Millicell® Plates

Millicell®-24 Cell Culture Insert Plate

• Millicell®-24 cell culture insert plates (PCF or PET membrane) — Merck cat. nos. PSHT 010 R5, PSRP 010 R5
• Tissue culture flasks
• Steriflip-GP or Stericup-GP filter units — Merck cat. no. SCGP 005 25 or SCGP U11 RE
• 0.02% EDTA — Merck cat. no. 20-307
• 1x Trypsin/EDTA — Merck cat. no. SM-2002-C or SM-2003-C
• Cell culture media — Merck cat. no. SLM-022-B
• Sterile 1 mL pipette tips, pipettors, microfuge/centrifuge tubes and basins
• Hemocytometer or other cell counter

1. Expand and cultivate cells in T-75 flasks in a cell culture incubator set at 37°C, 5–6% CO2 and 95% relative humidity. Allow cells to reach 80–90% confluence before detaching and passaging. Do not allow cells to become over confluent (>90%) as this will impact subsequent monolayer formation on the Millicell®-24 plates.
   
   Note: The number of cell passages can affect the formation of an optimal monolayer. It is therefore recommended that Caco-2 cells be subjected to no more that 20 passages before a new line is established. Similarly, MDCK cells should be subjected to no more that 40 passages.
   
   
2. Aspirate the media, rinse the cultivated cells in T-75 flasks with 5 mL 0.02% EDTA and incubate for 3–5 minutes. After aspiration of the 0.02% EDTA, add 1.5 mL of the trypsin/EDTA solution. Incubate at 37°C for approximately 5 to 15 minutes or until the cells detach and float. This can be confirmed by periodic visual inspection of the flasks.
   
   
3. Once the cells are detached, add fresh cell culture medium and mix until all cell clumps are dispersed. Count the cells using a hemocytometer or other cell counter to determine the cell number and pass cells accordingly to maintain stock of cell line. Mix cells frequently to ensure accurate counts.
   
   *The guidelines in this protocol are specific to Millicell®-24 cell culture plates. If using single-well inserts or Millicell®-96 cell culture plates, modifications must be made to these guidelines. Please refer to the recommended working volumes table to adjust liquid additions based on product selection.
   
   Note: To optimize the seeding density of a cell line, it is recommended that a range of cell concentrations across the plates be used in replicates of 6 or 8.
   
   
4. In sterile centrifuge tubes, dilute the cell solution with medium to enable the plating of a range of seeding densities. A good starting point to determine seeding density is calculated by multiplying the present cell density by the fold increase or decrease in surface area.
   
   Example: If the seeding density for the 9 mm Millicell® inserts (surface area=0.3 cm2) is 60,000 cells/well then multiply 60,000 by 2.3 to calculate the seeding density for Millicell®-24 cell culture insert plates (surface area=0.7 cm2).
   
   0.7 cm2/0.3 cm2=2.3
   60,000 cells/well x 2.3=138,000 cells/well
   
   A suitable range might therefore encompass 120,000–160,000 cells/well, depending on individual cell lines used. The following table lists Merck’s optimized densities (in terms of three different units) for seeding 3 day MDCK and 21 day Caco-2 cell monolayers.
   
   Note: If starting without any prior platform, initially bracket a range of seeding densities around the values listed in the “Cell Seeding Density Conversion” table.
   
   Note: Achieving a uniform cell suspension when initially plating the cells will promote a more consistent monolayer across the 24 wells. This may be particularly difficult when seeding multiple plates. Frequent mixing is recommended to minimize the risk of cells settling to the bottom, resulting in an inaccurate distribution of cells across the wells or plates.
   
   
5. Pipette 300 µL of cells at each seeding density into the appropriate filter wells of the Millicell®-24 plates (see “Range of Feeding Densities” figures). Pipette 28 mL of cell culture medium into the single-well feeder plate via the large access hole located at the lower right of the plate. Alternatively, disassemble the filter plate from the feeder plate. Place the filter plate on a sterile surface in a laminar flow hood and add medium directly to the feeder plate. Gently reassemble the two components and place in the cell culture incubator.
   
