BD BioCoat™ HTS Caco-2 Assay System

• I tried using the BD BioCoat™ HTS Caco-2 Assay System and got no differentiation. What did I do wrong? What should I look for?
• I tried using the BD BioCoat™ HTS Caco-2 Assay System and got no cell growth. What did I do wrong? What should I look for?
• I tried using the BD BioCoat™ HTS Caco-2 Assay System and found the monolayers too leaky to use. Where did I go wrong? What should I look for?
• I can't recover enough of my prospective drug compound to measure using the BD BioCoat™ HTS Caco-2 Assay System. What should I do?
• I am using ¹⁴C-labeled mannitol and inulin as permeability controls and get erratic readings. What should I look out for?
• I am using mannitol to measure barrier formation but can't detect it using our LCMS equipment. What's wrong?
• I've tried the system, but I really need to generate barrier function that is equivalent to the 21-day Caco-2 system.Is there any way to achieve this with the 3-day BD BioCoat™ HTS Caco-2 Assay System?
• I've tried the system, but I really need greater P-glycoprotein or active transporter activity. Are there modifications I can run to improve expression of these activities?
• During transport experiments, what drug concentrations should I use?
• I am having trouble with media contamination using the 3-day system. Is there anything to look out for?
• How can I reduce background binding of my target drug to the plastic components of the BD BioCoat™ HTS Caco-2 Assay System?

I tried using the BD BioCoat™ HTS Caco-2 Assay System and got no differentiation. What did I do wrong? What should I look for?

Did You make any of the following common mistakes when using the system?

1. Did you use the right medium for each step of the process? (Basal Seeding Medium, followed by Entero-STIM™ Medium)
2. Did you use the media past its stated shelf life? (3 months from date of shipment)
3. Did you seed the insert with enough cells? (For this technique, the seeding density is higher than normal- 200,000 cells/insert, so seeding brings the cell level close to confluence.)
4. Did you compromise your monolayer integrity with mechanical shearing of the cells? For best results, remove media with gentle pipetting, or by inverting the insert and letting the media drain into a waste vessel.
5. Did you keep your cells in Entero-STIM Medium more than the recommended time? (Barrier formation deteriorates 2.5 days after differentiation, unless fresh media is added.)

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I tried using the BD BioCoat™ HTS Caco-2 Assay System and got no cell growth. What did I do wrong? What should I look for?

Two things to check:

1. Did you remember to supplement the media with MITO+™ Serum Extender?
2. Did you check your cell stocks to make sure they're OK prior to use? (The figure inset shows what Caco-2 cells look like when they are grown to the recommended cell density of ~200,000 cells/cm².)

   

3. Are you aware it is difficult to microscopically examine Caco-2 cells on inserts? (You may want to fix and stain cells. See FALCON Technical Bulletins 405 and 406 for more information. To request copies, please call our Technical Service Department at (800) 343-2035, or e-mail us at: labware@bd.com.

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I tried using the BD BioCoat™ HTS Caco-2 Assay System and found the monolayers too leaky to use. Where did I go wrong? What should I look for?

Here are some things to look out for:

1. Did you culture the cells in Entero-STIM™ Medium longer than 2 days without a media change? For best results, do not allow cells to sit in Entero-STIM for more than 2 days without a media change.
2. Did you seed the inserts at the right cell density? (For this technique, the seeding density is higher than normal- ~200,000 cells/insert, so seeding brings the cell level close to confluence- see photo in A2.)
3. Did you over- or under-trypsinize the cells? (Cells should be in almost single cell suspensions to get good monolayer formation.)
4. If monolayer quality was measured via TEER, verify using the mannitol or other permeability test. (TEER is very sensitive to microscopic imperfections that have no effect on compound permeability. We recommend using the mannitol test if possible.)
5. Do you remember that permeability measurements with the BioCoat ™ Caco-2 Assay System will be slightly higher than measurements made with a 21-day Caco-2 assay? Nonetheless, the rank order of compound permeabilities is routinely preserved.
6. Do you have too much DMSO in your final transport buffer after addition of your prospective drug sample? For best results, keep final DMSO concentration to less than 0.5% during transport. Excess DMSO will permeabilize the Caco-2 cells. (Typical concentration range: 0.1-0.5% DMSO.)
7. Are you doing media changes using suction aspiration of the insert? Too much suction can dislodge both cells and the fibrillar collagen coating from the insert, increasing leakiness. We recommend gentle, slow pipetting of excess media from the inserts during changes. Media can also be removed by inverting the insert and emptying the waste into a suitable vessel.
8. Are you filling the feeder tray/24-well plate first, and filling the inserts last? This creates hydrostatic pressure, which could dislodge the fibrillar collagen coating and/or cell monolayer and increase leakiness. For best results, always fill the inserts first, then fill the bottom chamber(s).

