Cell culture I

Jean-Michel Escoffre

escoffre@ipbs.fr

I.U.T. Auch

2008 - 2009

History of cell culture

1885RouxCells canlive outsidethe body

1907HarrisonNerveoutgrowth

1952GeyEstablishedHeLa cells

1955EagleDefinedmedium

Late '50s and '60s Stoker, Dulbecco, GreenViral transformation

1961Hayflick/MoorheadFinite numberof divisions

1964LittlefieldSomatic cellhydrids

1965Ham Clonalgrowth ofmammalian cells

1986Martin/EvansMouse ES cells

1976SatoHormonesAnd growthFactors required inSF medium

1977WiglerAnd AxelTransfections

1998ThomsonAnd GearhartHuman ES cells

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Type of cells

Established cell lines Stern cellsPrimary cells

HeLa cellsPrimary cells

of Ormeau hematocytesMice stern cells

Established cell lines

� Proliferate indefinitely through random mutation or

deliberate modification

� Advantages:

� Many kinds of cell lines

� Generally easy to grow and manipulate

� Proliferate indefinitely

� Contact inhibition

� Limits:

� Ploidy problems

� Loss of biochemical properties of parent tissue

� Examples :

� HeLa, Henrietta Lacks, Human, Cervical cancer

� Sf-9, Spodoptera Frugiperda, Insect, Ovary

HeLa cells

Primary culture

� Remove from organ, trypsin digest and place in culture dish

� Advantages:

� Similar chromosome number as parent tissue

� Perform specialized biochemical properties as parent tissue

(Growth factors and hormones secretion…)

� Contact inhibition

� Limits:

� Limited lifespan

� Examples :

� MEF, Mouse Embryonic Fibroblasts

� ABAE, Adult Bovine Aortic Endothelial Cell

Primary cellsof Ormeau hematocytes

Stern cells

� Self-renewing cells that with proper growth conditions can

differentiate into different cell types with specific biological

functions

� Advantages:

� Can induce multiple cell types

� Potential for therapeutics

� Limits:

� Ethical considerations

� Most stern cells need to grow on feeder cells

� Examples :

� Human ES

Mice stern cells

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Cell culture medium

� Synthetic medium: Qualitative and quantitative compositions are well defined

� Complet medium:

� Ions

� Amino Acids

� Vitamins

� Decomplemented Fœtal Calf Serum:

� Heat-inactivation of complement and anitbodies

� FCS contributions:

� Albumin

� Transferrin: Mitotic activity

� Steroid hormone: Cell division

� Growth factors : cell growth

Water

68%

Inorganics

salts

11%

Serum

10%

Sugars

5%

Other

3%

Amino acids

2%Vitamins

1%

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Cell culture supports

� In adhesion

� Supports:

� Petri dishes

� Culture flasks

� Slide

� Microcarrier

� Synthetic matrix

� Pretreatments:

� Chemical (Fibronectine,

Polylysine…)

� Physical (Plasma)

� In suspension

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Incubator

� Temperature, 37°C

� 5% C02:

� CO2 + H20 � HCO3- + H+

� Prevent the cell death

� Medium pH : 7.2-7.4

� Phenol red : pH indicator

� 80% Humidity:

