In theory till infinity. We recommend to use the cells up to passage 60 and create your own stock in liquid N2. Until passage 60, we have proven that specific assays are functional and drug transporter expression is intact. Further, it is recommended that the end-user will also test its own assays both at low passage (i.e. x+3) and high passage (x+15). On top, the end-user can culture till higher than passage 60, and validate his/her own assays.
(x = passage number of ciPTEC when obtained by Cell4Pharma)
Yes, we have experience with Lenti-viral and retro-viral transfections. Also CRISPR/cas has been successfully demonstrated in ciPTEC. Transient expressions have been proven successful but with low efficiency (<10%).
Our GMO documentation on Drive: PERT negative, mycoplasm negative. Other test not performed by us. This should be shared with all customers that culture ciPTEC themselves!
ciPTEC is difficult to form tight monolayers. We have some data, but not very robust. The only success was gained when ciPTEC was cultured on hollow microfibres (see Jansen 2016). On Transwells, only a few times succeeded in our efforts. Feel free to optimize this!
The Vriend et al paper demonstrates functional transport (Pgp and MRP) of ciPTEC-OAT1 in the Organoplate. We know the RNA expression of the other transporters is intact (BCRP, OAT, OCT, etc). On the other side, the monolayers in the system are not as tight as you could expect with a MDCK or Caco cell line. The proximal tubular epithelium is just not that tight (also not in vivo!) and rather leaky. So, the platform might be unsuitable using the published conditions to analyse transepithelial transport. In our study, we used permeable substrates exposed both basolateral and apically, that are actively effluxed via Pgp/MRP only at the apical side.
In the paper by Vormann et al, you can see data using the Sigma-RPTEC line. This human renal PT cell does form a more tighter barrier, for which leakiness could be analysed using dextran-FITC upon toxicant exposures. However, OAT expression in this line is very poor and in our view useless to study organic anion compounds.
We do feel that the system with our ciPTEC could be further optimized in order to get tighter barriers. The leakiness assays are functional for ciPTEC as well, but so far much more moderate. Maybe using specific factors yet unexplored could improve the barrier
Yes, we ship the cells worldwide to any country on dry ice. Via FEDEX, DHL, or UPS.
Cells will be delivered on dry ice, frozen vial in culture medium. Upon delivery, the cells can be stored in liquid nitrogen, similar to commonly cultured cells. After thawing and culturing, we strongly recommend to split the cells and assure to create your own frozen batch in liquid nitrogen.
Culturing protocol and GMO information for your convenience. The culturing protocol describes all media supplements. Please pay attention to the fact you would need a 33C incubator. Also note the cells are immortalized, for which your local authorities might require specific permits. Other than that, common cell culture materials will be required.
We have not measured this volume. According to the protocol, cells need to be seeded at 33C, cultured for 1 day, and transferred to 37C for 7 days before performing assays. Why 1 day at 33C?
This protocol is a result of optimizing the monolayer required to mimic the in vivo situation. One day at 33 allows further division of the cells and when split 1:3, the cells will form a monolayer after 7 days, which is also convenient in a working week. Of course, the end-user is free to optimize and customize the protocol further for his/her purposes or convenience, but functionality and characteristics can differ.
Yes they are! Our GMO documentation on Drive: PERT negative, mycoplasm negative.
You can expect 5 x 10^6 ciPTEC cells in a confluent (100%) T75 culture flask
No. We are currently developing this cell-line. For co-development partnerships, please contact our service desk.
Ask our service desk or check out our list of assays in the resources tab. We do have SOP (standardized operating protocols) for many assays (culturing, MTT, transporter interactions)
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