Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. More cell culture techniques and best practices here. http://owl.li/dgS2Y

1. Watch the webinar
Cell Culture Best Practices
Busting Myths & Improving Outcomes
Gibco® Cell Culture
and
Timothy Fawcett, Ph.D.
BioSciConcepts/BioTechnical Institute of Maryland, Inc.

2. Agenda Overview
 Understanding the in-vitro system,
 Working with components of cell culture media & formulations, serum
performance characteristics,
 Aspects of cell culture productivity, inter-relationships between cell
growth cycle & its environment,
 Tips & techniques for maximizing recovery……and along the way we will
challenge lab-lores and bust some myths!
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4. Guidance on Good Cell Culture
Guidance has been prepared to promote an awareness of a
broad range of important issues in cell culture in workers
coming to use cells for their work for the first time, and to
remind others of the fundamental aspects of good practice in
cell and tissue culture.
Based on a report of the second European Centre for the Validation of Alternative Methods task force on
Good Cell Culture Practice; Coeche, et al, ATLA 33, 261-287, 2005.
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5. GCCP Guidance
Based Upon Six Operational Principles:
1. Establishment and maintenance of a sufficient understanding
of the in vitro system and of the relevant factors which could
affect it.
2. Assurance of the quality of all materials and
methods…reproducibility.
3. Documentation
4. Establishment and maintenance of adequate measures to
protect individuals and the environment from any potential
hazards.
5. Compliance with relevant laws and regulations, and with
ethical principles.
6. Provision of relevant and adequate education and training for
all personnel, to provide high quality work and safety.
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6. BHK-21 Confluent?
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7. Hepatocytes ?
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8. Will This Result in a Culture Change?
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9. Cell Culture Media:
Components & Characteristics
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10. Cell Culture: Media, Reagents
Cell culture media is a Components Commonly Added
to Culture Media
mixture of components
used to simulate the
natural environment of Inorganic Salts
the cell. − Bulk Ion Salts and Trace Elements
 Amino Acids
− Essential and Non-Essential
 Vitamins
 Sugars
 Phenol Red
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11. Media Components & Characteristics
 Cell culture media helps maintain correct osmotic pressure and
ionic equilibrium.
 Cell culture media must provide buffering capacity.
 pH needs to be maintained at pH=7.4
• The salts and amino acids in media provide some buffering capacity.
• H2CO2/NaH2CO3 system requires CO2 to maintain pH.
• If no CO2 is available use HEPES or NaH2PO4 buffer system.
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12. DMEM Formulation
Mole. Weight Conc. (mg/L)
Mole. Weight Molarity (mM)
Conc. (mg/L) Molarity (mM)
RGANIC SALTS INORGANIC SALTS Procure raw materials
ium Chloride (CaCl2) Calcium Chloride 111
(anhyd.) (CaCl2) (anhyd.) 200.00
111 1.80200.00 1.80
ic Nitrate (Fe(NO3)3-9H2O) Nitrate (Fe(NO3)3-9H2O)
Ferric 404 0.10404 0.000248
0.10 0.000248
ssium Chloride (KCl) Potassium Chloride (KCl)
75 400.00
75 5.30400.00 5.30
nesium Sulfate (MgSO4)Magnesium Sulfate (MgSO4)
120 97.67
120 0.813
97.67 0.813
um Chloride (NaCl) Sodium Chloride (NaCl)
58 6400.00
58 110.34
6400.00 110.34
um Bicarbonate (NaHCO3) Bicarbonate (NaHCO3)
Sodium 84 3700.00
84 44.10 QC test ALL raw materials
3700.00 44.10
um Phosphate (NaH2PO4-H2O)
