Animal Cell Culture: Definition, Types, Cell Lines, Procedures, and Applications

Typically, multicellular eukaryotes and their established cell lines provide the cells needed for animal cell culture.
Animal cell culture is a popular and commonly used method for isolating cells and cultivating them in sterile environments.
This method was first created as a lab procedure for specific investigations, but it has now been modified to keep living cell lines isolated from their original source.
The creation of fundamental tissue culture medium, which allows a range of cells to function under various circumstances, is what led to the development of animal cell culture techniques.
The identification of various cell activities and methods of action has been aided by in vitro growth of isolated cells from various species.
The most common uses of animal cell culture are in the fields of gene therapy, vaccine production, and cancer research.
Animal cells are more difficult to develop on artificial media than microbes are, thus they need more nutrients and growth hormones.
But because to developments in culture media, it is now feasible to cultivate both differentiated and undifferentiated cells on synthetic medium.
Animal cell cultures may be created using a variety of cell types, and complicated organs can also be employed to start in vitro organ cultivation.
Cells, tissues, or organs can all be employed in the culture process, depending on the technique's initial purpose.

Types of Animal cell culture

Depending on how many cell divisions occur during the procedure, animal cell cultures may be split into two main categories;

1. Primary cell culture

Primary cell culture is the initial culture made directly from animal tissue using enzymatic, chemical, or mechanical means of disintegration.
The primary cell culture's cells develop slowly and retain all the properties of the original tissue or cells.
These cultures have the same number of chromosomes as the original cells since they were taken straight from the source.
To retain and sustain the development of cells on an artificial growth medium at a certain condition, primary cell cultures are carried out.
To create new cultures that either expand endlessly or stop growing after a few subcultures, primary cell cultures can be subcultured.
Following primary cell culture, cell lines may be created as a result of the introduction of mutations into the cells.
The morphology of cells in primary cell cultures may vary, with epithelium type, epithelioid type, fibroblast type, and connective tissue type morphological structures being the most often seen ones.
Primary cell cultures are hard to get by and often last less time. Additionally, these are vulnerable to virus and bacterium infestation.
The main cell culture's rise in cell density may cause the substrate and nutrients to become depleted, which will have an impact on cellular activity.
Once they reach the confluence stage, primary cell cultures often need to be subcultured in order to maintain ongoing cell development.

Depending on the type of cells present in the culture, primary cell cultures can be further split into two classes;

a. Anchorage-dependent/Adherent cells

For adhesion and proliferation, the cells in the culture need a solid, physiologically inert surface.
Given that these cells are challenging to cultivate in cell suspensions, the surface should be stable and non-toxic.
Typically, these cells are extracted from organ tissues where they stay immobilised within the connective tissue.
Kidney cells and mouse fibroblast STO cells are examples of adherent cells.

b. Anchorage-independent/ Suspension cells

These cells don't need a firm surface to adhere to and can proliferate effectively in cell suspensions.
To create new subcultures, they can be continually developed on liquid media.
Since cells that remain in suspension inside the body are useful suspension cells, the capacity of cells to proliferate in suspension depends on the source of cells.
Blood cells that are vascular and remain suspended in the plasma are one example of suspension cells.

2. Secondary cell culture

After the main cell cultures are subcultured over time in new culture medium, secondary cell cultures are produced.
The secondary cell cultures' cells have a long lifespan because they have access to the right nutrients at regular intervals, which gives them a longer lifespan.
Since secondary cell cultures are easier to cultivate and preserve, they are preferred over primary cell cultures.
These are created by enzymatically treating adherent cells, washing them, and then resuspending them in specific quantities of new medium.
When the primary culture contains more cells than the medium can sustain for growth, secondary cell cultures are created.
Secondary cultures aid in preserving the ideal cell density required for sustained development.
Because of mutations and genetic modifications that might be introduced during the subculture process, the cells of the secondary cell culture could not resemble those on the mother tissue.
The cells may change because persistent culturing can occasionally produce immortal cells.
As the cells undergo transformation and have a lower susceptibility to infections, the danger of contamination by bacteria and viruses decreases.
The possibility that the cells can develop a propensity to differentiate over an extended period of time and produce abnormal cells is a significant drawback of secondary cell culture.

Cell Lines

A cell line is a collection of cells that develops from a primary culture that is made up entirely of one kind of cell. Although the genotype and phenotype of the cells can be changed, cell lines often exhibit functional characteristics that are similar to those of primary cells. Multiple cell lineages with the same or diverse traits make up a cell line.

Depending on how the cells develop, cell lines may be further split into two types;

a. Finite cell lines

Finite cell lines are cell lines in which the cultured cells proliferate just a finite number of times before ultimately dying.
Before they inevitably die and are no longer able to divide, the cells in the limited cell lines can divide somewhere between 20 and 100 times.
Cell lineage differences, species, culture conditions, and media are only a few of the variables that affect the number of cell divisions and longevity.
The cells of the finite cell lines develop on solid surfaces as adherent cells.

b. Continuous cell lines

Continuous cell lines are those that continue to develop throughout time through further subcultures.
The cells in the continuous cell lines multiply more quickly to create a distinct culture. The cells may divide endlessly and are immortal.
The cells in continuous cell lines are both tumorigenic and susceptible to genetic transformation.
After being exposed to chemical carcinogens or becoming infected with oncogenic viruses, normal primary cell cultures give rise to altered cells.
The cells can develop as suspensions on liquid medium and can proliferate to reach larger cell densities.
On the culture vessels, these cells can even grow on top of one another to create multilayered structures.

