Cell Culture Reagents and Assays

General

What is cell culture?

Cell culture is defined as the removal of cells from their natural environment and placed into a satisfactory in vitro environment in a laboratory for further study. It is a necessary technique across the world of cell biology research. The success of cell culture depends on the optimization of the culture conditions with appropriate levels of (1) gases (O₂, CO₂), pH, pressure, and temperature, (2) suitable media to provide the needed nutrients, minerals, salts, and amino acids, (3) effective growth factors to maintain phenotype and overall cell health, (4) proper cell attachment substrates for adherent cell cultures.

Can liquid cell culture media be frozen?

Freezing of liquid cell culture media is not recommended due to the fact that freezing typically causes precipitation and formation of insoluble salt complexes.

What is GlutaminePlus^™, and how does it compare to L-glutamine?

GlutaminePlus, a derivative of L-glutamine obtained by a chemical reaction of L-alanine with L-glutamine, is available in liquid form or as an ingredient in ready-to-use cell culture media (media with stable L-glutamine). GlutaminePlus is metabolized within the cells to yield L-glutamine plus the second amino acid. This results in more consistent delivery of L-glutamine to cells in culture and avoids toxic buildup of ammonia in cell cultures. This feature can be especially important for ammonia sensitive cell lines. L-glutamine is an essential amino acid and plays a major role for the growth and function of cells in culture. Although L-glutamine is stable in crystalline form, it has the tendency to degrade non-enzymatically and irreversibly in solution within a short time period. The rate of this L-glutamine breakdown is dependent on pH, temperature, and the presence of various anions. One of the byproducts, ammonia, may act as a toxin or growth inhibitor for the cultured cells. Because of its chemical instability and significance for cell growth, it is important that the delivery of L-glutamine be optimized to each specific cell culture application. A recommended way of achieving reliable delivery of L-glutamine to the cells in culture is the use of stable derivatives of L-glutamine in cell culture media.

Serum

What is the difference between fetal bovine serum (FBS) and fetal calf serum (FCS)?

There is no difference. These are different names for the same product. Serum derived from blood of bovine fetuses is referred to as fetal bovine serum or fetal calf serum.

After I heat inactivated my serum, some precipitates are visible. Why?

Heat inactivated serum, including fetal bovine serum (FBS) may contain some turbidity, flocculent material, or crystalline precipitate. This is a normal occurrence with serum products and in no way indicates that the quality of the product has been compromised. Commonly, this material is composed of fibrin that has converted from the soluble precursor form, fibrinogen, in serum. Bio-Techne collects and processes all sera rapidly at cold temperatures to yield the highest quality serum with excellent growth properties. This rapid cold processing allows some soluble fibrinogen to remain in the serum after filtration which may convert to fibrin upon thawing. Precipitates found in serum frequently also contain calcium complexes of inorganic serum components and proteins. Lipid serum components may also cause turbidity of the serum product. Incorrect thawing, frequent thaw-freeze cycles, heat inactivation, and extended storage at temperatures above freezing will result in a greater amount of precipitates. The presence of precipitates in serum does not alter the performance characteristics of the product when used as a growth supplement for cell culture. It is not recommended to filter the serum to remove these precipitates. Doing so may result in the loss of some serum nutrients and may clog the filter. Instead, if removal of the flocculence is desired, brief centrifugation of the serum in sterile tubes at 400 x g is recommended.

Can I still use my serum if it arrives partially thawed?

All Bio-Techne fetal bovine serum and other serum products are shipped frozen via overnight service and packaged in dry ice. Without a delay in shipping and at the receiving location, it should arrive frozen. If delays occur, and the serum becomes partially thawed, the serum can still be used. Thaw serum completely, mix gently, aliquot into single-use units if desired, and refreeze.

How should I thaw serum to ensure that the performance is not compromised?

Frozen serum should be thawed rapidly to avoid prolonged exposure of serum nutrients to higher salt concentrations during the thaw period. Thaw frozen serum at room temperature or in a 37 °C water bath. Periodically agitate the bottle during the thawing process to re-suspend the viscous solutes and to avoid the formation of salt, protein and lipid gradients that can lead to excessive precipitation. Promptly remove the serum from the water bath as soon as the serum is completely thawed. Thoroughly mix the thawed serum before it is added to a culture medium or is heat inactivated. Thawing of serum at temperatures above 37 °C is not recommended. This process may degrade heat labile nutrients, thus compromising the integrity and performance of the product, and can cause increased precipitate formation.

What procedure do you recommend for freezing serum?

Serum, including FBS, should be frozen as rapidly as possible to avoid prolonged exposure of serum nutrients to higher salt concentrations. Water is the first serum component to freeze, resulting in other serum components such as proteins and salts to accumulate at the bottom of the container at a higher concentration. Therefore, slow freezing will result in excessive formation of crystalline precipitates.

Does your fetal bovine serum contain any naturally occurring biotin?

Our FBS contains naturally derived biotin. We do not biotin-deplete nor measure the sera for biotin content.

