Cell culture is growing cells in a controlled environment outside their natural environment. Cell cultures study various biological processes, including cell growth and differentiation, gene expression, metabolism, signal transduction, protein folding and secretion, and many other aspects of biochemistry and molecular biology.
Cell culture is used in laboratories worldwide to study the cells' normal physiology and biochemistry, disease mechanisms (including cancer), and effects of drugs and virulent compounds. In addition to studying biological processes in cells, cell cultures are also used to produce pharmaceuticals and other therapeutic products.
An introduction to Cell culture
Cell culture is growing cells in a controlled environment outside their natural setting. This allows for the study and manipulation of cells for various purposes, such as research or drug development. You can create cell cultures from many different types of cells, including those from plants, animals, and bacteria. Here are a few things you need to know about cell culture:
Adherent and suspension cultures:
Adherent cultures refer to cell cultures grown on a solid substrate such as plastic or glass. These cells attach to the substrate and grow in two-dimensional monolayers. Adherent cultures are often used for experiments involving membrane proteins, signaling pathways, and gene expression analysis. Cells that adhere attach and grow on the surface of a cell culture vessel in one layer.
Suspension cultures refer to cells grown in liquid media and not attached to a substrate. These cells can be grown in liquid or semi-solid media, such as agar or gelatin. Suspension cultures are often used for experiments involving cell proliferation, drug testing, and gene expression analysis. Instead of forming monolayers on the surface of cell culture vessels, cells in suspension often form clumps, especially at higher densities.
Confluency and Cell growth:
Confluency refers to the percentage of cells that reach full coverage of the growth surface. It is an important factor in cell culture as it affects cell growth and differentiation rate. Higher confluency rates generally result in faster cell growth and greater differentiation potential. It is important to monitor confluency regularly to maintain optimal cell growth and differentiation.
Cell growth is the process by which cells increase in number. You can measure it in terms of cell number, size, or occupied surface area. Cell growth is affected by various factors, including nutrient availability, pH, temperature, and oxygen levels. Monitoring cell growth in culture allows researchers to assess the health of the cultures and adjust the environment accordingly. Cell growth slows when cells become too close together, and the cells may touch each other. If allowed to overcrowd, it can lead to cell death due to a lack of resources.
A cell population is in apoptosis when the natural cell death process predominates. This can occur due to environmental stress, nutritional deprivation, or when cells reach the end of their natural lifespan. Apoptosis is important for maintaining cell homeostasis and preventing uncontrolled growth, which could have consequences such as cancer or other diseases. Monitoring apoptosis in culture can help researchers control the health and size of their cell populations.
Primary and immortalized cells:
Primary cells are derived directly from tissue and can be used for more specialized studies. They are usually grown in monolayer culture, i.e., on a flat substrate such as plastic or glass but can also be grown as suspension cultures in liquid media. Primary cells have a limited lifespan, so they need to be replenished regularly, which makes them unsuitable for long-term experimentation.
On the other hand, Immortalized cells are derived from primary cells and can be maintained indefinitely with minimal maintenance. They are often used in drug development as they are more cost-effective and easier to maintain than primary cells. Immortalized cell lines come in various types, such as epithelial, endothelial, mesenchymal, and hematopoietic. They are usually grown in monolayer cultures but can also be grown in suspension cultures. Cells grown in culture can also be immortal by treatment with specific chemicals.
Both primary and immortalized cells can be used for various experiments, such as cell signaling, drug testing, and gene expression analysis. The choice between the two will depend on the type of experiment being performed and the resources available.
We can control the conditions of our cultures by changing the temperature, humidity, CO2 levels, and other aspects such as media composition or cell growth. It is important to use the right equipment, such as incubators, humidifiers, and CO2 controllers, to maintain optimal culture conditions. Here is an example of optimal cell culture conditions of mammalian cells:
Relative humidity: 95-100%
CO2 levels: 5-10%
Osmolarity: 280–320 mOsmol/kg
Non-mammalian culture and Mammalian culture:
Non-mammalian cells are derived from non-mammalian tissues and are used to study various biological processes such as cell signaling, drug testing, and gene expression analysis. These cells can be grown in adherent and suspension culture systems but have limited self-renewal capacity. Examples of non-mammalian cells include yeast, insect, plant, and bacterial cells.
Mammalian cells are derived from mammalian tissues and can study various biological processes, such as cell signaling, drug testing, and gene expression analysis. These cells have the capacity for self-renewal and can be grown in both adherent and suspension culture systems.
