F
LEXCELL
®
I
NTERNATIONAL
C
ORPORATION
2
INSTRUCTIONS
1.
Insert the BioFlex
®
Cell Seeder into a
BioFlex
®
Baseplate, similar to how the 25
mm equibiaxial Loading Stations are placed
in the baseplate.
Optional:
Add silicone
lubricant to the upper rim of a Cell Seeder
post to aid membrane gliding into the Cell
Seeder body.
2.
Place a BioFlex
®
plate into its respective
gasket positioned over the Cell Seeder,
ensuring that the wells in the plate align
concentrically over the posts on the Cell
Seeder.
3.
Using the FX-5000
™
Tension System, create
and start a regimen with the following
settings:
SHAPE: Static
MIN: 0.0
MAX: 15.0
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: BFlx Loading Station
(25mm)
4.
The recommended suspension volume when
using a 6-well, BioFlex
®
Cell Seeder for an
individual well is 1 mL medium plus cells.
This volume is large enough to allow
uniform distribution of the media-cell
suspension over the growth surface area
when dispensing cells and medium with a
1000 µL micropipette. Also, this volume is
small enough to retain the media-cell
suspension within the boundary of the Cell
Seeder form when handling the baseplate.
5.
Due to the limited suspension volume, it is
advised that the user observe the seeding
time required for the cells to adhere to the
membrane. A seeding time of at least two
hours is recommended, but the required time
is dependent on the type of cells being used.
6.
After the cells have adhered to the substrate,
remove the vacuum gradually. It is
recommended to create a slow vacuum
release regimen 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 medium to each
well to increase the volume for proper
aspiration of non-adherent cells. Then, insert
an aspirator tip near the side of each well
(adjacent to the seeded cells) and aspirate
the media containing non-adherent cells.
8.
Add 3 mL of fresh media to each well and
return the BioFlex
®
plate to the incubator
until you are ready to apply strain.
Table 1.
Sample regimen parameters for releasing the vacuum pressure slowly.
Step Shape Min Max Freq DC% dd:hh:mm:ss Back
To Repeat
1
Static
0.0
15.0
1.0
50.0
00:02:30:00
0
0
2
Static
0.0 13.0 1.0
50.0
00:00:00:06
0
0
3
Static
0.0
11.0
1.0
50.0
00:00:00:06
0
0
4
Static
0.0 9.0 1.0
50.0
00:00:00:06
0
0
5
Static
0.0
7.0
1.0
50.0
00:00:00:06
0
0
6
Static
0.0
5.0
1.0
50.0
00:00:00:06
0
0
7
Static
0.0
3.0
1.0
50.0
00:00:00:06
0
0
8
Static
0.0
1.0
1.0
50.0
00:00:00:06
0
0
In this example, we recommend a two hour adherence time plus a 30 minute set-up time (Step 1). Following
seeding, this example releases the strain by 1-2% every six seconds.
