flexcell BioFlex User manual

USER MANUAL
BioFlex®Cell Seeder
Optimize cell placement in each BioFlex®well!
10-31-17
Rev 1.0
Dynamic Culture for Tissue Engineering
™
Flexcell International Corporation 2730 Tucker Street, Suite 200 Burlington, NC 27215
800-728-3714 [phone] FAX: [phone] www.flexcellint.com
COPYRIGHT © 2017 FLEXCELL

INTERNATIONAL CORPORATION

FLEXCELL®INTERNATIONAL CORPORATION
1
INTRODUCTION
BioFlex
®
Cell Seeders (Fig. 1) confine cells during plating to the central area of a BioFlex
®
membrane. The central region of the membrane is positioned directly over the 25 mm diameter
cylindrical loading posts in the Loading Station
used during application of equibiaxial substrate
strain. Cells plated in this manner are subjected to defined strains when using the equibiaxial posts
in the Loading Stations (Fig. 2). The BioFlex
®
Cell Seeder
™
is inserted into the BioFlex
®
Baseplate
(Fig. 3), and the 6-well BioFlex
®
plate plus the gasket are placed on top of the Cell Seeder. Vacuum
draws the membrane into the defined central region of the membrane during cell plating and
adhesion. Once cells have adhered to the membrane, vacuum is released, and cells can be cultured
in the usual manner.
Figure 1. 6-well BioFlex
®
Cell Seeder Station in
a BioFlex
®
baseplate well.
Figure 2. The results of using a BioFlex
®
Cell
Seeder when plating cells to confine them to the area
directly above the 25 mm cylindrical loading posts
(
p
ur
p
le = cells stained with Cr
y
stal Violet stain).
Figure 3. BioFlex
®
Cell Seeders in a BioFlex
®
Baseplate.

FLEXCELL®INTERNATIONAL CORPORATION
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
1Static 0.0 15.0 1.0 50.0 00:02:30:00 0 0
2Static 0.0 13.0 1.0 50.0 00:00:00:06 0 0
3Static 0.0 11.0 1.0 50.0 00:00:00:06 0 0
4Static 0.0 9.0 1.0 50.0 00:00:00:06 0 0
5Static 0.0 7.0 1.0 50.0 00:00:00:06 0 0
6Static 0.0 5.0 1.0 50.0 00:00:00:06 0 0
7Static 0.0 3.0 1.0 50.0 00:00:00:06 0 0
8Static 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.