Technical library

After adding the Skeletal Muscle Cell Differentiation Medium; myoblasts will start to differentiate into myotubes and stop growing. The cells should not be split anymore. It is recommended rather to plate the SkMC into the needed vessels; (e.g. multiwell plates); prior to induction of differentiation and to perform studies directly on differentiated cells. If it is necessary for your tests to detach the myotubes and they are difficult to trypsinze; you can use a "rubber policeman".

Our SkMC are proliferating myoblasts that have retained the capacity to differentiate. Upon withdrawal of serum and growth factors differentiation is induced and the cells form multinucleated syncytia.

The PromoCell Basal Media must be stored between 4-8°C and should not be frozen; as this can lead to precipitations. The same is true after addition of the supplements: the complete medium has to be kept at 4-8°C. If you prefer to make up smaller volumes of complete medium; you can aliquot the Supplement Mix and refreeze those aliquots at -20°C until use. This way you can extend the period in which you can use the supplemented media.

We have obtained best results when the cells have reached 60-80 % confluency. At this point the Growth Medium is aspirated and replaced by Skeletal Muscle Differentiation Medium. After 2 - 8 days extensive formation of multinucleated syncytia can be observed. For a stable differentiation of SkMC switch back to Skeletal Muscle Cell Growth Medium after 5 days incubation in Skeletal Muscle Differentiation Medium.

To differentiate the SkMC into myotubes; we recommend to use cells that have undergone a maximum of 4-5 population doublings. For this; the cells (ideally in P2 or P3) are cultured to 60-80% confluency in Skeletal Muscle Cell Growth Medium (C-23060). Then; a change to (serum-free) Differentiation Medium (C-23061) is performed to induce the differentiation process (formation of multinucleated syncytia). After 5 days; switch back to the Skeletal Muscle Cell Growth Medium for another 8 days to complete the differentiation. This protocol leads to stable myotubes and some of the myotubes show spontaneous contractions.

Recommended procedure: - Thaw the vial according to our protocol - Seed one part of the cells (5;000 cells/cm²) directly into multiwell plates for subsequent differentiation induction; grow in Preadipocyte Growth Medium until they reach 100% confluency; then induce differentiation according to the recommended protocol - Seed the other part (5;000 cells/cm²) into a TC dish and expand them in Preadipocyte Growth Medium; trypsinize at subconfluence; if necessary divide the cell suspension again into two parts and use one part for differentiation tests (see above); the other part to continue the culture of undifferentiated HWP. - Differentiation capacity may decline after 1-2 passages in vitro; therefore best perform your differentiation tests at early passages. HWP at higher passages can still be used for studies that require undifferentiated HWP (e.g. proliferation assays).

The basic information we receive from the surgeons about the tissue donors usually includes age; gender; and ethnicity.

• For many of our donors of subcutaneous preadipocytes; we also have information on BMI; hair color; skin pigmentation; and; in some cases; smoking habits or known diseases (e.g. Diabetes). Most of our subcutaneous HWP donors are between ∼25-65 years old.
• The visceral HWP donors are mostly between ∼20-75 years old. For many cell lots we know the BMI; in some cases also the hair color; skin pigmentation; smoking habits; and/or known diseases (e.g. Diabetes or COPD).

If you are looking for particular specifications; please contact our Technical Customer Support; so that we can offer you appropriate HWP lots.

We recommend a seeding density for chondrocytes between 10.000 and 20.000 cells/cm². This means that a subconfluent T25-flask with approx. 900.000 cells/T25 flask (36.000 cells/cm² ) may be either split into 3 new T25 or seeded in one T75 flask or in one 100 mm petri dish. We do not recommend a specific type or brand for the culture of HCH.

Our HPAEC (C-12241) are harvested directly from the pulmonary artery. For their isolation; the vessel is explanted right after the position where the artery leaves the heart; including the bifurcation. HPAEC represent the innermost cell layer (i.e. the endothelial cells) of the pulmonary artery. We also supply pulmonary microvascular endothelial cells (HPMEC; C-12281) isolated from the capillaries of peripheral lung tissue.

The optimal calcium concentration for both proliferation and differentiation of keratinocytes depends on the species and also on the media formulation. To keep primary human keratinocytes in the proliferative status concentrations between 0.03 and 0.15 mM (PromoCell Keratinocyte Growth Medium 2: 0.06 mM) are generally used. Increasing the calcium above 1 mM will induce terminal differentiation and lead to the loss of proliferative activity.

It usually takes 5 to 8 days to grow our Normal Human Chondrocytes (C-12710) to subconfluency. The number of doublings (PDs) they undergo can be calculated from the number of seeded cells and the cell yield at subconfluency. Generally; when HCH are plated with 10;000 cells/cm² they perform between 1.5 and 2 doublings per passage.

The Chondrocyte Growth Medium SupplementMix consists of Fetal Calf Serum (final concentration 10 % v/v) which has specifically been tested for the culture of primary chondrocytes. FCS contains a variety of different growth factors which are however not analyzed in more detail. There are no further growth factors added to the SupplementMix.