Corning® FloWell™ 2W Plates User Guide - Corning Incorporated

Corning ® FloWell 2W Plates User Guide
Date Last Revised: 11/19/2012
Corning FloWell 2W plate is a multiple well cell culture
plate that contains 2 sets of 3 wells connected through
porous channels (Fig. 1A). This design allows continuous
fluid perfusion between connected wells for up to 3 days.
Flow direction and rate are driven primarily by hydrostatic
pressure. The Corning FloWell 2W plate is automation
friendly, as the dimensions are the same as the standard
6 well multiple well plate.
Corning FloWell plates can be used as cell feeding devices
for culturing cells that require frequent medium change,
such as some stem cells, neuronal and primary cells and fastgrowing
cancer cell lines. In this application, the middle well
from each set of 3 connected wells serves as the cell culture
well, respectively (Fig. 1B). The wells on either side of the
cell culture well are used as the medium supplying well
and waste well (Fig. 1B). With proper setup, medium flows
continuously from the medium wells to cell wells for up to
3 days. Concurrently, exhausted medium flows out of cell
culture wells, which can be beneficial to cell viability and
growth. This product is ideal for maintaining cell cultures
that require daily medium exchange, through the weekend
and holidays, saving both time and labor costs. By eliminating
the need for manual medium exchange, the product can
reduce the risk of contamination, cell loss and variability.
Corning FloWell plates can also be used in co-culturing
studies that investigate cell-to-cell communication with
the ability to spatially separate cells of different types.
Medium well Cell well
Flow direction Flow direction
Waste well
Porous materials
Figure 1A. Corning FloWell 2W Plate. Multiple well cell culture
plate with continuous fluid perfusion technology.
Figure 1B. Side View
Schematic Diagram of
Corning FloWell 2W Plate.
Typical view of the plate at
the beginning of the cell
culture process.

1 2 3
4 5 6
Figure 2. Well numbering
scheme for Corning FloWell
2W Plate.
The procedure below describes how to set up the plates for continuous cell feeding.
I. Coating Cell Culture Wells
Corning ® FloWell Plates are tissue culture treated (TCT). If a special surface coating is
required for cells to attach, the procedure outlined below provides general guidance. Only
the two wells in the middle column need to be coated (Fig. 2, wells 2 and 5). All steps should
be performed in a biological safety cabinet using aseptic techniques. This procedure may
require optimization depending on the material used to coat the wells (i.e., collagen, fibronectin,
Matrigel, gelatin, laminin or poly-ornithine).
1. Prepare coating solutions using procedures recommended by the material supplier or
procedures previously optimized for the cells to be cultured in 6 well plates.
2. Transfer 1.5 mL DPBS buffer to each of the medium wells (Fig. 2, wells 1 and 4) and
the waste wells (Fig. 2, wells 3 and 6).
3. Transfer 1.5 mL coating solution to each of the cell wells (Fig. 2, wells 2 and 5). It is
important to fill the cell wells with a volume equal to or slightly smaller than the volume
used for medium and waste wells. This will ensure the coating material does not flow
into the porous channels and reduces the risk of clogging the porous channels. This is
especially important when using coating materials that could form irreversible gels.
4. Incubate the plates for the required period of time and temperature (according to the
manufacturer’s instructions or as previously optimized for 6 well plates). Some frequently
used conditions identified from published literature include; 2 hours at room temperature,
1 hour at 37°C incubator, or overnight at 4°C.
5. For the best results, use the coated plates immediately. If plates are not used after the
coating procedure, they should be sealed and stored at 4°C. The length of time the coated
plates can be stored will depend on the coating materials used (follow manufacturer’s
recommendations of optimize for the particular cell type used).
II. Setting Up Cell Cultures
Depending on the cell lines to be cultured, starting volumes for each set of 3 connected wells
may vary. Therefore, determine the optimal conditions for each cell type to be cultured.
For medium wells (Fig. 2, wells 1 and 4), a minimum volume of 6.5 mL medium is recommended
in order to provide sufficient medium to cells cultured in the cell wells for up to 3 days.
For some cells, a larger starting volume for the medium well is needed to sustain the cell
growth. Do not fill the medium well with more than 9.5 mL per well to prevent overflow
during handling.
For cell wells (Fig. 2, wells 2 and 5), a minimal seeding volume of 2.5 mL medium per well.
For waste wells (Fig. 2, wells 3 and 6), 0.5 mL medium is recommended. This is required
to wet the waste wells and initiate fluid flow from the cell wells out into the waste wells.
1. Prepare cells (either passaged or from frozen vials) so that they are in growth medium
with desired cell seeding density.
2. If the Corning FloWell plate was pre-coated and stored at 4°C, allow the plate to reach
room temperature and remove the coating solutions or buffer from all wells.
3. Transfer 2.5 mL of cell suspension prepared in step 1 to each of the cell culture wells
(Fig. 2, wells 2 and 5).
4. Gently rock and swirl plate to ensure uniform cell distribution.
5. Aspirate 7 to10 mL pre-warmed growth medium with a 10 mL Stripette ® serological
pipet.
6. Dispense 3 mL growth medium to one waste well (Fig. 2, well 3). Then aspirate 2.5 mL
of the medium back from the same waste well. This step is intended to wet the bottom of
waste well, otherwise, a dry waste well may prevent medium flowing out of cell well. It is
important to make sure that the waste wells are wet when setting up the cultures.

7. Dispense the remaining growth medium from the pipet to one medium well (Fig. 2,
well 1). The total volume in the medium well should be in the range of 6.5 to 9.5 mL.
8. Repeat step 5 to step 7 for the other set of waste and medium wells on the plate (Fig. 2,
wells 6 and 4, respectively).
9. Place the plate in cell culture incubator for 72 hours.
10. Examine cell confluency and morphology daily to assess cell growth performance and
determine if the cultures are ready for harvest.
III. Medium Replenishment for Extended Cell Culture Growth
If extended growth beyond 72 hours is needed for the cells, follow the procedure below to
replenish the medium wells and discard exhausted medium.
1. Warm up growth medium to 37°C.
2. Remove Corning ® FloWell plate from the incubator and place into the biological
safety cabinet.
3. Aspirate and discard all the medium from the medium wells (Fig. 2, wells 1 and 4) and
add the desired amount of pre-warmed fresh growth medium (6.5 to 9.5 mL).
4. Aspirate and discard the exhausted medium in the waste wells (Fig. 2, wells 3 and 6).
5. Optional: During the culturing period, dead cells can accumulate in the cell wells (Fig. 2,
wells 2 and 5). If it is desired to remove the dead cells, a medium change step for the cell
wells can be included. In this case, simply aspirate the medium in the cell wells and add
2.5 mL pre-warmed fresh growth medium back into each cell well.
6. Place plate back into incubator.
IV. Cell Harvest
1. Aspirate growth medium from all wells.
2. Rinse cell wells with a minimum of 1.5 mL buffer.
3. Detach cells from the cell well following the normal procedure for harvesting cells from
a 6 well plate.
4. Collect cell suspension from the cell wells and process.

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