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.

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

Heat inactivated serum 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. It is best when sera is collected and processed 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.

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 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.

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 is unnecessary when sera is triple 0.1 µm filtered and tests 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?

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. 

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.