- 1 Introduction to Cell Culture – NL
- 2 Finite vs Continuous Cell Line
- 3 Culture Conditions
- 4 Cryopreservation
- 5 Morphology of Cells in Culture
- 6 Applications of Cell Culture
- 7 Related Cell Culture Basics Videos
- 8 Basic Process in Cell Culture in General
- 9 Preparation of Cells for Cell Culture
- 10 From Cell Culture Initiation to Passage
- 11 Processing after Cell Culture (Stock Preparation)
- 12 Mammalian cell tissue culture techniques protocol
- 12.1 Contents
- 12.2 1) Preparing an aseptic environment
- 12.3 2) Preparation of cell growth medium
- 12.4 3) Creating the correct culturing environment
- 12.5 4) Checking cells
- 12.6 5) Sub-culturing
- 12.7 6)Adherent subculture protocol (using dissociation reagent)
- 12.8 7) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture
- 12.9 8) Sub-culturing attached cell lines requiring trypsin
- 12.10 9) Sub-culturing of suspension cell lines
- 12.11 10) Changing media
- 12.12 11) Passage number
- 12.13 More useful protocols
- 13 Introduction to Cell Culture
- 14 Cell Culture – an overview
- 14.1 Publisher Summary
- 14.2 3Equipment
- 14.3 7.6Nanoemulsions in Cell Culture
- 14.4 Abstract
- 14.5 Introduction
- 14.6 Cell culture
- 14.7 22.214.171.124Micro-Scale Cell Culture
- 14.8 Purified environment
- 14.9 2Equipment
- 14.10 2.2.1Virus isolation in cell culture
Introduction to Cell Culture – NL
Cell culture is the process of removing cells from an animal or plant and allowing them to develop in a suitable artificial environment once they have been removed. Before culture, cells can be isolated directly from the tissue and disaggregated using enzymes and/or mechanical methods. Alternatively, cells can be isolated from an existing cell line or cell strain and cultivated in the same way that they were originally isolated. Primary culture refers to the stage of the culture after the cells have been separated from the tissue and have multiplied under the suitable circumstances until they have taken up the whole accessible surface area of the substrate (i.e., reachconfluence).
Upon the completion of the first subculture, the primary culture is known as a cell line or a subclone.
It is possible to transform a cell line into a cell strain by selectively cloning or using other methods to isolate a subpopulation of the cell line from the rest of the culture.
Finite vs Continuous Cell Line
A limited number of times in normal cells before they lose their capacity to multiply, which is a genetically regulated phenomenon known as senescence; these cell lines are referred to as finite. Although some cell lines become immortal naturally, some cell lines become immortal by a process known as transformation, which can occur spontaneously or be driven chemically or virally. The transformation of a finite cell line into a continuous cell line occurs when the cell line gains the ability to divide endlessly as a result of the transformation.
The culture conditions for each cell type differ significantly, but the artificial environment in which the cells are cultivated always consists of an appropriate vessel that contains the following components:
- A substrate or medium that provides the required nutrients (amino acids, carbohydrates, vitamins, and minerals)
- Growth factors
- Gases (oxygen and carbon dioxide)
- And other substances (such as oxygen and carbon dioxide). It is necessary to have a controlled physico-chemical environment (pH, osmotic pressure, temperature).
Adherent or monolayer culture is required for the majority of cells, which must be attached to a solid or semi-solid substrate (adherent or monolayer culture), while some can be grown floating in the solution (floating culture) (suspension culture).
If there is an excess of cells available after subculturing, they should be treated with a suitable protective agent (e.g., DMSO or glycerol) and kept at temperatures below –130°C (cryopreservation) until they are required again. Refer to theGuidelines for Maintaining Cultured Cells for further information on subculturing and cryopreservation of cells.
Morphology of Cells in Culture
It is possible to classify cells in culture into three fundamental types depending on the shape and appearance of the cells (i.e.,morphology). Bipolar or multipolar fibroblastic (or fibroblast-like) cells have elongated forms and grow connected to a substrate, whereas fibroblastic cells do not. A polygonal structure with more regular proportions characterizes epithelial-like cells, which develop in distinct patches linked to a substrate as they expand.
Lymphoblast-like cells are spherical in form and are often cultivated in suspension, without adhering to a surface, in order to maximize their growth potential.
Applications of Cell Culture
When it comes to cell culture, it is one of the most important tools in the field of cellular and molecular biology. It provides excellent model systems for studying the normal physiology and biochemistry of cells (for example, metabolic studies, aging), the effects of drugs and toxic compounds on the cells, as well as the mutagenesis and carcinogenesis of cells, among other things. It is also employed in the screening and development of pharmaceuticals, as well as the large-scale production of biological substances (e.g., vaccines, therapeutic proteins).
Related Cell Culture Basics Videos
An overview of the fundamental equipment needed in cell culture as well as the suitable laboratory set-up is provided in this video. Instructions and demonstrations on how to operate safely and aseptically in a cell culture hood are provided. It is the objective of this video to discuss the precautions you should take to avoid contamination of your cell culture. There is a demonstration of all of the fundamental procedures necessary to accomplish cell culture utilizing best-practice sterile methods.
Basic Process in Cell Culture in General
Return to the top of the Basic Knowledge page. The techniques and procedures used in cell culture differ based on the cell type and application. It’s important to remember that if cells are not treated in a manner that is proper for each activity, their traits may be altered. General cell culture techniques and procedures are introduced in this part, with crucial things to consider during the process.
