Fundamentals of cell culture: equipment, basic principles and experimental programs

2021-11-13 06:24:24 By : Ms. Jane Ni

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New to cell culture? Then don't look at it. Here, you will find a basic overview of everything about cell culture, from setting up a cell culture laboratory to understanding the basic principles and basic techniques. A good starting point for a springboard into the world of cell biology.

Cell culture refers to removing cells from animals or plants and then culturing them in an artificial environment for scientific research. The first cell culture technology was developed more than 100 years ago, and since then it has contributed to great breakthroughs in the scientific field. Today, it is a basic tool used in laboratories around the world to study the normal physiology and biochemistry of cells, the underlying mechanisms of diseases, and the effects of drugs and toxic compounds. It is also used for drug screening and development and large-scale manufacturing of biological compounds, such as vaccines and therapeutic proteins. 1, 2

In order to conduct research that requires cell culture work and perform basic cell culture protocols, several key equipment and some basic reagents are required, which are summarized in Table 1 and Table 2.

Table 1: Basic equipment required for cell culture. 3, 4, 5 The images were created using

Additional equipment includes a suction pump in a laminar flow hood, which can easily remove medium and reagents from cell culture vessels; an autoclave for sterilizing equipment and reusable glassware; syringes, needles and tweezers; Timer; several 70% ethanol spray bottles and paper towel rolls for disinfecting surfaces and equipment; tape and permanent markers for marking; pipe racks and trash cans. 4, 5

Table 2: Basic reagents required for cell culture. 2, 5

Please refer to the section on cell culture media below.

Phosphate buffered saline (PBS) for washing cells.

An enzyme used to separate adherent cells from the culture vessel used for culture, such as trypsin.

A reagent that lowers the freezing point of the medium and slows down the cooling rate to reduce the risk of ice crystal formation. Ice crystals can damage cells and cause cell death. Dimethyl sulfoxide (DMSO) is the most commonly used.

  Obtaining deionized and distilled water and ice is also important. The nature of the experiment to be performed will inform the reagents that need to be obtained.

Any cell culture setup requires several important design considerations. The most important aspect is the use of design to maintain a sterile and sterile environment to prevent cell contamination. First, a separate enclosed room or laboratory should be used, with an entry/exit point. The entrance and exit of the laboratory should be close to the handwashing sink filled with soap and disinfectant for hand cleaning. The dedicated cell culture lab coat and safety goggles should be stored at the entrance of the lab. Laminar flow hoods and incubators should be kept away from the entrance to minimize the risk of contamination. It is also important to place the fume hood and incubator away from any air conditioning equipment to prevent potentially contaminated airflow from entering the sterile working environment and incubator. There should be sufficient clean work surfaces, regular disinfection is required, and sufficient storage space to ensure that the surfaces are kept clean. All necessary equipment and consumables should be easy to use in the laboratory to prevent access. An ergonomic environment is very important for laminar flow hoods. It has enough space to place drawers or movable consumables trolleys so that incubators, microscopes and centrifuges can be conveniently used at work. 6

Cell culture laboratories have risks associated with handling and manipulating cells and tissues, as well as toxic, corrosive or mutagenic solvents and reagents. Therefore, compliance with standard microbiological practices and techniques is essential to always reduce risks and ensure safety. There are four ascending levels of biosafety control, called the biosafety level (BSL). When dealing with hazardous biological materials and preparations, each level has standard microbiological practices, safety equipment and facility safeguards. BSL-1 is the basic protection level common in most research and clinical laboratories. In these laboratories, the reagents used will not cause disease in normal and healthy humans​​. BSL-2 is suitable for medium-risk drugs that are known to cause human diseases of varying severity through ingestion or through transdermal or mucosal exposure. Most cell culture laboratories should achieve at least BSL-2, but the specific requirements depend on the biological materials used and the type of work performed. BSL-3 is necessary for pathogens that cause serious and potentially fatal infections, while BSL-4 is the highest level of containment used in laboratories that deal with infectious pathogens at high risk of causing life-threatening diseases to individuals. 4, 7

