07/07/2015

Cell Cultures Troubleshooting

Salomé Pires, Biophysics Unit, University of Coimbra
[email protected]

III Cell Culture and Tissue Training Course

Coimbra | 7th July 2015

Overview

 Good practices
 Problems subculturing adherent cells
 Problems subculturing suspension cells
 Slow cell growth
 No viable cells after thawing
 Golden rules

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Good practices
Before working
• Keep all work surfaces clean and tidy.
• Disinfect your gloves, cabinet, surfaces, all materials and equipment
before putting them into the cabinet.
• Examine cultures and media daily for evidence of gross bacterial or
fungal contamination.

During work
• Bottles with growth media and reagents are opened only inside the
hood and for the lowest time possible.
• To avoid cross contamination, cell lines should be manipulated one at a
time.

After completing work

• Work surfaces inside the hood must be sprayed with 70% ethanol;
the gloves must be discarded.
• Incubators, cabinet, centrifuges and microscopes must be cleaned
regularly.

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Cell growth and propagation

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Cell growth and propagation

That’s important to know cells normal growth conditions

Calculate the population

doubling time

Subculturing success depends on the time of

cell growth in which it is made
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Cell growth and propagation

To ensure that the characteristics of your cell line remain

constant:

 Same medium
 Same serum
 Same supplements
 Same subculturing regimen

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Cell growth and propagation

Pay attention to media and supplements formulations…

 Read descriptions
 Read formulations

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PROBLEMS WITH SUBCULTURING

ADHERENT CELLS

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If cells are difficult to remove

I. Inhibitors in medium have inactivated the dissociating agents

a) Rinse the cell monolayer with PBS before adding the
dissociating solution
II. The dissociation solution was too weak
a) Use higher enzymes concentration
b) Incubate cells at 37ºC
III. Cells have been confluent for too long
a) Subculture cells before they become confluent

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If cells form clumps after dissociation

I. The dissociation procedure was too harsh and genomic DNA

was released from lysed cells
a) Add DNAse (1 mg/ml in water) to the cell suspension to
break down DNA strands

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If cells form clumps after dissociation

I. The dissociation procedure was too harsh and genomic DNA

was released from lysed cells
a) Add DNAse (1 mg/ml in water) to the cell suspension to
break down DNA strands
II. Cells were centrifuged too hard or too long
a) Be sure to use gentle
centrifugation (10 minutes, 125 G)

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If cells do not reattach

I. The dissociation procedure was too long

a) Reduce the exposure time to the dissociation solution (e.g.
trypsin)
II. Insufficient serum or attachment factors were present in medium
a) Add attachment factors to the medium and/or use a protein-
coated flask

(e.g. colagen, poly-L-lysine,

fibronectin, gelatin, etc.)

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If cells do not reattach

I. The dissociation procedure was too long

a) Reduce the exposure time to the dissociation solution (e.g.
trypsin)
II. Insufficient serum or attachment factors were present in medium
a) Add attachment factors to the medium and/or use a protein-
coated flask
III. Trypsin was not inactivated
a) Be sure to use enough fresh medium to inactivate dissociation
enzymes

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New options

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PROBLEMS WITH SUBCULTURING

SUSPENSION CELLS

If viability is lower than expected

I. Cell suspension was left too long at too high cell concentration
a) Medium become yellow (acidic pH)
b) Completely change the medium by gently centrifugation and
resuspend in fresh medium

Fresh
medium
8 minutes, 125 G
Pellet

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If viability is lower than expected

I. Cell suspension was left too long at too high cell concentration
a) Medium become yellow (acidic pH)
b) Completely change the medium by gently centrifugation and
resuspend in fresh medium
II. Cell suspension diluted below the recommended cell density range
a) Recover cells by centrifugation and resuspend in fresh medium
III. The medium and/or supplements were faulty
a) Use recommended formulations

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SLOW CELL GROWTH

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Slow cell growth

Possible Causes
Growth medium is not correct

Serum in the growth medium is of poor quality

Cells have been passaged too many times

Cells were allowed to grow beyond confluency

Culture is contaminated with mycoplasma

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Growth medium is not correct