Preparation of Cells for Cell Culture
Drawing showing the flow diagram for a generalized cell culture procedure. It is possible to collect cells in two ways: either by acquiring them from a cell bank or by separating them from donor tissue. It is necessary to go through the steps of “thawing,” “cell seeding,” and “cell observation” when beginning a culture using cells received from a cell bank. When utilizing tissue that has been obtained from a donor, it is customary to remove any superfluous tissue that has been attached. When it comes to isolating cells from tissue, there are two main approaches: explant culture and the enzymatic technique.
To prepare the cell culture if an enzyme is employed, dilute the enzyme or halt the enzyme reaction using an enzyme reaction inhibitor before proceeding with the stages of” cell seeding ” and “cell observation” to complete the preparation of the cell culture.
The process of thawing frozen cryopreserved cells in order to begin a cell culture might be thought of as “waking up the cells.” After being moved from a liquid nitrogen tank *1or cryogenic deep freezer (-150 degrees Celsius) to a suitable cold storage container, such as a liquid nitrogen container, and delivered to the bench, frozen cells are defrosted in a 37°C water bath or melting device. *2. Before the ice is nearly completely melted, medium is swiftly added to dilute the cryoprotectant liquid (for example, DMSO), the cells are precipitated by centrifugation, and after discarding the supernatant, new medium pre-heated to 37°C is added to the cell suspension.
Using liquid nitrogen, there are two techniques to freeze and preserve cells: “vapor phase,” which involves using the cold gas from a liquid nitrogen tank, and “liquid phase,” which involves immediately immersing a frozen preserved object in the cold gas from a liquid nitrogen tank.
In order to avoid this, vapor phase storage is strongly suggested.” When liquid nitrogen is present in a vial that has been stored in the liquid phase, it is possible for the vial to explode if the vial is placed in a 37°C water bath or a melting equipment.
Before placing the lid in the 37°C water bath, it should be loosened once and retightened to ensure that it is completely sealed.
Calculate the quantity of new medium necessary to reach the desiredcell seedingdensity based on the observed cell counts and dilute the cell suspension appropriately in order to achieve the targetcell seedingdensity.
In order to determine the viability of the cells after they have been seeded in a fresh culture medium, use an optical microscope or other observation equipment in the following manner:
- Determine whether or whether there are any viable cells. Double-check to see that the cells are dispersed equally throughout the vessel. Make a visual examination for the existence of foreign things other than cells. Examine the morphology of the cells.
Determine whether any live cells are present. It’s important to make certain that the cells are spread uniformly throughout the tube. Make a visual examination for the existence of extraneous things other than cells; Take a look at the cell’s shape;
From Cell Culture Initiation to Passage
The cells are seeded into a fresh culture vessel, which is then put in a CO2incubator to begin their growth. In most cases, the following observations are carried out the following day *, using an optical microscope or other observation device:
- Ensure that the culture vessel does not include any extraneous things other than cells by visually inspecting it. In order to determine whether the culture is progressing normally, examine cell shape and condition. *In certain situations, depending on the culture conditions and cell type, they may be permitted to stand for up to two days.
Cells begin to develop in the CO2incubator after they have been thawed and planted in a culture vessel. A culture media that has been depleted of nutrients and loaded with metabolic products must be replenished with new medium to ensure that cells can continue to metabolize the nutrients in the medium. This is referred to as a “medium change” or a “medium replacement” process. Prior to changing the medium, check on the cells to ensure that they are growing regularly and that the culture is progressing normally.
Because certain cells may perish if they are not immersed in media, there are instances in which a tiny bit of the old medium is retained rather than being eliminated totally.
After changing the media, examine the cells to see if there is any evidence of damage.
Once the cells have begun to multiply, split them into other culture vessels before the present vessel gets completely crowded with cells. This is referred to as “passage.” “Confluent” refers to the state in which cells have expanded to the point that they completely fill the culture vessel. The majority of the time, it is advised that cells be passed when the area occupied by cells reaches roughly 70 to 80 percent of the vessel’s total volume. In the process of becoming confluent, cells come into touch with one another and perceive that they no longer need to expand any further, a phenomenon known as “contact inhibition,” and in the case of normal cells, this results in the cells ceasing to proliferate upon passage.
As a result, it is vital to study and monitor the progression of cells. The passage techniques used for suspension cells and adherent cells are different.
Passage of Suspension Cells
Centrifuge the cell cultures suspension into a tube to harvest the cells. Collecting the cells is easy. Remove the supernatant while keeping the cell pellet in place, and re-suspend in new medium to finish the experiment. Take a sample of the freshly prepared solution and stain it with the crucial dye trypan blue to determine the number of live cells. Consider cell dilution techniques, calculate the cell concentration, change the density of cells in suspension appropriately, and transfer to a new container.
Passage of Adherent Cells
Cells that have adhered to the surface of the culture vessel must be separated from the vessel’s surface in some way. Protease enzymes like as collagenase, dispase, and trypsin are commonly utilized in this process. Due to the fact that calcium and magnesium ions have been shown to hinder the action of trypsin, it is vital to thoroughly wash away any solution that contains these ions prior to using it. Collagenase and dispase, on the other hand, are active in the presence of calcium ions. The following is the basic method to be followed:
- Remove the supernatant from the old culture medium
- Cleanse with phosphate buffer or a new media if necessary
- Add the enzyme solution for cell dispersion
- Keep at a set temperature within the enzyme’s active range for a predetermined amount of time in order to facilitate the enzymatic process
- Utilize a microscope to determine the extent of cell dissociation
- Light mechanical perturbation, such as tapping, or other similar activities, can help to promote cell separation. If there is a stop solution for the enzyme(s) available, add it to the mixture to bring the process to a halt. If not, replace the medium with new media to dilute the enzyme and reduce activity. Remove cells from the media by pipetting it multiple times
- Single-cell suspension should result. Collect the medium, including the cells, centrifuge the medium to precipitate the cells, and discard the supernatant, which contains the enzyme and reaction stop solution. Tap the tube a few times to loosen the cell pellet
- Fill the tube with newly obtained cells with fresh medium
- Pipette the cells up and down to keep them suspended. Prepare the sample into a representative cell suspension in order to count the number of cells and density of cells
- Count the number of cells on a slide under a microscope using a hemocytometer or with an automatic cell counter. Identify the proper dilution for achieving the required cell density, then re-suspend the cells after adding the necessary amount of new media. In a fresh culture vessel, seed a predefined quantity of the cell suspension into the vessel. Make use of a microscope to see things
- Transfer to a humidified CO2incubator and heat to 37 degrees Celsius
- Repeat the process.
