Animal Cell Culture

• Tissue Culture is the term used to describe the process of removing tissues, cells or organs from either a plant or animal and their subsequent transfer into an artificial environment that encourages development.
• The environment is usually comprised of the right glass or plastic container for the culture that contains a semisolid or liquid medium that provides the necessary nutrients for growth and survival.
• The study of the whole organ or organ fragments that are intact to study their ongoing function or their growth is termed Organ Culture.
• After the cells have been removed from organ fragments, they are removed prior to, as well during cultivation thereby or during cultivation, thereby disrupting their normal relationship with their cells. This is referred to as or during cultivation, thereby disrupting their normal relationships with neighboring cells, it is known Culture.
• While animal cell cultures were first made successful through Ross Harrison in 1907, it wasn’t until mid 1940’s until the early 1950’s when a series of changes were made that made cell cultures widespread as a method for scientists.
• First, there was the development of antibiotics that made it easier to avoid many of the contamination problems that plagued earlier cell culture attempts. Second was the development of the techniques, such as the use of trypsin to remove cells from culture vessels, necessary to obtain continuously growing cell lines (such as HeLa cells). Third, using these cell lines, scientists were able to develop standardized, chemically defined culture media that made it far easier to grow cells. These three areas combined to allow many more scientists to use cell, tissue and organ culture in their research.
• In the 1960’s and the 1970’s, the commercialization process of this technology also had an effects on cell culture that continues until today. Companies, like Corning started to create and market disposable glass and plastic cells culture equipment, better filters and other materials, powdered and liquid tissues culture mediums, as well as the laminar flowhood. The end outcome for these, and many other technological advancements has resulted in an rise in the number of labs and companies that use cells in the present.

Types of animal cell culture

Based on the frequency of cell divisions Cell culture may be classified as primary cell culture or cell lines. Cell lines may undergo infinite or finite cell divisions.

1. Primary cell culture

• This is the culture of cells extracted directly from the host cell tissue.
• The cells separated from the parent tissue are then cultivated on an appropriate container, and the resulting culture is known as the primary culture.
• The cells in this culture are mostly heterogeneous cells. Most of them only divide for a short period of time. But, they are very like their predecessors.
• According to their source primary cells develop either in a monolayer that is adherent or as a suspension.

a. Adherent cells

• They are anchorage dependent, and they propagate in monolayers.
• These cells require to be fixed to solid or semi-solid substrate in order to increase their expansion.
• They adhere to the vessel of culture through the help of an extracellular matrix , which typically comes from organ tissues which are immobile and encased within a network of connective tissues. Epithelial cells and fibroblasts are examples of these types.
• The base of the vessel is covered by the same cell layer, typically only one cell thick is known as monolayer culture.
• The majority of cell lines that are continuous are developed in monolayers.
• Since they are single layers, the cells can be directly transferred to the cover slip for examination under microscope.

b. Suspension cells

• Suspension cells are not attached to the surfaces of vessels for culture.
• They are also known as anchorage independent cells or non-adherent ones which can be developed floating in the medium of culture.
• Hematopoietic stem cell (derived from spleen, blood as well as bone marrow) and cancer cells can be developed in suspension.
• The cells are growing much more quickly that they do not need regular renewal of medium, and are easily maintained.
• They are homogeneous and treatment with enzymes is not necessary for dissociation of cells. In addition, these cells have a short lag time.

Confluent culture and the necessity of sub-culture

After the cells have been isolated from the tissue and then proliferated in the right conditions, they take up all the substrate available i.e. reach confluence. After a couple of days it may become overcrowded for their containers and can cause harm to their growth. Usually, this is result in cell death when left for a prolonged period of time. Cells therefore need to be subcultured i.e. some cells are transferred to a new container with a fresh growth medium, which gives more room and nutrients for the continued development of both cells. Subculture helps keep cells in a healthy and developing condition.

A number in a passage refers to the amount of times a particular cell line has been sub-cultured. This is in contrast to the population doubling threshold, in that the exact number of cells involved in the experiment is irrelevant. It provides a general indication of how old cells are for different tests.

2. Secondary cell culture and cell line

• If a primary culture is sub-cultured, it’s known as secondary culture , or cells or sub-clones.
• The procedure involves removing the growth media , and then dissociating the cells that are adhered (usually through enzymes).
• Sub-culturing primary cells to different divisions results in the creation new cell lineages.
• In the course of time that follows, cells with the most capacity for growth prevail and result in some degree of genotypic as well as phenotypic consistency in the population. However, once they’re sub-cultured, they develop distinct from the cell that was originally.