Use pre-warmed growth medium as recommended by the supllier

Responsible for:
- Nutrients and metabolism
- Ionic balance
- pH control

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Growth medium constituents

Sodium - Regulating the CO2 levels

Bicarbonate - Maintain the optimal pH range

- Buffer medium pH
Hepes
- Present in certain formulations

Phenol Red - To monitor medium pH

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Growth medium constituents

Optimal pH at 7,0 – 7,4

Change Change Check CO2

Leave
today within 24-48h bottle

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Growth medium constituents

Sodium - Regulating the CO2 levels
Bicarbonate - Maintain the optimal pH range

- Buffer medium pH
Hepes
- Present in certain formulations

Phenol Red - To monitor medium pH

- Essential amino acid very unstable

L-Glutamine
used as energy source

Nonessential
amino acids

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Growth medium constituents

Sodium - Important for cellular metabolism

pyruvate

Other supplements non-available in the

base media and serum

- Hormones
- Growth factors
- Signaling substances
- Antibiotics
- Fetal bovine serum

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Slow cell growth

Possible Causes
Growth medium is not correct

Serum in the growth medium is of poor quality

Cells have been passaged too many times

Cells were allowed to grow beyond confluency

Culture is contaminated with mycoplasma

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FBS can be of poor quality

Provides:
- A source for amino acids, vitamins,
carbohydrates, lipids, growth factors, minerals
and tarce elements
- Support the growth of cells in culture

- Use serum from a different lot

- Check if heat-inactivation is needed
- Always test a sample before using a new lot
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Slow cell growth

Possible Causes
Growth medium is not correct

Serum in the growth medium is of poor quality

Cells have been passaged too many times

Cells were allowed to grow beyond confluency

Culture is contaminated with mycoplasma

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“Over-passaging”

Use healthy, low-passage-number cells

Continual passage will result in:

- Genotypic drift
- Phenotypic drift

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“Over-passaging”

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Adequate use

Controlled
Passage storage

Cryo-
Thaw Subculture
preserve
Optimal Optimal cell
resuscitation environment and
protocol subculture
protocol
Test cell identity
Test for mycoplasma
III Cell Culture and Tissue Training Course | Cell Cultures Troubleshooting Adapted from ECACC/Sigma Aldrich 30

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“Over passaging”

OVER Controlled
Passage storage

Cryo-
Thaw Subculture
preserve
Optimal Optimal cell
resuscitation environment and
protocol subculture
protocol
Test cell identity
Test for mycoplasma
III Cell Culture and Tissue Training Course | Cell Cultures Troubleshooting Adapted from ECACC/Sigma Aldrich 31

Slow cell growth

Possible Causes
Growth medium is not correct

Serum in the growth medium is of poor quality

Cells have been passaged too many times

Cells were allowed to grow beyond confluency

Culture is contaminated with mycoplasma

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Subculturing with high confluency

Passage cells when they are in the log-phase before they

reach confluency

III Cell Culture and Tissue Training Course | Cell Cultures Troubleshooting Adapted from ECACC/Sigma Aldrich 33

Slow cell growth

Possible Causes
Growth medium is not correct

Serum in the growth medium is of poor quality

Cells have been passaged too many times

Cells were allowed to grow beyond confluency

Culture is contaminated with mycoplasma

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Contamination

Discard cells, media and reagents.

Obtain new stock of cells and use fresh medium and reagents

Important questions:
 Is it confined to a single user or
widespread?
 Is it sporadic, repeated or
continuous?
 Single species or multi-specific?
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Contamination

Different sources:
- Cell lines
- Reagents
- Supplies (pipettes, culture vessels, tubes, etc.)
- Equipment (cabinet, incubator, centrifuge, etc,)
- Personnel

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Biological contaminants

Bacteria

Yeast

Molds

Viruses

Mycoplasma

Cross-contamination

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Bacterial contamination

Distinct changes:
- Turbidity
- Presence of particles in suspension
- Rapid pH decline

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Bacterial contamination
What to do?
- Eliminate a contamination from a cell line is time
consuming and does not always work
- Discard the culture and starting over is preferred

What if cells are unique and irreplaceable?

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Bacterial contamination

If cells are unique and irreplaceable…

1. Identify the contaminant
2. Select a suitable antibiotic for treatment
3. Culture cells for 1 to 2 weeks with antibiotic
4. Culture cells for 1 to 2 months without antibiotic
5. Test it for contamination using a very sensitive test

- Periodic retesting must be employed

NOTES:
- Validate the cured cultured to determine if it is sufficiently similar to the original

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Mycoplasma contamination
Distinct changes:
- Smallest self-replicating organism
- No visible signs of infection
- Very difficult to detect

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Why test for mycoplasma?

