Animal Cell Culture- Definition, Types, Cell Lines, Procedure, Applications

What is 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 in animal cell culture are usually obtained from multicellular eukaryotes and their established cell lines.
  • Animal cell culture is a common and widely used technique for the isolation of cells and their culture under artificial conditions.
  • This technique was developed as a laboratory technique for particular studies; however, it has since been developed to maintain live cell lines as a separate entity from the original source.
  • The development of animal cell culture techniques is due to the development of basic tissue culture media, which enables the working of a wide variety of cells under different conditions.
  • In vitro culture of isolated cells from different animals has helped in the discovery of different functions and mechanisms of operations of different cells.
  • Some of the areas where animal cell culture has found most applications include cancer research, vaccine production, and gene therapy.
  • The growth of animal cells on artificial media is difficult than growing microorganisms on artificial media and thus, require more nutrients and growth factors. 
  • However, advances in the 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 as complex structures like organs can also be used to initiate organ culture in vitro.
  • Depending on the purpose and application of the technique, cells, tissues, or organs can be used for the culture process.
Animal Cell Culture
Animal Cell Culture. Created with BioRender.com

Types of Animal cell culture

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

1. Primary cell culture

  • Primary cell culture is the first culture obtained directly from animal tissue via mechanical and chemical disintegration or enzymatic methods.
  • The cells of the primary cell culture are slow-growing cells that carry all the characteristics of the original tissue or cells.
  • Since these cultures are obtained directly from the origin, they have the same number of chromosomes as the original cells.
  • Primary cell cultures are performed in order to preserve and maintain the growth of cells on an artificial growth medium at a particular condition.
  • Primary cell cultures can be subcultured to obtained other cultures that either continue to grow indefinitely or die after a few subcultures.
  • The subsequent subculture of primary cell culture results in the introduction of mutations into the cells, which might result in cell lines.
  • The morphology of cells in the primary cell cultures might be different and varied, with the most commonly observed morphological structures being epithelium type, epithelioid type, fibroblast type, and connective tissue type.
  • Primary cell cultures are difficult to obtain and usually have a shorter lifespan. Besides, these are prone to contamination by bacteria and viruses.
  • The increase in cell numbers in the primary cell culture can result in exhaustion of the substrate and nutrients, which affects cellular activity.
  • Usually, primary cell cultures need to be subcultured in order to maintain continuous cell growth once they reach the confluence stage.

Primary cell cultures can be further divided into two groups depending on the kind of cells present in the culture;

a. Anchorage-dependent/Adherent cells

  • The cells in the culture require a stable biologically inert surface for adherence and growth.
  • The surface should be solid and nontoxic as these cells are difficult to grow as cell suspensions.
  • These cells are usually obtained from the tissues of organs where the cells remain immobilized within the connective tissue.
  • Examples of adherent cells include kidney cells and mouse fibroblast STO cells.

b. Anchorage-independent/ Suspension cells

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

2. Secondary cell culture

  • Secondary cell cultures are obtained after the primary cell cultures are subsequently subcultured over a period of time in fresh culture media.
  • The cells of the secondary cell cultures are long-lasting as these have a higher lifespan due to the availability of appropriate nutrients at regular intervals of time.
  • Secondary cell cultures are favored over primary cell cultures as these are more readily available and are easy to grow and preserve.
  • These are formed from the enzymatic treatment of the adherent cells followed by washing and resuspension of cells in particular volumes of fresh media.
  • Secondary cell cultures are prepared when the number of cells in the primary culture exceeds the capacity of the medium to support growth.
  • Secondary cultures help to maintain an optimal cell density necessary for continued growth.
  • The cells of the secondary cell culture might not resemble that on the parental tissue as mutations, and genetic alterations might be introduced during the subculture process.
  • The cells can be transformed as, in some cases, the continuous subculture can lead to immortal cells.
  • The risk of contamination by bacteria and viruses is less as the cells transform and become less susceptible to infections.
  • An important disadvantage associated with secondary cell culture is that the cells might develop the tendency to differentiate over a long period of time and result in aberrant cells.

