Technical library - RESOURCES

Adipogenic differentiation can be identified morphologically and without any staining by the formation of intracellular lipid vesicles.
In contrast, when MSC differentiate into bone cells, there is no significant change in morphology. It is recommended to perform Alkaline Phosphatase staining to detect osteoblastic differentiation or Alizarin Red S staining to show osteoblast mineralization.
Chondrogenic differentiation is generally performed as spheroids in 3-D cell culture and not in 2-D monolayer culture. Staining with Alcian Blue to visualize the differentiation process is indispensable.
Neurogenic differentiation can be detected using neuron specific markers (e.g. beta-3 tubulin, NeuN, MAP2) and by their typical neuronal morphology.

For differentiation protocols, please see attached Application Notes.

The human MSC derived from bone marrow, adipose tissue, and umbilical cord matrix are from different origins, but with comparable biological properties and function. Depending on the tissue of origin, they may have a higher preference for differentiation into one particular cell type and a lower preference for another one, but they all still retain the differentiation potential for the mesenchymal lineage.

MSC-BM: very good differentiation into bone cells, chondrocytes & fat cells
MSC-UC: very good differentiation into chondrocytes; but weaker potential into fat/bone cells
MSC-AT: very good differentiation into fat cells

These cells are frozen at the end of 2^nd culture. Thawing and seeding results in passage 2 (3^rd culture). We recommend that they be used for differentiation experiments not later than passage 5. The differentiation potential of hMSC in vitro is reduced with ongoing population doublings, meaning the earlier differentiation is induced, the higher the differentiation rates.

For optimal cell growth, the human CD34+ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO, SCF, flt3-ligand and IL-3. The strong expansion of CD34+ cells will persist for approx. 2 weeks.

The expansion factor for CD34+ cells is usually between 75 and 200-fold.

Mononuclear cells are mostly used in immunology, infection biology, hematology and cancer research to study subpopulations of blood cells. Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell. Please note: Depending on the conditions, the ratio of the subpopulations will gradually change, as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells, endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity, viability or metabolism).

The recommended plating density after thawing/subculture may vary depending on the cell type. It is specified on the Website (tab: additional Information) and in the Product Manual.

It actually depends on the culture conditions whether the cells remain in suspension or attach to the surface. When grown in our serum-free, xeno-free HPC Expansion Medium XF (C-28021), the cells remain in suspension.

The recommended plating density after thawing/subculture may vary depending on the cell type. Please refer to the data sheet for your cells under "Specifications".

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

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.

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

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.