Seeding and Feeding Guidlines*

Millicell-24 Cell Culture Insert Plate

Optimizing cell seeding protocols and densities is extremely important to ensure good cell attachment and growth in Millicell-24 cell culture insert plates. Initially testing a range of seeding densities is recommended. The following guidelines are designed to be carried out in a single device and can be performed using automation. Millicell-24 plates with a 0.4 µm polycarbonate (PCF) membrane or a 1.0 µm polyester (PET) membrane, which allows the cells to be visualized during the seeding and growth stages, were used in this protocol.

Materials and Reagents

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

Note: Although the following methods have been optimized for seeding and feeding adherent epithelial cell lines such as Caco-2 and MDCK, they contain the basic principles for handling the plate using any method or cell line.

Methods

Optimization of Seeding Density

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.
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.
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.
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 Millipore’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.
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.

Millicell-24 Culture Plate — Cell Seeding Density Conversions

	Cells per cm2	Cells per mL (0.4 ml.)	Cells per Well
21-day Caco-2 Seeding Density	85,700	150,000	60,000
3-day MDCK Seeding Density	714,000	1,250,000	500,000

Feeding

Note: Replacing the growth medium every other day is recommended for optimal cell growth. The most critical part of removing and replacing the medium in the Millicell-24 plate is to avoid damaging the monolayer of cells and the filter on which they are supported. This can be accomplished using a sterile 1 mL pipette tip and utilizing the apical assist feature.

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.
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.

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