cell culture - iut auch
TRANSCRIPT
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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
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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Type of cells
Established cell lines Stern cellsPrimary cells
HeLa cellsPrimary cells
of Ormeau hematocytesMice stern cells
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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
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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
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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
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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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%
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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Cell culture supports
� In adhesion
� Supports:
� Petri dishes
� Culture flasks
� Slide
� Microcarrier
� Synthetic matrix
� Pretreatments:
� Chemical (Fibronectine,
Polylysine…)
� Physical (Plasma)
� In suspension
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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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
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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Biosafety cabinet
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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Cell counting
� Manual counter
� Malassez
� Thoma
� Neubauer
� Modified Neubauer
� Bürker
� Nageotte
� Lemaur
� Fuchs Rosenthal
� Electronic counter
LemaurMalassez Nageotte NeubauerThoma
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How to culture cells
� Different types of cell
� Cell culture medium
� Cell culture supports
� Incubator
� Biosafety cabinet
� Cell counting
� Cell maintenance
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Cell maintenance
Remove the cell medium
Wash with PBS
Remove the PBSTrypsinize the cells
Resuspend with medium
Dilute the cells
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Cell culture as a research tool
� Model system
� Protein production
� Drug screening and development
� Cell-based manufacturing
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Cell culture as a research tool
� Model system
� Protein production
� Drug screening and development
� Cell-based manufacturing
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Model system
� Cell growth parameters
� Cell morphology
� Metabolic and genetic alterations
� DNA, RNA, proteins levels
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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
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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
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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
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Cell culture as a research tool
� Model system
� Protein production
� Drug screening and development
� Cell-based manufacturing
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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
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Protein production
Insect cell line
� Recombinant proteins
� Interferon
� EPO
� Interleukin 2
� Tissue plasminogeneactivator
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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
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Cell culture as a research tool
� Model system
� Protein production
� Drug screening and development
� Cell-based manufacturing
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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
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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
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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
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Cell culture as a research tool
� Model system
� Protein production
� Drug screening and development
� Cell-based manufacturing
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Cell-based manufacturing
Insuline
(Diabete)
TPA
(Heart attack)