Are there any differences in the cultivation protocol when cultivating MSCs compared to other cell types?

Yes, there are a few differences: We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding, as opposed to 16-24 hours after seeding for most other cell types/growth media, including MSC Growth Medium 2. IF MSC Growth Medium XF, MSC Neurogenic Differentiation Medium, MSC Adipogenic Differentiation Medium 2, or MSC Osteogenic Differentiation Medium are used, the TC vessels must be pre-coated according to the instruction manual. We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used, the contact time should not exceed 2 min.

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According to the product manual, Cryo-SFM should be stored at 4-8°C. However, since this solution is used to freeze cells in liquid nitrogen, we assume that storing Cryo-SFM once at -20°C should not have a negative impact on the product quality. After thawing, please store it at 4-8°C as recommended.

The supplement should be at room temperature when added to the MSC Adipogenic Basal Medium 2. It may also be beneficial to invert the tube a few times to bring precipitates back into solution. Please note: It is not recommended to filter the basal medium, supplements, or complete medium, as components that induce or promote differentiation may be removed, resulting in a low differentiation rate when using the medium.

Yes, there are a few differences:

We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding, as opposed to 16-24 hours after seeding for most other cell types/growth media, including MSC Growth Medium 2.
IF MSC Growth Medium XF, MSC Neurogenic Differentiation Medium, MSC Adipogenic Differentiation Medium 2, or MSC Osteogenic Differentiation Medium are used, the TC vessels must be pre-coated according to the instruction manual.
We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used, the contact time should not exceed 2 min.

Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media. Optionally, the precipitate can be removed by centrifugation under sterile conditions. We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.

Our HPAEC and HPASMC are isolated from the proximal pulmonary artery.

The basis for the intended use of our products is defined in our Terms & Conditions under the chapter “Use of Goods”.

The activation of macrophages as such is complete after 24 hrs. However, to maintain the activation status over a longer period of time (i.e., several days), fresh activation factors should be added with every medium change.

Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.

The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011), the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both, NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.

Yes, the NHEK-GM2 (C-12001, C-12003, C-12005, C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals, they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs. Conversely, NHEK-GM3 (C-12011, C-12013, C-12015, C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% - 90%), they grow slightly slower compared to Keratinocyte GM3, but also reach > 15 PDs.

The MSCs attach even without fibronectin coating, but the risk of detachment during differentiation is much higher. We therefore strongly recommend using fibronectin- or vitronectin-coated TC vessels.

PBMCs (= peripheral blood mononuclear cells) consist mainly of lymphocytes and monocytes. Cryopreservation causes the CD14+ monocytes to significantly lose their ability to attach to TC plastic. Therefore, the use of frozen PBMCs as starting material for DC generation is not possible because the purification step with Monocyte Attachment Medium will not work. You can either start with cryopreserved CD14+ monocytes (C-12909) or use freshly isolated mononuclear cells or fresh CD14+ monocytes. Please refer to the Product Manual of C-28050 or our Application Note [Generation of monocyte-derived Dendritic Cells] for the appropriate protocol.