2. Transfer an appropriate volume of lipoaspirate to a new sterile
container, and wash with an equal volume of warm HBSS
containing 1Â antibiotic-antimycotic mix three to four times
to remove excess blood cells. Rigorously shake the container
each time, and then allow phase separation for 3–5 min. The
adipose tissue will float above the HBSS now containing blood
cells. Carefully aspirate the HBSS (blood cells) with a 50-mL
pipette, and repeat the wash until the HBSS from the final wash
is clear.
3. Prepare a diluted collagenase solution from the 50% stock
solution prior to the enzymatic digestion step (final working
concentration of collagenase is 0.1%). Typically, the volume of
the enzyme solution required is half that of the lipoaspirate
volume. For example, if the lipoaspirate volume is 20 mL,
dilute 60 μL of the collagenase stock solution in 10 mL
HBSS containing typical concentrations of calcium and magnesium and 0.03% PVA. Next, add the diluted collagenase
solution to the washed lipoaspirate to have 0.1% final collagenase concentration, and mix it by shaking the bottle vigorously.
4. Place the lipoaspirate container in a 37
C shaking water bath at
~75 rpm for 40–60 min until the fat tissue appears smooth on
visual inspection.
5. During the collagenase treatment, prepare Ficoll-Paque gradients by dispensing 4 mL of Ficoll-Paque Premium 1.073 into
15-mL tubes (see Note 4). The gradients must be equilibrated
at room temperature (RT) before use.
6. After digestion, transfer the lipoaspirate container to the biosafety hood, and add 0.1 M EDTA stock solution to a final
concentration of 0.1 μM to stop collagenase activity. Next,
filter the digested lipoaspirate through a 1-mm sterile sieve
into 50-mL tubes (see Note 5).
7. Spin samples at 300 Â g in an appropriate centrifuge for 5 min
at RT. Thereafter, shake samples vigorously to disrupt the
pellet. This is done to complete the separation of the stromal
cells from the primary adipocytes.
8. Repeat centrifugation, and carefully remove top layer of oil and
fat, primary adipocytes (which will appear as a yellow layer of
floating cells), and the underlying layer of collagenase solution.
Leave behind small volume of solution above the pellet so the
cells of the stromal vascular fraction (SVF) are not disturbed.
The cell pellet usually includes a layer of dark red blood cells
and appears as a red/pink color.
9. Resuspend the cell pellet in 20 mL of HBSS containing
antibiotic-antimycotic mix, and centrifuge at 300 Â g for
5 min.
560
Melany Lo ´ pez and Ali Eroglu
container, and wash with an equal volume of warm HBSS
containing 1Â antibiotic-antimycotic mix three to four times
to remove excess blood cells. Rigorously shake the container
each time, and then allow phase separation for 3–5 min. The
adipose tissue will float above the HBSS now containing blood
cells. Carefully aspirate the HBSS (blood cells) with a 50-mL
pipette, and repeat the wash until the HBSS from the final wash
is clear.
3. Prepare a diluted collagenase solution from the 50% stock
solution prior to the enzymatic digestion step (final working
concentration of collagenase is 0.1%). Typically, the volume of
the enzyme solution required is half that of the lipoaspirate
volume. For example, if the lipoaspirate volume is 20 mL,
dilute 60 μL of the collagenase stock solution in 10 mL
HBSS containing typical concentrations of calcium and magnesium and 0.03% PVA. Next, add the diluted collagenase
solution to the washed lipoaspirate to have 0.1% final collagenase concentration, and mix it by shaking the bottle vigorously.
4. Place the lipoaspirate container in a 37
C shaking water bath at
~75 rpm for 40–60 min until the fat tissue appears smooth on
visual inspection.
5. During the collagenase treatment, prepare Ficoll-Paque gradients by dispensing 4 mL of Ficoll-Paque Premium 1.073 into
15-mL tubes (see Note 4). The gradients must be equilibrated
at room temperature (RT) before use.
6. After digestion, transfer the lipoaspirate container to the biosafety hood, and add 0.1 M EDTA stock solution to a final
concentration of 0.1 μM to stop collagenase activity. Next,
filter the digested lipoaspirate through a 1-mm sterile sieve
into 50-mL tubes (see Note 5).
7. Spin samples at 300 Â g in an appropriate centrifuge for 5 min
at RT. Thereafter, shake samples vigorously to disrupt the
pellet. This is done to complete the separation of the stromal
cells from the primary adipocytes.
8. Repeat centrifugation, and carefully remove top layer of oil and
fat, primary adipocytes (which will appear as a yellow layer of
floating cells), and the underlying layer of collagenase solution.
Leave behind small volume of solution above the pellet so the
cells of the stromal vascular fraction (SVF) are not disturbed.
The cell pellet usually includes a layer of dark red blood cells
and appears as a red/pink color.
9. Resuspend the cell pellet in 20 mL of HBSS containing
antibiotic-antimycotic mix, and centrifuge at 300 Â g for
5 min.
560
Melany Lo ´ pez and Ali Eroglu