Removing the old culture medium supernatant from the equation Cleanse with phosphate buffer or a new medium, if necessary. Add the enzyme solution for cell dispersion; mix well. In order to facilitate the enzymatic reaction, stabilize at a preset temperature that is within the enzyme’s active range for a predetermined period. Using a microscope, examine the extent of cell separation. Light mechanical perturbation, such as tapping, or other similar activities, can aid in cell separation and apoptosis.
- Add new media to dilute the enzyme and reduce its activity if this is not the case.
- Collect the medium, which includes the cells, centrifuge the cells to precipitate them, and then discard the supernatant, which contains the enzyme and reaction stop solution.
- Fill the tube with harvested cells with new media; repeat the procedure.
- To count the quantity of cells and density of cells in a sample, prepare it as a representative cell suspension.
Identify the proper dilution for achieving the required cell density, then re-suspend the cells after adding the necessary amount of new media; Place an exact amount of the cell suspension into a fresh culture vessel at a preset time; Examine things under a microscope; In a humidified CO2incubator, heat the mixture to 37 degrees Celsius.
Processing after Cell Culture (Stock Preparation)
When creating stocks, it is critical to use cells with the same properties as the original cells. These cells can be acquired through primary culture, purchased from a supplier, or transferred from another source.
This is due to the fact that cells are living materials, and their features may change over time. If the passing continues for an extended length of time, it is possible that they may become distinct from the initial cells. It is possible to construct a cell stock in the following ways:
Make the following observations with anoptical microscope or other observation instrument to support your conclusions.
- Check the culture vessel for extraneous things other than cells that may have gotten into it. Check to see if the cells are subconfluent and have not overproliferated before proceeding. Ascertain whether normal culture progression is occurring by examining the cell shape and state of the culture
Cells for stocks should be harvested using the passage process mentioned above.
Use just a modest amount of medium to resuspend the cells that were recovered by centrifugation since the density of the cell suspension has to be higher than that required for passage. Pipetting is used to suspend the cells, and a microscope or measurement device is used to count the number of cells or cell density in each suspension.
Fill up the gaps with an equal volume of 2x concentrated cryopreservation solution and re-suspend by pipetting the cells until they have reached the appropriate number of cells. Fill the cryotube with a preset amount of solution by pipetting or swirling it consistently during the process.
To maintain a freezing rate of -1°C per minute, the cryotube should be immediately put in a freezer container and stored in a deep freezer (-80° C). Alternatively, use a programmable controlled-rate freezer to freeze the food. The frozen cryotubes should be kept in the vapor phase within a liquid nitrogen storage tank or cryogenic deep freezer (-150 degrees Celsius) once the cells have been frozen. The processes, on the other hand, differ depending on the type of cryopreservation solution used.
One or two of the frozen vials should be thawed and cultured. Check to see whether the cells can continue to develop in the same manner and display almost comparable features to those observed earlier. Once this has been established, the cell culture should be stopped.
Start of Experiment
Prepared cell stocks should be thawed as needed for research purposes. After a given amount of time in culture, the cells should be discarded and a new batch of cells should be frozen and ready for use.
Mammalian cell tissue culture techniques protocol
Depending on the research activity, thaw created cell stocks. When the cells have been in culture for a particular amount of time, they should be discarded and a new batch of cells should be frozen and ready to use.
- Aseptic preparation
- Preparation of cell growth media
- Creation of the proper cell culture environment
- Checking of cells
- Compliance with subculture procedure Sub-culturing loosely attached cell lines that require cell scraping for sub-culturing
- Sub-culturing attached cell lines that require trypsin
- Sub-culturing suspension cell lines
- Changing medium
- Passage number
- Sub-culturing of suspension cell lines
1) Preparing an aseptic environment
Regulations for hoods
- Rigidity of the hood
All medium, supplements, and reagents must be sterile in order to avoid the development of microorganisms in cell cultures. In the event that any reagents or supplements are not given sterile, they will need filter sterilization. Watch the video procedure for ouraseptic approach to learn more about how to avoid infection.
2) Preparation of cell growth medium
Before beginning work, review the material provided with the cell line to determine what sort of medium, additives, and suggestions should be used and which should be avoided. Cells can be cultured in DMEM culture media or RPMI culture media supplemented with 10% Foetal Bovine Serum (FBS), 2 mM glutamine, and antibiotics if necessary, with the exception of some cancer cell lines (see table below). Before starting cultures, double-check that the cell line you’re using requires the specific culture medium and culture supplements that you’re using.