The following is a list of basic safety recommendations for cell culture laboratories. This list is not complete and should be supplemented with appropriate biosafety level recommendations. -Always wear appropriate personal protective equipment (PPE), including lab coats, gloves, and goggles. -Be sure to read the Material Safety Data Sheet (MSDS) of any substance you are using to ensure that proper safety precautions are taken when handling. -Disinfect all work surfaces before and after the experiment. -Clean laboratory equipment regularly, even if it is not contaminated. -Avoid generating aerosols and/or splashes. -Wash your hands after handling potentially hazardous materials and before leaving the laboratory. -Disinfect all potentially infectious materials before disposal. -Report any incidents that may lead to exposure to infectious materials to appropriate personnel (eg, laboratory supervisors, safety officials). -Do not eat, drink, smoke, touch contact lenses, apply cosmetics, or store food for human consumption in the laboratory.

For cell survival and proliferation, the culture environment must replicate the physiological environment of the cell as much as possible. The culture conditions that can be controlled include temperature, relative humidity, and CO2 levels, as well as factors related to the medium, such as nutrient composition, pH, osmotic pressure, and replenishment amount and frequency. These variables fluctuate over time, so they should be monitored. Table 3 highlights the best culture conditions for most mammalian cell cultures, but there are exceptions. 5

Table 3: Optimal cell culture conditions for most mammalian cells. 1, 2, 4, 8

Primary cell cultures are cells that are directly separated from intact or isolated tissue or organ fragments and grown in petri dishes. Once the primary culture is passaged for the first time, it is called a cell line. Primary cell lines have a limited lifespan and can only be subcultured 10-20 times before reaching a state of senescence (cell division stopped). 1

Some cell lines have no limit on their life span and have unlimited proliferation capacity. These cell lines are called continuous cell lines or immortal cell lines. The immortalization of cells can occur in many ways. Cancer cells have inherent mutations that allow cells to multiply without restriction in culture. Normal cells with a limited lifespan at the beginning can be transformed into immortalization through mutations in growth-promoting genes. Normal cells grown in culture can also be intentionally immortalized by chemical treatment or introduction of oncogenic viruses that activate growth-promoting genes. Figure 1 shows the evolution of the primary to transformed continuous cell line and the theoretical cell yield at each stage. 9

Figure 1: Evolution and theoretical cell yield from primary to continuous cell lines.

Adherent cells grow as a monolayer and attach to the surface of the cell culture vessel. When passing, they need to be separated from the surface using a separating agent. They reattach to the surface within a few hours after plating. Suspended cells do not form a monolayer on the surface of the cell culture container, but remain in suspension. Cells form clumps, especially at high densities. 1, 10

Mammalian cell culture is the most common, but it is possible to cultivate cells from a large number of organisms, such as plants, insects, bacteria, and yeast. Plant cell cultures are usually grown as cell suspension cultures in liquid media or as callus cultures on solid media. Cells derived from Drosophila melanogaster or Armyworm Spodoptera frugiperda are examples of insect cell lines that are used for biochemical analysis or recombinant protein expression, respectively. For bacteria and yeast, a small number of cells are usually grown on solid supports containing nutrients, usually gels such as agar, while large-scale cultures are grown in which the cells are suspended in nutrient broth.

Cell culture growth is usually divided into four stages (Figure 2). When the cells adapt to the culture conditions and do not divide, there will be a lag phase. The log phase occurs when cells are actively dividing. This is the best stage for cell experiments and data collection. When the cells reach the late logarithmic stage, they should be subcultured. This happened before overcrowding. When the cells are close to overcrowding, cell growth will slow down. This is called the stationary phase or plateau. Cells at this stage are at risk of cellular stress. When the natural process of cell death dominates, the cell population is considered to be in the death phase, also known as the decline phase. When plotting the cell count log over time, it will generate a sigmoidal curve, as shown in Figure 2. It is important to note that the time spent in each stage varies between cell lines and cultures. 11

Figure 2: The four stages of cell growth.

