Technical library

The following protocol is recommended for the thawing and recovery of PromoCell human Mononuclear Cells (hMNC):

1. Refer to the cell number stated in the lot-specific Certificate of Analysis (CoA) and calculate the volume of medium required to achieve a final cell concentration of 1×10⁶ hMNC/ml.
2. Transfer the calculated volume of culture medium (e.g. Mononuclear Cell Medium, C‑28030) into a suitable culture vessel and equilibrate for 30 minutes at 37°C and 5% CO₂.
3. Remove the cryovial from liquid nitrogen storage and immediately place it on dry ice, even during short transport.
4. Thaw the vial in a 37°C water bath for 2 minutes.
5. Disinfect the outside of the vial with 70% ethanol and open it in a laminar flow cabinet.
6. Using a 2 ml serological pipette, transfer the cell suspension into the pre-warmed medium without resuspending the cells.
7. Return the culture vessel to the incubator and culture the cells at 37°C and 5% CO₂.

Important notes

• Do not resuspend the cells during the thawing process, as this may promote cell clumping.
• For optimal recovery, leave the cells undisturbed for at least 18 hours after seeding.
• Perform the first medium change 18-24 hours after thawing.

For detailed instructions, please refer to the corresponding Instruction Manual.

Yes. In many cases, PromoCell can produce cell pellets from specific cell lots upon request.
 
Please note:

• Custom pellet production requires additional cell culture work and is therefore subject to an additional production fee on top of the standard pellet price.
• The typical lead time is approximately 3-4 weeks.
• If suitable pellets from the requested cell lot are already available in stock, no production fee will be charged.

Because availability and feasibility depend on the requested cell lot and cell type, we recommend contacting our Scientific Support team to discuss your requirements and obtain a quotation.

The suitability for re-freezing depends on the cell type.

• Human Mesenchymal Stem Cells (hMSC) and human placental pericytes (hPC‑PL) can be re-frozen. However, each additional freeze-thaw cycle may reduce cell recovery, viability, and proliferation capacity. As a result, the number of population doublings (PDs) specified in the Certificate of Analysis (CoA) can no longer be guaranteed. If re-freezing is required, we recommend doing so at a low passage number using Cryo‑SFM Plus (C‑29920), a defined, animal component-free, and protein-free cryopreservation medium.
• Human Mononuclear Cells (hMNC) and CD14⁺ monocytes can technically be re-frozen, but substantial cell damage and reduced recovery should be expected. Since these cells do not proliferate, re-freezing is generally not recommended.
• M1 and M2 macrophages should not be re-frozen.

After arrival, the NCCD reagent may be aliquoted and stored at -20°C for long-term storage.
For short-term use, the unopened vial may be stored at 2-8°C for up to 3 months.
Once diluted, the NCCD working solution can be stored at 2-8°C for up to 4 weeks, protected from light.
 
Important notes
The NCCD reagent is supplied as a 20× stock solution and must be diluted with PBS before use.
One 2 ml vial yields 40 ml of ready-to-use solution, sufficient to treat approximately 400 cm² of culture surface (equivalent to about seven 6-well plates).
 
Application
Use 100 µl of diluted NCCD solution per cm² of culture surface.
Incubate the closed culture vessel for at least 1 hour at room temperature

PromoCell has successfully cryopreserved intact tumorspheres using Cryo‑SFM Plus. Since neurospheres and tumorspheres are both three-dimensional cellular structures, Cryo‑SFM Plus is expected to be suitable for neurosphere cryopreservation as well.
 
Please note: Neurospheres have not been formally tested with Cryo‑SFM Plus. Therefore, we recommend validating the freezing and thawing conditions for your specific application.
For optimal results, neurospheres should be:

• Healthy and viable prior to freezing
• Preferably 100-200 µm in diameter
• No larger than approximately 200-300 µm in diameter

This size range helps ensure adequate cryoprotectant penetration and efficient post-thaw recovery.

HLA typing can be performed using various DNA-based methods, including sequence-specific primers (SSP), sequence-specific oligonucleotides (SSO), and next-generation sequencing (NGS).
At PromoCell, HLA typing is performed using intermediate-resolution (4-digit) PCR-RSSO analysis. In this method, the relevant HLA loci are amplified by PCR, and the resulting products are analyzed on the Luminex® platform using sequence-specific oligonucleotide probes.
This approach provides reliable HLA typing data for applications that require donor HLA information.
 
Further information: Additional details about PromoCell's HLA-typed primary cells are available on our HLA-Typed Human Primary Cells webpage.

Yes. In addition to the standard Endothelial Cell Growth Medium (C‑22010) and Endothelial Cell Growth Medium 2 (C‑22011), PromoCell HUVECs (single donor, pooled, and pre-screened) can also be cultured in:

• Endothelial Cell Growth Medium MV (C‑22020)
• Endothelial Cell Growth Medium MV2 (C‑22022)

While Endothelial Cell Growth Medium (C‑22010) remains the recommended standard medium for HUVEC culture, all four media support HUVEC growth. In general, low‑serum culture conditions are preferred for endothelial cells. Endothelial Cell Growth Medium (C‑22010) and Endothelial Cell Growth Medium 2 (C‑22011) therefore contain 2% FBS, whereas Endothelial Cell Growth Medium MV (C‑22020) and MV2 (C‑22022) contain 5% FBS. The choice of medium depends on the specific requirements of the application and the desired growth factor composition.
 
Please note: HUVEC 2 cell types (single donor HUVEC 2, C‑12206, and pooled HUVEC 2, C‑12208) are isolated directly in Endothelial Cell Growth Medium 2 and are therefore best cultured in Endothelial Cell Growth Medium 2 (C‑22011).

Yes. The isolation of human cancer cells from patient-derived xenograft (PDX) mouse models is a key application of the Primary Cancer Culture System (PCCS).
PCCS supports the selective recovery and expansion of malignant human cancer cells while minimizing the growth of non-malignant mouse cells, including mouse fibroblasts. This facilitates the establishment of human cancer cell cultures from PDX tissue and helps prevent overgrowth by murine stromal cells.

Yes. The Primary Cancer Culture System (PCCS, C‑28081) has been designed to support the growth of tumor cells and can help minimize fibroblast overgrowth in patient-derived organoid (PDO) cultures.
Please note: NCCD is not required for this application.

Freshly isolated monocytes readily attach when cultured in Monocyte Attachment Medium (C‑28051), whereas cryopreserved CD14⁺ monocytes (C‑12909) largely lose this attachment capability after thawing.
Monocyte attachment is important not only for establishing an adherent culture but also as a key step in the differentiation process from monocytes to macrophages.
As a result, cryopreserved CD14⁺ monocytes generally show reduced macrophage differentiation efficiency, leading to lower yields and less complete differentiation compared to freshly isolated monocytes.
For optimal macrophage generation, we therefore recommend using freshly isolated PBMC or monocytes in combination with Monocyte Attachment Medium (C‑28051).