Technical library

At manufacture, ECGS (Endothelial Cell Growth Supplement) is adjusted to a protein content of 3 mg/ml.

• For Human Endothelial Cells and Microvascular Endothelial Cells, the optimal amount of ECGS is 2 ml ECGS per 500 ml medium (0.4% v/v), corresponding to 6 mg extracted protein per 500 ml medium.
• In the SupplementPack and Medium Kit, ECGS is supplied together with heparin (ECGS/H) in a single vial. The ECGS/H supplement contains 22.5 mg/ml heparin and provides a final heparin concentration of 45 mg per 500 ml medium (90 µg/ml) when added as recommended.

The Growth Medium Kit is recommended when flexibility in supplement concentrations is required.
It allows you to adjust the final concentrations of growth supplements, making it more flexible than the “ready‑to‑use” Medium. For example, it can be used to prepare customized media, such as a starvation medium.
Please note that modifying supplement concentrations may affect cell growth and viability. It is therefore recommended to test in advance whether and for how long the cells tolerate the altered culture conditions.

The three components of the PromoCell DetachKit (HEPES BSS, Trypsin/EDTA, and TNS) have a shelf life of 12 months from the date of manufacture when stored at -20°C.

After thawing
Once thawed for use, all components should be stored at 4-8°C and can be used for up to 6 weeks. Repeated freeze-thaw cycles should be avoided, as they may affect reagent performance.

In general, no. PromoCell Normal Human Cells are cultured, adapted, and quality‑tested exclusively in PromoCell Growth Media.
Using other media, including in‑house formulations or commercially available alternatives, may lead to unsatisfactory results due to suboptimal nutrient and growth‑factor supply. Primary human cells have more demanding and specific requirements than standard cell lines.
PromoCell can only guarantee optimal cell growth and performance, as stated in the Certificate of Analysis, when the cells are cultured in the recommended PromoCell Growth Medium.

In general, no. PromoCell Normal Human Cells are optimized for use with PromoCell Growth Media, which have specialized and more complex formulations than standard media such as DMEM or RPMI 1640 supplemented with FBS.
Unlike immortalized cell lines, primary human cells have significantly higher and more specific requirements for nutrients, growth factors, and culture conditions. Classical media formulations typically do not support optimal growth or performance of PromoCell primary cells.

PromoCell cell pellets are prepared from more than 1×10⁶ cells.
Each pellet is dissolved in 200 µl RNAlater^TM. The exact cell number may vary slightly, but exceeds 1 million cells per pellet.

PromoCell Melanocyte Growth Medium M3 enables the serum‑, BPE‑, and PMA‑free cultivation of Normal Human Epidermal Melanocytes (NHEM) and does not require additional coating of standard tissue‑culture (TC) plastic.
 
Key considerations for optimal culture

• No coating required: NHEM can be cultured directly on standard TC‑treated plastic when using Melanocyte Growth Medium M3.
• Optimal confluency for passaging: To maintain the cells in a robust, adherent, and pro‑proliferative state, we strongly recommend passaging the cells at 70–90% confluency.
• Avoid high cell densities: High confluencies should be avoided, as elevated cell density can promote the formation of 3‑D spheroids. This phenomenon has been described in the literature for NHEM and may negatively affect monolayer culture and proliferation.

PromoCell strongly recommends using gentamicin at a final concentration of 50 µg/ml when isolating cancer stem cells (CSC) with the Primary Cancer Culture System.

Reason
Many commonly used antibiotics, such as penicillin or streptomycin, can inhibit the growth of primary cells, including primary cancer cells.
This inhibitory effect is particularly pronounced:
- at the clonal level
- at low cell density
Therefore, the use of antibiotics and antifungals should be approached with great caution in primary cancer cultures.

Recommendation
- Use gentamicin only (50 µg/ml) during isolation and culture.
- Avoid additional or alternative antibiotics whenever possible.

If antifungal protection is required
If the use of an antifungal agent is necessary, PromoCell recommends:
Caspofungin: 10 µg/ml (100× stock solution: 1 mg/ml in water)

Tumor‑associated macrophages (TAM) are an important component of the tumor microenvironment. Upon interaction with cancer cells, they typically adopt an anti‑inflammatory, pro‑tumor, and pro‑metastatic M2‑like phenotype.
For the isolation and culture of TAM from solid tumors, we recommend Primary Cancer Culture Medium supplemented with 250 ng/ml M-CSF, which supports TAM growth while reliably preventing stromal overgrowth.
The isolated TAM are non‑proliferating, adherent cells and can typically be cultured for at least two weeks.
 
Alternatively, the following media may be used:
- Macrophage Base Medium XF + 2% human AB serum
- M2‑Macrophage Generation Medium XF
However, please note that these media do not suppress fibroblast growth, resulting in a high risk of overgrowth by stromal cells.

The NCCD (Non‑Cancerous Cell Depletion) reagent is a non‑cytotoxic and essential functional component of the Primary Cancer Culture System (PCCS).
 
Purpose of NCCD
NCCD is required to 

• Selectively deplete non‑malignant cells
• Support the selective maintenance of malignant tumor cells

This selective pressure is a core principle of the PCCS and is necessary to ensure reliable enrichment of cancer cells.
 
Use in suspension cultures
NCCD is not dispensable, even if the tumor cells show a suspension growth pattern.
PromoCell recommends:

• Using NCCD throughout the entire culture period
• Applying NCCD both for adherent and suspension cultures

Omitting NCCD may allow non‑malignant cells to persist, which can compromise the selectivity and performance of the PCCS.