21. 70% Ethanol.
22. CD45-FITC, antihuman monoclonal antibody (Miltenyi
Biotec).
23. CD31 FITC, recombinant human antibody (Miltenyi Biotec).
24. Column buffer: DPBS (pH 7.2) containing 0.5% PVA and
2 mM EDTA.
25. Leibovitz’s L-15 Medium (Gibco).
26. CTS Knockout Dulbecco’s modified Eagle’s medium/Ham’s
F-12 mixture (DMEM/F-12; Gibco).
27. DMEM/F-12, HEPES (Gibco).
28. 50% (w/v) collagenase stock solution (0.5 g/mL). Weigh out
1 g of type I collagenase and dissolve it in 2 mL of HBSS
containing calcium and magnesium. Sterile filter using a 0.2μm syringe filter, aliquot into sterile microcentrifuge tubes, and
store at À20
C or below.
29. 1000Â (0.1 M) EDTA stock solution. Dissolve 0.42 g EDTA
tetrasodium salt in 10 mL DPBS, sterile filter using a 0.2-μm
syringe filter, and store at 4
C.
30. Red blood lysis buffer. Prepare by dissolving 155 mM NH 4 Cl
(Sigma), 10 mM KHCO 3 (Sigma), and 0.1 mM EDTA in
ultrapure water (pH 7.3), and sterilize by filtering through a
0.2-μm polyethersulfone (PES) membrane filter.
31. HBSS containing 0.03% PVA. Dissolve 0.03 g PVA in HBSS
containing calcium and magnesium (sterile filter using a 0.2-μ
m syringe filter, and store at 4
C).
32. Xeno-free-defined
cryopreservation
medium
without
penetrating cryoprotectants. Add 3 mM reduced glutathione,
5 mM ascorbic acid 2-phosphate, 0.25 M trehalose, 2% PVA,
5% ficoll, and 0.1 mM EGTA to HEPES-buffered DMEM/F12, and filter under the hood using 0.2-μm membrane filter.
33. Xeno-free-defined cryopreservation medium containing
2Â penetrating cryoprotectants (see Note 2). Add 10%
DMSO, 10% EG, 3 mM reduced glutathione, 5 mM ascorbic
acid 2-phosphate, 0.25 M trehalose, 2% PVA, 5% ficoll, and
0.1 mM EGTA to HEPES-buffered DMEM/F-12, and filter
under the hood using 0.2-μm nylon membrane filter.
3 Methods
3.1 Isolation
of Human ASCs from
Lipoaspirate
1. With the proper personal protective equipment (PPE) on, take
specimen container and spray down with ethanol. Place inside
biosafety hood (see Note 3).
Chemically Defined Cryopreservation of Human ASCs
559
22. CD45-FITC, antihuman monoclonal antibody (Miltenyi
Biotec).
23. CD31 FITC, recombinant human antibody (Miltenyi Biotec).
24. Column buffer: DPBS (pH 7.2) containing 0.5% PVA and
2 mM EDTA.
25. Leibovitz’s L-15 Medium (Gibco).
26. CTS Knockout Dulbecco’s modified Eagle’s medium/Ham’s
F-12 mixture (DMEM/F-12; Gibco).
27. DMEM/F-12, HEPES (Gibco).
28. 50% (w/v) collagenase stock solution (0.5 g/mL). Weigh out
1 g of type I collagenase and dissolve it in 2 mL of HBSS
containing calcium and magnesium. Sterile filter using a 0.2μm syringe filter, aliquot into sterile microcentrifuge tubes, and
store at À20
C or below.
29. 1000Â (0.1 M) EDTA stock solution. Dissolve 0.42 g EDTA
tetrasodium salt in 10 mL DPBS, sterile filter using a 0.2-μm
syringe filter, and store at 4
C.
30. Red blood lysis buffer. Prepare by dissolving 155 mM NH 4 Cl
(Sigma), 10 mM KHCO 3 (Sigma), and 0.1 mM EDTA in
ultrapure water (pH 7.3), and sterilize by filtering through a
0.2-μm polyethersulfone (PES) membrane filter.
31. HBSS containing 0.03% PVA. Dissolve 0.03 g PVA in HBSS
containing calcium and magnesium (sterile filter using a 0.2-μ
m syringe filter, and store at 4
C).
32. Xeno-free-defined
cryopreservation
medium
without
penetrating cryoprotectants. Add 3 mM reduced glutathione,
5 mM ascorbic acid 2-phosphate, 0.25 M trehalose, 2% PVA,
5% ficoll, and 0.1 mM EGTA to HEPES-buffered DMEM/F12, and filter under the hood using 0.2-μm membrane filter.
33. Xeno-free-defined cryopreservation medium containing
2Â penetrating cryoprotectants (see Note 2). Add 10%
DMSO, 10% EG, 3 mM reduced glutathione, 5 mM ascorbic
acid 2-phosphate, 0.25 M trehalose, 2% PVA, 5% ficoll, and
0.1 mM EGTA to HEPES-buffered DMEM/F-12, and filter
under the hood using 0.2-μm nylon membrane filter.
3 Methods
3.1 Isolation
of Human ASCs from
Lipoaspirate
1. With the proper personal protective equipment (PPE) on, take
specimen container and spray down with ethanol. Place inside
biosafety hood (see Note 3).
Chemically Defined Cryopreservation of Human ASCs
559