What Is 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.

Culture Conditions

In most cases, normal cells divide only a limited number of times before losing their capacity to proliferate, which is a genetically regulated occurrence known as senescence; these cell lines are referred to asfinite. Although some cell lines become immortal naturally, some cell lines become immortal by a process known as transformation. Transformation can occur spontaneously or be produced chemically or virally. The transition of a finite cell line into a continuous cell line occurs when the cell line has the ability to divide endlessly.

  • A limited amount of times in normal cells before losing their capacity to multiply, which is a genetically programmed occurrence known as senescence
  • These cell lines are referred to asfinite. Some cell lines, on the other hand, can become immortal through a process known as metamorphosis, which can occur spontaneously or be produced chemically or virally. Continuous cell lines are formed when a finite cell line undergoes transformation and gains the potential to divide endlessly.

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).

Cryopreservation

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.

Cell Culture – an overview

An overview of the fundamental equipment used in cell culture as well as the suitable laboratory set-up is provided by this video. Work in a cell culture hood is made safer and more aseptic by following the guidelines presented and shown. This video focuses on the precautions you should take to keep your cell culture from becoming contaminated. Everything need to do cell culture using best-practice sterile protocols is demonstrated, including all of the fundamental activities required to do so.

Publisher Summary

An overview of the fundamental equipment needed in cell culture as well as adequate laboratory set-up is provided in this video. It is explained and demonstrated how to operate safely and aseptically in a cell culture hood. This video focuses on the procedures you should follow to ensure that your cell culture does not become contaminated. Everything from setting up a cell culture to employing best-practice sterile practices is demonstrated.

3Equipment

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

7 forceps with a curved point Micropipettors made of parafilm Cell culture plate with 12 wells Eppendorf tubes are a kind of test tube. Container with a flat bottom and a cover Dish with rack and lid made of glass for staining (Research Products International Corp., Cat. No.- 144,200)

3.4For Visualization

G. RoshanDeen and Jan SkovPedersen published an article in Emulsions in 2016.

7.6Nanoemulsions in Cell Culture

Methods in Cell Biology, edited by Michael W. Hess and Thomas Seppi, published in 2010.

Abstract

Methods in Cell Biology, 2010; Michael W. Hess and Thomas S. Seppi

Introduction

Methods in Cell Biology, edited by Michael W. Hess and Thomas Seppi, was published in 2010.

Cell culture

(2011), in Comprehensive Biotechnology (Second Edition), by Y.-H. Lin and M.-H. Wu

5.12.4.4Micro-Scale Cell Culture

2016; J.Liu and L. Song, inReference Module in Biomedical Sciences (2016).

Purified environment

Methods in Enzymology, 2014; Donald L. Jarvis, in Methods in Enzymology, 2014;

2Equipment

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.

An Introduction to Cell Culture

Cell culture is an important tool for scientists because it allows them to investigate the physiology and biochemistry of both healthy and sick cells in the same way they would in the laboratory. Aside from being more cost-effective and simpler to deal with than complete organisms, cell cultures are also more reproducible, which makes them a popular choice for use in both research and large-scale manufacture of biological substances. Primal cell cultures are generated when cells from animal or plant tissue are harvested straight from the tissue and cultured in a culture dish.

  • Because most cells ultimately cease dividing (a natural process known as senescence), the life spans of primary cells and their subcultures are limited in length.
  • When this transition occurs, the cell line in question becomes continuous and may be maintained for an extended period of time.
  • Stem cells are in a category all their own.
  • Organisms and tissues can restore cells that have perished and repair damaged tissues as a result of this process.
  • Some investigations, however, necessitate the use of pluripotent cells, which are capable of differentiating into any of the cells that make up the body.
  • These induced pluripotent stem cells (iPSCs) have shown tremendous promise in the fields of regenerative medicine and autologous tissue transplantation, among other applications.
  • Introducing nucleic acids into cells allows scientists to investigate processes such as gene function and modification of gene expression, among other things.
  • We will not go into detail about this approach on this page, but you can find a lot more information on our transfection applications page.

We will also discuss the many types of cell culture medium, buffers, and reagents, as well as how mycoplasma infection can have a detrimental impact on your cell cultures.

Primary Cell Culture Basics

Primary cells are the most accurate representations of the tissue of origin. This means that they are removed straight from the tissue and treated in order to establish them in optimal culture conditions. Because they are produced from tissue rather than being changed, they are more akin to the in vivo condition and display normal physiology than synthetic alternatives. Therefore, they provide good model systems for investigating the normal physiology and biochemistry of cells (e.g., metabolism and ageing), as well as the impact of medications and hazardous substances on cells.

