What is the difference between DC Generation Medium XF (C-28052) and DC Base Medium XF (C-28054)?

DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs. DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines. Both, Base Medium XF and Generation Medium XF have a xeno-free formulation.

Why use ROCKi for organoid formation?

ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic. This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids: https://pubmed.ncbi.nlm.nih.gov/31871466/ https://pubmed.ncbi.nlm.nih.gov/35169685/ For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.

Can you control size / size distribution of organoids?

Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/ For best results we recommend the following: Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism. For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.

Technical library - RESOURCES

Q: In the past, we used MSC Growth Medium (C-28010) in our lab. Can I use MSC Growth Medium 2 (C-28009) instead? What is the difference?

MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation). You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow, adipose tissue, or umbilical cord) at 4,000 cells/cm².

Q: How do you set up high throughput systems with organoids? Only in 96 well?

It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells ( e.g ., Greiner Bio-One # 650185). A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.

Q: Can you drive organoid orientation (inward vs outward)?

Yes. The orientation can be triggered by the use or lack thereof of ECM. Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/ , where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM. * Please note that we have not tested this method in our labs and thus cannot guarantee it.

Q: For which cytokeratin are the keratinocytes tested?

PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.

The cells will die. Hematopoietic Progenitor Cell Expansion Medium XF always must be supplemented with Cytokine Mix E or an appropriate cocktail of cytokines.

DC Base Medium XF (C-28054) consists of 250 ml Basal Medium + SupplementMix. It does not include cytokines and must therefore be supplemented according to the user's individual needs.

DC Generation Medium XF (C-28052) is ready-to-use and consists of 250 ml Basal Medium + SupplementMix + appropriate cytokines.

Both, Base Medium XF and Generation Medium XF have a xeno-free formulation.

Our DC Generation Medium XF (C-28052) has a xeno-free formulation. It provides a complete media system (ready-to-use, all cytokines included) and shows efficient and reproducible in vitro maturation of moDCs from freshly isolated peripheral blood monocytes.

Yes, our Lymphocyte Separation Medium 1077 is endotoxin-tested. The specification for the product is < 10EU/ml.

MSC Growth Medium 2 (C-28009) is an optimized medium formulation with reduced serum content to allow for more standardized culture conditions (considerably lower lot to lot variation). You can replace MSC Growth Medium by MSC Growth Medium 2. Coating of culture vessels is not necessary. We recommend to plate the cells (hMSCs from bone marrow, adipose tissue, or umbilical cord) at 4,000 cells/cm².

It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g., Greiner Bio-One # 650185). A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.

ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic. This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:

For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.

Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/ For best results we recommend the following:

Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.

Yes. The orientation can be triggered by the use or lack thereof of ECM. Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/, where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM. * Please note that we have not tested this method in our labs and thus cannot guarantee it.

PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.

We guarantee > 500,000 viable cells per vial. To do this, we need to freeze more than 500,000 cells, as we do not yet know the viability after thawing at the time of freezing. For technical/organizational reasons, the initial cell number may also vary from lot to lot, so 2 lots with the same viability may not necessarily contain the same number of cells.

Yes, there is a reference (Campuzano et al.; J Immunol. 2020 Jun 15;204(12):3296) where bone marrow-derived macrophages from mouse were detached using our Macrophage Detachment Solution (40 minutes at 4°C).

Further Information

Campuzano et al.; J Immunol . 2020 Jun 15;204(12):3296