Frequently (and not so frequently!) asked questions.
Please review the list below. If you have any further questions, please don’t hesitate to contact us. For scientific questions and technical support, contact our scientists directly by email to firstname.lastname@example.org.
- Why is the recommended seeding density important?
- Which is the right incubation temperature for the cells?
- I want to grow primary epithelial cells not progenitor cells, can I use CELLnTEC’s Prime media with PCT factors?
- Why does CELLnTEC have different media for isolation / culture expansion and differentiation of primary cells?
- What concentration of calcium do CELLnTEC media contain?
- Which detachment enzyme should I use for adherent cells?
- What is the difference between 2D and 3D cell culture?
- At what temperature should I store the CELLnTEC media?
- Do I need to add any additional supplements?
- Is it necessary to use feeder layer cells, coated plates or conditioned medium with CnT-media?
- How long is the shelf life once the supplements are added to the medium?
- Why does CELLnTEC recommend to work without antibiotics / antimycotics for routine cultivation?
- Do you have suggested protocols?
- Can I have the formulation of the medium, as I am doing signaling experiments which may be affected by components of the medium?
- Can I have the formulation of the medium, as I need to know the components in order to publish my results?
- I have early passage cells growing, can I put them directly into the CELLnTEC medium?
- Why didn’t my cells grow after passaging them?
- I am developing therapies based on adult stem cells. Is the CELLnTEC medium produced as a therapeutic grade product?
- My previously isolated cells, growing in my current medium did not grow very well when switched to the CELLnTEC medium. What could be the problem?
- Why is my isolation efficiency very low compared to my current medium?
Why is the recommended seeding density important?
Answer: Cell density has a significant influence on the growth of cells in vitro. Cells secrete a variety of beneficial factors into the medium, and as such are able to condition the media in which they are growing. Increased cell-cell contact also directly stimulates a variety of membrane-bound receptors. When seeding cells, there is a key level at which the cell density is sufficient for this beneficial effect to function. All media and cells have a minimum cell density below which initial attachment and growth begins to decline. Accordingly, all CELLnTEC media have a recommended minimum seeding density at which extensive tests have confirmed that cells will attach and proliferate well. Conversely, too high seeding densities can also be detrimental, due to the need for frequent passaging, and the difficultly for colonies to form. Recommended seeding densities can be found in our datasheets and protocols.
Which is the right incubation temperature for the cells?
Answer: The cells should be cultivated at 37°C with 5% CO2. As epidermal keratinocytes are isolated from the skin, incubation at 35°C may be considered slightly more physiological. However the cells may grow slightly faster when cultivated at 37°C, compared to 35°C incubation.
I want to grow primary epithelial cells not progenitor cells, can I use CELLnTEC’s Prime media with PCT factors?
Answer: Yes. All primary cell culture involves the isolation of a population of proliferating cells. Prime media with PCT factors are designed to improve the isolation of progenitor cells, and are thus ideally suited to the establishment and extended maintenance of primary cell cultures.
Why does CELLnTEC have different media for isolation / culture expansion and differentiation of primary cells?
Answer: CELLnTEC’s unique Prime media are specifically designed to retain cells in a proliferative phenotype and delay differentiation. Thus they are not well suited to situations where cells should be induced to differentiate. For this reason, CELLnTEC provides optimzed versions of its media, which can be used when cells are induced to differentiate. Differentiation protocols describing the use of these media can be found in the resources section.
What concentration of calcium do CELLnTEC media contain?
Answer: CELLnTEC’s Prime media contain a low concentration of calcium ideally suited to the isolation and culture of epithelial cells. No further adjustments to the calcium level are required. For specific situations where the calcium level must be reduced still further, calcium-free versions of the media are available. All calcium free media require the addition of some calcium for cell growth and attachment.
Which enzyme should I use for detachment of the adherent cells?
Answer: For the detachment of the cells Accutase is recommended due to its gentle action. Trypsin / EDTA may also be used, but is harsher, and has a smaller time-window between successful detachment and over-digestion. The features of each are summarized below.
Accutase (Cat# CnT-Accutase-100) is a mixture of proteolytic and collagenolytic enzymes isolated from crustaceans. It is free of mammalian components. Accutase is the recommended detachment enzyme due to its gentle action that leaves most surface proteins intact, and the fact that it does not require a separate reagent to stop the reaction (simply dilute with medium immediately after detachment). Accutase is heat senstive, and must be stored at 4°C. Pre-warm only the amount required to 20°C just prior to use. Exposure to higher temperatures or repeated warming / cooling cycles may lead to reduced activity.
Trypsin / EDTA is a solution of proteolytic enzymes containing Trypsin, Chymotrypsin and Elastase. Trypsin is non-defined and of porcine origin. It may vary from lot-to-lot, and must be inactivated with a separate reagent immediately after cell detachment (preferably with an animal-component free inhibitor, alternatively with FCS). Trypsin is much more aggressive than Accutase, resulting in only a very small time window between succssful detachment and irreversible cell damage. If using Trypsin, extra special care must be taken to minimise the exposure time, and ensure rapid inactivation.
What is the difference between 2D and 3D cell culture?
Answer: 2D cell culture, also known as monolayer cell culture, involves the culture of cells in a single layer, submerged in culture medium. Cells are only exposed to culture medium, and can be either maintained in an “undifferentiated” proliferative phenotype, or induced to further differentiate. In contrast, 3D culture involves the culture of cells to form multi-layered cell structures. 3D culture may be used to maintain certain kinds of lower differentiated cells (for example dermal progenitors grown as spheres), or may also be used to create fully differentiated cell sheets, for example of the epidermis. 3D epidermal cultures allow the full spectrum of cell differentiation, extending from terminally differentiated non-proliferative cells on top, down to proliferating progenitors in the basal layer.
