Everything you need to know about cell culture

Cell culture Laboratory technique allows the growth and proliferation of cells, both prokaryotes, and eukaryotes, in a controlled artificial environment. Usually, we refer to in vitro cultivation of cells coming from multicellular eukaryotic organisms and, in particular, from mammals; however, even single-celled organisms (e.g., bacteria and yeasts) can be propagated by growing them in a soil that reproduces their natural growing environment. CCs are fundamental for the study and development of viral vaccines and, in general, of biotechnology products. With recombinant DNA technology, animals, enzymes, synthetic hormones, monoclonal antibodies, and anti-cancer agents are produced in cc.

Types:

The protocols for in vitro cell cultivation have been developed since the 1950s, initially for the study of embryonic development, and then in virology, as the culture of suitable cell types provide the ideal substrate for virus growth. Cultured cells can grow either in suspension or adherent to a surface (monolayer cc), in a medium or liquid growth medium. The flasks or culture plates containing the cells are kept in incubators at constant temperature and in a gaseous environment (for mammalian cells at 37 ° C and 5% CO 2). To avoid contamination by microorganisms, all CC manipulations are carried out under sterile conditions; antibiotics are often added to culture media. CC is said to be primary if they derive from the dissociation of ex vivo tissues, and secondary cc if they derive from previous cultures. Primary cells can be obtained from the blood (lymphocytes) or from tissue explants, dissociated with enzymes that break the extracellular matrix and that allow the isolation of individual cells that grow individually in culture.

Techniques for obtaining cells:

There are two types of cells:

- Free and circulating cells like blood cells

- The cells in cohesion with each other, constituting a tissue.

The techniques for obtaining these two classes of cells are different.

- Circulating cells are obtained by sampling and centrifugation.

- The cells organized into tissues require the implementation of more original techniques, divided into two groups: the dissection method and the enzymatic digestion method. 

Dissection Methods:

Carrel's method

consists of taking a piece of tissue that is reduced into a very small rectangle and deposited on the surface of a mixture of 2 drops of rooster plasma and two drops of 50% embryonic extract. After a 24-hour stay in the oven, the first cells are seen to migrate from the explant.

The dissection method

consists of cutting the tissue, which is further reduced using forceps, into fragments of approximately 1 to 4 mm3. These fragments are then placed in a culture flask containing a nutritive medium. The cells will migrate from different fragments and then multiply. This method is also called the explants method.

The Jensen method:

This technique is more rarely used. The one mm3 tissue fragments are placed on a disc of filter paper, which is placed on metal support in a petri dish. The cells that proliferate from the tissue fragment cross the paper's pores and attach themselves to the bottom of the box. 

The mechanical method:

This technique is applied for soft tissues like the thymus, the spleen. It consists of rubbing the fabric on a grid, then filtering and centrifuging. Tissues can also be shredded using a glass pipette by pipetting and repressing the tissue.

The enzymatic digestion method:

The enzymes used are proteolytic enzymes that digest the protein web that surrounds cells. Trypsin is often used at a concentration of 0.5 to 2.5 g / l in saline.

Cell division cycles:

 Most of the animal cells in culture, after passing a certain number of divisions, reach a state of senescence and stop multiplying, entering a state of quiescence. The number of division cycles (Hayflick limit) depends on the cell type, the species, and the age of the organism from which the cells derive. Primary cells that exceed this growth limit give rise to a stabilized or immortalized cell line, capable of dividing into culture indefinitely. Many cell lines derive from tumor explants, which had, therefore, already lost some limits to growth. Cell lines can also be obtained from primary cells if they can make them undertake a transformation in growth in culture (immortalization). This process, similar to the loss of growth limits found in tumors, it can occur spontaneously due to acquired mutations, or it can be induced by radiation, chemical mutagens, transforming viruses, or by transfection with particular oncogene DNA sequences. Foreign DNA can also be inserted into the cell by means of a virus; in this case, the method is called transduction, infection, or transformation.

Banks of cells:

 Thousands of cell lines, representing different human and animal tissues, are kept in cell banks, such as the ATCC (American Type Culture Collection) and the ECCC (European Collection of Cell Cultures).

Cell culture applications

Scientists have developed the cell culture process to grow microorganisms outside of their native environment. Several types of cells can be cultivated: unicellular microorganisms (bacteria, yeasts, etc.) and cells from multicellular organisms (plants and animals). By growing them in the laboratory, you can control their growth and obtain large amounts of microorganisms or useful substances.

There are several applications to cell culture, among others:

- allow researchers to better understand how cells work

- allow testing of drugs, beauty products or even checking the toxicity of certain chemicals and thus avoid testing on animals

- allow the production of certain vaccines whose viruses develop inside cells;

- Allow the production of tissue such as new skin for burn victims.

The processes used for cell culture

Cell culture is done in several stages. It is first necessary to obtain cells and install them in an appropriate culture medium in order to reproduce the living conditions which the cell knew in its original medium.