Types of Secondary cell culture and cell line

On the basis of the life span of culture, the cell lines are categorized into two types:

a. Finite cell lines 

• Cell lines that have a limit on the number of cell divisions with the capacity to live for a short time are referred to in the field of finite cells.
• The cells move through the blood multiple times before losing their proliferative capacity and grow, which is a genetically-determined phenomenon known as senescence.
• Cell lines derived from the primary cell cultures are cell lines that are finite.

b. Continuous cell lines 

• If a finite cell line undergoes transformation and gains the capacity to continue to divide it transforms into a continuous cell line. 
• The transformation or mutation could happen naturally or be either virally or chemically caused or result from the establishment of cell cultures in cancerous tissue.
• Cells grown in this manner can be sub-cultured and cultivated for a long time as cell lines that are permanent and remain immortal.
• They are less affixed to their environment, are fast growing, and less attentive to their nutrition requirements, and capable of growing to more cell numbers and are different in phenotypes compared to the original tissue. They also grow faster in suspension.
• They also tend to form on top of one another within multilayers of the surface of 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 among the very first continuous human cell lines that make use of cervical cancer.
• They are utilized for processes such as virus cultivation and preclinical drug testing.

b. HL 60 (Leukemia)

c. MCF-7 (breast cancer cells)

Cell strain

• The lineage of cells that originate from the primary culture is known as strain.
• They can be created from a primary culture or cell line generated by negative selection process or by the cloning cells with specific characteristics or features.
• A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.

Methods for Animal Cell Culture

Growth Requirements

• The media for cell culture are typically extremely complex and the culture conditions vary for every cell kind. The media usually contain amino acids and vitamins, as well as salts (maintain Osmotic pressure) and glucose and a bicarbonate buffer system (maintains the pH of between 7.2 between 7.4 and 7.4) and growth factors hormones, O2 as well as CO2.
• To ensure the highest growth, adding an amount of blood serum is often required along with several antibiotics such as streptomycin and penicillin are included to help prevent the spread of bacterial infections.
• Temperatures vary based on the type of cell host. Mammalian cells tend to be kept at 37oC to ensure optimal growth however, cells that originate from cold-blooded animals can tolerate greater temperature ranges (i.e. 15oC to 26oC). The cells that are active in growth of log phage are recommended that divide quickly during the course of the culture.

Primary cell culture

• Primary cell culture is created from tissue samples that are fresh.
• Tissue fragments from an organ can be removed in a clean manner which is usually done using a razor sharp and sterile and then dissociated through the proteolytic enzymes (such as trypsin) which break down between cells.
• The cell suspension that is obtained is then rinsed using an appropriate buffer (to get rid of the proteolytic enzymes employed).
• The suspension of cells is spread across the surface of flat like a bottle, or Petri dish. The small layer that adheres to the glass or dish is then covered with a suitable medium for culture and incubated at acceptable temperature.

Aseptic techniques

• Bacterial infections like Mycoplasma or fungal infection often occur in cell culture and pose a challenge to recognize and eradicate.
• So, all cell-culture work is performed in a clean environment using the right aseptic methods.
• Work must be conducted in laminar flows with the continuous unidirectional flow of HEPA filtering air over the area of work.
• All materials as well as the entire environment should be free of contaminants.

Cryopreservation

• If there is a surplus of cells are available due to sub-culturing, these should be treated by the proper protective substance (e.g., DMSO or Glycerol) and kept at temperatures that are below -130°C until they’re needed.
• This is a way to store cell stock and stops the loss of the original cell due to an unexpected failure of equipment and biological contamination.
• It also stops the development of finite cells and reduces the risk of changes in long-term cultures.
• In the process of thawing cells, the cell tube that has been frozen is quickly warmed up by soaking in warm water. It is then washed with serum and medium, later placed into the culture container after being suspended in the proper medium.

Applications of Cell Line

Cell culture has become one of the major tools used in cell and molecular biology. Some of

the important areas where cell culture is currently playing a major role are briefly described

below:

Model Systems

Cell cultures are a useful method of studying: 1)) the fundamental cell biology and biochemistry) the interaction between the disease-causing agents and the cells three) how drugs on cells 4.) the mechanism and triggers that lead to aging and five) nutrition research.

Toxicity Testing

Cells from culture are often employed in combination with animal experiments to examine the effects of novel chemical compounds, cosmetics and drugs on growth and survival in many cells. The most important of these are kidney- and liver-derived cells.