  • They are also more difficult to grow and maintain than a continuous cell line, therefore caution should be exercised while working with them.
  • Rather of starting with signaling investigations, researchers prefer to test the cells for sensitivity to common stimuli before beginning with them.
  • Primary cells utilized in research include epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells, and a variety of other cell types.
  • Through an in vitroprocess known as transformation, primary cells may be altered to allow for endless subculture indefinitely.
  • Genetic transformation can occur naturally or can be induced chemically or virally.
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Primary Cells vs. Cell Lines

Continuous cell lines have gained the potential to multiply forever (become immortalized) either via random mutation, as in transformed cancer cell lines, or through purposeful alteration, as in the case of the artificial production of cancer gene transcripts. Cell lines derived from continuous cultures are often more stable and simpler to work with than primary cells. They have virtually limitless expansion potential and are a quick and simple method to obtain basic knowledge about a subject.

Continuous cell lines have gained the potential to multiply forever (become immortalized) either via random mutation, as in transformed cancer cell lines, or through purposeful alteration, as in the case of the artificial expression of cancer gene expression.

This type of business has virtually limitless development potential and is a quick and simple method to obtain basic information.

Primary Cell Culture Applications

Increasingly, primary cell culture is becoming a major tool in cellular and molecular biology, serving as excellent model systems for studies of the normal physiology and biochemistry of cells (e.g. metabolic studies, aging, signaling studies), the effects of drugs and toxic compounds on cells, and the processes of mutagenesis and carcinogenesis. A vast number of biological molecules (such as vaccines and therapeutic proteins) are developed using this technique, which is also employed in drug screening and development.

  • Cell Culture in 3D: Because primary cells have not been transformed or immortalized, they closely mimic a biological model, produce more physiologically meaningful results, and may be utilized to represent 3D tissues. These cells may be used as a model system to investigate cell biology and biochemistry, the interaction between cells and disease-causing organisms (such as bacteria and viruses), the influence of medications, the process of aging, cell signaling and metabolic control, and many other topics. In many circumstances, the use of primary cells helps researchers to sidestep the difficulties (such as availability, expense, and ethical considerations) associated with the use of animal models.
  • Cancer Research: Primary cells can become malignant if they are exposed to radiation, chemicals, or viruses, among other things. As a result, the mechanism and etiology of cancer, as well as the changed signaling pathways, may all be investigated further. It can also be used to determine whether or not a medicine is effective against cancer cells. In this context, it is also possible to investigate the adverse effects of cancer therapies (chemotherapy and irradiation) on normal cells. Virology: It is possible to investigate the detection, isolation, growth, and development cycles of viruses. It is also possible to analyze the mechanism of infection using primary cells. Toxicology and drug screening: Primary cell cultures are used to investigate the cytotoxicity of novel medications (in order to determine the impact and safe dose) and/or drug carriers in order to determine their safety (nanoparticles). When used on an industrial scale, it may be used to synthesize or manufacture a wide range of biomolecules. This is very beneficial in the pharmaceutical business. Various research efforts involving the development of oncell-based medicinal products utilizing primary cells are now underway. Primary culture is used as a substitute for animal models in the testing of novel medications, cosmetics, and chemicals to see how they affect the body. They are also employed in the determination of the maximum permitted dose of new medications. Primarily, animal cells are used in the production of viruses, which are then used in the production of vaccines (such as those for deadly diseases like polio, rabies, chicken pox, measles, and hepatitis B that are produced using animal cell culture), thereby eliminating the need for the use of animal models. Genetic Engineering (GE) is a term that refers to the process of creating genetic material. Among other things, primary cell cultures are used to manufacture economically significant genetically designed proteins, such as monoclonal antibodies, insulin, hormones, and a variety of other proteins. Replacement Tissue or Organs: Primary cell cultures can be utilized to create replacement tissue or organs in the laboratory. A study is underway to determine if primary cells may be used in the restoration of injured tissue or the replacement of non-functional cells or tissues. Techniques for organ culture are being developed, and research is being carried out on both embryonic and adult stem cell cultures. Many different types of cells and organs may be formed from these cells as a result of their differentiation ability. We may be able to treat a wide range of medical diseases by manipulating the formation and differentiation of these cells in the future. A large number of primary cells have also been employed widely in 3D bioprinting procedures. Stem cell therapy involves the use of stem cells that have been extracted from bone marrow, blood, or embryonic tissue. It is possible to produce a patient’s own stem cells or stem cells obtained from a donor in vitro in order to generate sufficient cells that can be utilized to rebuild tissue or replace functionally defective cells. These are the kinds of areas that are now being investigated in order to develop treatments for genetic abnormalities, spinal cord injuries, degenerative illnesses, and cancer.