� Prevent cell plasmolysis

� Riks of contaminations: Use of fungicides/bactericides

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Biosafety cabinet

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Cell counting

� Manual counter

� Malassez

� Thoma

� Neubauer

� Modified Neubauer

� Bürker

� Nageotte

� Lemaur

� Fuchs Rosenthal

� Electronic counter

LemaurMalassez Nageotte NeubauerThoma

How to culture cells

� Different types of cell

� Cell culture medium

� Cell culture supports

� Incubator

� Biosafety cabinet

� Cell counting

� Cell maintenance

Cell maintenance

Remove the cell medium

Wash with PBS

Remove the PBSTrypsinize the cells

Resuspend with medium

Dilute the cells

Cell culture II

Jean-Michel Escoffre

escoffre@ipbs.fr

I.U.T. Auch

2008 - 2009

Cell culture as a research tool

� Model system

� Protein production

� Drug screening and development

� Cell-based manufacturing

Cell culture as a research tool

� Model system

� Protein production

� Drug screening and development

� Cell-based manufacturing

Model system

� Cell growth parameters

� Cell morphology

� Metabolic and genetic alterations

� DNA, RNA, proteins levels

Transfection

� Intoduction of foreign material into eukaryotic cells

� Genes

� Overexpression

� Protein production

� Messenger RNA

� Transient expression

� Protein activity

� Proteins

� Transient activity

� RNAi, morpholino…

� Downregulation

� Drugs

� Transient activity

Transfection

� Viral methods

� Adenovirus

� Retrovirus

� Lentivirus

� Baculovirus

� Chemical methods

� Calcium phosphate

� Dextran

� Cationic lipids/polymers (PEI, Fugene…)

� Liposome

� Peptides (SynB, Tat…)

� Physical methods

� Micro-injection

� Electroporation

� Sonoporation

� Magnetofection

mRNA

Protein

siRNA

Morpholino

Drugs

Gene

shRNA

Transient and stable expressions

� Transient expression

� No DNA insertion into host genome

� DNA lost after cell division

� Advantages

� Quick

� Easy

� Limits

� No control of protein expression

� Stable expression

� DNA inserted into host genome

� Selection pressure to maintain plasmid

� Advantages

� Cell lines

� Same protein level

� Limits

� Time consuming

� No control of DNA insertion

Cell culture as a research tool

� Model system

� Protein production

� Drug screening and development

� Cell-based manufacturing

Protein production

� Advantages� Large proteins (> 50 kDa)

� Correct glycosylation andpeptide signal

� Chaperones to help foldproteins

� High yield

� Cheap

� Limits� Low growth (10-12 days)

� Cell culture time (4-5 days)

� Time Consuming

Insect cell line

� Autographica californica multiple nuclear polyhedrosis virus (Baculovirus)

� Virus commonly infects insect cells of alpha looper (small beetle) or armyworms (and their larvae)

� Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insectcell

Protein production

Insect cell line

� Recombinant proteins

� Interferon

� EPO

� Interleukin 2

� Tissue plasminogeneactivator

Protein production

� Advantages

� Large proteins (> 50 kDa)

� Correct glycosylation andpeptide signal removal

� Generate authentic proteins

� Chaperones to help foldproteins

� Limits

� Selection time

� Cell culture time

� Time Consuming

� Cost

� Modest yields

Mammalian cell line

� Gene construct: Gene of interest, selection marker gene (NeoR, DHFR…), strong promoter (CMV, SV40…)

� Cell lines: CHO cells, HEK-293 cells and Cos cells

� Vectors: Electropororation, calcium phosphate, viral vector(retrovirus, lentivirus…)

� Expression: Transient, stable or episomal

Cell culture as a research tool

� Model system

� Protein production

� Drug screening and development

� Cell-based manufacturing

Drug screening/development

High Through Screening(HTS)

Chemical drugs, siRNA, ligand…

Cell assay to screenFor biological process, e.g.

Death cellsApoptosis

Cellularsignaling

Celldifferentiation

Cellproliferation

Cell culture

siRNA screening/development

Promoter Protein target eGFP

� Clone target protein

� Generation eGFP expression construct

� Generate stably transfected mammalian cell lines

� Visualize and quantify the inhibition of eGFP expression

using high troughput imaging

siRNA screening/development

Cells stablyexpressingtarget-eGFP

Advantages� Assay simplicity� Low reagent cost� Kinetic follow-up

Automated imagerQuantify inhibition of target-eGFP expression

with automated image analysis system on live cells

Add test siRNA

Plate cells(incubate overnight)

Different siRNA

siRNA concentration

Cell culture as a research tool

� Model system

� Protein production

� Drug screening and development

� Cell-based manufacturing

Cell-based manufacturing

Insuline

(Diabete)

TPA

(Heart attack)