   Note: Cells seeded onto the Millicell®-24 plate should be placed in an incubator that provides adequate humidity control. A cell culture incubator with electronic humidity control is recommended. If this is not possible, plates should be placed with a water pan in an incubator that will not be opened frequently.

1. Aspirate the medium from the feeder plate (basolateral) using a sterile 1 mL pipette tip via the large access hole or, after disassembly of the filter plate from the feeder plate.
   
   
2. Aspirate the medium from the filter wells using the apical assist feature. Use care not to contact the filter inside the wells when removing or adding medium. Add back 300 µL growth medium to the filter wells (at the apical assist) before adding back the 28 mL of medium to the feeder plate (basolateral).
   
   Note: If setting the filter plate down on the hood surface using the filter plate "feet", it is recommended to let the whole assembly sit idle in the hood for 15 seconds after removing it from the incubator. Disassembly of the plate after 15 seconds will minimize the potential for droplet formation on the underside of the membrane, which may contact the hood surface.
   
   Note: Evaluation of seeding densities is usually performed by testing the integrity of the monolayer with trans-epithelial electrical resistance (TEER) measurements, by evaluation of the transport of lucifer yellow (LY) or by using standard drug compounds.

• Millicell®-24 cell culture insert plate (PCF or PET membrane ) — Merck cat. nos. PSHT 010 R5, PSRP 010 R5
• Millicell® ERS System—Merck cat. no. MERS 000 01
• 24-well receiver plate—Merckcat. no. PSMW 010 R5
• 24-well feeder tray—Merck cat. no. PSSW 010 R5
• Lucifer Yellow, 100 µg/mL concentration
• Hanks balanced salt solution (HBSS)
• Radioactive drug transport:
• Wallac/Perkin Elmer 96-well flexible plate
• Microbeta® Trilux® Counter or equivalent
• Scintillation cocktail

1. At the end of the desired growth period, remove the plates from the incubator and allow them to equilibrate to room temperature (approximately 0.5 hours).
2. Measure the electrical resistance across the monolayer using the Millicell® ERS system. Position the probe such that the shorter prong is immersed in the media inside the filter well and the longer prong is placed through the basolateral access hole into the media in the growth plate.

1. Using the same methodology as when feeding, rinse the monolayer three times with 300 µL HBSS in the apical wells and 28 mL in the feeder tray.
2. Add 300 µL of LY solution to each well in the filter plate.
3. Add 600 µL HBSS to each well of a 24-well receiver tray.
4. Assemble the filter plate and 24-well receiver plate and incubate for 1–2 hours at 37°C.
5. Remove the filter plate from the receiver plate and place the receiver plate into a fluorescent plate reader. Determine the LY fluorescence using an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
6. Calculate the percent of LY rejection across the cell monolayer by measuring fluorescence in the receiver plate as compared to an ‘equilibrium’ standard.
   
   Note: The standard plate should consist of 4 wells with 600 µL HBSS (blank) and 4 wells with 200 µL LY (100 µg/mL) + 400 µL HBSS (equilibrium samples).

1. After the desired cell growth period, remove the Millicell®-24 plate from the incubator and determine the electrical resistance for each well (as described above). Wash the monolayer, exchanging the volume three times using sterile HBSS, pH 7.4. After washing, remove the buffer from the filter plate and feeder tray.
2. Transfer the filter plate to a 24-well transport analysis plate.
3. To determine the rate of drug transport in the apical to basolateral direction, add 300 µL of the test compounds to the filter well. Drug concentrations typically ranging from 10 µm to 200 µm may be used (achieve desired concentration using HBSS, pH 7.4 or an alternative buffer of desired pH).
4. Fill the wells of the 24-well receiver plate with 600 µL buffer.
5. To determine the rate of drug transport in the basolateral to apical direction, add 600 µL of the test compounds to the 24-well receiver plate.
6. Fill the filter wells (apical compartment) with 300 µL of buffer.
7. Combine the filter and receiver plates once all drugs and buffer have been added. Begin timing the experiment.
8. Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical incubation times are 1 to 2 hours. We recommend a 2-hour incubation for optimal results.
9. For LC/MS analysis: At the end of the incubation, remove a fixed volume (typically 50–100 µL) directly from the apical and basolateral wells (using the basolateral access holes) or by disassembling the plates. Transfer the volume to a clean plate.

