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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