lecture # 04

Mammalian Cell Culture

Dr E O Nna

Learning Objectives

Basic understanding of cell/tissue/ organ culture

Overview of historical development of cell culture

Areas of Application of Cell culture

Instruments & Materials required for cell culture

Basic cell culture techniques

Handling Infections/Contaminations

In vitro Cytotoxicity Assays

Safety, Bioethics and Validation

Advantages and Disadvantages of Tissue Culture

Basic Understanding of Tissue Culture

Tissue culture is a generic term for both organ and cell culture.

Organ culture implies a three-dimensional culture of undisaggregated tissue retaining some or all of the histological features of the tissue in vivo.

Cell culture refers to a culture derived from dispersed cells taken from original tissue, from primary culture, or from a cell line or cell strain by enzymatic, mechanical or chemical disaggregation.

Basically, tissue culture provides a method for studying the behaviour of animal cells free of systemic variations that might arise in vivo both during normal homeostasis and under the stress of an experiment.

Two more definitions to remember:

- Histotypic culture- cells of the same lineage that have been re-aggregated or grown to re-create a three-dimensional structure with tissue-like cell density e.g growing cells in a matrix (artificial scaffolds); corneal transplants.

- Organotypic culture –cells of different lineages grown in a three-dimensional matrix to create a ‘tissue equivalent’; e.g epidermal keratinocytes combined with dermal fibroblasts.

More Definitions

Primary Culture: It is that stage of the culture after isolation of the cells but before

the first subculture. Four key stages are involved:

- acquisition of the sample

- isolation of the tissue

- dissection and/ or disaggregation

- culture after seeding into the culture vessel

Cell Line: Once a primary culture is sub-cultured (or passaged), it becomes known

as a cell line. It consists of several cell lineages of either similar or distinct phenotypes.

Cell Strain: This refers to a cell lineage that has been selected either by physical cell

separation or by cloning. Such selection has certain specific properties identifiable in the

bulk of the cells in the culture.

Continuous cell line: This refers to a cell line that has transformed in vitro.

Continuous cell strain: This is a transformed cell line that has been selected by

physical separation or by cloning.

Passage number is the number of times that the culture has been sub-cultured.

Generation number is the number of doublings that the cell population has

undergone.

Finite cell line: refers to cell line with limited culture life spans.

Finite cell line Vs Continuous Cell line

Stain characteristicsGeneration no, tissue-specific markers

Control parameters

HighLowYield

Rapid ((TD of 12-24 h)Slow (TD of 24-96 h)Growth rate

Often LostMay be retainedSpecial functions e.g virus susceptibility, differentiation

Chromosomal, enzymic,antigenicTissue specificMarkers

HighLowCloning efficiency

Steady state possibleCyclicMaintenance

LowHighSerum requirement

Monolayer or suspensionMonolayerMode of growth

Reduced or lostYesDensity limitation of cell proliferation

NoYesContact inhibition

NoYesAnchorage dependence

Immortal, growth control altered, and tumorigenic

NormalTransformation

Aneuploid, heteroploidEuploid, diploidPloidy

Continuous Cell Line (Transformed)

Finite Cell LineProperties

Historical Development of Tissue Culture

Pluripotency of embryonal stem cells (ESC)

Selection of transformed cells in agar

1964

3T3 cells and spontaneous transformation1963

Establishment and transformation of BHK211962

Definition of finite life span of normal human cells.

Cell fusion-somatic cell hybridization

1961

Establishment of the first human cell line (HeLa from a cervical

carcinoma); Nuclear transplantation; Salk polio vaccine grown in

monkey kidney cells (1954);development of defined media (1955)

1952-55

Use of trypsin for generation of replicate subcultures

Virus plaque assay

1952

Growth of virus in cell culture1949

Cloning of the L-cell1948

Establishment of the first continuous cell line (the L-cell mouse

fibroblast)

1943

Introduction of use of antibiotics in tissue culture1940s

Subculture of fibroblastic cell lines

Differentiation in vitro in organ culture

1920s/30s

Trypsinization and subculture of explants1916

Frog embryo nerve fibre outgrowth in vitro [Harrison, 1907]1907

Historical Development of Tissue Culture (2)

Human Genome Project, genomics, proteomics; exploitation of tissue engineering2000+

Culture of human embryonic stem cells1998

Tissue-engineered cartilage1998

Culture of human adult mesenchymal stem cells1991

Industrial-scale culture of transfected cells for production of biopharmaceuticals1990s

Establishment of many specialized cell lines; production of recombinant tissue-

type plasminogen activator in mammalian cells (1984).

