Animal Cell Culture: Types, Cell Lines, Procedure, Applications, Advantages and Disadvantages

Introduction to Animal Cell Culture

Animal cell culture is a type of biotechnological technique where animal cells are artificially grown in a favorable environment.

The cells used are usually obtained from multicellular eukaryotes and their established cell lines.

Animal cell culture is a common and widely used technique for isolation of cells and their culture under artificial conditions.

This technique was developed as a laboratory technique for particular studies. It has since been developed to maintain live cell lines as a separate entity from the original source.

Development of animal cell culture is due to development of basic tissue culture media, which enables working of wide variety of cells under different conditions.

In vitro culture of isolated cells from different animals has helped in discovery of different functions and mechanisms of operations of different cells.

Some areas where animal cell culture has found most applications include:

• Cancer research
• Vaccine production
• Gene therapy

Growth of animal cells on artificial media is more difficult than growing microorganisms and thus requires more nutrients and growth factors.

However, advances in culture media have made it possible to culture both undifferentiated and differentiated cells on artificial media.

Animal cell cultures can be performed from different complexities of cells:

• Complex structures like organs can also be used to initiate organ culture in vitro.
• Depending on purpose and application, cells, tissues, or organs can be used for culture process.

Types of Animal Cell Culture

Animal cell cultures can be divided into two distinct groups depending on number of cell divisions occurring during the process.

• 1. Primary cell culture
• 2. Secondary cell culture

Primary Cell Culture

Primary cell culture is the first culture obtained directly from animal tissue via mechanical and chemical disintegration or enzymatic methods.

• Cells of primary culture are slow-growing cells that carry all characteristics of original tissue or cells.
• Since cultures are obtained directly from origin, they have same number of chromosomes as original cells.
• Performed to preserve and maintain growth of cells on artificial growth medium at particular condition.
• Can be subcultured to obtain other cultures that either continue to grow indefinitely or die after few subcultures.
• Subsequent subculture results in introduction of mutations into cells, which might result in cell lines.
• Morphology might be different and varied. Most commonly observed morphological structures are:
  • Epithelium type
  • Epithelioid type
  • Fibroblast type
  • Connective tissue type
• Difficult to obtain and usually have shorter lifespan.
• Prone to contamination by bacteria and viruses.
• Increase in cell numbers can result in exhaustion of substrate and nutrients, which affects cellular activity.
• Usually need to be subcultured to maintain continuous growth once they reach confluence stage.

Primary cell cultures can be further divided into two groups depending on kind of cells present:

Anchorage-Dependent or Adherent Cells

• Cells require stable biologically inert surface for adherence and growth.
• Surface should be solid and nontoxic as these cells are difficult to grow as cell suspensions.
• Usually obtained from tissues of organs where cells remain immobilized within connective tissue.
• Examples include kidney cells and mouse fibroblast STO cells.

Anchorage-Independent or Suspension Cells

• These cells can grow efficiently as cell suspensions and do not require solid surface for attachment.
• Can be grown on liquid media continuously to obtain fresh subcultures.
• Ability to grow as suspension depends on source of cells as cells that remain as suspensions in body are effective suspension cells.
• Examples include blood cells that are vascular and remain suspended in plasma.

Secondary Cell Culture

Secondary cell cultures are obtained after primary cell cultures are subsequently subcultured over period of time in fresh culture media.

• Cells are long-lasting as these have higher lifespan due to availability of appropriate nutrients at regular intervals.
• Favored over primary cultures as these are more readily available and easy to grow and preserve.
• Formed from enzymatic treatment of adherent cells followed by washing and resuspension in particular volumes of fresh media.
• Prepared when number of cells in primary culture exceeds capacity of medium to support growth.
• Help to maintain optimal cell density necessary for continued growth.
• Cells might not resemble parental tissue as mutations and genetic alterations might be introduced during subculture.
• Cells can be transformed as continuous subculture can lead to immortal cells.
• Risk of contamination by bacteria and viruses is less as cells transform and become less susceptible to infections.
• Important disadvantage is cells might develop tendency to differentiate over long period and result in aberrant cells.

Cell Lines

A cell line is a group of cells formed from subculture of primary culture consisting of pure culture of cells.

• Usually display functional features close to primary cells, but genotype and phenotype can be modified.
• Consists of several cell lineages with similar or different phenotypes.

Cell lines can be further divided into two groups based on growth patterns:

Finite Cell Lines

• Cells divide for limited number of times, after which they eventually die.
• Can divide 20 to 100 times before they die and cannot divide anymore.
• Number of division and lifespan depends on:
  • Cell lineage differences
  • Species
  • Culture conditions and media
• Cells grow as adherent cells on solid surfaces.

Continuous Cell Lines

• Exhibit indefinite growth via subsequent subcultures.
• Grow faster to form independent culture.
• Cells are immortal and can divide indefinitely.
• Can be transformed via genetic alterations and are also tumorigenic.
• Transformed cells are formed from normal primary cultures after treatment with chemical carcinogens or infection with oncogenic viruses.
• Capable of growing to prepare higher cell density and can grow as suspensions on liquid media.
• Can even grow on top of each other to form multilayered structures on culture vessels.

Common Examples of Cell Lines

• HeLa cell line - one of first continuous culture human cell lines with help of cells of cervical carcinoma. Used for processes like virus cultivation and preclinical drug evaluation.
• HL 60 - Leukemia cell line.
• MCF-7 - Breast cancer cells.

Procedure or Protocol of Animal Cell Culture

Growth Conditions

Animal cell culture requires specific culture media that are more complex than basic media used for microbial growth.

Important basic components of media are:

• Inorganic salts
• Nitrogen source
• Energy source
• Vitamins
• Fat and fat-soluble vitamins
• Growth factors and hormones
• In some cases, pH buffering systems and antibiotics are also added.