Primary Cell Culture Tips and Tricks

Primary cells can be cultivated in either suspension or adherent cultures, depending on their size. Some cells naturally exist in suspension, unattached to a surface, and this is how they evolved (for example those derived from peripheral blood). The ability to survive in suspension cultures has been developed in cell lines that have been genetically engineered to proliferate at a higher density than would be possible under adherent settings. Adherent cells (such as solid tissues) require a surface in order to develop correctly in vitro, but anchorage-dependent primary cells do not require a surface.

  1. The cell culture media is constituted of a baseline medium that has been supplemented with growth agents and cytokines that are appropriate for the cell type.
  2. Cells are cultured in a cell incubator.
  3. In order to accommodate different cell types, growth medium might have varying pH values, glucose concentrations, growth hormones, and the presence of other nutrients.
  4. gentamicin, penicillin, streptomycin, and amphotericin B are examples of antibiotics that may be used in combination.
  5. It is critical to maintain the viability of initial cells after isolation since, after a given number of population doublings, the majority of them undergo the process of senescence and cease to divide.
  6. growth medium, temperature, gas combination, pH, growth factor concentration, presence of nutrients, and glucose) are required for long-term survival of the cells.

Due to the fact that many growth factors used to supplement media are derived from animal blood (and therefore have the potential to be contaminated), it is suggested to avoid or eliminate the use of these components wherever feasible. It is also vital to employ aseptic techniques.

Cellular Confluence

Cellular confluence is a term that refers to the proportion of the culture vessel that is occupied by cells that have connected to it. 100 percent cellular confluence, for example, indicates that all of the surface area is totally covered by cells, whereas 50 percent confluence means that about half of the surface area is covered by cells. When it comes to primary cell culture, it is a critical and significant metric to watch and measure, since different cell types require different confluence end points, at which time they must be subcultured.

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Maintenance and Subculture

When separate cells are adhered to the surface of the culture plate, the maintenance phase of the cells begins to take place. Typically, attachment occurs within 24 hours of the start of the culture’s induction. A cell suspension should be subcultured when it has reached a certain percent of total cell density and is actively multiplying, as described above. It is important to subculture primary cell cultures before they achieve 100 percent confluence because cells that have reached 100 percent confluence may undergo differentiation and demonstrate slower proliferation after passing through the passaging process.

Sub-cultivation of monolayers necessitates the dissolution of both inter- and intracellular cell-to-surface connections during the process.

It is then necessary to count the cells after they have been dissociated and dispersed into a single-cell suspension.

Cell Counting

Hemocytometers, which employ the exclusion dyeTrypan Blue to estimate cell numbers and determine cell viability, are widely used in clinical laboratories for these purposes. A hemocytometer is a glass microscope slide that is rather thick and has a rectangular depression that provides a chamber for collecting blood samples. An engraved grid of perpendicular lines is laser-etched onto the chamber, which is then meticulously built by skilled craftsmen. It is possible to determine the area enclosed by the lines as well as the depth of the chamber.

Cryopreservation and Recovery

Cryopreservation is a technique for preserving structurally intact live cells by freezing them at extremely low temperatures. In order to limit cell damage and death during the cryopreservation and thawing of primary cells, it is necessary to perform both procedures. The use of a cryoprotectant, such as DMSO or glycerol, can be used to achieve this goal in the case of primary cells (at correct temperature and with a controlled rate of freezing). Most primary cells can be cryopreserved in a combination of 80 percent complete growth media supplemented with 10 percent FBS and 10 percent DMSO, which is ideal for most primary cells to use.

The frozen culture must be kept in the vapor phase of liquid nitrogen (-196 degrees Celsius) or below -130 degrees Celsius.

When initial cells are thawed, extra attention should be exercised to avoid centrifuging them (as they are extremely sensitive to damage during recovery from cryopreservation).

1When starting a culture of cryopreserved primary cells, it is critical to remove the spent media as soon as the cells have attached themselves to the culture medium (as DMSO is harmful to primary cells and may cause a drop in post-thaw viability).

