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Principles of Cell Culture

Hessah Alshammari

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Cell Culture in vi tro- A brief history

1885: Roux maintained embryonic chick cells alive in saline solution for

short lengths of time

1912: Alexis Carrel cultured connective tissue and showed heart muscletissue contractility over 2-3 months

1943: Earle et al. produced continuous rat cell line

1962: Buonassisi et al. Published methods for maintaining differentiatedcells (of tumour origin)

1970s: Gordon Sato et al. published the specific growth factor and mediarequirements for many cell types

1979: Bottenstein and Sato defined a serum-free medium for neural cells

1980 to date: Tissue culture becomes less of an experimental researchfield, and more of a widely accepted research tool

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Isolation of cell lines for in vi troculture

Resected Tissue

Cell or tissue culture in vi t ro

Primary culture

Secondary cultureSub-culture

Cell Line

Sub-culture

ImmortalizationSuccessive sub-cultureSingle cell isolation

Clonal cell line Senescence

Transformed cell line

Immortalised cell line

Loss of control

of cell growth

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Primary cultures

Derived directly from animal tissue

embryo or adult? Normal or neoplastic?

Cultured either as tissue explants or single cells

Initially heterogeneousbecome overpopulated withfibroblasts

Finite life span in vi t ro

Retain differentiated phenotype

Mainly anchorage dependant

Exhibit contact inhibition

Types of cell cultured in vi t ro

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Types of cell cultured in vi t ro

Secondary cultures

Derived from a primary cell culture

Isolated by selection or cloning

Becoming a more homogeneous cell population

Finite life span in vi t ro

Retain differentiated phenotype

Mainly anchorage dependant

Exhibit contact inhibition

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Types of cell cultured in vi t ro

Continuous cultures

Derived from a primary or secondary culture

Immortalised: Spontaneously (e.g.: spontaneous genetic mutation)

By transformation vectors (e.g.: viruses &/or plasmids)

Serially propagated in culture showing an increasedgrowth rate

Homogeneous cell population

Loss of anchorage dependency and contact inhibition

Infinite life span in vi tro Differentiated phenotype:

Retained to some degree in cancer derived cell lines

Very little retained with transformed cell lines

Genetically unstable

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Cell morphologies vary depending on cell type

Fibroblastic

Endothelial

Epithelial

Neuronal

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Cell culture environment (in vi tro)

What do cells need to grow?

Substrate or liquid (cell culture flask or scaffold material)

chemically modified plastic or coated with ECM proteins

suspension culture

Nutrients (culture media)

Environment (CO2, temperature 37oC, humidity)

Oxygen tension maintained at atmospheric but can be varied

Sterility (aseptic technique, antibiotics and antimycotics)

Mycoplasma tested

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Cell culture environment (in vi tro)

Basal Media

Maintain pH and osmolarity (260-320 mOsm/L). Provide nutrients and energy source.

Components of Basal Media

Inorganic Salts

Maintain osmolarity

Regulate membrane potential (Na+, K+, Ca2+)

Ions for cell attachment and enzyme cofactors

pH IndicatorPhenol Red

Optimum cell growth approx. pH 7.4

Buffers (Bicarbonate and HEPES) Bicarbonate buffered media requires CO2atmosphere

HEPES Strong chemical buffer range pH 7.27.6 (does not require CO2)

Glucose

Energy Source

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Components of Basal Media

Keto acids (oxalacetate and pyruvate)

Intermediate in Glycolysis/Krebs cycle

Keto acids added to the media as additional energy source

Maintain maximum cell metabolism

Carbohydrates Energy source

Glucose and galactose

Low (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media

Vitamins

Precursors for numerous co-factors B group vitamins necessary for cell growth and proliferation

Common vitamins found in basal media is riboflavin, thiamine and biotin

Trace Elements

Zinc, copper, selenium and tricarboxylic acid intermediates

Cell culture environment (in vi tro)

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Supplements

L-glutamine Essential amino acid (not synthesised by the cell)

Energy source (citric acid cycle), used in protein synthesis

Unstable in liquid media - added as a supplement

Non-essential amino acids (NEAA)

Usually added to basic media compositions

Energy source, used in protein synthesis May reduce metabolic burden on cells

Growth Factors and Hormones (e.g.: insulin)

Stimulate glucose transport and utilisation

Uptake of amino acids

Maintenance of differentiation

Antibiotics and Antimycotics

Penicillin, streptomycin, gentamicin, amphotericin B

Reduce the risk of bacterial and fungal contamination

Cells can become antibiotic resistantchanging phenotype

Preferably avoided in long term culture

Cell culture environment (in vi t ro)

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Foetal Calf/Bovine Serum (FCS & FBS)

Growth factors and hormones

Aids cell attachment

Binds and neutralise toxins

Long history of use

Infectious agents (prions) Variable composition

Expensive

Regulatory issues (to minimise risk)

Heat Inactivation (56oC for 30 mins)why?

Destruction of complement and immunoglobulins Destruction of some viruses (also gamma irradiated serum)

Care! Overdoing it can damage growth factors, hormones & vitamins

and affect cell growth

Cell culture environment (in vi t ro)

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How do we culture cells in the laboratory?

Revive frozen cell population

Isolate from tissue

Maintain in culture (aseptic technique)

Sub-culture (passaging)

Cryopreservation

Count cells

Containment level 2

cell culture laboratory

Typical

cell culture flask

Mr Frosty

Used to freeze cells

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Why passage cells?

To maintain cells in culture (i.e. dont overgrow) To increase cell number for experiments/storage

How?

70-80% confluency

Wash in PBS to remove dead cells and serum

Trypsin digests protein-surface interaction torelease cells (collagenase also useful)

EDTA enhances trypsin activity

Resuspend in serum (inactivates trypsin)

Transfer dilute cell suspension to new flask(fresh media)

Most cell lines will adhere in approx. 3-4 hours

Check confluency of cells

Remove spent medium

Wash with PBS

Resuspend in serum

containing media

Incubate with

trypsin/EDTA

Transfer to culture flask

Passaging Cells

70-80% confluence 100% confluence

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Passage cells

Resuspend cells in serum

containing media

Centrifuge &

Aspirate supernatant

Transfer to cryovial

Freeze at -80oC

Resuspend cells in

10% DMSO in FCS

Why cryopreserve cells?

Reduced risk of microbial contamination. Reduced risk of cross contamination with

other cell lines.

Reduced risk of genetic drift and

morphological changes.

Research conducted using cells at consistentlow passage.

How?

Log phase of growth and >90% viability

Passage cells & pellet for media exchange Cryopreservant (DMSO)precise mechanism

unknown but prevents ice crystal formation

Freeze at -80oCrapid yet slow freezing

Liquid nitrogen -196oC

Transfer to liquid

nitrogen storage tank

Cryopreservation of Cells

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Manual cell count (Hemocytometer)

Diagram represent cell count using hemocytometer.

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Automated cell count

Cellometer lets you:

View cell morphology, for visual confirmation after cell counting

Take advantage of 300+ cell types and easy, wizard-based parameter set-up

Save sample images with results securely on your computer, plus autosave

results on the network for added convenience and data protection

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The ideal growth curve for cells in culture

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ContaminationA cell culture contaminant can be defined as some element in the culture

system that is undesirable because of its possible adverse effects on either

the system or its use.1-Chemical Contamination

Media

Incubator

Serum

water

2-Biological Contamination

Bacteria and yeast

Viruses

Mycoplasmas

Cross-contamination by other cell culture

How Can Cell Culture Contamination Be Controlled?

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Thank you