We are trying to isolate adipocytes from human adipose tissue, using collagenase. We cut tissue into pieces and mince using a scalpel. After that we incubate tissue with 1-2 ml of 1.5mg/ml collagenase at 37 degrees for 45min-1 hour on a rotor. We then pass tissue/collagenase solution through a mesh (we have tried both cotton mesh and a 70um cell strainer) and centrifugate the solution at 300g for 5 minutes. We get the SVF pellet and the adipocyte ring, as we should.
We then isolate the adipocyte ring and wash a couple of times with PBS (have also tried without washing) and then we transfer the ring to a plate with DMEM, high glucose, 1% pen/strep, 10% FBS. The problem actually comes when we want to check the cells on the microscope.
There are a number of cells that seem to have the size of an adipocyte, but only a few, There are also bigger fat (oily) drops. Adipocytes tend to attach to these drops. There are also a lot of much smaller cells (they look like the size of a white blood cell or an endothelial cell). Adipocytes are always a few. All the other small cells seem too small to be adipocytes. However, incubation with collagenase seems to have worked fine and we get a really nice adipocyte ring.
We have tried to start with 500-2000 mg of adipose tissue and we only finally get very few adipocytes. Any ideas?
We were able to successfully isolate adipocytes but had to carefully optimise the protocol. Several parameters such as grade and brand of collegenase, incubation buffer and seperation method can qualitatively and quantitatively affect your yield. I can suggest you try to follow our protocol closely:
http://www.nature.com/ijo/journal/v31/n4/full/0803462a.html#bib14
Good luck.
Gulrez
Addition of adenosine during the isolation procedure might help retain the integrity of the adipocytes. You could refer to the following article for clarity:
http://www.jlr.org/content/47/8/1859.long
Thank you for your answers. We tried once more without a strainer. We minced tissue a little bit more and filtered only through a cotton mesh. Centrifugation was done at 100g for 5 minutes. We always get some oil after centrifugation. I guess this is due to adipocyte death..
The results seem to be a little bit better this time. We can actually see a lot of round cells (smaller and larger). I guess that they are probably all adipocytes of various sizes. Our adipocyte ring seems to be "of good quality". I don't think we have SVF cells in the white top ring. However, we will probably have to stain cells to be sure.....
This is the protocol we use for Lipolysis in vitro.
Important notes before starting:
- The mature adipocytes (single droplet fat cells) are extremely sensitive to temperature and strong light from the microscope can make them blow up as a balloon. Watch them shortly and with soft light.
- All solutions have to be warmed at 37 oC
- All the material (pipettes, tubes and tips) used before lysis the cells have to be siliconiced previously with Sigmacote (Sigma SL-2)
1. Rinse the adipose tissue in saline (0.85 % NaCl) previously warmed at 37 oC
2. Weight the tissue
