product in trials = product on market

Production process fixed

Each change requires FDA approval

Demonstrate product not changed

CO2 Problem

Notes


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Documentation

Extensive

Integrity must be validated

Electronic documents

Field compliance staff

Unannounced inspections

Criminal prosecution

Imprisonment (board of directors)

Other

Documents

Animal Cell Cultures