Examples of common Cell Lines

The following list includes some typical examples of cell lines:

a. HeLa cell line

With the aid of cervical cancer cells, HeLa cells are one of the first human cell lines to be continuously cultured.
These cells are utilised in procedures including preclinical drug testing and virus culture.

b. HL 60 (Leukemia)
c. MCF-7 (breast cancer cells)

Procedure or Protocol of Animal cell culture

1. Growth Conditions

The use of particular culture media that are more complicated and focused than the fundamental culture media used for microbial growth is necessary for animal cell culture.
Inorganic salts, nitrogen sources, energy sources, vitamins, fat-soluble vitamins, growth factors, and hormones are a few of the crucial fundamental elements of the media. Antibiotics and pH buffering devices are occasionally also included.
Because various species require different temperatures for cell growth and division, the growth temperature will vary depending on the origin of the cell.
Cold-blooded animals grow between 15°C and 25°C, and warm-blooded animal cells may be cultivated at 37°C as the ideal temperature.

2. Primary cell culture

Fresh tissues are taken from the organs using an aseptic razor, and these tissues are then used to create primary cell cultures.
In other instances, proteolytic enzymes or chemical disintegrators are used to remove the cells.
To get rid of the proteolytic enzymes, buffering liquid is used to wash the resulting cell suspension.
The cell suspension is put onto a flat surface, such as a sterile Petri plate or a culture dish.
An adequate culture medium is applied over the cells that may attach to the vessel's base, and they are then incubated at room temperature.

3. Cell thawing

The conserved cell culture may need to be employed in successive subcultures.
The growth medium where the cells are to be plated is warmed, and the water bath is heated to a temperature of 37°C.
The culture vessel is filled with the heated media. The frozen cells in the vial are then thawed in the water bath.
The via is cleaned on the exterior with 70% alcohol after defrosting. After being pipetted into the cell culture vessel, the cell suspension is gently stirred to combine everything.
After that, the medium is incubated for an overnight period under the normal growth conditions. The next day, the growing media is changed.

4. Trypsinizing Cells

Proteolytic enzymes are used in the trypsinization process to remove adherent cells from the surface of the culture medium. When the cells will be employed for passing through, counting, or other operations, it is done.
The cells are retrieved once the medium has been taken out. Phosphate buffer is then used to wash the cells.
The jar is then filled with warm trypsin-EDTA to cover the monolayer. To make sure the monolayer is coated, the jar can be shaken.
For 1-3 minutes, the vessel is heated to 37 °C in a CO2 incubator.
The flask is forcefully tapped on the side with the palm of the hand to aid in separation once the vessel has been taken from the incubator.

Applications of Animal cell culture

Several uses for animal cell culture include the following:

a. Production of vaccines

An essential method for the generation of viral vaccines is animal cell culture.
The method has been applied to the creation of recombinant vaccines for poliovirus and hepatitis B.
Viral vaccines are produced on a big scale or commercially using immortalised cell lines.

b. Recombinant proteins

Recombinant therapeutic proteins such cytokines, hematopoietic growth factors, growth factors, hormones, blood products, and enzymes can also be produced using animal cell cultures.
Baby hamster kidney and CHO cells are two of the popular animal cell lines utilised for the synthesis of these proteins.

c. Gene Therapy

The advancements in gene therapy depend heavily on the development of animal cell culture.
To correct these flaws and cure illnesses, cells with dysfunctional genes can be replaced by ones that operate.

d. Model systems

Cells grown by cell culture can be used as a model system for research into the biology of cells, host-pathogen interactions, medication effects, and effects brought on by changes in the composition of the cells.

e. Cancer Research

Since cancer cells may also be cultivated, animal cell culture can be utilised to explore the distinctions between cancer cells and normal cells.
The variations enable more thorough investigations of the probable origins and outcomes of various carcinogenic chemicals.
Certain chemicals, viruses, and radiation can be used to cultivate normal cells into cancer cells.
Studies on the effectiveness of cancer treatment methods and medications can also be conducted using cancer cells as test subjects.

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 benefits of using animal cells in culture:

Because it enables the manipulation of many physiological and physiobiological parameters including temperature, pH, and osmotic pressure, cell culture is preferable to other comparable biotechnological procedures.
Studies on cell metabolism and the understanding of cell biochemistry are made possible by animal cell culture.
Additionally, it enables the examination of how various substances, including proteins and medications, affect diverse cell types.
If only one cell type is employed, animal cell culture findings are consistently reproducible.
The method also makes it possible to distinguish between various cell types based on the presence of markers like molecules or through karyotyping.
Animals are not used in studies thanks to the utilisation of animal cells in testing and other procedures.
Animal cell culture may be utilised to produce huge amounts of proteins and antibodies without the need for a significant financial outlay.

Disadvantages of Animal cell culture

Animal cell culture has been employed as a cutting-edge technique, although there are certain drawbacks to this methodology.
It is a specialist approach that calls for aseptic settings and skilled staff. Due to the pricey equipment needed, the procedure is a costly operation.
The cell culture's second subculture might produce distinct characteristics from the initial strain.
The approach only yields a negligible quantity of recombinant proteins, which pushes up the cost of the procedure.
Mycoplasma contamination and viral infections are common and challenging to diagnose and cure.
Because of the prevalence of aneuploid chromosomal makeup, the cells produced using this method are unstable.