Frequently Asked Questions (FAQ)
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 scientistcellntec [dot] com. For all general inquiries contact infocellntec [dot] com.
- 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 Progenitor Cell Targeted (PCT) media?
- Why does CELLnTEC have different media for isolation / culture expansion and differentiation of primary cells?
- What concentration of calcium do CELLnTEC media contain?
- Which enzyme for the detachment of the adherent monolayer cells should be used?
- 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. 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. Find the recommended seeding densities in our protocol page of our resources section.
Which is the right incubation temperature for the cells?
Answer: The cells can be cultivated between 35 and 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 Progenitor Cell Targeted (PCT) media?
Answer: Yes. All primary cell culture involves the isolation of a population of proliferating cells. PCT media 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 PCT 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 non-PCT versions of its media, which can be used when cells are induced to differentiate. Differentiation protocols describing the use of non-PCT media can be found in the resources section.
What concentration of calcium do CELLnTEC media contain?
Answer: CELLnTEC’s PCT 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 for the detachment of the adherent monolayer cells should be used?
Answer: For the detachment of the cells Trypsin / EDTA, or Accutase can be used. Below please find a summarized overview of their properties.
Typsin / EDTA: is a solution of proteolytic enzymes containing Trypsin, Chymotrypsin and Elastase isolated from porcine pancreas that shows lot-to-lot variability in activity; is commonly used in cell culture; is available from different suppliers; has to be inactivated (e.g. FCS alone, FCS supplemented medium or trypsin inhibitor from soybean); compared to TrypLE or Accutase more cell culture experience of the user is necessary, because cells can be irreversibly damaged by too high trypsin concentrations or too long incubation times.
Accutase: is a mixture of proteolytic and collagenolytic enzymes isolated from crustacean; is mammary component free; for some cell types there is no need for inactivation or removal during passaging; is very gentle for cells, thus most of the surface proteins are intact after passaging; has to be stored at 4°C; Accutase is inactivated after 1 h at 37°C.
What is the difference between 2D and 3D cell culture?
Answer: 2D cell culture, also known as monolayer cell culture or adherent cell cultures, 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” specialized phenotype, or induced to further differentiate. In contrast 3D culture involves the culture of cells to form multi-layered, more differentiated cell structures. 3D culture may be used to maintain certain kinds of lower differentiated cells (for example 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. In this way, the 3D structures can closely mimic the structures see in vivo.
At what temperature should I store the CELLnTEC media?
Answer: CELLnTEC media are supplied as a kit including a 500 mL bottle of basal medium which must be stored at 4ºC in the dark, and a packet of frozen supplements that should be kept frozen at –20ºC until used. When the supplements are added to the basal medium this is to be stored at 4ºC in the dark. Do not freeze the basal medium or the supplemented medium because it may lead to irreversible coagulation of certain proteins.
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).
Supplements should be thawed and directly added to the basal medium by pipetting without any further dilution before addition to the medium.
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, plate coating or conditioned medium. For the growth of epithelial cells in CnT-medium it is not necessary to use any of the above mentioned techniques.
How long is the shelf life once the supplements are added to the medium?
Answer: The shelf life of the supplemented medium is 8 weeks when stored at 4ºC in the dark. Do not freeze the supplemented medium because it may lead to irreversible coagulation of certain proteins. 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 not thermo-stable which can lead to the use of ineffective concentrations of antibiotics / antimycotics and thus enhance the risk of development of resistant microorganisms.
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 scientistcellntec [dot] 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: No, cells grown in one medium are adapted to this medium and therefore it is necessary to 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.
For a one week approach the following dilutions can be used (new medium / old medium): 25 / 75%, 50 / 50 %, 75 / 25% and 100% new medium, in this case the change is made much quicker than in the two week approach and additional you should use a higher seeding density (10 to 20% more than normal) to be sure that enough cells will attach and survive to have the best density conditions for the further culture.
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: 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.
I am developing therapies based on adult stem cells. Is the CELLnTEC medium produced as a therapeutic grade product?
Answer: Basal media are manufactured in an ISO 9000 certified facility according to cGMP guidelines. However these media are only intended for research applications. When medium with greater certification is required (for example in phase I clinical trials), custom formulations with more highly certified components and expanded quality control procedures can be manufactured. In all cases, CELLnTEC must begin by working with the customer to determine the specifications needed for their application.
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 scientistcellntec [dot] com