Has this fetal bovine serum been tested for Bovine Spongiform Encephalopathy (BSE)?

We use a strict set of material sourcing and manufacturing policies to minimize the risk of BSE contamination. This includes only using material collected in U.S.D.A. approved slaughterhouses or in countries certified by the U.S.D.A. to be free of Foot and Mouth Disease (FMD), Bovine Spongiform Encephalopathy (BSE), and other exotic disease agents. We do not routinely perform BSE testing in our virus testing panel. For further information, please read our TSE/BSE policy at R&D Systems.

How should I determine which fetal bovine serum is most suitable for my cell culture system?

Bio-Techne requires all serum products to undergo rigorous testing to ensure consistent quality and proven performance even for the most sensitive cell culture systems. Because not all FBS needs are the same, Bio-Techne has developed a range of products that are tailored to specific cell culture requirements. Additional assays beyond the quality assurance and performance testing conducted on every FBS product, such as a hormone assay, are performed for some of our FBS products to address user-specific cell culture requirements.

How should serum be stored, and for how long?

Please refer to our Methods for Storage, Thawing, and Freezing of Serum Products protocol at R&D Systems.

Is serum light-sensitive?

Serum contains components that are affected by exposure to light. However, normal handling of serum under room lights should have little effect on the serum. The effect of light on the serum will depend upon the intensity of light, the wavelength of light and the duration of exposure. Therefore, high intensity lights and long-term storage under light should be avoided. Note that certain cell lines may be more sensitive to the effects of light on serum than other cell lines.

Why is serum heat inactivated?

Generally, the objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from the serum is not required for most cell cultures, but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if actually required for optimal cell growth. If heat inactivation is required, the process should be carefully controlled to avoid increased denaturation of serum proteins and formation of crystalline precipitates, potentially resulting in excessive loss of growth performance. Initially, heat inactivation was also used to inactivate microbial contaminants such as mycoplasma. Heat inactivation for this reason is unnecessary, since Bio-Techne FBS is triple 0.1 µm filtered and all other Bio-Techne manufactured sera must test negative for mycoplasma, bacteria and fungi. Sometimes, heat inactivation is performed to disrupt susceptible viruses. In most protocols for this application, prolonged heat inactivation is required. This is not recommended, since valuable components of the serum are rendered ineffective by this treatment.

Why do some cell culture media contain sodium pyruvate?

Sodium pyruvate is added to many low glucose and high glucose DMEM formulations. Sodium pyruvate can be used by cells as a readily accessible carbon source for energy production and other critical metabolic pathways, bypassing the need to produce it biosynthetically from glucose or amino acids. Some cell lines require the addition of pyruvate to the culture media since they lack the ability to convert glucose or amino acids into pyruvate.

Why is there lot-to-lot variability in serum?

Fetal bovine serum and other serum products contain a complex mixture of biological components, a majority of which have not yet been fully defined. The composition of these serum components naturally varies from lot-to-lot. The best serum lot for you is the one that works for your cells in your specific application. Bio-Techne serum manufacturing has a 40 year history of experience, which provides some of the most consistent serum on the market. We can help you select the serum lot that is ideal for your application by offering a variety of programs such as lot matching, free samples for lot prequalification, extended lot reserves, and free on-site serum storage, geared towards minimizing the impact of serum variability for our customers.

Extracellular Matrix Components

What is Laminin I?

Laminin I is a major component of extracellular matrix. Cultrex^™ Laminin I is purified from murine EHS sarcoma. It is composed of α1β1γ1 chains with a total MW of 800,000 Da. Cultrex Laminin I increases cell adhesion, migration, growth, differentiation, neurite outgrowth, protease production, and malignancy. The response is dependent on cell type.

What is the recommended working concentration for Cultrex Laminin I?

The recommended working concentration for thin coating is 0.05-10 µg/cm². However, conditions must be optimized for each cell line or model. Cultrex Laminin I should be used at 6 mg/mL for 3D culture applications.

Can Cultrex Mouse Laminin I be used on glass cover slips?

Coating glass cover slips with Cultrex Laminin I is quite common and has been published.

Basement Membrane Extracts

What are basement membrane extracts?

Cultrex^™ Basement Membrane Extracts are solubilized extracellular matrices purified from Englebreth-Holm-Swarm (EHS) tumor. They polymerize at 37 °C to form reconstituted basement membranes that are rich in extracellular matrix proteins, such as Laminin, Collagen IV, Entactin, and heparan sulfate proteoglycans. These specialized sheets of extracellular matrix provide structural support for cells and play an important role in establishing tissue organization by influencing cell adhesion, proliferation, migration, and differentiation.

What kinds of tumor cells or biopsy specimens grow in vivo with Cultrex BME?

Many cell lines and tumor biopsy specimens (usually cut into small fragments) have been found to grow in vivo when implanted with Cultrex BME. These include melanoma, intestinal, prostate, breast, lung, renal, and liver cancers as well as the 3T3 mouse embryonic fibroblast cell line.

Which matrix should I use for 3D culture or studying invasion?

Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex^™ Basement Membrane Extracts will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex^™ Laminin I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.

How will non-tumorigenic cells/tissues grow or differentiate when implanted in vivo in Cultrex^™ Basement Membrane Extracts?

Non-transformed cells mixed with Cultrex BME and implanted in vivo have been found to continue to survive and remain differentiated but generally do not grow. No normal tissues have been found to transform under these conditions. For example, Sertoli cells survive at least a week and retain their cord-like structures.

What is the difference between Cultrex Basement Membrane Extract (BME) and Cultrex 3D Culture Matrix

Cultrex 3D Culture Matrix was developed to provide the most standardized basement membrane extract for use in 3D Cultures. A special process is employed to reduce growth factors. This material is then incorporated in a 3D culture to validate efficacy. 3D Culture Basement Membrane Extract promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3) into acinar structures. The Cultrex 3D Culture Matrix is essentially the same as our standard Cultrex BME, but has been additionally qualified with the functional 3D assay as described above.

How do Cultrex^™ Basement Membrane Extracts promote cell differentiation?

All epithelial and endothelial cells are in contact with a basement membrane matrix on at least one of their surfaces. By providing them with their natural matrix in vitro as a substrate for the cells that provides biological cues, the cells can assume a more physiological morphology (i.e. correct shape) and begin expression of cell-lineage specific proteins. Two-dimensional plastic surfaces, in combination with serum-containing media, cause cells to flatten, proliferate and de-differentiate.

How should Cultrex^™ Basement Membrane Extracts be stored and handled?

BME should be stored at or below -20 °C for optimal stability. Preparation of working aliquots is recommended. BME should be thawed overnight on ice at 4 °C, however long term storage at 4 °C is not recommended. Freeze/thaw cycles and gel-liquid phase transitions should be avoided, since they can compromise product integrity.

3D Culture

What are 3D cultures?

3D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or tissue explants are placed within hydrogel matrices, such as Cultrex^™ Basement Membrane Extracts, that mimic in vivo cell environments and allow cells to proliferate in three dimensions. Download a copy of our Evolution of Cell Culture Model Systems eBook to learn more.

What are the different types of 3D culture?

The two principal methods for performing 3D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex^™ Basement Membrane Extracts or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.

What are the variables associated with 3D culture?

The major variables associated with 3D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.

What is the advantage of 3D culture over traditional 2D culture?

While 2D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3D environment, they assume biological and biochemical characteristics similar to what is observed in vivo. Download a copy of our Evolution of Cell Culture Model Systems eBook to learn more.

How can I harvest my organoids or 3D cultured cells for subsequent analysis?

Organoids or 3D cultured cells may be harvested using the Cultrex Organoid Harvesting Kit.

How should cells be cultured prior to setting up the 3D culture?

Cells need to be healthy and actively dividing in 2D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed for viability using trypan blue, and they should exhibit less than 5% staining.

What type of analysis is typically applied for organoid or 3D cell cultures?

Within the organoid, spheroid, or 3D culture, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. In addition, cells may be isolated from the 3D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA.

What is the Angiogenesis Tube Formation assay?

The tube formation assay is based on the ability of endothelial cells to form three-dimensional capillary-like tubular structures when cultured on a hydrogel of reconstituted basement membrane, such as Cultrex Basement Membrane Extract (BME).

Cell Invasion/Migration Assays

What is cell invasion?

Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Cultrex Cell Invasion Assays evaluate cell invasion based on the cells ability to traverse membranes that are coated with a layer of extracellular matrix proteins. The cells must traverse this barrier through a combination of protein degradation and cellular locomotion.

What is cell migration?

Cell migration is the movement of cells in response to a chemical stimulus; also known as chemotaxis. Cell migration assays evaluate cell migration based on the cells ability to traverse an uncoated membrane with 8 µm pores, in response to a chemotactic gradient. The cells must undergo cytoskeletal remodeling to fit into the pores and pull themselves through to the underside of the membrane.

Can the Calcein-labeled invasive cells be subcultured?

Calcein AM cytotoxicity should be determined empirically for each cell line or model. For best results, the cells should be removed from the cell dissociation solution and placed in fresh culture medium as soon as possible.

How do the Cultrex Cell Invasion Assays compare to wound healing assays?

Wound healing assays, also known as scratch assays, monitor cell migration laterally on a tissue culture treated plate. This is accomplished by generating a void in a cell monolayer by either removing cells or treating the surface of the plate to prevent cell growth in a designated area. The assay measures the ability of the cell monolayer to fill this void, and it may be conducted in the presence of extracellular matrix proteins. Since this assay is conducted within one chamber, there is no chemotactic gradient, and without the membrane, the cells are no longer required to change shape and squeeze through the pores. Another potential problem is that this assay does not control for differences in cell proliferation. While wound healing assays may be valuable for supplementing the Boyden chamber assay, it is not a replacement.

Which basement membrane protein is best for studying invasion?

Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex^™ Basement Membrane Extracts will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes, adipose cells, and migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.