Technical library - RESOURCES

The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time; increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop. Mycoplasma infections can also occur more easily; as they are often introduced along with contaminants such as bacteria and fungi. Last but not least; antibiotics are known to have negative effects on the metabolism of eukaryotic cells - more details on this topic can be found in our blog article "Antibiotics in cell culture: friend or enemy".

The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense. Leave the PBS on the cells after staining/washing and analyze the sample immediately; as the dye may bleed upon prolonged storage without embedding.

PromoCell's Normal Human Epidermal Keratinocytes (NHEK) from juvenile donors are isolated from both the epidermis of the outer and mucosal (i.e. inner) layers of the foreskin. Thus; each vial contains a mixture of mucosal and cutaneous keratinocytes.

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).

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.

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

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.

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.

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.

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.

To avoid the spreading of the matrix and to have nice drop-like domes; it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help. From our testing; we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered; but it was able to move on the bottom and the dome became misshaped overtime. However; the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user; we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.

Based on negative feedback we have received from customers using bead baths; we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells. If you don’t have a “normal” water bath but only a bead bath in your lab; thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand; and not in a floater; as described in the thawing protocol.