Cell Culture Techniques and Best Practices

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 5/24/2012 | Life Technologies™ Proprietary and confidential

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!

Watch this as a webinar

http://find.lifetechnologies.com/gibco/mythbusterswebinar/sldshr

3 5/24/2012 | Life Technologies™ Proprietary and confidentialWatch this as a webinar



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.




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.




BHK-21 Confluent?




Hepatocytes ?




Will This Result in a Culture Change?




Cell Culture Media: Components & Characteristics




Cell Culture: Media, Reagents

Cell culture media is a mixture of components

used to simulate the natural environment of

the cell.

Components Commonly Added to Culture Media

Inorganic Salts

− Bulk Ion Salts and Trace Elements

Amino Acids

− Essential and Non-Essential

Vitamins

Sugars

Phenol Red




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.




Mole. Weight Conc. (mg/L) Molarity (mM) INORGANIC SALTS Calcium Chloride (CaCl2) (anhyd.) 111 200.00 1.80 Ferric Nitrate (Fe(NO3)3-9H2O) 404 0.10 0.000248 Potassium Chloride (KCl) 75 400.00 5.30 Magnesium Sulfate (MgSO4) 120 97.67 0.813 Sodium Chloride (NaCl) 58 6400.00 110.34 Sodium Bicarbonate (NaHCO3) 84 3700.00 44.10 Sodium Phosphate (NaH2PO4-H2O) 138 125.00 0.906 OTHER COMPONENTS D-Glucose 180 4500.00 25.00 Phenol red 376.4 15.00 0.0398 AMINO ACIDS L-Arginine-HCl 211 84.00 0.398 L-Cystine 2HCl 313 63.00 0.200 L-Glutamine 146 584.00 4.00 Glycine 75 30.00 0.399 L-Histidine HCl-H2O 210 42.00 0.20 L-Isoleucine 131 105.00 0.802 L-Leucine 131 105.00 0.802 L-Lysine-HCl 183 146.00 0.798 L-Methionine 149 30.00 0.201 L-Phenylalanine 165 66.00 0.400 L-Serine 105 42.00 0.400 L-Threonine 119 95.00 0.078 L-Tryptophan 204 16.00 0.078 L-Tyrosine 2Na 2H20 261 104.00 0.398 L-Valine 117 94.00 0.803 VITAMINS D-Ca pantothenate 477 4.00 0.0083 Choline Chloride 140 4.00 0.0285 Folic Acid 441 4.00 0.00906 i-Inositol 180 7.20 0.04 Niacinamide 122 4.00 0.0328 *Pyridoxine HCl 206 4.00 0.0194 Riboflavin 376 0.40 0.00106 Thiamine HCl 337 4.00 0.0118

DMEM Formulation Mole. Weight Conc. (mg/L) Molarity (mM) INORGANIC SALTS Calcium Chloride (CaCl2) (anhyd.) 111 200.00 1.80 Ferric Nitrate (Fe(NO3)3-9H2O) 404 0.10 0.000248 Potassium Chloride (KCl) 75 400.00 5.30 Magnesium Sulfate (MgSO4) 120 97.67 0.813 Sodium Chloride (NaCl) 58 6400.00 110.34 Sodium Bicarbonate (NaHCO3) 84 3700.00 44.10 Sodium Phosphate (NaH2PO4-H2O) 138 125.00 0.906 OTHER COMPONENTS D-Glucose 180 4500.00 25.00 Phenol red 376.4 15.00 0.0398 AMINO ACIDS L-Arginine-HCl 211 84.00 0.398 L-Cystine 2HCl 313 63.00 0.200 L-Glutamine 146 584.00 4.00 Glycine 75 30.00 0.399 L-Histidine HCl-H2O 210 42.00 0.20 L-Isoleucine 131 105.00 0.802 L-Leucine 131 105.00 0.802 L-Lysine-HCl 183 146.00 0.798 L-Methionine 149 30.00 0.201 L-Phenylalanine 165 66.00 0.400 L-Serine 105 42.00 0.400 L-Threonine 119 95.00 0.078 L-Tryptophan 204 16.00 0.078 L-Tyrosine 2Na 2H20 261 104.00 0.398 L-Valine 117 94.00 0.803 VITAMINS D-Ca pantothenate 477 4.00 0.0083 Choline Chloride 140 4.00 0.0285 Folic Acid 441 4.00 0.00906 i-Inositol 180 7.20 0.04 Niacinamide 122 4.00 0.0328 *Pyridoxine HCl 206 4.00 0.0194 Riboflavin 376 0.40 0.00106 Thiamine HCl 337 4.00 0.0118

Procure raw materials

QC test ALL raw materials

Manufacture under cGMP conditions

QC Performance test

Release to customer

Quality and Reproducibility




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




MYTH…Adding more Glutamine is Good

+ FBS

+ L-glutamine

Efficient energy metabolism High growth yield

GlutaMAX™ is better!

Ammonia &pryyolidinecarboxylic acid




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




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

Cell Culture: Water




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.

Cell Culture: Serum




MYTH: All Sera is the same…..!