1980-87

Regulation of cell cycle; immortalization by SV401983

Regulation of gene expression, oncogenes, malignancy and transformation; matrix

from EHS sarcoma (Matrigel)

1980s

Totipotency of embryonal stem cells1976

Fibroblast growth factor; hybridomas- monoclonal antibodies1975

DNA transfer, calcium phosphate1973

Development of laminar-flow cabinets1970s

Rabies, Rubella vaccines in WI-38 human lung fibroblasts,

Serum-free cloning of Chinese hamster cells (1965); Heterokaryons (man-mouse

hybrids); Nerve growth factor and differentiation in rat hepatomas (1966);

Epidermal growth factor and lymphoblastoid cell lines (1967); Colony formation in

haematopoietic cells (1969)

1964-69

Areas of Application of Tissue Culture

The main drivers of tissue culture are cancer research, virology, genomics and

pharmacology. Freshney RI, 2005

Tissue Culture Equipment

Tissue Culture Equipment

Consumables or Refillables for Tissue Culture

Tissues culture flasks, dishes, slides, bottles and micro well plates

Culture media (balanced salt solutions with nutrients) e.g DMEM-F12, RPMI1640 etc

Trypsin/EDTA and /or cell scrappers

Glutamine

Antibiotics-Penicillin and Streptomycin

Antifungal- Amphotericin B

Serum- foetal calf serum (FCS), bovine growth serum (BGS) or treated serum e.g

Charcoal dextran treated serum

Steroids e.g hydrocortisone

Plastics- Falcon tube, Universal tube, 15ml tubes etc, pipettes, pipettes tips and bulbs

Disinfectants e.g Virkon

Trypan blue, dimethyl suphoxide (DMSO), Isopropanol (IPA), Ethanol (Molecular grade),

Phosphate buffered saline (PBS); ultrapure water, MTT reagent

Cell lines: mainly from ATCC – American type culture collection; ECACC – European

Collection of Animal Cell Cultures (now European Collection of Cell Cultures); ?????

ACCC- African Collection of Cell Cultures (Yes we can!!!).

Basic Cell Culture Techniques

Plating or Seeding of cells

Feeding cell cultures

Splitting cell cultures

Cell counting

Harvesting cell cultures for downstream uses

Freezing/ thawing cell cultures

Checking cell viability/Cytotoxicity testing

Detecting, preventing and/ or handling infections

Filling of liquid nitrogen freezers

Maintaining tissue bank log books

House keeping of tissue culture laboratory

Keratinocytes on MaxGel (human ECM)

Handling Infections (Contaminations) in Tissue Culture Laboratory

Most common contaminants are

- Bacteria, Fungi, Viruses and Mycoplasma

Routes of Contamination:

1. Technique: manipulation, pipetting, dispensing , thawing etc

2. Work surface e.g dust and spillage

3. Operator hair, hands, breath, clothing e.g aerosols from talking, coughing, sneezing

4. Materials and reagents e.g dirty or non sterile

5. Glassware and screw caps e.g dust and spores from storage, poor sterilization

6. Instruments and pipettes e.g ineffective sterilization

7. Culture flasks and media bottles in use e.g dust and spores from incubator or refrigerator

8. Equipment and Facilities

- Room air e.g turbulence, dust, aerosols etc

- laminar flow hoods e.g perforated filter, spillages, unchanged filter (when due)

- dry incubators e.g growth of moulds and bacteria on spillages

9. Other Equipment e.g Mites, insects or other infestations on wooden furniture or benches or

mice from animal house.

10. Importation of Biological materials e.g Calf serum from UK (Mad cow disease), contaminated

cell lines etc [http://www.mad-cow.org/]

Monitoring for Contaminations

Check for contamination macroscopically and microscopically

When contamination is suspected, carry out thorough cleaning of hoods and bench surfaces

using 70% alcohol. Examine the suspected flask or plate.

Record the nature of contamination in a log book

If contamination is new and not widespread, discard the culture, media and reagents used for

that particular culture.

If contamination is new and widespread, discard all media, stock solutions and reagents.

If same kind of contamination has occurred before, check stock solutions for contamination

- by incubation alone or in nutrient broth

- by plating out the solution on nutrient agar

- 100ml of solution can be incubated, filtered through 0.2µm filter and plated on nutrient agar

• If contamination is widespread, multi-specific and repeated, check

- the laboratory’s sterilization procedures

- the packaging and storage practices

- the integrity of the aseptic room and laminar-flow hood filters

• Do not attempt to decontaminate cultures unless they are irreplaceable.

Don’t culture bacteria or fungi in the

same hood or lab where mammalian

cells are handled!!

Visible Microbial Contamination

Characteristic features of microbial contamination in cell cultures are:

• A sudden change in pH, usually a decrease with most bacteria infection; very little

change with yeast until contamination is heavy.

• Cloudiness in the medium; sometimes with a slight film or scum on the surface or spots

on the growth surface that dissipate when the flask is moved.

• Under a low-power microscopes, spaces between cells will appear granular for bacteria

infection; yeasts appear as separate round or ovoid particles that may bud off smaller

particles; fungi produce thin filamentous mycelia.

• Under high-power microscopy, individual bacteria can be resolved as rods or cocci.