Obtain cells

Obtaining cells differ according to whether they come from unicellular or multicellular living beings. Cells from single-celled beings (yeast, bacteria, etc.) are directly taken from various media and transferred to an appropriate culture medium. On the other hand, in the case of organisms formed from several cells, specialists can use isolated cells; for example, those of blood. However, they will usually use linked cells found in different tissues of the body. In this case, the first step in cell culture will be the separation of these cells. A sampling of cells using a swab and transfer to a culture medium

Find an appropriate support and culture medium

Depending on the type of cell cultivated, the cells collected must be placed in a culture medium that meets their needs. A culture medium is therefore defined as a medium in which all the elements necessary for the growth of cells placed in culture are found. The composition of the culture media varies, but they contain, among other things, water, mineral salts, amino acids, glucose, gases, etc. The culture media can be liquid or solid. Generally, it is estimated that a liquid culture medium promotes the rapid growth of microorganisms, while a solid medium is practical for listing and identifying them.

Solid culture medium

Reproduce the conditions of the cells' original living environment

Cell culture requires the reconstruction of the original conditions of the cell environment. To do this, one must control the temperature, pressure, humidity, pH, the composition of nutrients and minerals, etc. culture medium. This can promote normal growth and efficient cell reproduction. Incubator to control the surrounding conditions of the culture medium (temperature, pressure, humidity, amount of light)

Work in a sterile environment

All the procedures necessary for cell culture require work in a sterile medium, that is to say, a medium free of any living microorganism. To do this, many treatments exist to sterilize equipment useful for work.

Treatment and description Possible inconvenience

Flame treatment

The material is heated over a flame. Microorganisms die due to heat. The material can melt under the heat of the flame.

Dry heat oven treatment

The material is heated inside an oven, which kills microorganisms. Material that does not withstand heat can break.

Chemical treatment

The material is soaked in a solution or exposed to a gas, which kills microorganisms. The substances used are often harmful to human health and difficult to handle safely.

Steam Treatment 

This treatment is generally carried out in an autoclave, which is to say a device that has an airtight chamber in which the material to be sterilized is placed. The high pressure and heat kill microorganisms. Equipment that does not withstand humidity cannot be steam-sterilized.

Radiation treatment

The material is exposed to radiation (X-rays, UV rays, gamma rays, etc.), which kill microorganisms. Exposure to radiation can be harmful to human health. 

Growth and conservation of cell cultures

During cell culture, the growth rate of cells is not constant. It is rather carried out according to a curve in which we can distinguish 4 phases.

1. The adaptation phase. There is practically no cell growth since the cells adapt to and settle in their new environment.

2. The rapid growth phase. The cells divide quickly because they consume most of the nutrients in the culture medium.

3. The stationary phase. The number of cells is constant since there are as many cells that die as new ones are produced. This is explained by a depletion of nutrients, an accumulation of waste, and a lack of available space.

4. The decline phase. Nutrients and space are too scarce to maintain the maximum number of cells. This number is decreasing.

The maximum possible number of cells is reached at the end of the rapid growth phase. When the stationary phase is reached, it may be useful to stop the culture and keep it for analysis or later use. The cultures can be frozen to preserve them. It is also during this phase that the cells must be transplanted (transfer them) to a new culture medium since the nutrients in the initial medium are being depleted, which will trigger the decline phase.

Stem cell culture

Our body contains two types of cells: specialized cells and stem cells. Specialized cells fulfill specific roles in our organism. When you put such a cell type in culture, you get cells that have exactly the same specialization as the original cell. Thus, a muscle cell will only produce muscle cells. To get around this fact, stem cells can be used.

Principle of production of an organ from specialized cells

Stem cells are cells that do not play a particular role in the body. They have the capacity to divide almost indefinitely, and, under special conditions, they can transform into specialized cells. Due to these two features, stem cells make it possible to produce replacement tissues and organs. In adults, some stem cells are found in the blood and bone marrow. The first cells of an embryo also stem cells, which will later specialize in giving the different specialized cells of the body. Stem cells can, therefore, be obtained from an embryo, an umbilical cord, or a placenta.

Embryonic stem cells that can develop into various types of specialized cells

The cultivation of stem cells raises a lot of controversies since certain moral principles can be transgressed by this practice. For example, if you consider that an individual's life begins as soon as an egg is fertilized by a sperm; the use of embryos for the cultivation of stem cells becomes unacceptable. Ethical standards, that is to say, rules that seek to uphold certain moral principles, have therefore been imposed by the Canadian Scientific Research Institute in order to regulate stem cell research.

- The embryos used must not have been obtained as a result of commercial transactions.

- A pregnant woman who gives her embryo must do so without having been forced to do so.

- A pregnant woman who donates her embryo should know that it will be used for stem cell research.

- The umbilical cord and placenta can be used for stem cell research if both parents give their consent.

- Human stem cells taken from an adult must have been obtained with the consent of the donor.

Despite fears related to the use of stem cells, the fact remains that their culture can cure certain forms of cancer, including lymph nodes and blood. In addition, due to stem cell research, we hope one day to cure diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and several heart diseases.

Author: Vicki Lezama