3. Cut the tissue in small fragments (two or three per pad) and put in 50 ml tube whit 3 ml Buffer A (Kerbs-Ringer HEPES, 1 mM KH2PO4, 1% BSA) containing 1 mg/ml collagenase (Sigma C-6885)
4. Incubate 1 h in 37 °C whit shaking (the best place is the bacteria incubator)
5. Check time to time the liberation of the fat cell by increasing the turbidity
6. Spin 2 min. 1200 rpm in swing arm rotor centrifuge at ~30 oC
7. Remove the infranatant using a siliconiced glass Pasteur pipette connected to the vacuum
8. Add 10 ml of Buffer B (Kerbs-Ringer HEPES, 100 µM KH2PO4 ,1% BSA)
9. Spin 2 min. 1200 rpm
10. Remove the infranatant and add 10 ml of Buffer B
11. Filter the suspension through nylon mesh (70 µm, Falcon 2350)
12. Wash the tube and nylon mesh with 10 ml of Buffer B
13. Spin 2 min. 1200 rpm
14. Remove carefully the infanatant whit aspiration
15. Wash one more time with 10 ml of Buffer B
16. Resuspend the cells in 1ml of Buffer B
17. Wash two times more with Buffer B
18. Count the cell number and dilute to 0.7x106 cell/ml with Buffer B
19. Add into the tube 500 µl of Buffer C (Kerbs-Ringer HEPES, 100 µM KH2PO4 with 4% BSA w/o Adenisine) with the appropriated treatments (CL316,243, Isoproterenol, etc) and 2 unit/ml adenosine deaminase (final concentration of adenosine deaminase 1U/ml)
Buffer A (Kerbs-Ringer HEPES, 1 mM KH2PO4, 1% BSA): 1 mM KH2PO4, 125 mM NaCl, 5m M KCl, 2.5 mM MgCl2, 2.5 mM CaCl2, 25 mM HEPES/NaOH pH 7.4, 2 mM Glucose, 0.2 mM Adenosine, 1% BSA)
5 ml 0.1 M KH2PO4
50 ml 1.25 M NaCl
5 ml 0.5 M KCl
5 ml 0.25 M MgCl2
5 ml 0.25 M CaCl2
50 ml 0.25 M HEPES pH 7.4
5 ml 0.2 M Glucose
5 µl 20 mM Adenosine
5 g BSA
Up to 500 ml with H2O and filter
Buffer B (Kerbs-Ringer HEPES, 100 µM KH2PO4 1% BSA): 100 µM KH2PO4, 125 mM NaCl, 5m M KCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 25 mM HEPES/NaOH pH 7.4, 0.2 mM Adenosine, 2 mM Glucose, 1% BSA)
0.5 ml 0.1 M KH2PO4
50 ml 1.25 M NaCl
5 ml 0.5 M KCl
5 ml 0.25 M MgCl2
5 ml 0.2.5 M CaCl2
50 ml 0.25 M HEPES pH 7.4
5 ml 0.2 M Glucose
5 µl 20 mM Adenosine
5 g BSA
Up to 500 ml with H2O and filter
Buffer C (Kerbs-Ringer HEPES, 100 µM KH2PO4, 4% BSA, w/o Adenosine): 100 µM KH2PO4, 125 mM NaCl, 5m M KCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 25 mM HEPES/NaOH pH 7.4, 2 mM Glucose, 4% BSA)
0.5 ml 0.1 M KH2PO4
50 ml 1.25 M NaCl
5 ml 0.5 M KCl
5 ml 0.25 M MgCl2
5 ml 0.25 M CaCl2
50 ml 0.25 M HEPES pH 7.4
5 ml 0.2 M Glucose
20 g BSA
Up to 500 ml with H2O and filter
Buffer D (Kerbs-Ringer HEPES, 100 µM KH2PO4, w/o BSA): 125 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 100 µM KH2PO4, 2.5 mM MgCl2, 25 mM HEPES/NaOH pH 7.4, 0.2 mM Adenosine, 2 mM Glucose)
0.5 ml 0.1 M KH2PO4
50 ml 1.25 M NaCl
5 ml 0.5 M KCl
5 ml 0.25 M MgCl2
5 ml 0.25 M CaCl2
50 ml 0.25 M HEPES pH 7.4
5 ml 0.2 M Glucose
5 µl 20 mM Adenosine
Up to 500 ml with H2O and filter
Stock solutions:
· 0.1 M KH2PO4 (x100 or x1000)
1.361 g/100 ml
· 1.25 M NaCl (x10)
36.525 g/500 ml
· 0.5 M KCl (x100)
3.728 g/100 ml
· 0.25 M MgCl2 (x100)
5.083 g/100 ml
· 0.2.5 M CaCl2 (x100)
3.676g/100 ml
· 0.25 M HEPES pH 7.4 (x10)
29.79 g/500 ml and adjust pH to 7.4 with NaOH
· 0.2 M Glucose (x100)
3.604 g/100 ml
· 20 mM Adenosine (x100,000)
0.534 g/100 ml
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