Sterilization of culture medium and supplements is essential. When at all feasible, purchase sterile reagents and utilize them solely under aseptic conditions in a culture hood to guarantee that they remain sterile during the whole process. DMEM media is used as a general example.
|DMEM – Remove 50 ml from 500 ml bottle, add other constituents.||450 ml|
|10% FBS||50 ml|
|2mM glutamine||5 ml|
|100 U penicillin / 0.1 mg/ml streptomycin||5 ml|
3) Creating the correct culturing environment
The majority of cell lines will grow in culture flasks without the need of specific matrixes or other additives. Some cells, particularly initial cells, may, however, need the formation of specific matrixes such as collagen in order to facilitate cell attachment, differentiation, or cell proliferation, whilst others will not. In order to obtain further information on the cells you are growing, we propose that you consult relevant literature. The following is an illustration of endothelial and epithelial cells in action: Flasks should be coated with 1 percent gelatin if they are to be used for human cells.
- It is not necessary to use specific growth media or other materials to grow most cell lines in culture flasks. While certain cells, particularly initial cells, may require growing on specific matrixes such as collagen to encourage cell attachment, differentiation, or proliferation in order to function properly, others, including stem cells, will not require such support. In order to obtain further information about the cells you are growing, we propose that you consult relevant literature. An illustration of endothelial and epithelial cells may be found below. Fill flasks with 1 percent gelatin to use with human cells. Flasks can also be coated with 1 percent fibronectin if you’re working with other cell types, such as BAECs.
4) Checking cells
The majority of cell lines may be grown in culture flasks without the need of specific matrixes or other additives. Some cells, particularly initial cells, may, however, need the formation of specific matrixes such as collagen in order to facilitate cell attachment, differentiation, or cell proliferation, while others will not. We urge that you look into the cells you are growing more in the appropriate literature. An illustration of endothelial and epithelial cells may be found below: Fill flasks with 1 percent gelatin to culture human cells.
- In great numbers (connected lines), they are separating from the skin and appearing shrivelled and grainy/dark in color. They appear to be in a state of quiescence (i.e., they do not appear to be growing).
Cell splitting and cell passaging are other terms for the same thing. To ensure that cells are ready for an experiment on a specific day, split ratios or seeding densities can be utilized. Split ratios or seeding densities can also be used to retain cell cultures for future use or as a backup. Adherent cell lines are seeded based on flask surface area, so seeding densities for suspension cell lines will be calculated as cells/mL, whereas suspension cell lines are seeded based on volume, so seeding densities for adherent cell lines will be calculated as cells/cm 2.
If a high split ratio is applied, it is possible that slow-growing cells will not grow.
It is possible to divide adherent cell lines using cell line-specific split ratios or seeding densities (cells/cm 2), as follows:
- It should take 1 to 2 days for a 1:2 split to reach 70-80% confluence and be ready for experimentation. It should take 2 to 4 days for the 1:5 split to reach 70-80% confluence and be ready for an experiment. It should take 4 to 6 days for the 1:10 split to become 70-80% confluent and suitable for sub-culturing or plating.
Split ratios are calculated depending on the surface area of the flask, for example: 1 x 25 centimeters 2flask 3 × 25 cm would be obtained from a 1:3 split. 1 x 75 cm or 2 x 75 cm flasks The following cell densities (cells per milliliter of suspension) should be used to sustain suspension cell lines:
- Ideally, 2e5 will be ready for an experiment within 3-4 days. It should be possible to do an experiment with 1e6 in 1-2 days.
The use of a lower than normal seeding density/split ratio is advised if cells are going to be left unattended for an extended length of time (such as weekends or bank holiday weekends).
6)Adherent subculture protocol (using dissociation reagent)
Using a lower than normal seeding density/split ratio is advised if cells will be left unattended for prolonged periods of time (e.g., bank holiday weekends).
- Remove the cell culture media and dissociation reagent from the refrigerator and place them in an incubator set at 37 degrees Celsius, allowing them to reach room temperature. – It is important not to keep media in the incubator for any longer than is absolutely required since the media components will deteriorate with time. Start by turning on your biological safety cabinet and doing a simple clean within it. – Before placing any media bottles, pipettes, and centrifuge tubes in the biological safety cabinet, spray them with ethanol to disinfect them. Remove the conditioned medium from under the biological safety cabinet and gently wash the cell monolayer with room temperature DPBS in the biological safety cabinet. – Carefully apply DPBS to the side of the flask so that adhering cells are not violently dislodged. Then, using an autoclaved serological pipette, carefully remove the DPBS and pour in the pre-warmed dissociation reagent (Trypsin-EDTA) before placing it in an incubator for 2 minutes (dissociation times can vary between cell lines). Check the flask on a regular basis to ensure that all cells have separated from the flask surface.- Not all cells will require trypsinization, and it might be hazardous to certain cells if it is performed on them. It should be noted that trypsin can also cause the transient internalization of several membrane proteins, which should be taken into mind when designing experimental procedures. It is typically possible to use other procedures, such as gentle cell scraping or the use of a very mild dissociation reagent (Versene), in these types of cases, to get the same results. Following complete cell detachment, neutralize the dissociation reagent with serum containing growth medium suitable for the cell line in culture. Transfer the cell suspension to a centrifuge tube and spin it down. Wash the flask with sterile medium and transfer it to a centrifuge tube, making sure that all of the cells have been extracted from the flask. Centrifuge the cell suspension at room temperature for 5 minutes at 1000 rpm for 5 minutes. To count cells, discard the supernatant and gently flick the cell pellet (to break up the pellet), then resuspend cells in sterile medium to a volume appropriate for counting
- Utilizing a hemocytometer technique, consult the Abcamcounting cell. Determine the proper flask and density for the cell suspension based on the count and viability statistics, for example T175, 30mL at 2e4 cells/cm 2. – Label the culture flask with all of the relevant information, such as the cell line and passage number
- Incubate the freshly planted cultures in a 37 o C/5 percent CO 2air humidified incubator as soon as possible.
7) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture
- When you’re ready, gently pour out the medium from the flask containing the needed cells into a trash container (which should contain approximately 100 mL of 10% sodium hypochlorite), being cautious not to increase the danger of contamination with any drops. Replace this immediately by gently putting an equivalent volume of pre-warmed new culture medium into the flask
- This should be done immediately. Scrape the cells from the bottom of the flask into the medium with a cell scraper, being careful not to damage them. Before continuing, verify the base of the flask to ensure that all of the cells have been removed
- Otherwise, proceed as directed. Serological pipettes are used to extract the exact amount of cell solution needed for the desired split ratio. If the ratio is 1:2, remove 50 ml from 100 ml and place it in a new flask. 1.15 divide 100 ml into two equal halves and transfer 20 ml to a fresh flask 10 mL from a 100 mL flask is divided in half and transferred to a new flask. Fill the new flasks to the needed volume (while taking into consideration the split ratio) with pre-warmed fresh culture medium, for example, in a 25 cm flask. 2flasks (about 5-10 mL each) 75 cm about 10-30 mL in 2flasks, each 175 cm 2flasks containing around 40-150 mL
8) Sub-culturing attached cell lines requiring trypsin
It is important to note that not all cells will require trypsinization, and that trypsinization can be hazardous to some cells. It has also been shown to cause transient internalization of several membrane proteins, which should be taken into mind when designing experimental procedures. The use of alternative procedures such as gentle cell scraping or the use of a very weak detergent may frequently be substituted for traditional treatments in these situations.
- As soon as you are ready, gently pour out the medium from the flask containing the needed cells into a trash container (which should contain approximately 100 mL of 10% sodium hypochlorite), taking care not to increase the danger of contamination with any drips. Pour/pipette enough sterile PBS into the flask to give the cells a good wash and to remove any FBS that may have remained in the remaining culture media using aseptic procedures. Pour the PBS back into the flask gently a few times to rinse the cells, then carefully pour or pipette it back into the waste container. This step may be repeated one or two more times if necessary (some cell lines take a long time to trypsinize and will require additional washes to remove any residual FBS that could interfere with trypsinization)
- Using a pipette, add enough trypsin EDTA to completely cover the cells at the bottom of the flask
- Repeat steps 1 and 2 if necessary. for example, in 25 cm Approximate volume of 1 mL in a 75 cm flask about 5 mL in 2flasks, 175 cm about 10 mL each flask
- Gently roll flask to ensure that trypsin is in touch with all cells. Place the flask in an incubator set at 37°C. Different cell lines need different timeframes for trypsinization to be effective. The cells must be checked every few minutes to avoid over-trypsinization, which can cause severe damage to the cells. As soon as the cells have detached (the flask may require a few gentle taps), add some culture media to the flask (the FBS in the media will inhibit trypsin activity)
- Using this cell suspension, pipette the required volume of cells into new flasks at the required split ratio. Following that, the flasks should be topped up with culture medium to the desired volume, for example, 25 cm3. 2flasks (about 5-10 mL each) 75 cm about 10-30 mL in 2flasks, each 175 cm 2flasks (about 40-150 mL) Allow cells to recuperate and settle for the next day. Change the medium to ensure that any remaining trypsin is removed
9) Sub-culturing of suspension cell lines
- Check the cell line’s specifications for the required split ratio or subculturing cell densities
- If necessary, make adjustments. Using a pipette, carefully remove the needed amount of cell suspension from the flask and transfer it to a fresh flask. e.g. Take 50 mL of cell suspension from 100 mL of cell suspension to make a 1:2 split. Take 20 mL of cell suspension from 100 mL of cell suspension to make a 1:5 split
- Fill the new flask halfway with the needed amount of pre-warmed cell culture medium. e.g. For a 1:2 split, start with 100 mL of fresh medium and add 50 mL of cell suspension to make 50 mL of cell suspension. For a 1:5 split, start with 100 mL of fresh medium and add 80 mL to 20 mL cell suspension.
10) Changing media
If cells have been growing well for a few days but have not yet reached confluence, they will require a medium change in order to replenish nutrients and maintain the proper pH level in the culture. Positive growth promoting factors are produced by cells and released into their media; hence, doing a half media change can be advantageous in replenishing nutrients given by the media while also maintaining these positive growth factors. In order to change culture medium, it is necessary to warm it up at 37°C in a water bath or incubator for at least 30 minutes.
11) Passage number
The number of sub-cultures that the cells have gone through is represented by the passage number. It is important to keep track of your passage number and not let it go too high. This is done in order to avoid the use of cells that have undergone genetic drift or other changes.
More useful protocols
- Methods of cell counting with a hemocytometer
- Cryopreservation of mammalian cell lines
- Aseptic approach View all of the protocols.
Introduction to Cell Culture
Cell culture is the process of growing cells from an animal or plant in a controlled setting, usually in an artificial environment. Cells are harvested either directly from the organism and disaggregated before culture, or from a cell line or cell strain that has already been created and is being used in the experiment. The kind of cell influences the culture conditions, but each culture must have an appropriate vessel with a substrate or medium that offers nutrients (such as amino acids, carbohydrates, vitamins, and minerals), growth factors, or necessary hormones for cultivating cells.
Cell culture applications
- Molecular and cellular biology rely on it as a significant reliable and reproducible tool. Research into normal cell homeostasis, cell biochemistry, metabolic processes, mutagenesis and illnesses, as well as the impact of compounds
- It serves as a model system for the study of illnesses and the screening of drugs.
