Download Keeping Cells Happy – Topics in Cell Health Maintenance

Keeping Cells Happy – Topics in Cell
Health Maintenance and Viability
S te v e n B u d d , M . S . , M . B . A
P r o d u c t L i n e B u s i n e s s S p e c i a l i st , ATC C
S e p te m b e r 2 9 , 2 0 1 6
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Outline
Cell health topics
 Overview
 Complete growth media
 Cryopreservation
 Cell growth and propagation
 Contamination and aseptic technique
 Cell authentication
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Overview of cell health
Cell culture practices that involve good cell
health techniques cover a wide range of topics,
including:
 Measuring and monitoring growth
 Good aseptic technique
 Addressing cryopreservation issues
 Cell line authentication
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Complete growth media
 Classical cell culture media
 Media ingredients
 Additives
 Animal sera
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Classical cell culture media
Eagle’s Minimum Essential Medium (EMEM; ATCC® 30-2003™)
 Early formula for mouse L cells
 Amino acid composition to match mammalian cells
Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC® 30-2002™)
 Twice the amino acid concentration as EMEM, four times the
vitamin concentration
Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC® 30-2005™)
 Additional amino acids as DMEM, suited for lymphocytes and
hybridomas
Kaighn’s Modification of Ham’s F-12 (ATCC® 30-2004™)
 Designed for primary cells with or without serum
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Classical cell culture media
DMEM/ F12 Medium (ATCC® 30-2006™)
 Mixture of DMEM and Ham’s F-12, used in a wide range
of cell types
 Proliferation assays
McCoy’s 5A (ATCC® 30-2007™)
 Hepatoma cells and primary cells
RPMI-1640 (ATCC® 30-2001™)
 Ideal for Lymphocytes
Leibovitz’s L-15 (ATCC® 30-2008™)
 For use without CO2
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Media ingredients
Sodium bicarbonate
H2CO3
H2O + CO2
H2O + CO2
NaHCO3
H+ + HCO3H+ + 2HCO3-
HEPES buffer
 Can buffer without CO2 enrichment
 Good for working under the hood
Phenol Red
 Monitors pH of media
 Yellow = acidic
 Purple = basic
 May mimic action of steroid hormones.
Sodium Pyruvate
 Helps maintain metabolism
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Additives
Nonessential Amino Acids
 Can be added to reduce the metabolic burden on
cells
L-Glutamine (ATCC® 30-2214™)
 Present in ATCC classical cell culture media
 Relatively stable in bottles kept at 4°C - 8°C
 Glutamine degradation increases ammonia
toxicity
 Generally not recommended to “spike” media
with L-Glutamine
Antibiotics and Antimycotics
 Penicillin-Streptomycin, Gentamicin Sulfate
 Amphothericin B
 Generally not recommended
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Animal sera
Fetal Bovine Serum (ATCC® 30-2020™)
Fetal Bovine Serum, Embryonic Stem Cell
Qualified (ATCC® SCRR-30-2020™)
 Very rich in growth factors, most common
choice
 Heat inactivation: Not Advised
Calf Bovine Serum (ATCC® 30-2030™)
 Lower concentrations of growth factors,
good for contact inhibition studies
Horse Serum (ATCC® 30-2040™)
 Collected from closed herds, lot-to-lot
consistency, no bovine viruses
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Media usage considerations
Maintain cells in the same media
Media variability
 Possible osmotic shock
When transferring to new media:
 Use 1:1 mix (50% old, 50% new media)
 1:2 mix
 1:3 mix
 1:7 mix
ATCC® Animal Cell Culture Guide
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Cryopreservation procedure
 Overview
 Cryoprotectants and media preparation
 Freezing cells in a controlled-rate chamber
 Long-term storage
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Cryopreservation principles
Ideal
Negative effect
on viability
High
Ice
formation
Solute
concentration
Low
0.1
1.0
Rate of cooling (°C/min)
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 High levels of ice formation and increased solute concentration have a
negative impact on cell viability
 Optimal cooling rate for cell viability is 1°C/min to 3°C/min
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Cryoprotectants
Cell type
Cryoprotectant
Temperature
Number of cells
Animal cells
DMSO (5-10%) or
Glycerol (5-10%)
-140°C
106 to 107/mL
Bacteria
Glycerol (5-10%)
-80°C
107/mL
Yeast
Glycerol (10%)
-140°C
107/mL
Protozoa
DMSO (5-10%) or
Glycerol (10-20%)
-140°C
105 to 107/mL
Plant cells
DMSO (5-10%) and
Glycerol (5-10%)
-140°C
3% to 20% cell volume
Animal viruses (free)
None
-80°C
NA
Animal viruses
(infected cells)
DMSO (7%)
-10°C
106/mL
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Media preparation
Classical cell culture media – DMEM, EMEM,
RPMI-1640 (for suspension cells)
 5-10% DMSO
 20% fetal bovine serum (FBS) or bovine
serum albumin (BSA)
• Additional cryprotectant properties
• Necessary for post-thaw cell survival
ATCC Serum-free Freezing Media (ATCC® 302600™)
 All in one media
 10% DMSO with proteins and additives for
cell survival
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Freezing cells
-70°C
Controlled rate freeze chamber
-1°C/min cooling rate
A few hours to 24 hours
-140°C
Liquid nitrogen tank
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Freezing cells
CoolCell® LX (ATCC® ACS-6000™)
 Reliable -1°C/min cooling rate
 4 hours in -70°C freezer
 Comfortable to touch
 No alcohol use or maintenance
 Can be used with all cell types
Insulated
polyethylene
material
Thermoconductive
alloy
• Verified use with organoids
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Low temperature storage
For the best security, always store your cells in
liquid nitrogen freezers