Cancer Research

Because both normal cells as well as tumor cells are able to be created in a culture and manipulated in a laboratory, the fundamental distinctions in them could be investigated. Additionally, it is possible, with the help of radioactive substances, chemicals and viruses to change normal cells into cancer-causing cells. This means that the mechanisms which cause this change can be examined. Cancer cells grown in culture are also used as a testing system for determining the right treatments and methods of selectively killing cancerous forms.

Virology

One of the initial and principal uses of cell cultures is the reproduction of viruses in cell culture (in instead of animal) to aid in the development of vaccines. Cell culture is also employed in the identification or isolation of viral infections and for fundamental research into the ways they spread and infect organisms.

Cell-Based Manufacturing

Although cultured cells are able to be used in the production of various useful products, three areas that are the most popular. One is massive production of viruses that are used in the production of vaccines. This includes vaccines against rabies, polio the chicken pox, the hepatitis B as well as measles. The second is the massive production of cells genetically engineered to make proteins that are of commercial or medicinal significance. This includes monoclonal antibodies insulin, hormones and more. Thirdly, are the cells used to organs or replacement tissues. Artificial skin used for treating ulcers and burns is the very first commercially available product. 

The testing process is currently underway on artificial organs like the pancreas, kidney and liver. The possibility of a supply of tissue and cells for replacement could result from the research that is currently being conducted using both embryonic stem cells and adult stem cells. These cells have the ability for differentiation into range of different types of cells. It is expected that understanding how to control the growth of these cells can provide solutions to many medical diseases.

Genetic Counseling

Amniocentesis is a diagnostic procedure which allows doctors to take out and study fetal cells taken from women who are pregnant it has given doctors an important tool to aid in the diagnosis early of fetal problems. The cells are then checked for abnormalities on their chromosomes, genes and chromosomes using the karyotyping method, chromosome painting and other molecular techniques.

Genetic Engineering

The capability to transfect or change the cultured cells’ genes with DNA (DNA as well as genes) is a key instrument for molecular biologists who want to examine the cellular effects of the expression of these genetic material (new protein). These methods are also able to make these new proteins in large quantities in cells that are cultured to further research. Insect cells are employed as miniature cell factories to produce huge amounts of proteins they produce after infecting themselves with genetically engineered Baculoviruses.

Gene Therapy

The ability to engineer genetically cells is also a reason for their application to treat diseases using gene therapy. Cells can be taken from patients lacking an active gene, and the damaged or absent gene could be substituted. The cells could be grown time in a culture before being transplanted into the patient. Another option is to put the gene that is missing into the viral vector, and then “infect patients with this virus, hoping that the gene that is missing will appear in cells of the patient.

Drug Screening and Development

Cell-based tests have become increasingly vital for companies in the field of pharmaceuticals, and not only for cytotoxicity testing, as well as for high-throughput testing of compounds that might have potential as drugs. At first, these tests were conducted in plates that had 96 wells, but more and more are comprised of 384 and 1536 well plates.

Advantages of Animal cell culture

Here are a few of the benefits of using animal cell culture:

1. Cell culture is superior to similar methods of biotechnology because it allows for the alteration of various physiological and physiobiological conditions such as pH, temperature and Osmotic pressure.
2. Animal cell culture is a great way to study issues that study cell metabolism and helps to understand the cell biochemistry.
3. It also permits observation of the effects of different chemicals like drugs and proteins on various cell types.
4. The results of animal cell culture are consistent regardless of the type of cell that is utilized.
5. This technique can also facilitate the identification of various cells on the basis in the appearance of marker, such as molecules, or through the karyotyping.
6. The usage of animal cells for testing and other purposes is a barrier to the animal experiments.
7. Animal cell cultures can be utilized to produce massive amounts of antibodies and proteins that otherwise would require an investment of a substantial amount.

Disadvantages of Animal cell culture

Although the culture of animal cells is used as an advanced technology but there are some drawbacks that are associated with this method.

• It is a highly specialized method that requires experienced personnel as well as the right conditions for aseptic treatment. It is a costly procedure because it requires expensive equipment.
• Subcultures of the cell line could result in different properties when different from that of the initial strain.
• The process produces a tiny amount of recombinant proteins which increases the cost of the procedure.
• Mycoplasma-related contamination and viral infections are common and are hard to identify and manage.
• The cells produced by this technique lead to instability due to the occurrence of aneuploidy chromosomal constitution.

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