Qualified FBS - US

Certified FBS - US

FBS – New Zealand

FBS – Australian

Chicken serum

Horse serum Goat serum

Lamb serum

NB calf serum

Porcine serum

Rabbit serum

Charcoal Stripped FBS

Dialyzed FBS

ESC Qualified FBSMSC Qualified FBS

Ultra-low IgG FBS

Qualified FBS–USDA, SA

Que Sera Sera…..?




Robust /Not Sensitive Finicky/Sensitive

HEK293

HeLaCHO

3t3

Fibroblast

Embryonic

stem cells

Mesenchymal

stem cells

Adipose-derived

stem cells

Jurkat

Primary

fibroblasts

HUVEC

A549

MCF-7

Primary

Neurons

But we work with a broad range of cells…..




• 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

Use a serum that meets your research needs




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




A549 cells grow consistently in Gibco® Certified, US

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0.90

Passage 1 Passage 2 Passage 3 Passage 4 Passage 5

Via

ble

Cell

s/m

L (

x1

06)

A549

Qualified, USDA

Certified, US

Competitor P

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Via

ble

Cell

s/m

L (

x1

06)

MCF-7

Qualified, USDA

Certified, US

Competitor P

MCF-7 cells grow consistently in Gibco® Certified, US

‘Consistency of cell growth over time & passages’ is the # 1 need for sera - according to researchers*

* Percepta Life Sciences quantitative market research for Sera VOC Need-Gaps, Nov 2010 among US & EU researchers.




HEK293 cells grow consistently in Gibco® USDA Qualified

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0.50

1.00

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2.00

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3.00

3.50

4.00

4.50


Via

ble

Cell

s/m

L (

x1

06)

HEK293

Qualified, USDA

Certified, US

Competitor P

‘Consistency of cell growth over time & passages’ is the # 1 need for sera - according to researchers




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




Gibco Qualified ESC FBS Performance Comparison

•Pluripotency Gene• Transcription factor essential

for self-renewal

• Controls multiple key transcription factors

• GIBCO® ES Cell FBS lots are selected for high expression

•Differentiation Gene• Expressed at early stages of

development

• Indicator of early differentiation

• GIBCO® ES Cell FBS lots are selected for low expression

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Control gibco ES Cell FBS Qualified by New Assay

gibco Non-ES Cell FBS

Competitor M Competitor S

Leve

l of

Plu

rip

ote

ncy

Expression of Pluripotency Gene

0.00

0.50

1.00

1.50

2.00

2.50

Control gibco ES Cell FBS Qualified by New Assay

gibco Non-ES Cell FBS

Competitor M Competitor S

Leve

l of

Dif

fere

nti

atio

n

Expression of Differentiation Gene




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®


http://www.invitrogen.com/imatch



Cell Culture Media: Effect Of Environmental Factors




Effect of Light on Media Performance

120%

0%

20%

40%

60%

80%

100%

0.5

hours 2.25

hours

4.25

hours

6.25

hours

Light

No Light

Sp2 Growth as a Percent of Control




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

Freeze serum only one time

Do not freeze basal media

PROTECT FROM LIGHT

Minimize the time media is at 37oC.

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 .




“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




MYTH – Nothing’s as Good as Trypsin to Detach Adherent Cells

Mean Time Required for Cell Release

0

5

10

15

20

25

30

35

40

MDCK PK-15 A549 MSC SFM MSC+10%FBS

Cell Lines

Tim

e f

or

ce

ll r

ele

as

e (

Min

ute

)

Trypsin-EDTA

1x TrypLE

1x TrypLE Diluted

10x TrypLE

HyQTase

Accutase

TrypZean

• Compared TrypLE™ Select 10x with Trypsin and other cell dissociation products

• Tested with 5 strongly adherent cell types

TrypLE™ Select 10x – superior performance




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




Cell Culture: Tips & Tricks




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.




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




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




Resources & Sampling Offers

Glutamax www.lifetechnologies.com/glutamaxTrypLE www.lifetechnologies.com/trypLEGibco FBS Samples www.lifetechnologies.com/fbsGibco ES Cell FBS www.lifetechnologies.com/escfbs Cell Culture Basics Videos www.lifetechnologies.com/cellculturebasics


http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/media-supplements/GlutaMAX-Media.html?CID=mythbusters-slideshare-52012





http://www.invitrogen.com/gibco-cell-culture-basics

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/reagents/Trypsin/TrypLE-Express.html?CID=mythbusters-slideshare-52012







http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/fbs.html?CID=mythbusters-slideshare-52012





http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Stem-Cell-Research/Embryonic-Stem-Cells/ESC-Stem-Cell-Culture-Media-Reagents/es-cell-qualified-fbs.html?CID=mythbusters-slideshare-52012






http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics.html?CID=mythbusters-slideshare-52012





http://www.invitrogen.com/site/us/en/home/Global/forms/pdf-request-form.html?CID-mythbusters-slideshare-52012



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Cell Culture Techniques and Best Practices

Education

Transcript of Cell Culture Techniques and Best Practices