An important indicator for describing monolayer cell culture is confluence. It is the percentage of the surface area of ​​the culture vessel covered by a layer of cells when viewed through a microscope. For example, when half of the surface area of ​​the culture vessel is covered by cells, the population is considered to be at 50% confluence. The schematic in Figure 3 shows an example of the confluence of different cells. Cell density is used to describe cells growing in suspension. 2, 5

Figure 3: Schematic diagram showing 4 fields of view under a cell microscope depicted at 20%, 50%, 80%, and 100% confluence. This image was created using

Thousands of mature cell lines are used in laboratories around the world, which can be purchased from commercial or non-profit suppliers (cell banks). Obtaining cell lines from a reputable supplier is essential because the cells are validated and contamination-free. Obtaining cell lines from other laboratories has a high risk of contamination and lack of cell line validation, so this is not recommended.

When selecting the appropriate cell line for the experiment, the criteria in Table 4 should be considered. The cell line selected depends to a large extent on the nature and requirements of the experiment to be performed.

Table 4: Criteria to consider when selecting cell lines. 1, 2

Do I need to use a species-specific cell line? If not, non-human and non-primate cell lines usually need to reduce biosafety restrictions, which may be advantageous.

Use the appropriate cell line for your experiment. For example, cell lines derived from liver and kidney may be more suitable for toxicity testing.

Limited cell lines are more functionally related because they have not undergone immortalization, but immortalized cell lines are generally easier to maintain and clone.

The transformed cell line has a higher growth rate and higher plating efficiency, which is advantageous, but the cells have undergone permanent genetic changes. Will this affect your experiment?

Is growth rate, cloning efficiency, or saturation density important to your experiment? Do you need adherent cells or suspension cells? For example, if you want to express recombinant protein in high yield, you can choose a fast-growing cell line that can grow in suspension.

Table 5 lists 20 commonly used cell lines and their characteristics, including species, tissue origin and morphology. The morphology of the cells is often described as fibroblasts, epithelial cells or lymphoblasts, which indicates the origin and physical appearance of the cells. Fibroblasts are bipolar or multipolar and have a slender shape; epithelial cells are polygonal and usually more regular in size, and lymphoblasts are spherical and usually grow in suspension. 1, 5 Table 5: 20 commonly used cell lines and their characteristics.

Adhesion, adaptable to suspension

Cell culture is an important tool in laboratories around the world. However, the cell line may be misidentified or contaminated by other cells. This invalidates the published data and wastes laboratory time and resources. Due to the severity of this problem and the need to ensure valid and reproducible results, journals, institutions, and funding agencies now recommend or require cell line certification. Cell line identification can be achieved by genetic analysis using polymorphic short tandem repeat (STR) loci. Cells should be identified after receiving a new cell line, and periodically during subculture. 12

The cultured cells are fed with liquid medium. There are four key components to consider when preparing the culture medium for your cells. See Table 6 for details. The specific details of your cell culture media requirements can be found in the data sheet. When cultivating a new cell line, be sure to check the detailed information on the culture conditions in the data sheet.

Table 6: Key components of mammalian cell culture media. 1, 13, 14

The basal medium is a mixture of nutrients and salt. There are a variety of formulations, including Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI). They can be purchased in liquid or powder form from commercial sources.

Glutamine is an essential amino acid necessary for cell growth. You can buy basal media containing glutamine, glutamine-free or stable dipeptide glutamine substitutes. It is important to ensure that your culture contains glutamine under normal growth conditions.

Cells are usually grown in a basal medium supplemented with animal serum. This is called the "complete" medium. Serum provides the growth factors and nutrients needed by the cells. Fetal bovine (fetal bovine) serum is the most commonly used. In most cases, serum is added so that the final volume in the complete medium mixture is between 5% and 20%.

The cell culture medium is usually supplemented with an antibiotic combination of penicillin and streptomycin as a measure to prevent bacterial growth. Using aseptic technique, healthy cell cultures can be grown without the use of antibiotics.

All cell culture laboratories are implementing several basic cell culture programs. It is important to be familiar with and understand these agreements.

Consistent use of aseptic techniques helps to ensure the sterility of all media and culture vessels, thereby reducing the exposure of cells to contaminants and maintaining the health, vitality and purity of the culture. Strict aseptic technique is a necessary prerequisite for the success of cell culture to keep the culture free from microbial contamination and cell cross-contamination. Aseptic technique includes handling, reagents and workplace, summarized in Table 7. 15, 16

Table 7: Summary of aseptic techniques required when using cell culture.

