Flexcell® ThermaCol® Kit

Flexcell ThermaCol Kit
Collagen gel solution with rapid gelation at 37 C

.

• All components in one kit for creating a 3D cell-seeded bioartificial
  type I collagen gel.

• Kit contains atelopeptide and telopetide-containing type I collagen,
  5X MEM, fetal bovine
  serum, 20 mM hepes, and 0.1 M NaOH.

• Available in three sizes: mini, midi, and maxi.

Flexcell  Thermacol  Kit

3D Culture of Cells
THERMACOL  KIT MATERIALS (CAT.NO.THERMAKIT-100A)

A Mini-Thermacol  Kit has approximately enough solution to make BATs
in eight 6-well linear
Tissue Train  culture plates, a Midi kit has enough for sixteen
6-well plates, and a Maxi kit for
thirty-two 6-well plates.
PROTOCOL FOR PREPARATION OF 1 MLTHERMACOL  COLLAGEN GEL FOR GENERAL USE
1) In a 1.5 mL centrifuge tube, combine
a. 160 uL reagent A
b. 80 uL reagent B
c. 20 uL reagent C
2) In a second 1.5mL centrifuge tube, combine1:
a. 630 uL Collagel
b. 70 uL Thermacol
c. 40 uL reagent D.
3) After mixing well, ensure that pH of Collagel/Reagent D mixture
is 7.0 – 7.2. If not, add 2.5uL of eagent D,

mix well, and test again. Repeat until pH is in desired range.
4) Combine the two tubes, mix well by pipetting.
5) This may be used to resuspend your cell pellet or other constituents
for plating. See figure 1 for kinetics of gelation at 37^。C.
1 This is to produce a 10% Thermacol/Collagel gel. The ratio can be
adjusted (0-20%) to produce gels that form at a different rate and have different physical properties.
See figure 1.

EXAMPLE OF USE:PROTOCOL FOR POURING BIOARTIFICIAL CELLPOPULATED HYDROGELS
(BAT) IN FLEXCELL LINEAR TISSUE TRAIN  CULTURE PLATES

1) Trypsinize cells by aspiration of general growth medium (GM),
washing the monolayer once with 1X DPBS,

and addition of 2.0 mL 0.05% Trypsin with EDTA followed by
incubation at 37 ^。C 5% CO2 for 5 minutes. Next,

resuspend cells with an additional 8.0 mL of growth media followed by
centrifugation to remove

trypsin containing medium and a final resuspension in 10 mL GM.
2) Count cells. A concentration of 1.0x103 cells/μL of Thermacol-media
matrix is recommended;

however, this Thermacol /cell ratio will require optimization depending
on cell type and application.
3) Sediment the desired amount of cells that will be used for seeding
a single Tissue Train culture

plate for linear BATs. Example: 1.8x106 total cells are recommended
one 6-well Tissue Train  linear BAT plate batch (200 μL/BAT).
4) Position Tissue Train plate with gasket on top of a Trough
Loader  Loading Station  and
ensure proper seating to achieve a sealed vacuum.
5) Initiate a maximum elongation (~90 KPa) static pull down regimen
using the Flexcell FX-5000 Tension System. Note: For standard Thermacol ,
2 hours of gelation time is recommended.

It is also recommended that the vacuum be released slowly by 2%
elongation of maximum every 6 seconds until 0% elongation is achieved.
6) Preparing the Thermacol -media matrix:


a. Note: Keep all above reagents at 4^。C or prepare on ice.
b. In a 1.5 mL tube (not supplied), combine reagents A, B and C.
c. In a 5.0 mL tube (not supplied), combine Collagel  with Thermacol?
(90% Collagel to 10% Thermacol then add reagent D and mix well. A thoroughly mixed
solution should become light peach in color.
d. Remove 10 μL of the Reagent D-Collagel  mixture and apply to pH paper
(not supplied) to ensure

pH is neutral (7.0-7.2 is ideal). Note: If not add 2.5 μL Reagent D and
check again (repeat until pH is in desired range).
e. Add Reagent A, B, C mixture to the Thermacol  mixture and mix well
by pipetting.
7) Working quickly, resuspend the cell pellet in the complete Thermacol?
-media matrix and mix well by pipetting.
8) Micropipette 200 μL cell constituted Thermacol -matrix into each
trough of a Tissue Train  plate:
a. Wet underside of the anchors first, then move the pipet along the trough,
depositing and spreading

the gel-cell matrix.
b. Pipet the mixture on the top of the anchor tabs and push anchors into gel.
c. When all BATs are poured the tab should be in the middle of the gel. Avoid air bubbles,

aspirate via micropipette if necessary.
9) Place baseplate into 37 ^。C 5% CO2 incubator for 2 hours of gelation time.
10)Near the end of the static pull down (approximately 10 minutes remaining) while the vacuum
is still engaged, transfer baseplate to a tissue culture hood and add 4.0 mL

complete growth media making sure to pipet very gently.
Media addition is done this way to prevent

undue motion to the gelling BATs.
11)Once vacuum has released, remove plates and place in incubator at
appropriate growth conditions.

It is recommended that the growth media be changed every 3 days.

RECOMMENDED REAGENT STORAGE CONDITIONS
Reagent Storage Temperature
Collagel? 4^。C
Thermacol? 4^。C
Reagent A 4^。C
Reagent B ‐20^。C
Reagent C 4^。C
ReagentD 4^。C