Basic cell culture equipment
The particular equipment used in a cell culture laboratory varies depending on the sort of research being undertaken; nonetheless, all cell culture labs share the same general goal: to be free of dangerous microorganisms (pathogens). The enlarged list that follows corresponds to the equipment and supplies used by the vast majority of cell culture facilities, which enables for more efficient and accurate work to be accomplished.
|– 70% ethanol antiseptic||– Cell incubator|
|– Aspiratory pump||– Centrifuge|
|– Autoclave||– Fridge/freezer|
|– Cell counter||– Gloves|
|– Cell culture flasks||– Media, sera, cell media additives|
|– Cell culture-grade Petri dishes||– Pipettes of various capacities|
|– Cell culture-grade tubes of various sizes||– Serological pipettor|
|– Cell culture hood||– Sterile filters|
|– Cell culture microwell plates||– Waste container|
*Please keep in mind that the particular cell culture equipment required will vary depending on the cell type and the study’s objectives.
Cell culture safety
Work in a cell culture laboratory is associated with a variety of risk factors and hazards, including exposure to toxins and mutagenic reagents. The human/animal material may contain viruses and other biological agents that are potentially harmful. When working with human or animal material, it is critical to adhere to general safety guidelines for laboratory practices when performing manipulations. 1. Before entering and before leaving the laboratory, wash your hands thoroughly. 2. Put on protective clothing (gloves, closed shoes, lab coat).
- There will be no eating, drinking, or smoking.
- There is no or very little aerosol production.
- Decontaminate all surfaces before and after the experiment to ensure no contamination occurs.
- Work in accordance with the facility’s policies and procedures.
7. Ensure that all waste is disposed of in an appropriate manner. 8. Access is restricted to only authorized personnel. 9. Avoid the use of pointed objects. 10. Always make sure that all samples are clearly labeled. 11. Notify the safety officer of any incidents that occur.
Aseptic techniques required while working with cell culture
It is critical to maintain a contamination-free environment in cell culture in order to achieve success (bacteria, fungi etc) Microorganisms are prevented from entering the cell culture using aspetic procedures. A series of processes is used to ensure the sterility of cell cultures. When working with cell culture, it is necessary to use aseptic procedures.
|Slow/careful handling.||Pre-sterilisation of all reagents/equipment||Cell culture hood works properly|
|Sterilization of all items before starting.||No contamination in reagents (expiration date, appearance normal).||Frequent de-contamination (hood, fridge etc)|
|Sterile pipettes||Work area: sterile and tidy|
|No touching of sterile items to non-sterilized surfaces|
Cell culture environment
Cell culture is a fantastic tool because it allows for the simple control and modification of all physiochemical and physiological cell variables, such as temperature, osmotic pressure, pH, gas, hormones, and nutrients, in a controlled and reproducible environment. The environment in which cells are cultured
|Contains nutrients, growth factors, and hormones||Average pH for mammatian cells is pH 7.4||Depends on body temperature of the host||Controlled by media|
|Sera source of growth, tipids,hormones||Mammalian cell lines 36-37°C||Organic or C02 bicarbonate buﬀer systems are popular|
|Insert cell lines 27-30°C||Can impact pH|
|4-10% C02 is most common|
Media supplements for cell culture
Media supplements aid in the optimization of cell development for specific purposes based on the tissue or cell type that is being used. The benefits of utilizing media supplements like as growth factors or cytokines are that they can boost cell viability and proliferation while also keeping cells healthy for a longer period of time. Proteintech thermostable FGF (HZ-1285) is an example of a growth factor that plays an important role in a variety of biological functions both in vivo and in vitro.
|The stability of FGFbasic-TS and FGF basic (E. coli-derived) in xeno-free, chemically defined cell culture media at 37˚C. The protein concentration was determined by ELISA each day for 3 days. After one day of incubation at 37˚C, FGF basic was undetectable, while FGFbasic-TS was present at levels of 60%, 35%, and 20% of its starting concentration at days 1, 2, and 3.|
It is critical for the development of in vitro cultures to have growth factors present. Certain cells can give rise to a number of lineage-specific cell types, depending on the environment in which they are raised. See the list below for a concise overview of the essential growth factors required for normal cell growth, metabolism, cell development in culture, and the process of cell differentiation.
|Supplement||Cat no||Involved in/used for|
|Fibroblast growth factor (FGF)||HZ-1285||Embryonic development, neuron differentiation, and the proliferation of cells of mesodermal origin and many cells of neuroectodermal, ectodermal, and endodermal origin.|
|Bone morphogenetic protein 2 (BMP-2) and bone morphogenetic protein 4 (BMP-4)||HZ 1128 HZ-1045||Bone formation and regeneration.Uses as differentiationfactor of pluripotent stem cellsand promotes osteogenic differentiation of mesenchymal stem cells|
|Hepatocyte growth factor (HGF)||HZ-1084||Epithelial-mesenchymal transition.Acts as a mitogen for many cell types including hepatocytes|
|Granulocyte-macrophage colony-stimulating factor (GM-CSF)||HZ-1002||Promoting growth and differentiation of hematopoietic progenitor cells.Used to support the in vitrocolony formation of granulocyte-macrophage progenitors.|
|Activin A||HZ-1138||Regulating cell proliferation.Used to maintainstem cell pluripotency and self-renewal in vitro|
|Beta-nerve growth factor (beta NGF)||HZ-1222||Differentiation and survival of neurons|
|Stem cell factor (SCF)||HZ-1024||Promoting survival, expansion,and differentiation of hematopoietic stem cells.|
|Transforming growth factor beta (TGF-beta)||HZ-1011 HZ-1092 HZ-1090||Regulating cell growth, proliferation,and differentiation.Widely used, e.g.,in undifferentiated embryonic stem cellcultureand induced pluripotent stem cells|
|Vascular endothelial growth factor (VEGF121 and VEGV165)||HZ-1204 HZ-1038||Angiogenesis and vasculogenesis.Used in endothelial cell culture.|
|Wnt3a||HZ-1296||Regulation of neuronal stem cell self-renewal.Used in expansion of neural stem cells|
Cell Culture – an overview
Dwight E.Lynn’s entry in the Encyclopedia of Insects (Second Edition), published in 2009.