1. Artursson, P., Karlsson, J. (1991) Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Comm. 175:880–85.
2. Artursson, P. (1990) Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. J. Pharm. Sci. 79:476–82.
3. Artursson, P., Palm, K., and Luthman, K. (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46:27–43.
4. Bailey, C.A., Bryla, P., Malick, A.W. (1996) The use of intestinal epithelial cell culture model, Caco-2, in pharmaceutical development. Adv. Drug Deliv. Rev. 22:85–103.
5. Arena, A.A., et.al. (2005) Development of a higher throughput, permeability model system using MDR1-transfected MDCK cells in 24- and 96-well formats. 2005 American Association of Pharmaceutical Sciences Annual Meeeting, Nashville, TN.

• 24-well tissue culture plates (3 plates for each compound)
• 12 mm Millicell®-HA cell culture inserts. Merck cat. no. PIHA 012 50
• MDCK epithelial cells
• T-75 tissue culture flasks
• MEM media with 10% fetal bovine serum and gentamycin, 1 µg/mL
• Hanks' Balanced Salt Solution (HBSS)
• Trypsin-Versene™
• Sodium Fluorescein, 1mg/vial

1. Grow MDCK cell line in T-75 culture flask with MEM (10% FBS, 1 µg/mL gentamycin) and pass at weekly intervals using trypsin-versene.
2. Place inserts into a 24-well culture plate containing 0.5 mL media per well. One 24-well plate can be used to test a compound at six concentrations in quadruplicate.
3. Seed 1.5×105 cells per insert; confluency should occur in 3 days. Test for monolayer before proceeding. During this incubation period the media should be changed daily.
4. When cultures are confluent, remove media and rinse inserts with HBSS.
5. Place inserts into a new 24-well plate containing 0.5 mL HBSS and a specific amount of the test compound. Incubate at 24°C for 15 minutes.
6. Decant solution and wash inserts three times, each with 1 mL HBSS.
7. Place inserts into a new 24-well plate containing 0.5 mL HBSS. Add 0.5 mL HBSS containing 0.02% sodium fluorescein to the apical side of the inserts. Incubate at 24°C for 30 minutes.
8. Remove inserts and fix for examination via light and electron microscopy.
9. Dilute sodium fluorescein solution in each well to 3 mL and measure in a spectrophotometer at 490 nm.

1. Viewing from below the plate (through transparent PET or CM membranes)
   Millicell® devices using PET or CM membrane have been designed to allow visualization of cells from below using an inverted microscope. For viewing live cells, microscopic observations can be made through the receiver or plastic plate containing media. In order to focus on the cells, the microscope objective (typically 5–20X) must have an appropriate working distance. (For objective specifications, visit the websites listed in the Microscope Objective Information section.) Fixed cells that do not require to be visualized in media can be viewed directly without the receiver plate. However, care should be taken not to contaminate the objective with liquid residue (media, mounting fluid) on the membrane.
2. Viewing from above the plate (Millicell® 6-well inserts, Millicell®-24 cell culture insert plates)
   Some cell culture platforms can allow the cells to be viewed in a conventional microscope directly from above using low magnification. Cells can be visualized through the lid to maintain sterility or with the lid removed for fixed cells or when maintenance of sterility is not required. Working distances of the objective must be longer when reading from above compared to when reading from below. If using immunofluorescence, it is recommended to use a mounting fluid that contains an anti-fade additive to prevent photobleaching.
3. Visualizing membranes on microscope slides (for higher magnification or withobjectives with short working distances [less than 2 mm])
   The membrane can be removed from each well for microscopic evaluation. This allows for higher magnification examination and storage of the slides for future use.

