(i.e., À7
C). While the program is running, fill a Styrofoam
cup with liquid nitrogen to prechill forceps for seeding (see
Note 9).
12. Upon cooling to À7
C and following the soak time, seed
extracellular ice by touching the prechilled forceps to the powder end of each straw (or to the wall of each cryovial).
13. Reactivate the cooling program to complete the subsequent
steps.
14. Upon cooling to À70
C, remove and plunge sample holders
into liquid nitrogen without raising sample temperature.
15. End the cooling program, and allow controlled rate freezer to
return to RT before turning off.
3.4 Thawing
Human ASCs
1. To thaw ASCs frozen in 0.5-cc straws:
(a) Remove a straw from liquid nitrogen, and thaw its content
at RT for ~3 min by horizontally placing it on a Kimwipe
under the hood.
(b) Wipe down the outside of the straw with 70% ethanol, and
aseptically cut off both ends.
(c) Release cells into a 15-mL tube by pushing on cotton end
with a long blunt needle.
2. To thaw ASCs frozen in cryovials:
(a) Remove a cryovial from liquid nitrogen and hold in air for
20 s. Then, partially immerse it in a water bath at 37
C,
and swirl until ice melts (see Note 10).
(b) Wipe down the outside of the cryovial with 70% ethanol.
(c) Gently mix cell suspension under the hood, and transfer
to a 15-mL centrifuge tube.
3. To dilute cryoprotectants, add an equal amount of defined
cryopreservation medium without penetrating cryoprotectants
(1:1 dilution), gently swirl the tube, and hold at RT for 5 min.
4. Repeat the 1:1 dilution step by adding an equal amount of
defined cryopreservation medium without penetrating cryoprotectants, gently swirl the tube, and hold at RT for 5 min.
5. For the final dilution step, add excess amount (e.g., 8–10 mL)
of plain HEPES-buffered DMEM/F-12, gently mix, and wait
for 5 min.
6. Pellet ASCs by centrifugation at 300 Â g for 10 min.
7. Aspirate supernatant, resuspend ASCs in appropriate medium,
and determine cell number and viability.
564
Melany Lo ´ pez and Ali Eroglu
C). While the program is running, fill a Styrofoam
cup with liquid nitrogen to prechill forceps for seeding (see
Note 9).
12. Upon cooling to À7
C and following the soak time, seed
extracellular ice by touching the prechilled forceps to the powder end of each straw (or to the wall of each cryovial).
13. Reactivate the cooling program to complete the subsequent
steps.
14. Upon cooling to À70
C, remove and plunge sample holders
into liquid nitrogen without raising sample temperature.
15. End the cooling program, and allow controlled rate freezer to
return to RT before turning off.
3.4 Thawing
Human ASCs
1. To thaw ASCs frozen in 0.5-cc straws:
(a) Remove a straw from liquid nitrogen, and thaw its content
at RT for ~3 min by horizontally placing it on a Kimwipe
under the hood.
(b) Wipe down the outside of the straw with 70% ethanol, and
aseptically cut off both ends.
(c) Release cells into a 15-mL tube by pushing on cotton end
with a long blunt needle.
2. To thaw ASCs frozen in cryovials:
(a) Remove a cryovial from liquid nitrogen and hold in air for
20 s. Then, partially immerse it in a water bath at 37
C,
and swirl until ice melts (see Note 10).
(b) Wipe down the outside of the cryovial with 70% ethanol.
(c) Gently mix cell suspension under the hood, and transfer
to a 15-mL centrifuge tube.
3. To dilute cryoprotectants, add an equal amount of defined
cryopreservation medium without penetrating cryoprotectants
(1:1 dilution), gently swirl the tube, and hold at RT for 5 min.
4. Repeat the 1:1 dilution step by adding an equal amount of
defined cryopreservation medium without penetrating cryoprotectants, gently swirl the tube, and hold at RT for 5 min.
5. For the final dilution step, add excess amount (e.g., 8–10 mL)
of plain HEPES-buffered DMEM/F-12, gently mix, and wait
for 5 min.
6. Pellet ASCs by centrifugation at 300 Â g for 10 min.
7. Aspirate supernatant, resuspend ASCs in appropriate medium,
and determine cell number and viability.
564
Melany Lo ´ pez and Ali Eroglu