At what temperature should I store the CELLnTEC media?
Answer: the Prime media are supplied fully supplemented, and frozen. They must be stored at -15º to -25ºC until ready for use. Thaw the medium overnight at 4ºC just before use. Once thawed, store the medium at 4ºC, and ensure that it remains protected from light. Several media components are very light sensitive.
Do I need to add any additional supplements?
Answer: No, the medium is supplied with all required supplements for cell cultivation. Antibiotics are not supplied but are available from CELLnTEC and may be added if desired (but we recommend working without antibiotics / antimycotics for routine cultivation of primary cells).
Is it necessary to use feeder layer cells, coated plates or conditioned medium with CnT-media?
Answer: No, the CnT-media are designed to be used without feeder layer cells, or conditioned medium. When isolating fresh cultures, plate coatings (such as collagen I) may improve initial cell attachment in the first passage or two. Please see our protocols for specific recommendations.
How long is the shelf life once the supplements are added to the medium?
Answer: The shelf life of the supplemented medium is 6 weeks when stored at 4ºC in the dark. Minimize all light exposure – most culture media are quickly degraded by any light exposure.
Why does CELLnTEC recommend to work without antibiotics / antimycotics for routine cultivation?
Answer: Primary cells are sensitive to treatment with antibiotics / antimycotics. The supplementation of the medium with antibiotics / antimycotics can cause slower proliferation rates and can have influence on the differentiation behavior of cells. All commonly used antibiotics are quickly degraded by the temperatures used during cell culture which can lead to the use of ineffective concentrations of antibiotics / antimycotics and thus enhance the risk of development of resistant microorganisms.
Cells are especially sensitive to the effect of antibiotics during differentiation in both 2D and 3D culture.
For isolation of primary cells antibiotics / antimycotics are often needed because the tissue is not sterile, thus we recommend the use of antibiotics / antimycotics up to passage 2.
Do you have suggested protocols?
Answer: Yes, CELLnTEC provides a wide range of protocols, for example for isolation, passaging / freezing, transfection and differentiation. They are available on our resources section.
Can I have the formulation of the medium, as I am doing signaling experiments which may be affected by components of the medium?
Answer: To find out the majority of components in these media, please see the component list in the resources section of the CELLnTEC website. While complete formulation is a trade secret, elements of the formulation relevant to your research can be disclosed. Please forward a description of your situation and your specific questions to email@example.com.
Can I have the formulation of the medium, as I need to know the components in order to publish my results?
Answer: You can publish in scientific journals without knowing the medium formulation. There is an extensive publication list on the CELLnTEC website which provides numerous examples of CELLnTEC media in the literature. In addition, there is also a component list in the resources section of the CELLnTEC website, which lists the majority of components in the media.
I have early passage cells growing, can I swap them directly into the CELLnTEC medium?
Answer: It depends on the medium you are currently using. It is often the case that cells that have adapted to one medium to not transfer well to another.
In these cases, cells must be weaned away from the old medium to the new medium. This is best done in a step-wise decreasing dilution of the old medium over a period of one to four weeks, depending on the severity of the transition and the proliferation rate of the cells.
For a four week approach this can be done with the following dilutions (new medium / old medium): 20 / 80%, 40 / 60%, 50 / 50%, 60 / 40%, 80 / 20% and 100 % new medium.
For a two week approach the following dilutions can be used (new medium / old medium): 25 / 75%, 50 / 50 %, 75 / 25% and 100% new medium.
Please keep in mind that changing medium sometimes can lead to changes in cell morphology or growth behavior of the cells.
Why didn’t my cells grow after passaging them?
Answer: Overdigestion or failure to quickly stop the digestion reaction are the most common reasons.
A very common problem researcher’s encounter when switching to a serum free or low serum medium is that the trypsin is not deactivated with the addition of medium. Over digestion with trypsin can irreversibly damage the cells, thus detachment must be closely monitored under the microscope. CELLnTEC recommends using a milder enzyme such as Accutase for the passaging of cells. A protocol is found on our resources section.
Answer: the Prime media are completely free of all anima, and human-derived components. This makes them a good candidate for use in cell therapy applications. When necessary, they can be manufactured with additional QC tests, to increase their suitability for clinical use.
My previously isolated cells, growing in my current medium did not grow very well when switched to the CELLnTEC medium. What could be the problem?
Answer: Primary cells can quickly adapt to specific cell culture media. Hence when switching a cell culture to a different medium, there is a need to wean your cells off the old medium. This is accomplished with a step-wise serial dilution of the old medium with CELLnTEC medium over a period of one to four weeks, depending on the severity of the transition and the proliferation rate of the cells. See detailed protocols listed above.
Why is my isolation efficiency very low compared to my current medium?
Answer: There are a multitude of potential explanations for such a situation, in variably we need a full description of the procedure you followed. Key questions include what is your current medium? Does it contain FBS or BPE? Was the CELLnTEC medium tested in side by side comparison to your current medium? Did you use trypsin in your isolation protocol? In this case low isolation efficiency could be due to over-trypsinization, previously the trypsin reaction was inhibited by proteins from FBS or BPE. Other reasons for over- trypsinization include high trypsin concentrations or a too long trypsin treatment. Refer to the specific FAQ above where we explain in detail the different options of detaching cells. For more detailed info, email firstname.lastname@example.org