Technical library

Yes. Pre‑coated culture vessels are required when culturing human mesenchymal stem cells (hMSCs) in PromoCell Mesenchymal Stem Cell Growth Medium XF (C‑28019).

Reason
MSC Growth Medium XF is a xeno‑free culture system that contains all necessary growth factors and supplements, but does not include attachment or spreading factors. Therefore, cells require an external coating to ensure proper adhesion.

Recommended coatings
Culture vessels must be pre‑coated according to the manufacturer’s instructions using one of the following options:
- Human fibronectin: 1 µg/cm²
- Human vitronectin: 0.5 µg/cm²
Alternatively, bovine fibronectin may also be used.

No, it is not necessary to use coated flasks, therefore we don´t recommend their usage in the Instruction Manual. However, for special applications, some of our customers use collagen-coated dishes.

Yes, our MSC Growth Medium XF (# C-28019) contains phenol red. The concentration is confidential.

But we also supply a phenol red-free variant: Mesenchymal Stem Cell Growth Medium XF, prf (# C-28018).

Problems in obtaining RNA with good yield and purity from mononuclear cells (hMNCs) are quite common. The reason for this is the large amount of free genomic DNA usually contained in MNC preparations. This DNA originates mostly from granulocytes which underwent lysis during the isolation of the MNC. The granulocytes are gone in the final MNC preparation, but their genomic DNA - released during cellular lysis - is still there "sticking" to the MNCs. Solution: Remove DNA prior to RNA purification by a DNase digestion step. Most commercial systems include the option for such a DNase digest.

For optimal post‑thaw recovery, please follow the protocol below when freezing normal human cells.
Cell freezing protocol
1. Detach the cells using your standard detachment procedure.
2. Count the cells and ensure a viability greater than 70% before freezing.
3. Centrifuge the cells and resuspend them in a suitable freezing medium at a density of 0.1–10 × 10⁶ cells/ml.

• We recommend Cryo‑SFM Plus, a defined, animal‑component‑free and protein‑free cryopreservation medium.
• The freezing medium should be pre‑cooled to 4°C. Do not exceed room temperature.

4. Aliquot the cell suspension into cryovials.
5. Cool the cells slowly to −80°C at an approximate rate of −1°C per minute.

• We recommend using a controlled‑rate freezing container such as Mr. Frosty™ (Nalgene) and placing it in a −80°C freezer overnight.

6. Transfer the frozen vials to liquid nitrogen for long‑term storage.
 

Primary cells (primary culture) are freshly isolated cells that are plated in a tissue culture vessel for the first time. This initial culture stage is referred to as passage 0 (P0).
Normal cells are primary cells that have been subcultured at least once. From this point onward (i.e. passage ≥ 1), they should correctly be termed normal cells.
 
In summary, the distinction is purely based on culture history and passage number, not on cell origin or biological characteristics.
In everyday laboratory practice, however, the term “primary cells” is often used interchangeably with “normal cells".
 

Adult stem cells are stem cells isolated from postnatal tissues that have retained the capacity for self‑renewal as well as for differentiation into multiple cell lineages (multipotency).

PromoCell does not determine the number of passages but instead we calculate the population doublings (PD) that can be performed with the cells. The term passage only describes the process of detachment and replating and does not take into account different split ratios. The optimal split ratio is calculated from the actual cell yield after trypsinisation and the recommended plating density. In most of our cell types, the split ratio is usually between 1:3 and 1:6. Using 1:4 splits (i.e. increasing the growth surface by factor 4 each time), 15 doublings are achieved after 6-8 passages. For recommended plating densities, please view the respective Manual, section "Specifications".

PromoCell guarantees 15 population doublings (PD) for most normal human cells, unless otherwise stated in the Certificate of Analysis (CoA).
This guarantee applies when the cells are cultured using the recommended PromoCell growth media and PromoCell detachment reagents.

Additional information
Further details can be found in the Instruction Manual for the respective cell type under “Specifications” or on the corresponding product page in our webshop (see the “Product Description” tab).

If cells do not detach or detach very slowly during subculture, this is usually caused by suboptimal subcultivation conditions or handling.

Possible reasons and solutions

• Cells are too confluent → Trypsinize cells at a subconfluent stage
• Washing step was omitted → Perform a washing step with HepesBSS or PBS to remove residual medium before adding trypsin
• Trypsin solution is too cold → Use pre‑warmed trypsin (room temperature or 37°C)
• Trypsin solution is too old → Check the expiry date and use a fresh solution
•  Incubation time too short →  Use proper incubation time until 80% have detached 

If the issue persists, PromoCell Scientific Support will be happy to assist you in identifying the cause and optimizing your subculturing protocol.
 
Please note: Some epithelial cell types adhere very firmly and need prolonged incubation times. To avoid irreversible damage by over-trypsinization, the use of accutase is recommended. Accutase is very gentle to the cells, preserves the surface receptors and doesn’t affect the cellular viability during extended contact with the cells.