Temperature for growth depends on source of cell:

• Warm-blooded animal cells can be cultured at 37°C as optimal temperature.
• Cold-blooded animals grow between 15°C-25°C.

Primary Cell Culture Procedure

• Primary cultures obtained from fresh tissues removed from organs with help of aseptic razor.
• In some cases, cells removed by use of chemical disintegrators or proteolytic enzymes.
• Cell suspension obtained is washed with buffering liquid to remove proteolytic enzymes.
• Suspension is poured onto flat surface which can be culture vessel or sterile Petri plate.
• Cells that can adhere to base are overlaid with appropriate culture medium and incubated at room temperature.

Cell Thawing

• Used for subsequent subcultures when preserved cell culture has to be used.
• Steps include:
  • Water bath heated to 37°C.
  • Growth media where cells are to be plated is warmed.
  • Warm medium added to culture vessel.
  • Vial with frozen cells placed in water bath until thawed.
  • After thawing, vial washed with 70% alcohol on outside.
  • Cell suspension pipetted into vessel and swirled gently to mix.
  • Medium incubated overnight under usual growth conditions.
  • Growth medium replaced next day.

Trypsinizing Cells

Trypsinization is method of separating adherent cells from surface of culture vessel with help of proteolytic enzymes.

It is done when cells are to be used for passaging, counting, or other purposes.

• Medium removed and cells recovered.
• Cells washed with phosphate buffer.
• Warm trypsin-EDTA added to vessel so as to cover monolayer.
• Vessel rocked to ensure monolayer is coated.
• Vessel incubated in CO2 incubator at 37°C for 1-3 minutes.
• Vessel removed and flask firmly tapped on side with palm to assist detachment.
• Once dislodged, cells resuspended in appropriate growth medium containing some amount of serum.
• Cells separated with help of syringe needles by disrupting cell clumps and used accordingly.

Applications of Animal Cell Culture

Production of Vaccines

• Important technique used for development of viral vaccine production.
• Used for development of recombinant vaccine against hepatitis B and poliovirus.
• Immortalized cell lines are used for large-scale or industrial production of viral vaccines.

Recombinant Proteins

• Can be used for production of recombinant therapeutic proteins like:
  • Cytokines
  • Hematopoietic growth factors
  • Growth factors
  • Hormones
  • Blood products
  • Enzymes
• Common animal cell lines used are baby hamster kidney and CHO cells.

Gene Therapy

• Development of animal cell culture is critical for advances in gene therapy.
• Cells with faulty genes can be replaced by functional gene to remove defects and diseases.

Model Systems

• Cells obtained from culture can be studied as model system for studies related to:
  • Cell biology
  • Host-pathogen interactions
  • Effects of drugs
  • Effects due to changes in cell composition

Cancer Research

• Can be used to study differences in cancer cells and normal cells as cancer cells can also be cultured.
• Differences allow more detailed studies on potential causes and effects of different carcinogenic substances.
• Normal cells can be cultured to form cancer cells by use of certain chemicals, viruses, and radiation.
• Cancer cells can also be used as test systems for efficiencies of drugs and techniques used in cancer treatment.

Production of Biopesticides

• Animal cell lines like Sf21 and Sf9 can be used for production of biopesticides due to faster growth rate and higher cell density.
• Organisms like baculovirus can be produced through animal cell culture as well.

Advantages of Animal Cell Culture

• Superior to other biotechnological approaches as it allows alteration of physiological conditions like temperature, pH, and osmotic pressure.
• Enables studies related to cell metabolism and understanding biochemistry of cells.
• Allows observation of effects of various compounds like proteins and drugs on different cell types.
• Results are consistent if single cell type is used.
• Enables identification of different cell types on basis of presence of markers like molecules or by karyotyping.
• Prevents use of animals in experiments.
• Can be used for production of large quantities of proteins and antibodies, which would otherwise require large investment.

Disadvantages of Animal Cell Culture

• Specialized technique that requires trained personnel and aseptic conditions.
• Expensive process as it requires costly equipment.
• Subsequent subculture might result in differentiated properties compared to original strain.
• Produces minuscule amount of recombinant proteins, which further increases expenses.
• Contamination with mycoplasma and viral infection occur frequently and are difficult to detect and treat.
• Cells produced lead to instability due to occurrence of aneuploidy chromosomal constitution.

Summary

Animal cell culture is a biotechnological technique where cells from multicellular eukaryotes and established cell lines are artificially grown in favorable environment using complex media with salts, nutrients, vitamins, growth factors and hormones at 37°C for warm-blooded cells. Primary cell culture is first culture directly from tissue via mechanical or enzymatic disintegration, slow-growing with same chromosome number, existing as anchorage-dependent adherent cells like kidney and STO cells requiring solid surface, and anchorage-independent suspension cells like blood cells growing in liquid. Secondary cultures are subcultures of primary with longer lifespan and less contamination but with mutations and differentiation risk. Cell lines are pure subcultures, finite lines dividing 20-100 times as adherent cells, and continuous lines like HeLa cervical carcinoma, HL 60 leukemia and MCF-7 breast cancer that are immortal and tumorigenic. Procedure includes primary isolation, thawing at 37°C, and trypsinization with trypsin-EDTA. Applications include viral vaccine production including hepatitis B and polio, recombinant proteins using BHK and CHO cells, gene therapy, model systems for cell biology and drug testing, cancer research, and biopesticide production using Sf21 and baculovirus. Advantages include control of pH and temperature, metabolic studies and protein production without animal use, while disadvantages include need for trained personnel, high cost, low yield, mycoplasma contamination and aneuploidy instability.

References

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