Growth of Animal Cells in Culture, General Procedure for Cell Culture

Animal cells are grown artificially in a favorable environment in the form of animal cell culture.

• The cells needed for animal culture should come from multicellular eukaryotes and their established cell lines.
• The technique of animal cell culture is used to separate and cultivate cells from animals under artificial conditions.
• A cell line can be maintained as a distinct entity from its original source using this technique, which was developed for specific laboratory studies.
• To develop animal cell culture techniques, basic tissue culture media were developed in order to enable the work of so many different types of cells under different conditions.
• Cells isolated from various animals have been cultured in vitro to help discover different functions and mechanisms of behavior.
• Cancer research, vaccine production, and gene therapy are some of the areas where animal cell cultures find the most application.
• Cells grown on artificial media require more nutrients and growth factors than microorganisms, since they grow more slowly.
• The artificial media now offer both the possibility of culturing differentiated cells and those that are undifferentiated.
• In vitro animal cell cultures can be initiated using different types of cells, and in vitro organ cultures can be initiated using complex structures like organs.
• The culture process can use any type of tissue, cell, or organ depending on the purpose and application.

How to prepare for cell passage

• Sterilizing the culture hood requires the application of UV radiation first.
• If you are starting out with media previously stored at 4°C, let it warm up to room temperature first.
• Swish 70% ethyl alcohol over the hood.
• After rinsing them with alcohol, keep all necessary objects in the culture hood.
• To discard the spent media from the culture flask, discard should be available in the hood.
• Under a microscope, check to see if cells are confluent. Make sure the cells are healthy.
• Confluent cells should never be split. Stress will be caused to the cells. Handle the cells carefully and gently since they are very fragile.
• Hands should be cleaned with 70% ethyl alcohol.
• It is necessary to pre-warm Trypsin/EDTA at 37 °C for cell splitting.
• To remove the spent media, use a serological pipette to remove the confluent flask.
• Using phosphate buffer saline, wash your monolayer of cells once.
• Next, add one mL of Trypsin/EDTA solution to the monolayer and let it stand.
• Trypsin solution should be removed.
• For approximately one and a half minutes, place the flask in a BOD incubator at 37°C.
• Gently tap the flask on the side to dislodge cells.
• After dislodging the cells, 2-3 mL of 10% complete DMEM medium should be used to flush them off.
• Observe at 1500 rpm for 5 minutes the centrifuged tube containing the cells.
• It will form a cell pellet.
• Pour 2 ml of DEMEM media over the pellet and dissolve it.
• A hemocytometer should be used to count the cell number and transfer the cells to the new flask to maintain the cell line.
• If the flask is contaminated, check it after 24 hours.
• This flask should be split at regular intervals in order to maintain the cell line for a long time (up to 30 passages).

Types of cell culture

Primary cell culture

Cells are typically propagated by culturing the tissues directly and growing until they occupy the whole surface of the culture medium (confluence).

To perform primary culture, it is first necessary to separate the organ from the tissue and then divide the cells from the tissues. A trypsin solution can be used for this.

• Cells are then grown in freshly prepared cell-specific medium, and incubated in an incubator under conditions that support cell growth.
• Primary cultures are known as cell lines or subclones after the first subculture.

Secondary cell culture

• Secondary culture refers to the process of isolating and culturing cells from primary cultures at confluence in new media.
• Cell splitting is another name for secondary culture or subculture.
• Cells can expand more easily in subcultures due to fresh nutrients.
• Cells are separated from primary cultures using trypsin/EDTA.
• It is a serine protease that digests extracellular proteins or matrix proteins in order to free cells.
• An EDTA chelating agent can chelate calcium ion, which plays a role in adhesion between cells.

Cell line

Subcultured cells derived from primary cells constitute a cell line. Cell lines can have altered genotypes and phenotypes, but they usually exhibit similar properties compared with primary cells. There are several cell lines, each with a different phenotype or phenotypes similar to each other.

Based on the growth patterns of the cells, the cell lines can be further classified into two groups;

Finite cell lines

• A cell line with a limited number of cell divisions will eventually die, while a cell line with an unlimited number of cell divisions will divide indefinitely.
• Cells within finite lines of cells can divide from 20 to 100 times before dying and not being able to divide again.
• Number and duration of cell divisions are influenced by many factors, including cell lineage, species, culture conditions, and media.

Continuous cell line

• Their growth rate increases so quickly that they form independent cultures. Cells can divide indefinitely if they are immortal.
• Genetic alterations could also result in the transformation of cells from continuous cell lines into tumors.
• Infected with oncogenic viruses or exposed to chemicals that cause cancer, primary cell cultures become transformed.
• A suspension of cells can grow in liquid media and is capable of forming higher cell density.
• Even by growing on top of each other, cells can form multilayered structures in culture vessels.

Finite vs continuous cell line

• The finite cell line consists of cells that divide for a limited period and then lose their proliferation capability (senescence).
• Those cells that can multiply indefinitely are transformed, such as those created by chemical or viral means. They are called continuous cell lines.
• Anchorage dependence and anchorage dependence are characteristics of cells with finite growth rates and contact inhibition.
• Continually growing cells do not suffer from contact or anchorage inhibition, nor do they grow slowly.
• The cells of a fixed cell line grow as monolayer cultures, meaning they cover the bottom of the culture vessel uniformly.
• Continous monolayers of cells can be grown in suspension cultures (suspension cultures, not attachment cultures).

The process of freezing cells in liquid nitrogen

• Upon trypsinization, a pellet forms on the confluent culture flask and dissolves in DMEM.
• Cell density can be determined by counting the cells.
• After that, place 2-3 million cells in each cryovial.
• This type of media consists of DMEM media with 10% FBS and 10% DMSO. Dimethyl sulfoxide (DMSO) acts as a cryoprotectant, preventing ice crystals from forming.
• Cryovial should be cooled to -80°C and then cooled in liquid nitrogen the following day.
• Using this method, our cell line can be preserved for a very long time.

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