Biology Reference
In-Depth Information
3.3. Production of Cell-Populated Collagen Matrix
The following volumes are sufficient to produce six cell-populated collagen
lattices using 2.5-cm diameter lattice inserts (
see
Note 12
).
1.
Measure out 18.5 mL of the collagen solution into a 50-mL conical tube and
place on ice.
10
5
cells/mL
2.
Prepare a suspension of dermal fibroblasts at a concentration of 2.5
×
in DMEM with 10% NBCS.
3.
Add 5.6 mL of cold (2-8
C) premix solution to the tube containing the collagen.
Mix well by swirling (
see
Note 13
).
°
4.
Immediately place 1 mL of the neutralized collagen solution into each of six 2.5-cm
diameter, 3-
m pore size culture inserts (Corning Costar, Cambridge, MA) (
see
Note 14
) and ensure that the filter surface is completely covered. Allow this acel-
lular layer to gel at room temperature (
see
Note 15
).
µ
5.
Add 2 mL of the fibroblast suspension to the remainder of the neutralized
collagen solution, swirl to mix (
see
Note 13
) and immediately add 3-mL
aliquots of the cellular collagen solution on top of the collagen gels in each of the
culture inserts. Allow the collagen to gel without any disturbance or vibration
(
see
Note 15
).
6.
Add sufficient DMEM with 10% NBCS to cover the top of the collagen gels and
incubate at 37
C, 10% CO
2
until the lattices have contracted away from the sides
of the culture insert (
see
Note 16
).
°
7.
Replace the medium with fresh DMEM with 10% NBCS at 2-3 d intervals.
4. Notes
1.
Different collagen preparations (such as acid extracted rat tail yendon collagen)
may be used, but these need to be carefully screened for gelation properties as
well as cellular interactions and possible toxicity.
2.
Rat tail tendon may also be used in place of the bovine digital extensor tendon.
3.
As an alternative to grinding, either tendon can be sliced into pieces approx 1-3-mm
thick. It is easier to achieve this if the tendon is partially frozen and a number-22
scalpel blade is used.
4.
As an alternative to the Belco vessel, a conical flask and large magnetic stir bar
may be used for volumes around 2 L. If the mixing is too vigorous then small
insoluble particles can be produced which are difficult to remove at later stages.
5.
The final PBS:water wash should have negligible UV absorbance at 280 nm. If
this is not the case, repeat the wash step until the UV absorbance is negligible.
6.
The material should separate into three layers, an upper layer, a middle layer
that contains gelatinous particles and a lower layer containing the insoluble ten-
don pieces.
7.
If air bubbles have been introduced at the precipitation step, this can cause
someofthe precipitated collagen to float at the end of the centrifugation step.
This material may be removed with a sterile instrument and added to the rest of
the precipitate.
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