Cell Lines

A cell line is a group of cells that are formed from the subculture of primary culture consisting of a pure culture of cells. Cell lines usually display functional features that are close to the primary cells, but the genotype and phenotype of the cells can be modified. A cell line consists of several cell lineages with similar or different phenotypes.

Cell lines can be further divided into two groups based on the growth patterns of the cells;

a. Finite cell lines

  • Finite cell lines are cell lines where the cells in the culture divide for a limited number of times, after which they eventually die.
  • The cells in the finite cell lines can divide from 20 to 100 times before they eventually die and cannot divide anymore.
  • The number of cell division and lifespan depends on a number of factors like cell lineage differences, species, culture conditions, and media.
  • The cells of the finite cell lines grow as adherent cells on solid surfaces.

b. Continuous cell lines

  • Continuous cell lines are cells that exhibit indefinite growth via subsequent subcultures.
  • The cells in the continuous cell lines grow faster to form an independent culture. The cells are immortal and can divide indefinitely.
  • The cells in the continuous cell lines can be transformed via genetic alterations and are also tumorigenic.
  • The transformed cells are formed from the normal primary cell cultures after treatment with chemical carcinogens or by infection with oncogenic viruses.
  • The cells are capable of growing to prepare higher cell density and can grow as suspensions on liquid media.
  • These cells can even grow on top of each other to form multilayered structures on the culture vessels.

Examples of common Cell Lines

The following are some of the common examples of cell lines;

a. HeLa cell line

  • HeLa cells are one of the first continuous culture human cell lines with the help of cells of the cervical carcinoma.
  • These cells are used for processes like virus cultivation and preclinical drug evaluation.

b. HL 60 (Leukemia)

c. MCF-7 (breast cancer cells)

Procedure or Protocol of Animal cell culture

1. Growth Conditions

  • Animal cell culture requires the use of specific culture media that are more complex and specific than the basic culture media used for microbial growth.
  • Some of the important basic components of the 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.
  • The temperature for the growth depends on the source of the cell as different organisms require different temperatures for cell growth and division.
  • Warm-blooded animal cells can be cultured at 37°C as the optimal temperature, whereas cold-blooded animals grow between 15°C-25°C.

2. Primary cell culture

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

3. Cell thawing

  • In the case of subsequent subcultures, the preserved cell culture might have to be used.
  • The water bath is heated to a temperature of 37°C, and the growth media where the cells are to be plated is warmed.
  • The warm medium is added to the culture vessel. The vial with the frozen cells is then placed in the water bath until thawed.
  • After thawing, the via is washed with 70% alcohol on the outside. The cell suspension is pipetted into the cell culture vessel and swirled gently to mix everything.
  • The medium is then incubated overnight under the usual growth conditions. The growth medium is replaced the next day.

4. Trypsinizing Cells

  • Trypsinization is the method of separating adherent cells from the surface of the culture vessel with the help of proteolytic enzymes. It is done when the cells are to be used for passaging, counting, or other purposes.
  • The medium is removed, and the cells are recovered. The cells are then washed with phosphate buffer.
  • Warm trypsin-EDTA is added to the vessel so as to cover the monolayer. The vessel can be rocked to ensure that the monolayer is coated.
  • The vessel is incubated in a CO2 incubator at 37°C for 1-3 minutes.
  • The vessel is removed from the incubator, and the flask is firmly tapped on the side with the palm of the hand to assist detachment.
  • Once the cells are dislodged, they are resuspended in an appropriate growth medium containing some amount of serum.
  • The cells are then separated with the help of syringe needles by disrupting the cell clumps and used accordingly.