1. Remove the membrane from the well using a sharp scalpel to make a small incision in the edge of the membrane. Carefully cut along the inside of the well wall for approximately one quarter of the well diameter. Using forceps (Merck cat. no. XX62 000 06), carefully hold the membrane while continuing to cut around the well diameter to remove membrane. Alternatively, a cork borer may be used to remove the membrane. Note: Use care to prevent membrane from curling.
2. Place the membrane disk, cells facing up, onto a microscope slide.
3. Add 50 µL mounting fluid to the membrane disk and allow it to wet out in order to prevent bubbles under the disk.
4. Slowly lower a cover slip onto the membrane at an angle to allow air bubbles to be removed.

• Nikon Instruments: www.nikoninstruments.com
• Olympus Corporation: www.olympusamerica.com
• Carl Zeiss: www.zeiss.com/miscroscopy

• Millicell® Cell Culture Inserts
• Phosphate Buffered Solution (PBS)
• Glutaraldehyde
• Osmium tetroxide
• Sodium cacodylate
• Sucrose, reagent grade
• Calcium chloride
• Lead nitrate
• Sodium citrate/Sodium hydroxide
• Uranyl acetate
• Ethanol
• Flat embedding trays
• Embedding Resin (i.e. EPON812)
• Fine forceps — Merck cat. no. XX66 000 06
• Diamond Knife
• Cork Borer
• Durapore® Filter Disk — Merck

1. Wash cells briefly (2 times for 5 minutes each) at room temperature with phosphate buffered solution without fixative.
2. Fix cells in 2% glutaraldehyde in 100 mm sodium cacodylate buffer, pH 7.5, at room temperature from 15 minutes to 2 hours.
3. Wash cells (2 times for 5 minutes each) in 100 mm sodium cacodylate buffer at room temperature. Note: At this point, cells can be stored in the above buffer with 7 g sucrose/100 mL buffer at 4°C.
4. Fix cells in 1% osmium tetroxide in either 100 mm sodium cacodylate or suitable phosphate buffer.
5. Dehydrate cells in the following concentrations of ethanol:
   
   

   Ethanol Concentration Kit (%)	Time (minutes)
   30	15
   50	15
   70	15
   95	15
   100	3 x 15

    
    
   Note: Dehydration of Millicell®-HA units should be performed in a metal pan that will be used as the embedding tray due to the tendency of the cellulosic membranes to be less rigid during the dehydration process. Attempts to transfer the membranes during these steps could lead to mechanical damage to the cells.
   
   
6. For infiltration, EPON812, an EDPON substitution, or LX112 is suitable for both devices (do not use Spurr’s). The following is a general infiltration scheme:
   
   

   Ethanol Concentration Kit (%)/Tray (% Plastic)	Time (minutes)
   75/25	30 on a shaker
   50/50	30 on a shaker
   0/100	30 each/3x on a shaker
   0/100	Overnight

   
   
   Note: It is not necessary to use any other agent, such as propylene oxide, with plastic. Propylene oxide will dissolve the cellulosic filters. In addition, the standard inversion/rotation of specimens used in these steps is not advised since either (1) damage to the cell layer or (2) stretching of the cellulosic filter may occur. Mild shaking on a gel shaker apparatus is sufficient for successful infiltration.
   
   Note: Before the next step the membrane must be detached from the surrounding plastic ring. Sometimes this will occur without manipulation since the EPON may loosen the membrane-to-ring bond. If this does not occur, use a sharp scalpel or a cork borer and cut the membrane. It may also help to cut the membrane over a 47 mm filter support disk. Under no circumstances should the membrane be left attached to the ring during polymerization.
   
   
7. Transfer to fresh plastic and polymerize at 68°C overnight.

1. Nitrocellulose (HA), polycarbonate (PC) and polyethylene terepthalate (PET) membrane: These membranes can be sectioned in any plane without difficulty.
2. CM (Biopore) membrane: The Biopore membrane must be processed in one of two ways based on the final thickness of the section.