Sodium Phosphate (NaH2PO4-H2O) 125.00
138 138 0.906
125.00 0.906
HER COMPONENTS OTHER COMPONENTS
lucose D-Glucose 180 4500.00
180 25.00
4500.00 25.00
nol red Phenol red 376.4 15.00
376.4 0.0398
15.00 0.0398
NO ACIDS AMINO ACIDS
Manufacture under
ginine-HCl L-Arginine-HCl 211 84.00
211 0.398
84.00 cGMP conditions
0.398
ystine 2HCl L-Cystine 2HCl 313 63.00
313 0.200
63.00 0.200
utamine L-Glutamine 146 584.00
146 4.00584.00 4.00
ine Glycine 75 30.00
75 0.399
30.00 0.399
stidine HCl-H2O L-Histidine HCl-H2O
210 42.00
210 0.2042.00 0.20
oleucine L-Isoleucine 131 105.00
131 0.802
105.00 0.802
ucine L-Leucine 131 105.00
131 0.802
105.00 0.802
sine-HCl L-Lysine-HCl 183 146.00
183 0.798
146.00 QC Performance test
0.798
ethionine L-Methionine 149 30.00
149 0.201
30.00 0.201
henylalanine L-Phenylalanine 165 66.00
165 0.400
66.00 0.400
erine L-Serine 105 42.00
105 0.400
42.00 0.400
hreonine L-Threonine 119 95.00
119 0.078
95.00 0.078
yptophan L-Tryptophan 204 16.00
204 0.078
16.00 0.078
rosine 2Na 2H20 L-Tyrosine 2Na 2H20
261 104.00
261 0.398
104.00 0.398
aline L-Valine 117 94.00
117 0.803
94.00 Release to customer
0.803
AMINS VITAMINS
a pantothenate D-Ca pantothenate477 4.00477 0.0083
4.00 0.0083
ine Chloride Choline Chloride 140 4.00140 0.0285
4.00 0.0285
c Acid Folic Acid 441 4.00441 0.00906
4.00 0.00906
sitol i-Inositol 180 7.20180 Quality and Reproducibility
0.047.20 0.04
inamide Niacinamide 122 4.00122 0.0328
4.00 0.0328
idoxine HCl *Pyridoxine HCl 206 4.00206 0.0194
4.00 0.0194
flavin Riboflavin 376 0.40376 0.00106
0.40 0.00106
mine HCl Thiamine HCl 337 4.00337 0.0118
4.00 0.0118
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13. Media Components & Characteristics
 Glutamine, a major catabolite for fast growing cells-Source of ATP
 Short half-life: 3 weeks at 4oC, 5 days at 37oC
 The rate of decomposition is pH dependent and temperature
dependent
 In rapidly growing cultures, glutamine levels can go to zero in 48
hours
 Media
 serves as a carbon source for purine biosynthesis.
 is a donor of primary amino groups, for the synthesis of pyrimidines and
asparagine
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14. MYTH…Adding more Glutamine is Good
+ L-glutamine
+ FBS
 Efficient energy metabolism
 High growth yield
GlutaMAX™ is better!
Ammonia &
pryyolidine
carboxylic
acid
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15. Adding GlutaMAX™ is BETTER
• L-glutamine and GlutaMAX supplemented into DMEM
• Left at 37⁰ C for 7 days
• Ammonia measured each day via HPLC
• Glutamine breakdown was rapid compared to GlutaMAX
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16. Cell Culture: Water
Cell culture must be of the highest quality
with little or no bio-burden present.
− Seasonal variation in water quality can be a potential problem
− Dissolved gasses, rust inhibitors and organics can be present in water
− Water used for cell culture must be highly purified
− Usually multiple approaches to purification are taken
− Water For Injection …e.g. Gibco® WFI
> same as in the cGMP / ISO controlled manufacturing process
> saves steps, time – already triple filtered
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17. Cell Culture: Serum
 Serum for cell culture
 A biological product isolated and processed under very stringent
QC & manufacturing conditions
 Provides additional nutrients and growth/attachment factors
 Serum is undefined ~10,000 components
 When serum has been added to basal media it is termed
COMPLETE MEDIA
 Animal Sera Provides: Growth Factors, Proteins, Attachment
Factors, Hormones, Lipids and Minerals and Other Nutrients.
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18. Que Sera Sera…..?
Certified FBS - US FBS – Australian
Horse serum Goat serum
MYTH: All Sera is the same…..!
