flexcell HT Cell Seeder User manual

Culturing Cells in a Mechanically Active Environment
™
Flexcell International Corporation 437 Dimmocks Mill Road, Suite 28 Hillsborough, NC 27278
800-728-3714 [phone] FAX: [phone] www.flexcellint.com
COPYRIGHT © 2012 FLEXCELL

INTERNATIONAL CORPORATION
HT Cell Seeder™
User Manual
R EVISION 1.2
03/25/14

FLEXCELL
®
INTERNATIONAL CORPORATION

HT  C ELL S EEDER U SER I NSTRUCTIONS P AGE 1 OF 3
I NTRODUCTION
The HT Cell Seeder
™
(Figure 1) confines cells during plating to the area of the HT BioFlex
®
membrane
that is directly over the Loading Station
™
. Thus, it prevents cells from being subjected to undefined
strains when using the 24-well equibiaxial Loading Stations
™
during strain application (Figure 2). The
HT Cell Seeder
™
is inserted into the 24-well HT Baseplate (Figure 3), and the HT BioFlex
®
plate plus
the gasket is placed on top of the HT Cell Seeder
™
. The HT Cell Seeder
™
is only required for seeding
cells onto the membrane for a minimal period of time. Afterwards, cell feedings and experiments can be
conducted as normal.
Figure 1. 24-well HT Cell Seeders
™
.
Figure 2. A) A cylindrical loading post used when applying equibiaxial strain under the well of an HT
BioFlex
®
plate. Cells seeded on the membrane in areas outside of the loading post will be subject to
undefined strains. B) The results of using an HT Cell Seeder
™
when plating cells to confine them to the
area directly above the loading post (blue = Crystal Violet stain; bar = 1 mm).
A B

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®
INTERNATIONAL CORPORATION

HT  C ELL S EEDER U SER M ANUAL P AGE 2 OF 3
Figure 3. HT Cell Seeders
™
in a 24-well HT Baseplate.
I NSTRUCTIONS
1. Insert the HT Cell Seeder
™
into a 24-well HT Baseplate, similar to how the cylindrical loading
stations are placed in the baseplate.
2. Place an HT BioFlex
®
plate and its respective gasket over the HT Cell Seeder
™
, ensuring that the
wells in the plate align with the posts on the HT Cell Seeder
™
.
3. Using the FX-5000
™
Tension System, create and start a regimen with the following settings:
SHAPE: Static
MIN: 0.0
MAX: 8.0
FREQ: 1 Hz (this value must still be entered even though the regimen is static)
DURATION: Equal to the seeding time (see step 5 below) plus the time needed to plate
the cells into the well.
PLATFORM: HT 24-Well Plate (Cylindrical LS)
NOTE: The membrane may be subjected to strains up to 1-2% (10 to 20 microns per 1 millimeter) when
seeding, due to the inherent strain caused when using the HT Cell Seeder
™
.
4. The recommended suspension volume when using an HT Cell Seeder
™
for an individual well is
175 µL. This volume is large enough to allow uniform distribution of the media-cell suspension
within the well when dispensing with a 1000 µL micropipette (Figure 4). Also, this volume is
small enough to prevent the media-cell suspension from spilling out of the HT Cell Seeder
™
when handling the baseplate.

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®
INTERNATIONAL CORPORATION

HT  C ELL S EEDER U SER M ANUAL P AGE 3 OF 3
Figure 4. Suspension volume within the HT Cell Seeder
™
.
5. Due to the limited suspension volume, it is advised that the user try to limit the seeding time
required for the cells to adhere to the membrane. We recommend a seeding time of at least two
hours, but the time that will be required is dependent on the type of cells being used.
6. After the cells have adhered, remove the vacuum gradually. You can create a slow vacuum
release regimen if needed that reduces the vacuum a set percent every “n” seconds. An example
regimen is outlined below in Table 1.
7. Slowly add 1 mL of fresh media to each well to increase the volume for proper aspiration of non-
adherent cells. Then, insert an aspirator tip near the side of the plate well (instead of the cell
seeder well) and aspirate the media containing non-adherent cells.
8. Add 1 mL of fresh media to each well.
Table 1. Sample regimen parameters for releasing the vacuum pressure slowly.
Step Shape Min Max Freq DC% dd:hh:mm:ss Back To Repeat
1Static 0.0 8.0 1.0 50.0 00:02:30:00 0 0
2Static 0.0 6.0 1.0 50.0 00:00:00:06 0 0
3Static 0.0 4.0 1.0 50.0 00:00:00:06 0 0
4Static 0.0 2.0 1.0 50.0 00:00:00:06 0 0
5Static 0.0 1.0 1.0 50.0 00:00:00:06 0 0
In this example, we are assuming a two hour seeding time plus a 30 minute set-up time (Step 1). Following
seeding, this example releases the strain by 1-2% every six seconds.