According to Dwight E.Lynn’s Encyclopedia of Insects (Second Edition), BiswanathMaity,. Rory A. Fisher’s paper was published in Methods in Cell Biology in 2013.
Dishes for cell culture that have been treated weight coverslips (about one and a half pounds) (round 12 mm preferred) Media for cell culture that is sterile Forceps Micropipettes that are sterile Aspirator with a vacuum In a sterile cell culture environment Incubator with 5 percent CO at 237 degrees Celsius.
3.2To Section Tissues
Fine scissorForcepsDissection microscopeFine scissorForceps CryostatMicrotome
3.3For Fixation and Staining of Cells, Tissues
Dissection microscope with fine scissor and forceps CryostatMicrotome
Microscope with 488 and 546 filters, 63mm objective, and mercury light for studying fluorescence in living organisms (immunofluorescence) An immunoperoxidase light microscope with objectives of 20x, 40x, and 63x was used. and properties of nanoemulsions (URL: and properties of nanoemulsions G. RoshanDeen and Jan SkovPedersen published an article in Emulsions in 2016.
7.6Nanoemulsions in Cell Culture
For in vitro experiments or the production of biological substances such as recombinant proteins or antibodies, cell cultures are commonly employed. In order to enhance cell development, it is common practice to supplement the culture media with blood or a certain quantity of specified molecules. Even after years of research, it is still difficult to augment the medium with lipophilic compounds since this reduces the quantity of accessible material for absorption by the cells. Nanoemulsions are capable of solubilizing and transporting lipophilic compounds into cell cultures.
Because of their nanoscale size, the nanoemulsion droplets are easily taken up by the cells, resulting in increased bioavailability of the lipophilic chemical in the solution.
The whole chapter may be found at URL: Microscopy of Model Systems.
Methods in Cell Biology, edited by Michael W. Hess and Thomas Seppi, published in 2010.
Cell culturesystems are essential tools for fundamental research as well as a wide range of clinical in vitro investigations, and they are becoming increasingly popular. Conventional 2D cell cultures, on the other hand, do a poor job of simulating the circumstances seen in the actual organism. This shortcoming has the potential to substantially impair the reliability and importance of the data collected through such methods. So we present here a comparative study on selected 3D and 2D cell cultures of U87-MG human glioblastoma cells that were processed using high-pressure freezing and freeze substitution, as well as conventional chemical fixation and Tokuyasu cryo-section immuno-labeling, as well as conventional chemical fixation and Tokuyasu cryo-section immuno-labeling.
- The reference models for static 2D culture systems were cell cultures in dishes and on coverslips, which were also employed in this study.
- Read the entire chapter.
- A large range of clinical in vitrostudies rely on the use of cell culture systems, which are crucial for fundamental research.
- A fundamental disadvantage of such procedures is that they can’t be used to gather reliable and statistically significant data.
- Pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures were all used to construct three-dimensional cultures.
- Our discussion will center on the practicality of the cell culture methods tested for state-of-the-art electron microscopy, as evidenced by morphological and immuno-cytochemical findings in the cells.
- in Cell Culture (website address)
It is the cultivation of nucleated (eukaryotic) cells under well regulated circumstances in a laboratory that is referred to as cell culture. In order to isolate infectious pathogens that require living host cells for multiplication, cell culture is the only method available. The introduction of molecular diagnostic assays based on nucleic acid detection has resulted in cell culture being used less frequently for routine clinical diagnostic purposes. This is due to the long turnaround times (ranging from days to weeks), the high cost, and the requirement for significant technical expertise to perform cell culture and interpret the results of these tests (Table 1-1).
Canine distemper, canine adenovirus infections, parvovirus infections, rabies, and feline viral and chlamydial respiratory tract illness are among the vaccines for dogs and cats that are being developed in cell culture.
Knowledge of cell culture procedures can assist veterinary physicians in submitting the best specimens possible, as well as understanding laboratory turnaround times, potential difficulties, and how to read and interpret data in the laboratory. Read the entire chapter here: URL: Enterovirus
The year 2021 is the year of Zubair Anwar, who will be teaching a Reference Module in Biomedical Sciences.
When it comes to EV diagnosis, cell culture is no longer employed. However, it can be used for the verification of PCR-negative specimens in the event of the novel variation, which includes the identification of more than one virus strain in a single test. The collection of specimens, processing of samples, and methods used in viral isolation are all detailed in the World Health Organization (WHO) recommendations (World Health Organization, 2004;Chen et al., 2014). There are several strains of EVs that may be extracted from cell cultures derived from mammalian cells and certain other cell lines such as monkey kidney cells and human fibroblasts (World Health Organization, 2004;Schmidt et al., 1975).
- The CPEs were discovered in cell culture after 2–5 days after injection and were determined to be active.
- It has been shown that EV 71 does not develop well in cell cultures (Jaramillo-Gutierrez et al., 2013), but EVD68 needed a lower incubation temperature than is typically used for EVs (Jaramillo-Gutierrez et al., 2013).
