At what passage are PromoCell Normal Human Cells upon arrival?

The passage number varies depending on the cell type. Please refer to the Manual for your cells under "Specifications". You will also find the information in the Certificate of Analysis (CoA). Further Information Download Certificates of Analysis

In which passage do you test the cell type-specific markers?

Cell type-specific markers are usually determined one passage after thawing. Example: After thawing, HUVEC are in P1. Markers (CD31 expression, Dil-Ac-LDL uptake) are tested in P2. Please note: Cells that are frozen directly after isolation (blood cells) are tested directly after thawing with no further culturing step.

What is the expected differentiation rate when using PromoCell Mesenchymal Stem Cell Osteogenic Differentiation Medium with hMSC-BM?

The differentiation rate into the osteogenic lineage is 70-100%.

Does PromoCell offer a special 3-D system to block de-differentiation or does PromoCell have special culture conditions that stabilize the chondrocyte phenotype?

PromoCell Chondrocytes (HCH) can be expanded in normal monolayer culture using our Chondrocyte Growth Medium (C-27101). The Medium consists of an optimized formulation and is supplemented with 10 % FCS. De-differentiation of chondrocytes is a known phenomenon observed during in vitro -culture after a period of approx. 2 weeks, but in vitro- culture is needed to expand the cells. Once a suitable cell number is obtained, the monolayer system can be changed to a more complex 3-D system either by culturing the cells on substrates like alginate beads, gels, or degradable polymer scaffolds or by using 3-D spheroid culture. Using appropriate conditions, re-differentiation is triggered and the cells start producing cartilage-specific ECM again. PromoCell does not supply a special culture system but we use 3-D spheroid culture and Alcian Blue staining to characterize our chondrocytes during quality control. The protocol is very similar to the one we use for chondrogenic differentiation of MSC.

At what passage do PromoCell supply the cell pellets?

The majority of PromoCell cell pellets (C-14**) are prepared 1 passage after thawing the cryopreserved cells. Examples: HUVEC and HUAEC correspond to P1 after thawing; therefore the pellets are frozen in P2. SMC or keratinocytes or epithelial cells correspond to P2 after thawing; therefore the pellets are frozen in P3. In contrast, our blood cells are cryopreserved directly after cell isolation. These pellets are prepared after thawing the cryovials with no further cultivation step.

How long does it take to detach primary cells with accutase (C-41310)?

Most primary cells detach within 5-10 min at 37°C. Inactivation isn't required but we recommend to centrifuge the cell suspension to remove accutase and EDTA before replating the cells.

What's the difference between visceral and subcutaneous HWP? When should I use which cell type?

PromoCell Preadipocytes are isolated from subcutaneous or visceral fat. Subcutaneous fat is found just underneath the skin and it is not related to the obesity-linked diseases. Its accumulation represents the normal physiological buffer for excess energy intake. When the storage capacity of subcutaneous fat is exceeded or the generation of new adipocytes is impaired, visceral fat starts to accumulate. It is located in the abdomen and around internal organs (e.g. kidney, heart, or bladder) and it is linked to hypertension, diabetes, and cardiovascular disease. Adipocytes from subcutaneous and visceral fat differ in several functions, like their response to insulin and other hormones or their lipolytic activity. It highly depends on the scientific problem being addressed, which of the two cell types are best suited for your experiments.

Is it feasible to store the melanocytes for a second time in liquid nitrogen after subculture, similar to cell lines?

Upon arrival, you can either defrost the cells immediately and seed them in a TC vessel or store the vial in liquid nitrogen until needed. It is possible to re-freeze normal human cells but PromoCell does not recommend it, because each freezing cycle leads to a loss in proliferation potential. If you want to freeze them, please use our Cryo-SFM Plus (C-29920). It is also recommended to freeze the cells at an early passage.

Could you please let me know whether you have used the baculovirus expression system to produce components of your media?

Baculovirus is generally used in conjunction with insect cells (Sf-9, Sf-21) to produce recombinant proteins (cytokines, growth factors). None of our Specialized Media (Media for Primary Human Cells; Blood and Stem Cell Media, Cancer Cell Media) contain recombinant proteins produced in insect cells. This also applies to our Cryo-SFM Plus Freezing Medium.

How do I best store PromoCell cell pellets? What is their shelf life?

PromoCell cell pellets (C-14**) can be stored indefinitely at -20°C and are stable for up to 1 month at 4°C and up to one week at room temperature. Please note: The samples do not freeze at -20°C.

At what passage are PromoCell Mesenchymal Stem Cells / Pericytes upon arrival?

Our Human Mesenchymal Stem Cells and Human Pericytes are cryopreserved at the end of secondary culture (P1). After thawing, they are in P2.

Technical library - RESOURCES

At PromoCell we guarantee for our primary human cells ≥ 500,000 viable cells after thawing. For this, we dispense > 500,000 cells per cryovial before cryopreservation as there will always be a certain percentage of dead cells after freeze/thaw. In order to know the number of cells that survived the procedure, we defrost a representative number of vials per lot during QC, determine the cell viability using an electronic counting device and then calculate the number of viable cells that can be recovered after thawing. Both numbers - the calculated number of viable cells and the viability - can be found on the lot-specific Certificate of Analysis (CoA) that can be downloaded from our website. Example: When the CoA indicates 600,000 viable cells and a viability of 80%, this means that the vial actually contains 750,000 cells (viable + dead), 80% thereof (600,000) were viable after thawing in our QC. We do not indicate the total number of cells per vial but just the number of expected viable cells which can be recovered when the recommended thawing protocol is used. You don't have to calculate any viabilities by yourself. When the recommended plating density for your cell type is 5,000 - 10,000 cells/cm², then the 600,000 viable cells can be plated e.g. in a T75 (corresponding to 8,000 cells/cm²) or in a T75 + a T25 (corresponding to 6,000 cells/cm²).