4 Notes
1. GMP-grade collagenase is available for clinical applications
(e.g., Serva collagenase NB 6, cat. no. 17458).
2. To reduce osmotic stresses, it is recommended to first resuspend a cell pellet in xeno-free-defined cryopreservation
medium without penetrating cryoprotectants and then dropwise add equal volume of xeno-free-defined cryopreservation
medium containing 2Â penetrating cryoprotectants to have
final xeno-free-defined cryopreservation medium consisting
of 5% DMSO, 5% EG, 3 mM reduced glutathione, 5 mM
ascorbic acid 2-phosphate, 0.25 M trehalose, 2% PVA, 5%
ficoll, and 0.1 mM EGTA in HEPES-buffered DMEM/F-12.
3. Working with human tissues and cells: all work with human
cells and tissues should be done according to Biosafety Level
2 (BSL-2) practices and containment. It is necessary to obtain
approval from the institutional biosafety committee and
develop standard operating procedures based on guidelines
for BSL-2. Extreme care must be taken to avoid aerosolproducing procedures, spilling, and splashing when working
with any of these materials. Pathogens should be presumed
in/on all equipment and devices that come into direct contact
with any of these materials. All human material should be
decontaminated by autoclaving or disinfection before
discarding.
4. To preferentially isolate mesenchymal stem cells, a density gradient separation using Ficoll-Paque Premium 1.073 has been
suggested [22, 23], although other formulations also give
satisfactory results.
5. To avoid spilling of the digested lipoaspirate, the metal sieve
with 1-mm pores could be placed on top of a sterile funnel that
in turn could be placed on top of a 50-mL tube.
6. When aspirating, the tip of the pipette should aspirate from the
top so that the oil is removed as thoroughly as possible.
7. Cooling and warming rates can be better controlled when
using a controlled rate freezer. Importantly, a controlled rate
freezer allows deliberate seeding of extracellular ice at a programmed temperature close to the freezing point of a given
cryopreservation medium. Although not systematically compared, we also obtained satisfactory viability rates after freezing
of human ASCs in Mr. Frosty freezing container that provides
cooling rates around 1
C/min upon placing in a À80
C
freezer. It is important to fill Mr. Frosty with 250 mL isopropanol and ensure that it is at RT before transferring to a À80
C
freezer. After overnight cooling to À80
C, samples should be
Chemically Defined Cryopreservation of Human ASCs
565
1. GMP-grade collagenase is available for clinical applications
(e.g., Serva collagenase NB 6, cat. no. 17458).
2. To reduce osmotic stresses, it is recommended to first resuspend a cell pellet in xeno-free-defined cryopreservation
medium without penetrating cryoprotectants and then dropwise add equal volume of xeno-free-defined cryopreservation
medium containing 2Â penetrating cryoprotectants to have
final xeno-free-defined cryopreservation medium consisting
of 5% DMSO, 5% EG, 3 mM reduced glutathione, 5 mM
ascorbic acid 2-phosphate, 0.25 M trehalose, 2% PVA, 5%
ficoll, and 0.1 mM EGTA in HEPES-buffered DMEM/F-12.
3. Working with human tissues and cells: all work with human
cells and tissues should be done according to Biosafety Level
2 (BSL-2) practices and containment. It is necessary to obtain
approval from the institutional biosafety committee and
develop standard operating procedures based on guidelines
for BSL-2. Extreme care must be taken to avoid aerosolproducing procedures, spilling, and splashing when working
with any of these materials. Pathogens should be presumed
in/on all equipment and devices that come into direct contact
with any of these materials. All human material should be
decontaminated by autoclaving or disinfection before
discarding.
4. To preferentially isolate mesenchymal stem cells, a density gradient separation using Ficoll-Paque Premium 1.073 has been
suggested [22, 23], although other formulations also give
satisfactory results.
5. To avoid spilling of the digested lipoaspirate, the metal sieve
with 1-mm pores could be placed on top of a sterile funnel that
in turn could be placed on top of a 50-mL tube.
6. When aspirating, the tip of the pipette should aspirate from the
top so that the oil is removed as thoroughly as possible.
7. Cooling and warming rates can be better controlled when
using a controlled rate freezer. Importantly, a controlled rate
freezer allows deliberate seeding of extracellular ice at a programmed temperature close to the freezing point of a given
cryopreservation medium. Although not systematically compared, we also obtained satisfactory viability rates after freezing
of human ASCs in Mr. Frosty freezing container that provides
cooling rates around 1
C/min upon placing in a À80
C
freezer. It is important to fill Mr. Frosty with 250 mL isopropanol and ensure that it is at RT before transferring to a À80
C
freezer. After overnight cooling to À80
C, samples should be
Chemically Defined Cryopreservation of Human ASCs
565