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I can't recover enough of my prospective drug compound to measure using the BD BioCoat™ HTS Caco-2 Assay System. What should I do?

Here are some suggestions:

1. Are you using too short an incubation time? Typically we recommend incubation times of 30 minutes to 2 hours. The optimal time should be determined by each user.
2. You may need more cell monolayer surface area to accurately measure your compound. In this case we recommend using the BD BioCoat™ Fibrillar Collagen 1.0µm Cell Culture Insert designed for 6-well plates. For more information, contact our Technical Service Department at labware@bd.com.
3. Make sure the plastic components of the system are not absorbing your target compound via nonspecific binding.

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I am using ¹⁴C-labeled mannitol and inulin as permeability controls and get erratic readings. What should I look out for?

Since these compounds have low permeability, be suspicious of any readings below 500dpm (~450cpm). Counting background and trace radioactive contaminants in these products (most have stated purity specifications of >95% radiochemical purity) can easily account for 200-300dpm per experiment. Breakdown products of inulin (e.g., [¹⁴C]glucose) may be transported across the monolayer and account for additional background. When using radioactivity, it is also important to use a high quality, high efficiency Liquid Scintillation Cocktail. (For most experiments, we recommend Beckman's Ready Safe LSC Cocktail.)

For best results, we recommend using at least 1.5x10⁶dpm/ml.

As an alternative, the fluorophore lucifer yellow may also be used as a permeability marker at working concentrations of 10^-5M. Lucifer yellow is available from Molecular Probes (Eugene, Oregon, http://www.probes.com/).

For additional information on excitation and emission wavelength spectra for lucifer yellow, refer to Molecular Probes' Product Information Sheet (http://www.probes.com/pis/mp00453.pdf).

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I am using mannitol to measure barrier formation but can't detect it using our LCMS equipment. What's wrong?

LCMS detection requires analytes capable of generating molecular ions for efficient detection. Mannitol is not charged and is not detected efficiently using LCMS. We recommend either switching to lucifer yellow for a negative (no transport) control, or use an alternate, LCMS-compatible control. Although lucifer yellow may be used with LC columns, we recommend running experiments with prospective drugs first to simplify detection in LCMS. Afterward, lucifer yellow may be added to the insert to verify that the barrier is still intact using a fluorescence plate reader. This modification will extend LC column life.

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I've tried the system, but I really need to generate barrier function that is equivalent to the 21-day Caco-2 system. Is there any way to achieve this with the 3-day BD BioCoat™ HTS Caco-2 Assay System?

If desired, 10% fetal bovine serum can be added to the Basal Seeding Media (in addition to the MITO+™ Serum Extender) during the initial 1 day growth phase. The slight addition of serum assists cells to divide and cover the insert more efficiently. Follow the manual for all subsequent steps (do not add serum during the differentiation step). This modification will provide barriers that are nearly equivalent to the 21-day system.

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I've tried the system, but I really need greater P-glycoprotein or active transporter activity. Are there modifications I can run to improve expression of these activities?

Several customers have had success extending the system to 5 days, with useful increases in P-glycoprotein and active transporter activities. An outline of 2 alternate customer modifications are shown below:

Modification I

• Day 1, seed cells as described in the manual.
• Day 2, switch to supplemented Entero-STIM differentiation media as described in the manual.
• Day 3, replenish with fresh supplemented Entero-STIM differentiation media.
• Day 4, replenish with fresh supplemented Entero-STIM differentiation media.
• Day 5, system is ready for transport experiments.

Modification II

• Seed cells as described.
• Change to supplemented Entero-STIM differentiation media after 2.5 days.
• Wait an additional 2.5 days- system is now ready for transport experiments.

Modification II is particularly suited for setting up experiments over a weekend.

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During transport experiments, what drug concentrations should I use?

For starters, we recommend using drug concentrations at 100-200µM. Effective compound concentrations can vary due to inherent compound permeabilities and potential interactions with transporter system. These optimal compound concentrations may have to be determined experimentally.

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I am having trouble with media contamination using the 3-day system. Is there anything to look out for?

Reduce media volume in the feeder tray to 25-30 ml. In many cases, this fix has eliminated contamination problems.

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How can I reduce background binding of my target drug to the plastic components of the BD BioCoat™ HTS Caco-2 Assay System?

At present, we know of no way to do this while maintaining the integrity of the Caco-2 System. However, pretreatment of the system with MITO+™ Serum Extender may reduce or eliminate nonspecific protein binding, thereby improving the recovery of hydrophobic, proteinaceous drug prospects.

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REFERENCES

¹Chong, S. et al., Pharm. Res., Vol. 14, No. 12, 1835-1837 (1997)

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