• Take care to differentiate microbial infection from precipitates of media constituents;

Clumps of bacteria may be confused with precipitates of proteins.

• Mycoplasma infection is not obvious by microscopy; it requires techniques such as

PCR, ELISA , Fluorescent staining, immunostaining, autoradiography or microbiological

assay. Usually there are always signs of deterioration in the cell culture e.g diminished

rate of proliferation, reduced saturation density and agglutination during growth in

suspension.

• PCR for Mycoplasma is designed to detect the 16s rDNA which contains regions with

conserved sequences- very sensitive, specfic, low cost, labour, time; objectivity of

interpretation, reproducibility and documentation of results.

Eradication of Contamination

For bacteria, fungi and yeast, the most reliable method of eliminating a

microbial infection is to discard the culture and medium and reagents especially

where the cells are replaceable.

The same rule applies of Mycoplasma-discard by autoclaving or incineration.

Where cells are irreplaceable, decontamination can be attempted using agents

such Mycoplasma including Kanamycin, Gentamycin,Tylosin, UV light etc.

Contaminated cultures must be treated in isolation.

For viral infections, there are no reliable means of decontamination except to

discard and dispose the cells.

Cases of persistent and / or cross contamination in a laboratory require stringent

measures, improvement in aseptic procedures and thorough investigation.

Be proactive e.g quarantine all new cell lines into your laboratory until you are

sure they are uncontaminated; don’t share media or other solutions among cell

lines or among operators, check cell cultures regularly for contamination; new cell

lines should be characterized by DNA profiling; don’t attempt decontaminating cells

unless they are irreplaceable; don’t maintain all cultures routinely in antibiotics.

In Vitro Cytotoxicity Testing

Protein stain updateCellular protein contentSulforhodamine B assaySRB

Metabolism of fluorescein diacetateMetabolic deathFlourometric microculture cytotoxicityFMC

ATP levelsMetabolic deathAdenosine triphosphate ATP

Dye reductionMetabolic death3-(4,5-dimethylthiazol-2-yl)- 2,5-

diphenyltetrazolium bromide

MTT

morphologyCell membrane integrityTumour response to antineoplastic

compounds

TRAC

Dye exclusion, morphologyCell membrane integrityDifferential staining cytotoxicityDiSC

Dye exclusionCell membrane integrityTrypan blueTB

Measured byTechnologyNameAbbreviation

useful for screening drugs e.g antineoplastic agents

assessing cell viability

Safety, Bioethics and Validation

Laboratory safety

Risk assessment

Standard Operating Procedures (SOPs)

Safety Regulations in the country e.g National code of conduct for biomedical research

in Nigeria, 2007

General safety

- Operator

- Equipment

- Glassware and Sharp items

- Chemical toxicity

- Gases

- Liquid Nitrogen

- Burns ( provisions for First Aid)

- Ingestion

Fire

Ionizing radiation and disposal of radioactive waste

Biohazards/ levels of biological containment e.g. Class III,Class II and Class I

Bioethics and Validation

Bioethics:

Human Biopsy Material

Genetic Manipulation

Disposal of Bio-hazardous Waste

Fumigation

Animal tissues

Human tissues

Validation:

Authentication: is the cell line what it is claimed to be? E.g DNA

profiling

Provenance: what has happened to the cell line since its original

isolation e.g maintenance records, contamination checks, genetic

modification if any etc

Contamination: is the cell line free from all known forms of microbial

contamination?

Advantages of Tissue Culture

Cytotoxicity and screening of pharmaceutics, cosmetics etcReduction of animal use

Available with microtitration and roboticsMechanization

Ability to define dose, concentration (C ) and time (T)Control of C x T

Reduced volumes, direct access to cells, lower costReagent saving

Quantification is easyReplicates and Viability

Origin, history, purity can be authenticated and recordedValidation and accreditation

Can be stored in liquid nitrogenPreservation

Cytology and immunostaining easily performedCharacterization

Availability of selective media, cloningCell line homogeneity

Regulation of matrix, cell-cell interaction, gaseous diffusionMicroenvironment

Control of hormones & nutrient concentrationsPhysiological conditions

Control pH, temperature, osmolality & dissolved gasesPhysico-chemical environment

AdvantageCategory

Disadvantages of Tissue Culture

Markers not always expressed, Histology may difficult to

re-create and atypical; geometry and microenvironment

change cytology

Identification of cell type

Dedifferentiation, Adaptation, Selective overgrowth,Phenotypic instability

Heterogeneity, variabilityGenetic instability

Capital equipment for scale-up, medium, serum,

disposable plastics

Quantity and Cost

Workplace, Incubation, pH,Containment and disposal of

biohazards

Environmental Control

Sterile handling, chemical contamination, microbial

contamination, persistent/cross contamination

Necessary Expertise

ExamplesCategory

For Further Reading

Freshney R I (2005). Culture of Animal Cells. A manual of BasicTechnique. 5th Edition; Wiley, USA. ISBN-13 978-0-471-45329-1