Choosing a cell line:
When choosing a cell line for culture, it is important to consider several factors, such as the type of experiment being performed, the resources available, and the desired outcome. Different cell lines have different properties and should be chosen accordingly. For example, immortalized cells are often preferable when long-term experiments are required, as you can maintain them indefinitely with minimal effort. On the other hand, primary cells can provide more accurate results as they are derived from living tissues and have not been subjected to genetic manipulation.
Pick a cell line compatible with your experiment that will lead to the desired results. Carefully monitor your cultures and adjust environmental conditions to maintain optimal cell growth and differentiation.
Finite cell lines are more functionally relevant as they have not been subjected to genetic manipulation and can provide more accurate results. They are, however, limited in their self-renewal capacity and may not be suitable for long-term experiments.
Cell lines that have been transformed display enhanced growth rates and higher plating efficiency because of their manipulation with oncogenic viruses or genetic engineering. This makes them ideal for high-throughput drug screening and gene expression analysis experiments.
Cell culture media:
Cultured cells are fed with liquid media. The right media should be chosen depending on the cell type, requirements, and the experiment performed. Different media types are available such as basal medium, glutamine, animal serum, and antibiotics.
The cells in your body need a variety of nutrients and minerals to function properly. This mixture is known as basal medium. Glutamine is an amino acid that provides energy to the cells and helps maintain their health. You can add animal serum to the basal medium as a source of growth factors and other nutrients. Antibiotics are used to inhibit bacterial growth and prevent contamination.
Careful consideration should be taken when selecting media, as certain components can affect the cells' growth and differentiation. It is important to check the medium's compatibility with the cells before using it in experiments.
Cell line authentication:
Cell line authentication verifies that a cell line is true to its source and free from contamination. Cell lines can be contaminated by other cell types or different species, which can lead to unexpected results in experiments. Authentication involves testing the DNA fingerprint of the cells and comparing it to a reference sample. This ensures that the cells are of the correct species and are free from contamination. Authentication is crucial in cell culture as it ensures reliable results and reduces the risk of experimental failure.
Cell lines can be authenticated by studying their STR loci, which are Short Tandem Repeats that vary from individual to individual. Comparing the cell line's STR loci to a reference sample will determine its origin and verify that it is free from contamination.
The aseptic procedure is a set of guidelines for setting up and maintaining cell cultures without contamination. It involves following rigorous cleaning protocols, properly handling materials, sterilizing all equipment with disinfectants, and avoiding cross-contamination. The aseptic technique is essential to obtain successful results from cell culture experiments.
The aseptic procedure encompasses handling, reagents, and workplace hygiene. It is crucial to adhere to an aseptic technique to maintain a clean working environment and sterilize all equipment before use. Always wear gloves when handling cultures to prevent contamination. Pay close attention to the details, as any mistake can seriously affect your experiment.
Primary cell isolation:
Primary cells are derived directly from the patient's body and are not immortalized. They have limited replicative potential and a short lifespan, making them difficult to work with. Complex biological samples, including bone marrow, lymph nodes, tissues and blood, and spleen, are sources of primary cells.
Primary cell isolation is the process of separating viable cells from a complex sample. This involves enzymatic digestion, centrifugation, and fluorescence-activated cell sorting. Once isolated, the cells can be cultured in vitro to study their properties and behavior. Isolated primary cells can also be used for drug discovery, gene therapy, and regenerative medicine.
The tissue being worked with should be cut into manageable pieces 2-4 mm in size to successfully isolate primary cells. This can be done best with sterile scissors or a scalpel. To start, add the tissue pieces to a suitable balanced salt solution or buffer on ice and then let it sit for 2-3 washes. The samples are then incubated with enzymes such as collagenase or disposed of to break down the connective tissue and allow for cell separation. The solution is then centrifuged, and cells are separated based on density.
Once isolated, the cells can be cultured in vitro for further experimentation and study. Careful attention must be taken to ensure that the cells are not contaminated or overgrown with other cell types.
Procedures for cryopreservation and thawing:
Cells can be cryopreserved to extend their lifespan and maintain them for future use. Samples must be cooled rapidly to subzero temperatures in liquid nitrogen. This causes the cell's membrane lipids to change their structure and solidify, forming a protective coating around the cell and preventing it from damaging during freezing. Cells can be stored long-term at temperatures below -130 °C.
A cryoprotective agent, such as DMSO or glycerol, is commonly added to the cell suspension before freezing to prevent ice crystals from damaging cells. The freezing process begins by setting the temperature to the desired state below zero°C. The specific conditions vary depending on which cell line is used, but it typically involves cooling at a rate of -1°C per minute.
When ready to use, the frozen cells must be thawed quickly in a warm buffer and resuspended in a fresh growth medium. The cells must then be cultured as usual, checking for any changes that may have occurred during storage.
