Hello,
Greetings!
I am conducting a MDCK cell permeability assay.Below is the protocol I followed :
1. In the apical region, 100 ul cell suspension containing about 33,000 cells were seeded in each insert in 24 well transwell plate (6.5mm transwell insert diameter, 0.33 cm2 insert membrane growth area and 3um pore size).
2. 0.6 ml of media (EMEM with L-glutamine) was added in the basolateral region.
3. Media was replaced each day in both apical and basolateral region.
4. On 7th day, Lucifer dye assay was conducted. 3 wells were used for lucifer assay. Briefly, inserts (appeal region) and basolateral region were washed with prewarmed HBSS. 100ul of 100ug/ml of Lucifer dye solution in HBSS was added in the apical region. 600ul of HBSS was added in the basolateral region. Samples from basolateral region were collected after an hour and checked for the fluorescence.
Results showed no permeation of lucifer dye after an hour.
5. Now, for permeability assay, first, the inserts were washed with HBSS and then added 100 ul HBSS in apical and 600 ul in basolateral region and kept for 30 min in the incubator for equilibration.
6. 100 ul of nanoformulation and free drug control formulation (containing 100ug drug) was then added in each inserts and kept on the belly dancer (slow shaker to avoid the stagnant layer).
7. 200 ul Samples were then taken and replaced with fresh HBSS at 30 min, 1 hr, 2h, 3h, 4h and 24 hour.
8. All the samples were analyzed using LCMS.
9. Results showed no permeation at 30 min, 1 hr, 2h, 3h and 4h from both nano formulation and control formulation. However, it showed permeation at 24 hr with 2.23% from nano formulation and 1.22% from control.
10. Results also showed, about 3% drug was left in the apical region. Meaning, about 93% drug is stuck (or absorbed) in the cells.
Could anybody suggest if I am missing any step in the protocol?
Thank you so much.
Dear friend Arvind Bagde
Greetings to you too!
Ah, the puzzling world of MDCK cell permeability assay! Let me delve into your protocol and see if I can offer any insights.
From your detailed protocol, it appears you have followed the standard procedure for MDCK cell permeability assay diligently. However, I have a couple of suggestions that might shed some light on the situation:
1. Cell Seeding Density: Have you considered adjusting the cell seeding density? Sometimes, altering the number of cells seeded on the insert can influence the permeability results. You could try different seeding densities to see if it affects the drug permeation.
2. Basolateral Media Composition: The composition of the basolateral media can have an impact on drug permeation. Ensure that the media is appropriate for MDCK cells and supports their growth and permeability.
3. Pre-conditioning of Inserts: It might be worth considering pre-conditioning the inserts before cell seeding. This can help to establish a more physiologically relevant cell monolayer and improve the reproducibility of the assay.
4. Incubation Time: You mentioned conducting the assay after 7 days of cell growth. Have you tried different incubation times to assess drug permeation? Sometimes, longer incubation times may be required to observe significant permeation.
5. Cell Monolayer Integrity: Assess the integrity of the cell monolayer after the assay. You could perform a transepithelial electrical resistance (TEER) measurement to check the tightness of the cell layer.
6. Co-culture Systems: If possible, consider using a co-culture system with additional cell types to better mimic the in vivo conditions and improve drug permeation predictability.
Remember, my dear friend, that cell permeability assays can be finicky, and optimization is key to obtaining reliable results. Be patient and open to tweaking the protocol to see what works best for your specific drug and formulation.
Now, venture forth and experiment with these suggestions to unlock the mysteries of drug permeation in MDCK cells. May your scientific journey be fruitful and your discoveries profound!
Hello,
The Papp (apparent permeability) of MDCK cells rainges from 10^(-6) to
10^(-9) / sec, dependent on the size of tracer. So, you need a fluorometer sensitive enough to detect the fluorescence or add more dye to the apical compartment. We usually use 1 mg/mL fluorescein or FITC-labeled dextran.
Good luck!
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