Lamb serum
Ultra-low IgG FBS
Qualified FBS - US
NB calf serum
Rabbit serum
FBS – New Zealand
Dialyzed FBS
MSC Qualified FBS
Charcoal Stripped FBS ESC Qualified FBS
Porcine serum
Qualified FBS–USDA, SA Chicken serum
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19. But we work with a broad range of cells…..
Robust /Not Sensitive Finicky/Sensitive
CHO A549 Jurkat
HeLa Adipose-derived Embryonic
MCF-7 stem cells stem cells
HEK293 3t3 Primary
Fibroblast Mesenchymal
fibroblasts
stem cells
HUVEC
Primary
Neurons
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20. Use a serum that meets your research needs
• Lowest BSE risk/least viral load
• Lowest endotoxin levels
• Need for biochemical & hormonal profile certification
• Sensitive, finicky cell lines or general cell culture
• Specific applications and assays, need for OBS…..?
Choose the right sera for your specific research
Not all sera is qualified and validated for the right applications
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21. Sera Experimental Design
• Gibco® Certified, Qualified grade FBS – various lots
• Versus a competitor
• Tested with several human cell lines
HEK293: Human Embryonic Kidney - epithelial
HeLa (Henrietta Lacks): epithelial - cervix cancer
A549: epithelial- lung
MCF-7: epithelial - breast cancer
Jurkat: lymphoblast - cancer
• Measured cell growth and viability over 5 passages
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22. ‘Consistency of cell growth over time & passages’ is the # 1 need
for sera - according to researchers*
A549 MCF-7
0.90 2.00
0.80 1.80
1.60
0.70
Viable Cells/mL (x106)
Viable Cells/mL (x106)
1.40
0.60
1.20
0.50 Qualified, Qualified,
USDA 1.00 USDA
0.40 Certified, US Certified, US
0.80
0.30
Competitor P 0.60 Competitor P
0.20 0.40
0.10 0.20
0.00 0.00
Passage 1 Passage 2 Passage 3 Passage 4 Passage 5 Passage 1 Passage 2 Passage 3 Passage 4 Passage 5
A549 cells grow consistently in Gibco® MCF-7 cells grow consistently in Gibco®
Certified, US Certified, US
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* Percepta Life Sciences quantitative market research for Sera VOC Need-Gaps, Nov 2010 among US & EU researchers.

23. ‘Consistency of cell growth over time & passages’ is the # 1 need
for sera - according to researchers
HEK293
4.50
4.00
3.50
Viable Cells/mL (x106)
3.00
2.50 Qualified, USDA
2.00
Certified, US
1.50
1.00 Competitor P
0.50
0.00
Passage 1 Passage 2 Passage 3 Passage 4 Passage 5
HEK293 cells grow consistently in Gibco® USDA Qualified
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24. Gibco® ES Cells Qualified FBS
validated with qRT-PCR Assay
 Quantitative assay from Gibco® vs. traditional methods for other brands
 Improved lot-to-lot consistency of GIBCO® ESC FBS qualities
 Detection in variation of differentiation and pluripotency
 Fast and accurate quantitation of gene expression
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25. Gibco Qualified ESC FBS Performance Comparison
Expression of Pluripotency Gene
1.80
•Pluripotency Gene 1.60
Level of Pluripotency
1.40
• Transcription factor essential 1.20
for self-renewal 1.00
0.80
• Controls multiple key 0.60
transcription factors 0.40
0.20
• GIBCO® ES Cell FBS lots are 0.00
selected for high expression Control gibco ES Cell gibco Non-ES Competitor M Competitor S
FBS Qualified Cell FBS
by New Assay
Expression of Differentiation Gene
2.50
•Differentiation Gene
Level of Differentiation
2.00
• Expressed at early stages of 1.50
development 1.00
• Indicator of early differentiation 0.50
• GIBCO® ES Cell FBS lots are 0.00
selected for low expression Control gibco ES Cell gibco Non-ES Competitor M Competitor S
FBS Qualified Cell FBS
by New Assay
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26. Sera: Lab-Lores ?