- Read the entire chapter.
- Lin and M.-H.
126.96.36.199Micro-Scale Cell Culture
As a general rule, cell culture is considered to be a procedure in which cells are grown outside of a live creature under carefully regulated circumstances (e.g., temperature, pH, nutrient, and waste levels). It has a long history of usage in a wide range of biological applications, including the study of cell biology and physiology, drug screening, toxin testing, and various cell-based assays, among others. Currently, the most extensively utilized cell-culture technique is the use of multiwell microplates or Petri dishes as culture containers for the cultivation of cells.
- The latter is attributable to the conventional static cell-culture format as well as the presence of chemical gradients in the culture system in question.
- A cell-culture system may be reduced to the size of a micro-scale device, thanks to recent advancements in microfabrication and microfluidic technology.
- This paves the way for the creation of a cellular microenvironment in vitro that is more like the one found in the body.
- Particularly relevant in situations where resources are restricted (for example, drug testing/screening), this is a useful feature to have.
- More importantly, because of the reduced size of the miniaturized cell-culture scale, the chemical gradient phenomena that exist in culture settings or cultivated constructions may be significantly reduced, allowing for more control over the cell’s microenvironment.
Microfluidic-based cell-culture systems fabricated in polydimethylsiloxane (PDMS) using well-developed soft lithography techniques have piqued the interest of biologists primarily because of the ease with which they can be constructed and because the inherent nature of PDMS makes it suitable for use in cell-culture systems.
- Recent years have seen a flurry of activity in the development of different microfluidic-based cell-culture systems for a variety of purposes, including drug or toxin testing/screening and cell-based bioassays.
- Its characteristics include the ability to maintain homogeneous and stable culture conditions, as well as effective medium pumping methods and cell/scaffold loading, which allows for more precise and high-throughput cell-culture-based tests to be performed.
- However, despite the fact that microfluidic-based cell-culture systems offer enormous potential as platforms for a wide range of applications, the adoption of such developing technologies has not yet resulted in an evolutionary shift away from conventional techniques of cell culture.
- Some of the technological concerns that must be addressed include the avoidance of liquid evaporation in the microfluidic system as well as the development of detection systems that are effective and high-throughput in their reading out of the results of a cellular test.
- Photos of a perfusion-based, 3D cell-culture platform and (II) photographs of the full experimental setup are shown in Fig.
- (the platform is integrated with a hand-held controller) (b) Schematic representation of a microfluidic system for cell-based, high-throughput screening, which includes an upstream concentration-gradient generator and downstream parallel cell-culture chambers.
- Development of a perfusion-based 3-D cell culture platform and its use for high-throughput drug testing were the focus of this research in 2008.
(b) Adapted from Ye N, Qin J, Shi W, and colleagues High content screening of cells using an integrated microfluidic device was published in 2007. 7th Lab on a Chip (pages 1696–1704) Read the entire chapter. Design of a Cell Therapy Facility is the URL.
2016; J.Liu and L. Song, inReference Module in Biomedical Sciences (2016).
It is necessary to operate in an aseptic atmosphere in order to protect against germ contamination and the impact of potentially dangerous variables when performing cell cultureoperations. Cell culture room design necessitates a clean working environment that is free of pollution and filled with fresh, dry air. The room is placed on the inner side of the laboratory and has Class 10,000 grade purification, which allows one to stay away from the turbulence generated by people and maintain the space tidy and clean, which is important.
- Special autoclaved lab coats are required for cell cultivation in order to maintain sterility (Fig.
- Read the entire chapter.
- It is necessary to operate in an aseptic atmosphere in order to protect against germ contamination and the impact of toxic substances when performing cell culture operations.
- With Class 10,000 grade purification, it is positioned on the inner side of the laboratory where one may stay away from turbulence created by people, allowing for the space to be kept neat and clean at all times.
- To guarantee sterility in cell culture, it is necessary to wear autoclaved lab coats (Fig.
- A panoramic view of the cell culture facility is shown in Figure 8.
- Proteomics and Protein Part A: Methods in Enzymology
Cell culture viability (e.g., class II biological safety cabinet) Incubator and/or incubator shaker (must be capable of maintaining a temperature of 28°C). Microscope with compound lenses centrifuge (low-speed) on a surface that has been refrigerated High-speed centrifuge that is kept chilled Water bath (60 degrees Celsius) Water bath (temperature of 30°C) Aide à la pipette Six-well cell culture plates with a micropipettor T-flasks for cell culture Shaking flasks for cell culture (Erlenmeyer) Glass test tubes with a diameter of 1275mm.
Parafilm Read the entire chapter.
The authors, XinZhang and Yi-WeiTang, published in Advances in Clinical Chemistry in 2020.
2.2.1Virus isolation in cell culture
Previously, cell culture was widely utilized in clinical diagnostics for the isolation and identification of viruses, as well as for other purposes. In terms of viruses that may cause gastroenteritis, only norovirus has been shown to be incapable of being produced in cell culture. The capacity to replicate rotaviruses, enteric adenoviruses, and astroviruses in cell culture has tremendously aided the advancement of research into these pathogens. The separation of astroviruses and adenoviruses from cell cultures is frequently coupled with immunofluorescence detection of the viruses using particular antibodies.
Despite the fact that growth in cell culture may be used to detect many distinct viruses from clinical specimens, it is typically believed to be too time-consuming and sluggish to make a significant contribution to useful AGE treatment.
Centrifugation-enhanced rapid cell culture, which may be used with standard cell lines, such as SuperE -Mix, can be used for the detection of enteroviruses.