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General questions
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 Which plates do you use for your CacoReady plates?
Standard 24-well CacoReady™ plates are COSTAR plates.
Standard 96-well CacoReady™ plates are from Millipore (BD plates have also been validated)
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Are the plates pre-coated with collagen?
Only the 24-well format, not the 96-well format.
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Do you provide CacoReady plates in 6-well formats?
Not routinely, but we could produce them upon request. However, a test should be performed in house first to confirm the behaviour of Caco-2 cell barrier. We do not recommend this format, as handling is much more time consuming (no automation possible, no tools for easy handling), and also reagents consuming (very high amount of compounds required due to bigger volumes of compartments). Finally, the “chopstick” electrode system to measure TEER is not accurate enough for this size of Transwell because the surface is too large.
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Can we have easy access to the compartment under the cells?
CacoReady™ plates are integrated Transwell plates specifically designed for transport studies. Therefore, it is possible to get samples from basal compartment, either directly in 24-well format, or after removing upper insert compartment in 96-well format.
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Are cells cultured in individual wells or is it possible to replace the medium in all wells at once?
Cells are grown on top of individual inserts (although integrated, ie you can remove all inserts at once as they are fused in one piece). During CacoReady™ production, “reservoir” basal plates are used (on top of which sit the inserts), in order to change basal medium in one go.
However, end-users receive the plates with independent although integrated wells and Transwells, as required for absorption assay (“Ready-to-use” concept). They can certainly use a manifold (aspiration system specifically designed for Transwell plates) and a multi-channel pipette to perform medium changes or washes, etc.., in all wells column by column. This way, handling the plates becomes very user friendly. Please refer to the user manual for more details.
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Logistics
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Don’t you run the risk of overflowing during shipping of the plates?
No. Our exclusive solid shipping medium has been designed to solve problems of overflowing and mechanical stress during Transwell plates transportation. We ship the cells embedded in this medium which remains solid at room temperature. It maintains the cells alive and differentiated. The only restriction is the shipping temperature: CacoReady™ plates have to be shipped and stored between 10 and 30ºC. Outside this range, biological properties and viability of Caco-2 cells can be irreversibly damaged. During summer days, we do include a cool pack inside the box, as well as heaters in winter time.
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Don’t you run the risk of damaging the cells with such shipping conditions and the use of this solid transport medium?
Looking at TEER values, paracellular (Lucifer yellow, inulin, sucrose, manitol) as well as transcellular (propanolol, caffein, testosterone) transport properties of Caco-2 cells that have been under shipping medium for 1-4 days and evaluated at day 21, we find no difference with the cells cultured without the shipping medium evaluated on same day. It looks like this shipping medium allows cells to maintain some kind of “resting” status, with just basal cell functions, in such a way that no change of medium is required, nor CO2, nor 37ºC incubation temperature. We start to observe irreversible damages (decreasing barrier status) of the cells when immobilized for more than 4 days in the shipping medium. We also observed that days 20-22 (first Monday-to-Wednesday following reception) were the best days for permeability assay (cells are fully recovered from shipment, and they are fully differentiated as well).
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When do I have to change the medium once I receive the plates?
Plates are shipped on Mondays from the production laboratory in Spain. If you receive the cells on a Friday, you need to change the medium the same day. The cells will be OK with this fresh medium until the following Monday. If you receive the plates before Friday, just unpack the plates as stated in the User Manual, and keep them at room temperature (not in the fridge, and not in an incubator), until Friday morning (week of reception). On Friday morning (week of reception), proceed with melting the shipping medium as stated in the User Manual.
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The melting process
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How long does it take before melting of the shipping medium is complete?
The melting is complete after 4 hours at 37ºC (in a CO2 incubator). Do not let the plates melt for a shorter period as shipping medium may damage the barrier if changed when not completely melted.
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I bought 4 plates. How should I proceed regarding melting shipping medium?
You can melt all plates at once (put them altogether in the incubator). However, once you start taking out the melted shipping medium from 1 plate (after 4hrs incubation), make sure you leave the other plates in the incubator while doing so.
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Cell production
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Which cell line do you use?
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What is the initial seeding density of the cells?
The one recommended by ECVAM Workshop Report 46.
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What are the characteristics of the medium?
DMEM, 1 g/l glucose, 10% FCS, 1% glutamine 200 mM, 1% Pen (10,000 U/ml)-Strep (10 mg/ml).
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What kind of certificate of analysis do you provide?
Each batch has to fulfil our quality controls, which are TEER values (>1000 Ohm x cm2) and Apparent Permeability of Lucifer Yellow (before plate is shipped). The values of quality controls obtained for each batch are indicated on the Technical sheet received by the end-user, together with the plate(s). Quality controls are further evaluated in-house at day 21 on control plates..
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Do you test the level of mycoplasma in your cultures?
Indeed, like for any cell production...
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Do you characterize the activity of the Pgp transporter?
Not for each batch. CacoReady™ system has been characterized for post-conditioning Pgp activity, using markers such as cyclosporin A, verapamil, rhodamin, vincristin, digoxin, and terfenadin. This was done once, during the validation phase of the product. Some of the Papp values obtained for these standard compounds are indicated in the technical sheet (see graph).
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