 Concerns about variability when using FBS
− Unique Lot Matching Service for Gibco® Sera: Gibco® iMATCH™
> proprietary algorithm based on performance & test variables to offer closest
match to previous lots
> check www.invitrogen.com/imatch
 Concerns about Flocculence
− Only a coagulation of proteins, look like strings, not abnormal
 Sera (or Media) - cannot be sterile
− manufactured & tested for sterility
 FBS will influence or differentiate stem cells
− ES Cell Qualified FBS validated with qRT-PCR assay from Gibco®
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27. Cell Culture Media:
Effect Of Environmental Factors
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28. Effect of Light on Media Performance
120%
100%
80%
60%
Light
40% No Light
20%
0%
0.5 2.25 4.25 6.25
hours hours hours hours
Sp2 Growth as a Percent of Control
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29. Critical Parameters for Optimal Media Performance
 Recognize there is a natural decay rate of unstable compounds
in media
 Maintain correct pH, affects the stability of some compounds Qu i c k T i m e ™ a n d a
d e c o m p re s s o r
a re n e e d e d t o s e e t h i s p i c t u re .
 Freeze serum only one time
 Do not freeze basal media
 PROTECT FROM LIGHT
 Minimize the time media is at 37oC.
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30. “Trypsinization”, Detachment of Adherent Cells
 Trypsin
− Traditional, contains animal products.
− Cleaves arg and lys amino acids
− Needs to be quenched using serum or soybean trypsin inhibitor
− A mixture of enzymes
− Degrades itself
 TrypLE™ Express
− A recombinant fungal trypsin-like enzyme
− High purity
− Lower cell toxicity
− Room temperature stable
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31. MYTH – Nothing’s as Good as Trypsin to Detach
Adherent Cells
• Compared TrypLE™ Select 10x with Trypsin and other cell dissociation products
• Tested with 5 strongly adherent cell types
Mean Time Required for Cell Release
40
Trypsin-EDTA
35 1x TrypLE
Time for cell release (Minute)
1x TrypLE Diluted
30
10x TrypLE
25 HyQTase
Accutase
20 TrypZean
15
10
5
0
MDCK PK-15 A549 MSC SFM MSC+10%FBS
Cell Lines
TrypLE™ Select 10x – superior performance
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32. Using TrypLE™ Select 10x is BETTER
• Removing cells from plastic surface is ROUGH on cells
• TrypLE™ Select 1x or 10x is GENTLE
• TrypLE™ Select 1x or 10x is AOF
• TrypLE™ Select 1x or 10x is STABLE at room temperature
• Can be used on ALL CELL TYPES
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33. Cell Culture:
Tips & Tricks
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34. Tips: Freezing and Thawing
 Freeze cells slowly, 1oC /minute.
 Thaw as rapidly as possible to 37oC.
− Use a cup of 37oC water to move the vial of cells from freezer to 37oC for
rapid thawing.
− Place cells SLOWLY into pre-warmed media.
 Freeze early passage, healthy, well fed cells.
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35. Tips: Freezing & Thawing
 Use Conditioned Media
− Conditioned media is media that has been used to grow cells.
− Has unknown factors from paracrine growth.
− Great for getting cells through difficult times or when growing cells at very low density.
 Use a Styrofoam tray when organizing cells
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36. Tips: Efficiency & Reduced Risk of Contamination
 Aliquoting sera from 500 ml or liter bottles
− Will a 50 ml easy-to-use bottle be efficient…?
− Consider Gibco® 50ml One Shot™ FBS bottles
> easy to use, quicker to thaw
> reduced risk of contamination
 Also consider using Lab Armor™ Beads with Gibco® products
− efficient and reduced contamination risk compared to water baths
+ = Peace of Mind!
50 ml One Shot™ FBS Lab Armor™ Beads
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37. Resources & Sampling Offers
Glutamax www.lifetechnologies.com/glutamax
TrypLE www.lifetechnologies.com/trypLE
Gibco FBS Samples www.lifetechnologies.com/fbs
Gibco ES Cell FBS www.lifetechnologies.com/escfbs
Cell Culture Basics Videos www.lifetechnologies.com/cellculturebasics
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38. THANK YOU
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