Mycoplasma can:
- Affect the rate of cell growth – but not always*
- Induce morphological changes – but not always*
- Cause chromosomal aberrations (Paton et al Nature 1965, 207 (992): 43)
- Influence amino acid and nucleic acid metabolism
(*MacPherson, J Cell Sci 1966 I,145)

- Induce cell transformation (Zhang et al Proc Soc Exp Biol Med 1997 214 (4):359)
- INVALIDATE RESEARCH!!

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Mycoplasma detection

Mycoplasma-free Cells infected with mycoplasma

cultured cells

 Mycoplasmas have no cell wall – most antibiotics act on cell wall.

 No antibiotic will kill Mycoplasma – just slow its growth

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Tests for mycoplasma

Regulatory authorities currently only recognize
 Culture Isolation
 Indirect DNA Stain

Other Methods for Mycoplasma Testing:

• Enzymatic: Myco Alert (Lonza)
• Colorimetric: Plasmotest (Invivogen), MycoProbe (R&D)
• Fluorescence: Mycoplasma Stain kit (Sigma)
• PCR (Stratagene): Mycoplasma Plus PCR Primers
• PCR (Sigma): LookOut and VenorGeM
• RT PCR: Sensitive – large capital outlay

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NO VIABLE CELLS AFTER

THAWING

No viable cells after thawing

Possible Causes
Cells were stored incorrectly

Home made freezer stock is not viable

Cells were thawed incorrectly

Glycerol used in freezing medium was stored in light

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Cells were stored incorrectly

Obtain new stock and store in liquid nitrogen.

Keep cells in liquid nitrogen until thawing

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No viable cells after thawing

Possible Causes
Cells were stored incorrectly

Home made freezer stock is not viable

Cells were thawed incorrectly

Glycerol used in freezing medium was stored in light

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Home made freezer stock is not viable

Ensure that…

 Freeze cell at a density recommended by the supplier

 Use low-passage cells to make your own freezer stocks

 Follow procedures for freezing cells exactly as recommended by

?
the supplier
- DMSO (5%)
I. Composition of the freezing medium - Medium (95%)
II. Cryoprotectant
III. Number and concentration of cells in the vial

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No viable cells after thawing

Possible Causes
Cells were stored incorrectly

Home made freezer stock is not viable

Cells were thawed incorrectly

Glycerol used in freezing medium was stored in light

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Cells thawed incorrectly

Make sure that…

 Thaw the frozen cells quickly

 Dilute them slowly using pre-warmed growth medium
 Plate thawed cells at high density to optimize recovery
 Use the medium recommended by the supplier

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No viable cells after thawing

Possible Causes
Cells were stored incorrectly

Home made freezer stock is not viable

Cells were thawed incorrectly

Glycerol used in freezing medium was stored in light

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Glycerol stored in light

In case of using glycerol as cryoprotectant…

It is
converted to Toxic to
Glycerol acrolein
cells

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GOLDEN RULES

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 Clearance and segregation

 Only one cell line in a cabinet at any time
 Regularly refresh spent media pots
 Regularly check for Mycoplasma and Identity
 Work “clean to dirty”
 Establish a “quarantine regime”
 Bank and cryopreserve your cells properly
 Aliquot prepared medium and reagents into usable volumes
 If in doubt....throw it out!
 Adequate air changes between cell lines
 Cell counting is the norm...not the exception!
 Use valid cell stocks
 Understand the biology – plot a growth curve
 Regularly check morphology and photo-document

III Cell Culture and Tissue Training Course | Cell Cultures Troubleshooting Adapted from ECACC/Sigma Aldrich 55

Bibliography

 ATCC® Animal Cell Culture Guide, 2014

 R. Ian Freshney,; Culture of Animal Cells – A Manual of Basic Techniques, 5th Edition,
New Jersey, 2005
 Cell Culture Basics Handbook, Invitrogen,
 www.invitrogen.com/cellculturebasics
 Fundamental Techniques in Cell Culture, 2010, ECACC/Sigma Aldrich
 www.hpacultures.org.uk
 Peyton Hughes et al., The costs of using unauthenticated, over-passaged cell lines:
how much more data do we need?, BioTechniques 43:575-586, 2007.
 Thomas Hartung et al., Good Cell Culture Practice, ATLA 30, 407-414, 2002.

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Thanks for your attention!!

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