Applications of Animal cell culture

The following are some of the applications of animal cell culture;

a. Production of vaccines

  • Animal cell culture is an important technique used for the development of viral vaccine production.
  • The technique has been used for the development of a recombinant vaccine against hepatitis B and poliovirus.
  • Immortalized cell lines are used for the large-scale or industrial production of viral vaccines.

b. Recombinant proteins

  • Animal cell cultures can also be used for the production of recombinant therapeutic proteins like cytokines, hematopoietic growth factors, growth factors, hormones, blood products, and enzymes.
  • Some of the common animal cell lines used for the production of these proteins are baby hamster kidney and CHO cells.

c. Gene Therapy

  • The development of animal cell culture is critical for the advances in gene therapy.
  • Cells with faulty genes can be replaced by a functional gene in order to remove such defects and diseases.

d. Model systems

  • Cells obtained from cell culture can be studied as a model system for studies related to cell biology, host-pathogen interactions, effects of drugs, and effects due to changes in the cell composition.

e. Cancer Research

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

f. Production of Biopesticides

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

Advantages of Animal cell culture

The following are some of the advantages of animal cell culture;

  1. Cell culture is superior to other similar biotechnological approaches as it allows the alteration of different physiological and physiobiological conditions like temperature, pH, and osmotic pressure.
  2. Animal cell culture enables studies related to cell metabolism and understand the biochemistry of cells. 
  3. It also allows observation of the effects of various compounds like proteins and drugs on different cell types.
  4. The results from animal cell cultures are consistent if a single cell type is used.
  5. The technique also enables the identification of different cell types on the basis of the presence of markers like molecules or by karyotyping.
  6. The use of animal cell culture for testing and other processes prevents the use of animals in experiments.
  7. Animal cell culture can be used for the production of large quantities of proteins and antibodies, which would otherwise require a large investment.

Disadvantages of Animal cell culture

Even though animal cell culture has been used as a technologically advanced method, there are some disadvantages associated with this approach.

  1. It is a specialized technique that requires trained personnel and aseptic conditions. The technique is an expensive process as it requires costly equipment.
  2. The subsequent subculture of the cell culture might results in differentiated properties as compared to the original strain.
  3. The method produces a minuscule amount of recombinant proteins, which further increases the expenses of the process.
  4. Contamination with mycoplasma and viral infection occur frequently and are difficult to detect and treat.
  5. The cells produced by this technique lead to instability due to the occurrence of aneuploidy chromosomal constitution.

References

  1. Kaur, Gurvinder, and Jannette M Dufour. “Cell lines: Valuable tools or useless artifacts.” Spermatogenesis vol. 2,1 (2012): 1-5. doi:10.4161/spmg.19885
  2. Yao, Tatsuma, and Yuta Asayama. “Animal-cell culture media: History, characteristics, and current issues.” Reproductive medicine and biology vol. 16,2 99-117. 21 Mar. 2017, doi:10.1002/rmb2.12024
  3. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 6.2, Growth of Animal Cells in Culture. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21682/
  4. Hudu, Shuaibu Abdullahi et al. “Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.” Journal of clinical and diagnostic research : JCDR vol. 10,3 (2016): DE01-5. doi:10.7860/JCDR/2016/15837.7460
  5. Verma, Anju et al. “Animal tissue culture principles and applications.” Animal Biotechnology (2020): 269–293. doi:10.1016/B978-0-12-811710-1.00012-4
  6. Gilgenkrantz S. Regard sur soixante années de culture de cellules HeLa [Sixty years of HeLa cell cultures]. Hist Sci Med. 2014 Jan-Mar;48(1):139-44. French. PMID: 24908793.
  7. Lucey BP, Nelson-Rees WA, Hutchins GM. Henrietta Lacks, HeLa cells, and cell culture contamination. Arch Pathol Lab Med. 2009 Sep;133(9):1463-7. doi: 10.1043/1543-2165-133.9.1463. PMID: 19722756.
  8. https://www.biologydiscussion.com/cell/cell-lines/cell-lines-types-nomenclature-selection-and-maintenance-with-statistics/10517
  9. https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/animal-cell-culture/essential-protocols-for-animal-cell-culture/essential-protocols-for-animal-cell-culture#Maintaining-cell-cultures.

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