Biology Reference
In-Depth Information
year if stored in a sterile condition at 2-8
C. In order to minimize the
bioburden, sterile technique should be used throughout the process and all open
transfers of the collagen solution should take place in a tissue-culture hood.
1.
°
Dissect out the digital extensor tendon from calf hooves (
see
Note 2
) by making
two lateral incisions and peeling back the dorsal flap of skin to expose the com-
mon digital extensor tendon.
2.
Cut the tendon from the surrounding tissue and sheath. Place the tendons in ice
water until all are collected. The tendons can be stored at -70
°
C until used.
3.
Grind the tendon with an equivalent volume of ice (
see
Note 3
). Wash the tendon
pieces three times in three volumes of ice water and store at -70
°
C until used.
4.
Take 150 g of tendon pieces and add to 15 L of cold (2-8
C), sterile PBS:water in
a Belco (Model 7764-00110) top stirring reaction vessel. Mix the solution (set
motor to setting 10) for 2 h at 2-8
°
°
C (
see
Note 4
).
5.
Aspirate off the buffer and repeat the washing step three more times (
see
Note 5
).
6.
Add 15 L of cold (2-8
C), sterile 0.5
M
acetic acid to the Belco vessel and mix
gently (motor setting 5) for 72 h at 2-8
°
°
C.
7.
Decant the acetic acid into 250-mL centrifuge bottles and spin at 25,000
g
for
30 min at 4
°
C in a Beckman J2-21 centrifuge, or equivalent.
8.
Very carefully decant just the uppermost layers and combine them in another 15 L
Belco vessel (
see
Note 6
).
9.
Pass the solution through an open mesh 5-6
µ
m filter in order to remove any
tendon particles.
10.
Make the solution 0.9
M
with respect to NaCl by
slowly
adding one-third the
volume of sterile, room temperature 3.6
M
NaCl with continuous, gentle mixing
(motor speed 5). After the NaCl has been added, continue mixing for 30 min.
11.
Decant the solution into 500-mL centrifuge bottles and collect the precipitate by
centrifugation at 10,000
g
for 30 min at 4
°
C.
12.
Discard clear supernatants (
see
Note 7
) and combine the precipitates in a 15 L
Belco flask.
13.
Add 2 L of cold, sterile 0.5
M
acetic acid to the Belco flask and mix at 2-8
°
C
until the precipitate has completely dissolved.
14.
Repeat the precipitation step once more by making the solution 0.9
M
NaCl.
Redissolve this precipitate completely in 1
M
acetic acid by vigorous mixing.
15.
Transfer the collagen solution to 1 meter lengths of sterilized dialysis tubing.
16.
Place the dialysis tube sections in 20 vol of 8.75 m
M
acetic acid at 2-8
°
C.
17.
Saturate the solution with chloroform (approx 0.5 mL/L) and mix the acetic acid
solution gently at 2-8
°
C for 24 h (
see
Note 8
).
18.
Discard the acetic acid/chloroform solution to waste and replace with 20 vol of
fresh, cold 8.75 m
M
acetic acid. Mix for 24 h.
19.
Repeat
step 17
twice.
20.
Using sterile technique, spray one end of the dialysis tubing liberally with 70%
ethanol solution. Use a sterile wipe to remove excess, cut open the dialysis tube
and carefully poor into a suitable, sterile, screwcap container (
see
Note 9
).
Search WWH ::
Custom Search