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Low temperature storage
Mammalian cells
Long-term storage should be below -140°C
 -140°C for an indefinite length of time
 -80°C for less than 1 year
Vials should be stored in a liquid nitrogen unit
above the volume of liquid at the bottom of the
tank
This temperature should be between -140°C
and -180°C
-150°C
-178°C
-196°C
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Cell growth and propagation
 Population doubling level
 Measuring cell viability and growth
• MTT Assay
• XTT Assay
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Population doubling level
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Growth and viability
 Quantitative evaluation of cell proliferation rate and response to external
factors that affect cell viability
 Uses tetrazolium salts in a colorimetric method for evaluating cell
populations
 MTT Cell Proliferation Assay (ATCC® 30-1010K™)
• Tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium
bromide)
 XTT Cell Proliferation Assay (ATCC® 30- 1011K™)
• Tetrazolium XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium)
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Population doubling level
MTT Reaction
XTT Reaction
MTT salt is reduced within cellular
matrix to Formazan, lysed with
detergent to solubilize crystals
XTT salt is reduced to Formazan at cell
membrane with PMS agent
Media turns PURPLE
Media turns ORANGE
Detergent
Reagent
Formazan MTT
NADH NAD+
NADH NAD+
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Growth and viability
Determining Optimal Cell Counts
 Plate, in triplicate, a serial dilution of 1 x 106 to 1 x 103 cells per mL (96-well plate)
MTT Assay
XTT Assay
Add MTT Reagent
Add XTT Reagent + Activation Agent
Incubate 2-4 hours – add Detergent
Incubate 2-4 hours
Incubate 2-4 hours or overnight
 Determine optimal number of cells to use
 Repeat assay with experimental factors – compare absorbance at optimal cell
volume
 Plots absorbance versus cell number
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Growth and viability
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Contamination and aseptic technique
 Sources of contamination
 Aseptic technique
 Mycoplasma contamination
 Cross-contamination
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Sources of contamination
Types
 Microbial – Bacteria, mycoplasma, fungi,
viruses
 Cellular – Cross-contamination
Signs (bacteria and fungi)
 Turbid media
 Rapid decline in pH – color change
 Morphological changes
 Filamentous structures
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Sources of contamination
Contaminated Resources
 Cell Lines
 Media, Sera, Reagents
 Nonsterile equipment
Personnel and equipment
 Poor culturing practices
 Dust and aerosol
Physical Causes
 Improperly maintained autoclaves/ovens
 Faulty laminar flow
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Aseptic technique
Make it difficult for microorganisms to invade
culture vessels
 Sealed cultured vessels
 Vented cap flasks
Disposable aspirators and pipette tips
Cell culture hoods with good laminar flow
 Do not use as a storage area!
Spray media bottles / reagents with alcohol
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Aseptic technique
Use small volumes of reagents at a time
Use clean lab coats and protective clothing
AVOID USING ANTIBIOTICS IN MEDIA!
 Can contribute to chronic contamination
 Rarely completely prevents contamination
 Toxic to cells
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Mycoplasma contamination








Not easily detected, cannot be seen by microscopy
Chromosomal aberrations
Disruption of nucleic acid synthesis
Changes in membrane antigenicity
Inhibition of cell proliferation and metabolism
Decreased transfection rates
Changes in gene expression profiles
Cell death
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Mycoplasma contamination
PCR-based kit
Detects any of the 60 most
common mycoplasmas
Kit Components/Procedure
 Buffer
 Mix of primers specific to
mycoplasma
 Positive control
• DNA from mycoplasma
Collect/pellet cells
Cell lysis
Touchdown PCR
Run gel/stain
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Cross-contamination
Leads to the replacement of the original cell
line with the contaminant
Causes
 Multiple cell lines under the hood at the
same time
 Failure to change out pipettes
 Receiving cell lines from other labs
20% of scientific publications use misidentified
cultures
50% of preclinical research is not reproducible
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ATCC Short Tandem Repeat (STR) Profiling
Ensures your cells are what you think they are
 STR profile of your cell line
 Comparison of your cells against ATCC STR
Profile database
 Electropherograms supporting the allele calls
at each locus
 Comprehensive interpretation of results
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Summary points
Cell culture
• Select appropriate media for your cells
• Understand issues/considerations for adding
additional ingredients
• Consistently use same media whenever possible
Cryopreservation
• Use a reliable rate-controlled cooler
• Keep mammalian cells at -140°C for long term
• Keep cells in the vapor phase in liquid nitrogen
tanks
Contamination
control
• Follow aseptic technique
• Don’t rely on antibiotics/antimycotics
• Test for mycoplasma
• Make sure to authenticate cell lines
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Disclaimers
© 2016 American Type Culture Collection. The ATCC
trademark and trade name, and any other
trademarks listed in this publication are trademarks
owned by the American Type Culture Collection
unless indicated otherwise.
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