First, move the frozen vial into a 37 °C water bath to thaw the cells. Once thawed, immediately centrifuge and discard any remaining cryoprotectant, then resuspend cells in a fresh growth medium. Cells should be used within 1-2 hours of thawing and monitored carefully for any signs of damage or contamination.
Mycoplasma cell infection testing:
Mycoplasma is small bacteria that can contaminate cell cultures. They cannot be seen with the naked eye and do not respond to traditional antibiotic treatments.
Mycoplasma infection of cells can cause various issues, including reduced growth rate, altered cell morphology, and death. To prevent this, it is important to regularly test for mycoplasma contamination. You should test new cell lines for mycoplasma and repeat this practice every 10 passages.
Mycoplasma testing can be performed using PCR or a commercial mycoplasma detection kit. The most accurate test is PCR, which amplifies the DNA of specific mycoplasma species and provides results quickly. Mycoplasma kits use a combination of different reagents that react with mycoplasma in the sample to indicate contamination.
If an infection is found, it should be treated immediately with antibiotics or antibacterial agents designed specifically for mycoplasma. If possible, the infected cell line should also be discarded and replaced with a new one. All contaminated material should be disposed of properly to avoid spreading the infection.
Cell counting is an important procedure that allows researchers to accurately measure the number of cells in a culture. It is necessary for experiments that require precise cell numbers, such as drug testing and cell sorting. A hemocytometer is used to count cells, which is essentially a small, flat microscope slide with a counting chamber etched into it. The sample is placed in the chamber and can be seen through a microscope.
The technique involves diluting the sample and then placing a known volume of it in the hemocytometer chamber. This allows for an accurate count of how many cells are in the chamber, which can then be used to calculate the total number of cells in the sample. Cell counting can also measure cell viability or the number of live cells in a culture.
In cases where cell numbers fall outside the desired range (i.e., too few or too many), you must adjust the culture. This can involve diluting or concentrating the sample, adding more cells, or removing some. The proper technique must ensure accurate results and minimize damage to delicate cells.
For example, add 5 mL of the cell suspension to a hemocytometer chamber. In each of the 16 squares, an average of 10 cells can be seen. This means that 80 cells were counted in total, giving us a cell concentration of 1x10^6 cells/mL (80 x 10^6 = 800). The result is accurate regardless of how many individual cells are present in the 5 mL sample.
Cell transfection is a technique used to introduce genetic material into eukaryotic cells. It involves using a vector, or carrier molecule, to deliver the desired DNA into the cell's nucleus. This allows researchers to study gene expression and activity in vivo and explore how certain proteins interact with each other and influence cellular behavior.
Transfection is often performed using viral vectors, such as plasmids and retroviruses. Plasmid DNA is inserted into the cell's nucleus, while retroviral vectors can integrate foreign DNA into the host genome. Other transfection methods include electroporation and chemical-mediated transfection, which involve subjecting cells to an electrical current or specific chemical to create a temporary opening in the cell membrane, allowing DNA to enter.
The success of transfection depends on several factors, such as the vector used and the type of cell being transfected. Proper selection and optimization of these components are essential for successful transfection and for obtaining accurate results. After the DNA has been introduced, researchers can then analyze gene expression or protein activity to understand the effects of the transfection.
Once the cells have been grown and expanded in culture, they should be subcultured or passaged. Subculturing is splitting a cell population to reduce its density and maintain healthy cell growth. It also allows for efficient medium exchange and helps prevent cell death due to overcrowding or nutrient depletion.
Passage cells when they are 80-90% confluent for adherent cultures and at the highest density for suspension cultures. Counting the number of cell passages for every cell line is crucial. This way, you can monitor the cells' growth and health. Cell damage or death can be caused by too frequent or infrequent passaging, so it is important to find the right balance.
You should not experiment on cells passed through too many times, as they can become senescent and lose their physiological relevance. Typically, cells should be used within 10-15 passages before discarding them.
Once the cells have been passaged, they can be used in experiments or further studied. Careful consideration must be taken when selecting media and following all the necessary protocols to ensure successful cell culture. Briefly, the subculturing protocol for adherent cells is as follows:
1. Rinse the cells with a PBS solution to remove non-adherent cells.
2. Detach the adherent cells using trypsin/EDTA or another appropriate buffer, then neutralize the solution with a growth medium.
3. Centrifuge at low speed for 5 minutes and discard the supernatant.
4. Resuspend the cells in a fresh growth medium.
5. Plate the cells at an appropriate density.
6. Incubate the culture and observe for cell attachment, then change the medium every other day or as needed.
7. Subculture when the cells are 80-90% confluent.
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