Primary Cell Culture Troubleshooting

  • By freezing live cells at extremely low temperatures, cryopreservation can be used to retain their structural integrity. In order to limit cell damage and death during the cryopreservation and thawing of primary cells, it is necessary to perform both processes. The addition of a cryoprotectant, such as DMSO or glycerol, is necessary in the case of primary cells (at correct temperature and with a controlled rate of freezing). Almost all primary cells may be preserved in a combination of 80 percent complete growth media supplemented with 10% FBS and 10% DMSO, which is suitable for cryopreservation. To avoid the production of ice crystals within the cells, the freezing procedure must be carried out at a modest rate of -1 °C per minute. Ideally, the frozen culture should be kept in the vapor phase of liquid nitrogen (-196 degrees Celsius) or at a temperature below – 130 degrees Celsius. Thawing cryopreserved cells is a simple procedure that may be completed in 1 to 2 minutes by immersing the frozen cells in a 37 °C water bath. When initial cells are thawed, extra attention should be exercised to avoid centrifuges (as they are extremely sensitive to damage during recovery from cryopreservation). It is beneficial to plate cells immediately after thawing because it permits cultures to connect to the cells for the first 24 hours after thawing is complete. If you are starting a culture of cryopreserved primary cells, it is critical to remove the spent media as soon as the cells have been attached to the culture medium (as DMSO is harmful to primary cells and may cause a drop in post-thaw viability).

Mammalian cell tissue culture techniques protocol

For the purpose of growing cell lines, the following guidelines are provided. In order to assure sterility, all cell culture procedures must be carried out in a microbiological safety cabinet using aseptic technique. This protocol is available for download as a PDF. ​

Contents

  1. Aseptic preparation
  2. Preparation of cell growth media
  3. Creation of the proper cell culture environment
  4. Checking of cells
  5. Sub-culturing
  6. Compliance with subculture procedure Sub-culturing loosely attached cell lines that require cell scraping for sub-culturing
  7. Sub-culturing attached cell lines that require trypsin
  8. Sub-culturing suspension cell lines
  9. Changing medium
  10. Passage number
  11. Sub-culturing of suspension cell lines

​1) Preparing an aseptic environment

Regulations for hoods

  • (a) Keep the hood sash in the right position to promote laminar air flow
  • (b) Keep the area free of debris.

Autoclaving

  • Autoclaving

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.

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.

Media Measure
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.

  1. Prepare a 10mL coating solution containing 1 percent gelatin or 1 percent fibronectin by diluting it with distilled water and filtering it through a fine mesh strainer. This method coats around 5 flasks in a single batch. Fill the flask halfway with coating solution. Rock the flask back and forth to ensure that the bottom is equally distributed. Allow for 15-30 minutes of incubation in an incubator. Before looking at the cells, aspirate the coating solution and wash it with sterile dH 2 O.

4) Checking cells

Cells should be examined under a microscope on a daily basis to assess their health, growth rates, and confluency ( percent surface area covered with cell monolayer). Membrane-bound adherent cells should be mostly adhered to the bottom of the flask, exhibit adherent morphology (which will vary depending on the cell type), and refract light around their perimeter (refer to Abcam cell line data sheet images). Suspension cells should have a circular shape and refract light around their membrane, according to the literature.

The hue of the medium that contains phenol red should be pink or orange (media color may change depending on CO 2environment).

A pale yellow color in the medium would imply acidity and a fall in pH, which are frequently linked with contamination or sick cells, according to the manufacturer. If any of the following conditions are met, cells are discarded:

  • 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).

5) Sub-culturing

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:

  • For example, split ratios are calculated based on the surface area of a flask, as follows: One 25-centimeter square. 2flask 3 x 25 cm would be produced if the ratio was one-third. 1 x 75 cm or 2 x 75 cm flasks. The following cell densities (cells per milliliter) should be used to sustain suspension cell lines:
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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)

At roughly 80% confluence (i.e., when the cell monolayer covers 80% of the flask surface), the cells should still be in their log phase of growth and will require subculturing to complete the process. It is not suggested to allow cells to grow very confluent since this may have a detrimental impact on gene expression and cell survival in the laboratory. ​

  1. 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
  2. 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
  3. 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

  1. 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
  2. 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
  3. 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.

  1. 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)
  2. Using a pipette, add enough trypsin EDTA to completely cover the cells at the bottom of the flask
  3. 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
  4. 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)
  5. 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​

  1. Check the cell line’s specifications for the required split ratio or subculturing cell densities
  2. 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
  3. 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. Remove the old culture medium from the flask and replace it with the required amount of fresh pre-warmed culture media before returning the flask to the incubator. ​

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.

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