· Handle gently and carefully.

· Disinfect all items before starting.

· Sterile pipettes, pipette tips and plastic utensils.

· It is forbidden to bring sterile items into contact with unsterilized surfaces (including gloved hands touching one's skin, clothes or hair).

· Pre-sterilization of all reagents and equipment.

· There is no visible contamination in the reagents.

· Dispense reagents into smaller volumes to prevent contamination of the entire inventory.

· Having your own working inventory can eliminate the risk of contamination from shared inventory.

· Check that the culture hood is working properly.

· The work area is always kept sterile and tidy.

· Frequently clean and decontaminate fume hoods, incubators and refrigerators.

The basic outline of the separation of primary cells requires the use of sterile scissors or a scalpel to mince or cut the separated tissue into pieces of 2-4 mm. Add tissue pieces to appropriate buffer or balanced salt solution on ice and wash 2-3 times. Add the dissociation enzyme according to the protocol and incubate. Disperse the cells by gently pipetting. The cell suspension was filtered through a fine mesh and washed 2-3 times. The cells are resuspended in the culture medium and seeded. 18

Table 8: Enzymes commonly used in tissue dissociation protocols for cell separation. 18

Hydrolyzed collagen is widely used to separate cells from animal tissues.

Used in conjunction with collagenase and catalyzes the hydrolysis of 1,4-β-D-glycosidic bonds.

Add to cell suspension to minimize cell clumps due to DNA released by damaged cells.

Used to digest tissues containing a lot of elastin.

A serine protease specific for peptide bonds, usually combined with other enzymes (such as elastase and/or collagenase) for tissue dissociation.

It is important to always handle cells gently, as vigorous or rough handling can cause cell damage or death. Never transfer the culture medium or washing buffer directly onto the cells, always add it gently to the side of the container to avoid damaging the cells. When resuspending the pellet or grinding to mix the cells, do so gently.

In short, the subculture protocol for adherent cells is as follows: remove the medium and wash the cells once with PBS. Add a separating agent, such as trypsin (which breaks down the protein that allows the cells to adhere to the container), and incubate the cells at 37 °C until they are completely separated. Depending on the cell line, separation may take 1-20 minutes. Monitor the cells under a microscope to determine when detachment has occurred. The cells are pelleted by adding complete medium (serum inactivates trypsin because it contains protease inhibitors) and spinning in a centrifuge (3-5 minutes at 150–300 xg) to inactivate trypsin. live. Remove the medium (liquid on top of the pellet), gently resuspend the cells in fresh medium, and spread the cells in a new culture vessel at the desired density. For suspension cells, trypsin is not required. Collect the cells and centrifuge (150–300 xg for 3-5 minutes) to form a pellet, remove the medium and resuspend the cells in PBS for a washing step. After another round of centrifugation, the buffer was removed, the cells were resuspended in fresh medium and re-seeded at the desired density. 5

Cell lines are a precious resource, so keeping inventory for long-term storage is essential. Cryopreservation refers to the process of cooling and storing cells at extremely low temperatures to maintain their viability. The battery is suitable for long-term storage at temperatures below -130 °C.

The best way to cryopreserve cultured cells is to store them in liquid nitrogen in a complete medium in the presence of a cryoprotectant (such as DMSO). Cryoprotectants can lower the freezing point of the culture medium and allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death. Cells should be cryopreserved at a high concentration (such as 90% confluence) and passages as early as possible. In short, cryopreservation includes washing and pelleting cells, resuspending them in complete medium containing DMSO (eg 5% DMSO), and transferring the cell suspension to a 1 mL sterile cryotube. Since the cells must be frozen slowly, the cryotube is placed in a rate-controlled freezer or freezer-freezer. The cryogenic freezer container is stored at -50 to -80 °C for 24 hours, and then the cryotube is transferred to liquid nitrogen storage. 2, 8

The exact freezing conditions depend on the cell line used. It is very important to check the specific conditions of the cell line, otherwise your frozen stock solution may not produce viable cells when it is thawed and re-cultured.

