5. When in vitro expanding, trypsinize ASCs upon reaching 80%
confluency. To do so, first replace culture medium with versene. Next, aspirate versene, and add an appropriate volume of
defined trypsin-EDTA solution at RT. Observe detachment of
ASCs under a microscope. Do not wait until complete detachment of ASCs. Facilitate detachment by tapping the culture
dish. After approximately 10 min, add an equivalent amount of
soybean trypsin inhibitor to stop trypsin activity. Complete cell
detachment by aspirating and forcefully releasing cell suspension on culture surface. Finally, transfer cell suspension in a
centrifuge tube, and pellet the cells at 300 Â g for 10 min.
6. Discard supernatant, and resuspend the ASC pellet in xenofree-defined cryopreservation medium containing no
penetrating cryoprotectants. Perform cell count and centrifuge
at 300 Â g for 10 min.
7. Discard supernatant and resuspend the ASC pellet in fresh
xeno-free-defined cryopreservation medium containing no
penetrating cryoprotectants.
8. Add dropwise an equal volume of the xeno-free-defined cryopreservation medium containing 2Â penetrating cryoprotectants while swaying the tube to gently mix the cell suspension
(final penetrating cryoprotectant concentrations: 5% DMSO
and 5% EG).
9. Allow the cells to equilibrate with 1Â cryopreservation
medium at RT for 10 min and load the cells into 0.5-cc straws
or cryovials during the equilibration period as instructed next.
10. Gently mix the cell suspension and aseptically load 0.5-cc
straws under the hood in the following order:
(a) Connect a 1-mL syringe to the cotton-plugged end of a
0.5-cc straw via short silicon tubing.
(b) Aspirate 1-cm column of 1Â cryopreservation medium.
(c) Aspirate 1-cm air column.
(d) Aspirate 7–8-cm cell suspension column.
(e) Finally, aspirate 2–3-cm air column. This should result in
wetting white powder at the cotton-plugged end with the
first 1-cm column of the cryopreservation medium.
(f) Seal both ends of the straw with double impulse sealer.
(g) When using cryovials, mix and dispense 1 mL cell suspension in each cryovial.
11. At the end of the equilibration time, place two straws (or two
cryovials) in each sample holder, introduce the sample holders
into controlled-rate freezer, and reactivate the freezing program to cool the samples from 0
C to the seeding temperature
Chemically Defined Cryopreservation of Human ASCs
563
confluency. To do so, first replace culture medium with versene. Next, aspirate versene, and add an appropriate volume of
defined trypsin-EDTA solution at RT. Observe detachment of
ASCs under a microscope. Do not wait until complete detachment of ASCs. Facilitate detachment by tapping the culture
dish. After approximately 10 min, add an equivalent amount of
soybean trypsin inhibitor to stop trypsin activity. Complete cell
detachment by aspirating and forcefully releasing cell suspension on culture surface. Finally, transfer cell suspension in a
centrifuge tube, and pellet the cells at 300 Â g for 10 min.
6. Discard supernatant, and resuspend the ASC pellet in xenofree-defined cryopreservation medium containing no
penetrating cryoprotectants. Perform cell count and centrifuge
at 300 Â g for 10 min.
7. Discard supernatant and resuspend the ASC pellet in fresh
xeno-free-defined cryopreservation medium containing no
penetrating cryoprotectants.
8. Add dropwise an equal volume of the xeno-free-defined cryopreservation medium containing 2Â penetrating cryoprotectants while swaying the tube to gently mix the cell suspension
(final penetrating cryoprotectant concentrations: 5% DMSO
and 5% EG).
9. Allow the cells to equilibrate with 1Â cryopreservation
medium at RT for 10 min and load the cells into 0.5-cc straws
or cryovials during the equilibration period as instructed next.
10. Gently mix the cell suspension and aseptically load 0.5-cc
straws under the hood in the following order:
(a) Connect a 1-mL syringe to the cotton-plugged end of a
0.5-cc straw via short silicon tubing.
(b) Aspirate 1-cm column of 1Â cryopreservation medium.
(c) Aspirate 1-cm air column.
(d) Aspirate 7–8-cm cell suspension column.
(e) Finally, aspirate 2–3-cm air column. This should result in
wetting white powder at the cotton-plugged end with the
first 1-cm column of the cryopreservation medium.
(f) Seal both ends of the straw with double impulse sealer.
(g) When using cryovials, mix and dispense 1 mL cell suspension in each cryovial.
11. At the end of the equilibration time, place two straws (or two
cryovials) in each sample holder, introduce the sample holders
into controlled-rate freezer, and reactivate the freezing program to cool the samples from 0
C to the seeding temperature
Chemically Defined Cryopreservation of Human ASCs
563