10. Aspirate supernatant without disturbing the cell pellet (see
Note 6).
11. To eliminate red blood cells, resuspend the cell pellet in 20 mL
of red blood cell lysis buffer. Incubate at RT for 10 min.
12. Centrifuge at 300 Â g for 10 min and aspirate cell lysis buffer.
13. Resuspend SVF in HBSS and pass through a 40-μm cell
strainer.
14. Adjust the volume of cell suspension such that each FicollPaque gradient tube receives 9 mL cell suspension (total volume per gradient: 13 mL).
15. Hold a gradient tube containing 4-mL Ficoll-Paque at a 45
angle, and slowly add 9 mL cell suspension to form a layer
without any mixing. Proper layering is critical for successful cell
separation.
16. Centrifuge at 400 Â g for 30 min.
17. Aspirate the top HBSS fraction (~8 mL) above the white band
of cells found at the gradient interface and discard.
18. Carefully aspirate the white band of cells (2–5 mL), and release
into a 50-mL tube containing 25 mL HBSS.
19. Centrifuge at 300 Â g for 10 min.
20. Repeat the washing step by resuspending the cell pellet in fresh
HBSS to eliminate Ficoll-Paque taken while aspirating the
white band of cells at the interphase.
21. Centrifuge at 300 Â g for 10 min, resuspend the cell pellet in
HBSS, and determine cell count and viability using trypan blue
staining or similar approaches. After this step, the isolated cells
can be first subjected to sorting (optional) and then be cryopreserved or expanded in vitro.
3.2 Magnetic Cell
Sorting (Optional)
To further enrich ASCs, the cells collected by density gradient
separation can be subjected to magnetic sorting and negative selection. To this end, unwanted endothelial cells (CD31
+ ) and leukocytes (CD45
+ ) can be labeled first with FITC-conjugated antiCD31 and anti-CD45 antibodies, and then with MACS antiFITC microbeads. When applied to MACS LD columns placed
on a MidiMACS separator, the magnetically labeled CD31
+ (endothelial cells) and CD45
+ (leukocytes) are retained in the columns,
while unlabeled CD31
À CD45
À ASCs are passed through and collected in a tube for subsequent use.
1. Wipe down the MidiMACS separator with 70% ETOH, and
introduce under the hood.
2. Resuspend approximately 10
7 cells collected by density gradient separation in 100 μL of column buffer, and label with antiChemically Defined Cryopreservation of Human ASCs
561
Note 6).
11. To eliminate red blood cells, resuspend the cell pellet in 20 mL
of red blood cell lysis buffer. Incubate at RT for 10 min.
12. Centrifuge at 300 Â g for 10 min and aspirate cell lysis buffer.
13. Resuspend SVF in HBSS and pass through a 40-μm cell
strainer.
14. Adjust the volume of cell suspension such that each FicollPaque gradient tube receives 9 mL cell suspension (total volume per gradient: 13 mL).
15. Hold a gradient tube containing 4-mL Ficoll-Paque at a 45
angle, and slowly add 9 mL cell suspension to form a layer
without any mixing. Proper layering is critical for successful cell
separation.
16. Centrifuge at 400 Â g for 30 min.
17. Aspirate the top HBSS fraction (~8 mL) above the white band
of cells found at the gradient interface and discard.
18. Carefully aspirate the white band of cells (2–5 mL), and release
into a 50-mL tube containing 25 mL HBSS.
19. Centrifuge at 300 Â g for 10 min.
20. Repeat the washing step by resuspending the cell pellet in fresh
HBSS to eliminate Ficoll-Paque taken while aspirating the
white band of cells at the interphase.
21. Centrifuge at 300 Â g for 10 min, resuspend the cell pellet in
HBSS, and determine cell count and viability using trypan blue
staining or similar approaches. After this step, the isolated cells
can be first subjected to sorting (optional) and then be cryopreserved or expanded in vitro.
3.2 Magnetic Cell
Sorting (Optional)
To further enrich ASCs, the cells collected by density gradient
separation can be subjected to magnetic sorting and negative selection. To this end, unwanted endothelial cells (CD31
+ ) and leukocytes (CD45
+ ) can be labeled first with FITC-conjugated antiCD31 and anti-CD45 antibodies, and then with MACS antiFITC microbeads. When applied to MACS LD columns placed
on a MidiMACS separator, the magnetically labeled CD31
+ (endothelial cells) and CD45
+ (leukocytes) are retained in the columns,
while unlabeled CD31
À CD45
À ASCs are passed through and collected in a tube for subsequent use.
1. Wipe down the MidiMACS separator with 70% ETOH, and
introduce under the hood.
2. Resuspend approximately 10
7 cells collected by density gradient separation in 100 μL of column buffer, and label with antiChemically Defined Cryopreservation of Human ASCs
561