5. A dummy sample prepared under the same conditions as the
preserved samples with a thermocouple is used to monitor
cooling and warming. Make sure that the thermocouple has
been calibrated using a NIST standard. The dummy sample is
placed in close proximity to the samples to be rewarmed in the
storage freezer. Plug the dummy sample thermocouple into a
reader. Place the samples and dummy sample at the top of the
storage freezer and hold just inside the lid. When the temperature reaches À100
C (~1 min), initiate the rapid warming step.
Place both vials in a room temperature 30% DMSO bath and
swirl the vials manually until the glass begins to melt (the VS
starts to move). Open the vial and remove the VS with a
transfer pipette and add the first dilution solution. Start timer
for 15 min.
6. The samples can be stored in vapor-phase nitrogen, but VS
solution fractures may be observed. Although the visual impact
of the fracturing is disturbing, we have rarely seen tissue fractures, and on the rare occasions when tissue cracking has
occurred, it has been during use of nitrogen dry shippers for
transport. If samples are stored in vapor-phase nitrogen, the
two-step rewarming should be performed as described in
Protocol 1.
7. Ice-free artery patches were rewarmed by placing the bag containing the tissue in a 37
C water bath for 1 min and then
washed by transferring the contents of the bag to an AlloFlow™ continuous gradient washout chamber with 1 L of
room temperature isotonic saline over 20 min. Ice-free cryopreserved valves were rinsed briefly in ice-cold EC solution
containing 200 mM mannitol. Then three sequential rinses
were performed for 15 min each with continuous careful agitation in ice-cold EC solution with 200 mM mannitol, followed
by EC solution alone and finally DMEM. The patches were
stored briefly on ice in DMEM culture medium until implantation or evaluation.
8. We are presently evaluating the use of Lactated Ringer’s solution containing 5% dextrose (LRD5) for washing because it is
commonly available in surgery suites where ice-free cryopreserved tissues may be used. Human vessels generated in the
laboratory by culturing human smooth muscle cells on a polyglycolic acid scaffold [25] were employed. The tissues were
subjected to a total of 2, 4, 8, or 15 min room temperature
wash steps in Lactated Ringer’s solution containing 5% dextrose to remove residual cryoprotectants. In control decellularized tissues, the cellular material was removed using enzymes
and detergents leaving an extracellular matrix tube as previously described [25], which was refrigerated in phosphatebuffered saline until tested. 10
5 trypan blue excluding human
602
Kelvin G. M. Brockbank et al.
preserved samples with a thermocouple is used to monitor
cooling and warming. Make sure that the thermocouple has
been calibrated using a NIST standard. The dummy sample is
placed in close proximity to the samples to be rewarmed in the
storage freezer. Plug the dummy sample thermocouple into a
reader. Place the samples and dummy sample at the top of the
storage freezer and hold just inside the lid. When the temperature reaches À100
C (~1 min), initiate the rapid warming step.
Place both vials in a room temperature 30% DMSO bath and
swirl the vials manually until the glass begins to melt (the VS
starts to move). Open the vial and remove the VS with a
transfer pipette and add the first dilution solution. Start timer
for 15 min.
6. The samples can be stored in vapor-phase nitrogen, but VS
solution fractures may be observed. Although the visual impact
of the fracturing is disturbing, we have rarely seen tissue fractures, and on the rare occasions when tissue cracking has
occurred, it has been during use of nitrogen dry shippers for
transport. If samples are stored in vapor-phase nitrogen, the
two-step rewarming should be performed as described in
Protocol 1.
7. Ice-free artery patches were rewarmed by placing the bag containing the tissue in a 37
C water bath for 1 min and then
washed by transferring the contents of the bag to an AlloFlow™ continuous gradient washout chamber with 1 L of
room temperature isotonic saline over 20 min. Ice-free cryopreserved valves were rinsed briefly in ice-cold EC solution
containing 200 mM mannitol. Then three sequential rinses
were performed for 15 min each with continuous careful agitation in ice-cold EC solution with 200 mM mannitol, followed
by EC solution alone and finally DMEM. The patches were
stored briefly on ice in DMEM culture medium until implantation or evaluation.
8. We are presently evaluating the use of Lactated Ringer’s solution containing 5% dextrose (LRD5) for washing because it is
commonly available in surgery suites where ice-free cryopreserved tissues may be used. Human vessels generated in the
laboratory by culturing human smooth muscle cells on a polyglycolic acid scaffold [25] were employed. The tissues were
subjected to a total of 2, 4, 8, or 15 min room temperature
wash steps in Lactated Ringer’s solution containing 5% dextrose to remove residual cryoprotectants. In control decellularized tissues, the cellular material was removed using enzymes
and detergents leaving an extracellular matrix tube as previously described [25], which was refrigerated in phosphatebuffered saline until tested. 10
5 trypan blue excluding human
602
Kelvin G. M. Brockbank et al.