Cell density plays an important role in cell growth in vitro. At adequate densities, cells benefit from factors released into the medium and from cell–cell interactions, which support attachment and proliferation. If cells are seeded too sparsely, these effects may be limited and growth can be reduced. Very high seeding densities can also be suboptimal, as they may restrict colony formation or require frequent passaging. For guidance, recommended seeding densities for CELLnTEC media are provided in the relevant datasheets and protocols.
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.
CELLnTEC’s proliferation 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 optimized 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.
CELLnTEC’s proliferation media are specifically designed to retain cells in a proliferative phenotype. When cells are proliferating fast, response to certain stimuli can be weakened. For this reason, CELLnTEC provides optimized versions of its media for homeostatic maintenance of the cells with minimal growth stimulation.
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). Since Accutase is much gentler, it can be left longer on the cells (10 – 20 minutes) if required for complete detachment. Accutase is heat sensitive and must be stored at 4°C. Pre-warm only the amount required to room temperature 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 successful detachment and irreversible cell damage. If using Trypsin, extra special care must be taken to minimize the exposure time and ensure rapid inactivation.
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.
It is important to follow the recommended thawing procedure. Please find a detailed protocol in the resources section.
No, the CnT-media are designed to be used without feeder layer cells or conditioned medium. Plate coatings (such as collagen I) may improve cell attachment and can be tested whether it brings a benefit in a specific application but is not generally required.
The shelf life of the supplemented medium is 6 weeks when stored at 2-8ºC in the dark.
Primary cells can be 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. 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 CnT-GAB-5 up to passage 2.
Yes, CELLnTEC provides a wide range of protocols, for example for isolation, passaging / freezing, transfection and differentiation. They are available on our resources section.
The formulation of our media is proprietary and cannot be disclosed.
The CnT-Prime and CnT-NX media share the following formulation elements:
These media do not contain cholera toxin or phenol red.
Should you have additional specific question about particular parts of the formulation relevant to your research, please send us a description of your planned experiments and your specific questions to support@cellntec.com.
It depends on the medium you are currently using. It is often the case that cells that have adapted to one medium do not transfer well to another.
In these cases, it can be required to gradually switch the medium by stepwise changing the ratio of old medium and new medium over the course of several medium changes, depending on the severity of the transition and the proliferation rate of the cells.
Please keep in mind that changing medium sometimes can lead to changes in cell morphology or growth behavior of the cells.
The optimal approach is to use cells isolated in CELLnTEC medium. For several cell types, primary cells isolated in CnT media are available in our catalog.
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. After detachment with either trypsin or Accutase, it is important to centrifuge the cells and re-suspend with fresh culture medium. A protocol can be found in our resources section.
Reduced attachment, survival, or proliferation after thawing can have multiple causes. It is often influenced by factors such as storage and transport conditions, thawing procedures, or early post‑thaw culture steps. For example, slow or sub‑optimal thawing may result in prolonged exposure to cryoprotectants, and variability in handling or pre‑thaw storage conditions can also play a role. To preserve cell quality, it is important that cells are stored at ≤ −80 °C, ideally in liquid nitrogen, until thawing. A detailed cell‑thawing protocol is available in our resources section.
For applications progressing toward clinical use (e.g. ATMP development), we recommend using CELLnTEC’s Higher Certified (HC) Media. HC Media are specifically designed to support translational workflows from early research through to clinical phases, without requiring changes in medium formulation. Standard research-grade CnT media may be appropriate for early discovery work, but for regulated applications (e.g. when working under GLP or GMP), HC versions are the appropriate choice. For more details, please refer to our Higher Certified (HC) Media page.
Primary cells can quickly adapt to specific cell culture media. Hence when switching a cell culture to a different medium, there can be a need to wean your cells off the old medium. This is accomplished with a stepwise serial dilution of the old medium with CELLnTEC medium over a period of several medium changes, depending on the severity of the transition and the proliferation rate of the cells.
Yes, we strongly recommend culturing primary cells in CnT proliferation media before transitioning them to 3D models, in accordance with our protocols. CELLnTEC media are based on a shared core formulation and are specifically optimized to support a seamless transition from proliferation media to application‑specific media (e.g. for 3D model generation). As a result, model formation is most robust when cells are first expanded and well adapted in the corresponding CnT proliferation medium.
Primary cells can be used for a few passages only, before they start to show signs of senescence. For experiments, in which longer expansion of epithelial cells is required, consider to use CnT-NX-EX Epithelial Extended Proliferation Medium.
For long-term cell lines, it is recommended to limit the total number of passages (e.g. ~10–20) and to use consistent passage numbers across experiments to avoid variability from accumulating genetic changes.
Generally, we recommend to expand the cells from the initial vial for 1–2 passages before freezing working stocks. For many protocols, the cells from these stocks are expanded for one more passage before they are used for an assay or to seed 3D models.
Re-freezing medium aliquots is possible and done by some users. No impact on medium performance has been observed from one additional freeze-thaw cycle. However, suitability should be experimentally verified in the specific application by the user.
Before splitting into aliquots, we recommend to make sure that the medium is thawed completely according to the thawing instructions (see protocol section), mixed well, and that there are no undissolved precipitates. Avoid additional freeze-thaw cycles. Follow thawing instructions also for thawing of individual aliquots. Total storage duration at 2-8°C should not exceed six weeks.