To recover the cell line from liquid nitrogen storage, the frozen vial is transported to the cell culture area in a portable liquid nitrogen container or dry ice. DMSO in the cryotube will be toxic to cells once thawed, so to ensure high cell viability, the cells must be quickly thawed in a 37 °C water bath and immediately transferred to a petri dish with pre-warmed culture medium. The medium is diluted with DMSO so that it is no longer at a toxic concentration. Once the thawed cells are multiplied and passaged twice, they can be used in experiments. It is a good practice to freeze more cells as soon as possible and replace vials taken from long-term storage. 2

A major problem in cell culture is mycoplasma infection. This bacterial infection can change cell behavior and metabolism, and adversely affect cells. Regular mycoplasma testing is very important, especially for continuous cell lines. It is good practice to test for Mycoplasma upon receipt of new cell lines, thawing and culture stocks, just before freezing, and when testing the cell stocks in culture every 4-6 weeks. A simple and reliable method to detect mycoplasma is to stain the cell sample with Hoechst 33258 (a fluorescent dye that specifically binds to DNA) and then observe it under a fluorescence microscope. Amycoplasma cells showed clear and clean nuclear Hoechst staining, while cells infected with mycoplasma also showed a filamentous staining pattern outside the nucleus, that is, bacterial DNA (Figure 4). 5, 19, 20

Figure 4: Schematic diagram showing the field of view of unstained (top) cells and cells stained with Hoechst 33258 that are not contaminated with mycoplasma (bottom left) and contaminated with mycoplasma (bottom right). Uncontaminated cell cultures show nuclear staining, while contaminated cell cultures show nuclear staining and cytoplasmic staining. Cytoplasmic staining indicates the presence of mycoplasma bacterial DNA. 5 This image was created using

Despite the recent development of automated cell counters, manual cell counting using a hemocytometer is still the most commonly used method. The hemocytometer consists of a thick glass microscope slide and two etched grids of vertical lines to form a chamber. Thin cover glass is also provided. Be sure to clean the hemocytometer and cover glass before use, and place the cover glass above the chamber. Add 10 – 20 μl of cell suspension to one of the two chambers on the microscope slide under the coverslip. Use an inverted phase contrast microscope with a magnification of 20 times to count the cells in each of the four outer squares, as shown in Figure 5. Your goal should be to count 100-200 cells per square to get accurate results. If you have too few cells, resuspend your cells with less medium next time. If you need to count too many cells, resuspend your cells in a larger volume of culture medium. After counting each corner, add the counts and divide by four. Your cell concentration will be your count x 104 cells/mL. To calculate the total number of cells, multiply the concentration by the volume of the cell suspension. For example, a 5 mL cell suspension with a concentration of 80 x 104 cells/mL has a total of 400 x 104 cells or 4 x 106 cells or 4 million cells. 4, 5, 21, 22

Figure 5: Counting cells with a hemocytometer. The top panel shows the aerial and side view schematics of the hemocytometer. The lower left panel shows what the counting chamber looks like under the microscope. The red box highlights the quadrant to be calculated. The lower right panel is an example of how to count cells within the object limit.

Cell transfection refers to the delivery of nucleic acid (DNA or RNA) to cultured cells. The most commonly used reagents are cationic lipids, which can combine with nucleic acids to form positively charged complexes and allow DNA/RNA to interact with negatively charged cell membranes. This results in the efficient entry of nucleic acids into cells through endocytosis. This is commonly referred to as lipofection or lipofection. Alternatively, the nucleic acid can be delivered to the cell by electroporation, co-precipitation of DNA and calcium phosphate, or polybrene/DMSO shock. The delivered DNA usually does not integrate into the host genome and is therefore transient. In order to achieve stable gene expression or knockdown, stable cell lines can be designed. This requires DNA vector design and antibiotic selection, where the DNA has been stably integrated into the genome. 23, 24

The lipofection protocol is very simple. Essentially, a solution containing the appropriate concentration of DNA/RNA for transfection is prepared, and a solution containing lipofection reagent is prepared. The two solutions are mixed together and incubated for a period of time according to the protocol. The solution is then added to 60-80% confluent cells, and the experiment can be performed as early as 6 hours after transfection. Transient transfection usually lasts up to 72 hours. 25

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