Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants

Materials and Reagents

1. Pipettes tips (Dutscher, ClearLine^®, catalog numbers: 037660CL (10 µl); 032260CL (200 µl); 027120CL (1,000 µl))
   
2. 50 ml conical tubes (Corning, Falcon^®, catalog number: 352070 )
   
3. 20 ml syringe (Terumo, catalog number: SS-20ES1 )
   
4. Sterile individually packaged 5 ml graduated pipettes (Corning, Falcon^®, catalog number: 357543 )
   
5. Sterile 150 x 20 mm cell culture polystyrene Petri dish (Thermo Fisher Scientific, Thermo Scientic^TM, catalog number: 168381 )
   
6. 10 ml syringe (Terumo, catalog number: SS-10ES1 )
   
7. Sterile individually needle 21 G x 1^1/2” (Terumo, catalog number: NN-2138R )
   
8. 15 ml conical tubes (Greiner Bio One International, catalog number: 188271 )
   
9. 0.22 µm syringe filter (Dutscher, catalog number: 051732 )
   
10. 96 wells microplate clear flat bottom (Thermo Fisher Scientific, Thermo Scientic^TM, catalog number: 269620 )
    
11. 50 ml syringe (Terumo, catalog number: SS-50L1 )
    
12. Filtration unit for sterilization, Stericup 500 ml (Merck, catalog number: SCGPU05RE )
    
13. Clinicell^® 25 cassette (Mabio International, catalog number: 00109 )
    
14. RNA lysis buffer (Quiazol, QIAGEN, catalog number: 79306 )
    
15. Free glycerol reagent (Sigma-Aldrich, catalog number: F6428 )
    
16. Wako NEFA (SOBIODA, catalog numbers: W1W434-91795 and W1W436-91995 )
    
17. Lactate FS (Diasys Diagnostics, catalog number: 1400199109 )
    
18. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
    
19. Glucose GOD FS (Diasys Diagnostics, catalog number: 1250099100 )
    
20. Gentamycin (Sigma-Aldrich, catalog number: G1272 )
    
21. ECBM buffer (PromoCell, catalog number: C-22210 )
    
22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
    
23. HEPES 1 M (PAA, catalog number: S11-001 )
    
24. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    
25. Krebs Ringer powder (Sigma-Aldrich, catalog number: K4002 )
    
26. (-)Isoproterenol hydrochloride (Sigma-Aldrich, catalog number: I6504 )
    
27. Human alpha-ANF(1-28) (R&D Systems, catalog number: 1906/1 )
    
28. ECBM medium (see Recipes)
    
29. BSA free fatty acid 20% (see Recipes)
    
30. Adipose tissue explant media (ATEM) (see Recipes)
    
31. Krebs-Ringer Bicarbonate HEPES 0.1% BSA (KRBHA) (see Recipes)
    
32. Isoproterenol stock solution (see Recipes)
    
33. ANP stock solution at 200 µM (see Recipes)
    

Equipment

1. P20 pipetman (Gilson, catalog number: F123600 )
   
2. P200 pipetman (Gilson, catalog number: F123601 )
   
3. P1000 pipetman (Gilson, catalog number: F123602 )
   
4. Laminar flow hood (Faster, model: BHA36 )
   
5. Sterile steel stainless scissors, 16 cm, straight (Dutscher, catalog number: 005055 )
   
6. Sterile stainless steel dressing forceps 11.5 cm, straight (Dutscher, catalog number: 711202 )
   
7. Stainless steel round-point needle 14 G x 6” (Dutscher, catalog number: 075515 )
   
8. Refrigerated tabletop centrifuge for 15 and 50 ml conical tube (Eppendorf, model: 5810 R )
   
9. 37 °C, 5% CO₂ water jacketed incubator (Thermo Fisher Scientific, Thermo Scientic^TM, model: HeraCell^TM 150i )
   
10. Pipettor PipetGirl (Integra Biosciences, catalog number: 155 021 )
    
11. Benchtop dry bath (Thermo Electron LED, catalog number: D-63505 )
    
12. Autoclave
    
13. -20 °C freezer
    
14. 100 ml beaker (Corning, PYREX^®, catalog: 1000-100 )
    
15. 1 L beaker (Corning, PYREX^®, catalog: 1000-1L )
    
16. Microplate spectrophotometer (Labsystem, model: iEMS Reader MF )
    
17. pH-meter (Mettler-Toledo, model: EL20 )
    

Software

1. Excel 2013
   
2. GraphPad Prism6 software
   

Procedure

Notes:

1. Pre-warm KRBHA buffer and ATEM (see Recipes) to 37 °C prior to the experiment
   
2. This protocol was performed in sterile conditions under a laminar flow hood.

1. Adipose tissue explant preparation (Figure1)
   1. Cut adipose tissue into pieces of around 1-2 cm³ and put it in a 50 ml conical tubes (Figure 1A). Do not exceed 25 ml of AT explant for each 50 ml conical tube (this amount is sufficient to perform 4-5 x Clinicell AT explant)
      
   2. Cut with scissors for 2 min (Figure 1B).
      
   3. Add 20 ml of KRBHA, close the tube, and mix gently by inverting the tube 3-4 times.
      
   4. Centrifuge the tube at 100 x g for 2 min (Figure 1C).
      
   5. Remove the infranatant by using a 14 G stainless cannula and a 20 ml syringe (Figures H-I).
      
   6. Repeat Steps A2-A6 until AT explant is able to be pipetted with a 5 ml plastic pipet.
      
   7. When AT explants are sufficiently cut, replace the KRBHA by 20 ml of ATEM (see Recipes).
      
   8. Close the tube, mix gently by inverting the tube and leave it for 15 min at 37 °C in the benchtop dry bath.
      
   9. Remove the infranatant by using the 14 G stainless cannula and a 20 ml syringe (Figures G-I).
      
      
      Figure 1. Preparation of adipose tissue explants. A. AT pieces before mincing; B. First 2 min mincing; C. AT explants after a first mincing, washing and centrifugation; D-F. Each photograph shows the aspect of the adipose tissue explant after 2 min mincing with scissors and after ATEM addition and 100 x g centrifugation. G-I. AT explants after last ATEM wash, 15 min rest and infranatant removal using a 14 G stainless cannula and a 20 ml syringe.
      
   
   
2. Clinicell filling (Figure 2)
   1. Under the hood, take a 150 mm sterile Petri dish and use the lower part.
      
   2. Prepare 10 ml of pre-warmed ATEM in a 50 ml conical tube for each Clinicell. For lipolysis with isoproterenol and ANP, the final concentrations used are typically 1 and 0.5 µM respectively. Taking into account that the volume of explant represents 1/3 of the total cassette volume, the real concentration of the lipolytic drug in the ATEM should be then 1.33 and 0.667 µM for isoproterenol and ANP respectively.
      
   3. Fill a 10 ml syringe connected with a 21 G needle.
      
   4. Open the Clinicell by one side (be cautious to keep the cap sterile) and open a half turn the cap of the other side in order to fill the Clinicell with AT explants (Figure 2B).
      
   5. Take the AT explant under the hood and aspirate the ATEM with the14 G stainless cannula and a 20 ml syringe.
      
   6. Pipette 3.5 ml of AT explant with a 5 ml pipette connected to a pipettor (Figure 2B).
      
   7. Completely fill the Clinicell with the ATEM using the 10 ml syringe connected to the 21 G needle by one side and by tilting it lightly (Figures 2E-2F). When the level of media is nearly to the top of the other side (Figure 2G), close this side and completely fill the cassette and close the second side (Figure 2H).
      
   8. Gently mix AT explant and media by tilting the Clinicell by rotating movement and lay it horizontally in the 150 mm Petri dish (Figure 2I).
      
   9. Put all the system in the incubator (37 °C, 5% CO₂) and leave it for the time you need. Typically 4 h incubation time is sufficient to study short term lipolysis. The incubation time could be extended to 48 h for studying long term effect on adipose tissue stroma cells.
      
      
      Figure 2. Clinicell cassette filling. A. Disposition of the Clinicell before filling; B-D. Filling of the Clinicell with AT explants with a 5 ml pipet; E-H. Complete filling of the Clinicell with ATEM with a 10 ml syringe connected to a 21 G needle; I. Disposition of the Clinicell in the Petri dish before incubation in the incubator.
      
      
3. Explant and media recovery (Figure 3)
   1. Prepare a 15 ml conical tube, a 10 ml syringe and the stainless cannula.
      
   2. Take the Clinicell out of the incubator and transfer under the laminar flow hood. After tilting vigorously (Figure 3B) open one side, put it vertically above the 15 ml conical tube, then open the second side and let the explant and the media pour out (Figures 3C-3E).
      
   3. Centrifuge the tube at 100 x g for 2 min.
      
   4. Recover the conditioned media by using the 10 ml syringe connected to the stainless steel cannula (Figure 3F). When done, vertically return the syringe and cannula, aspirate 1 ml of air, discard the cannula, connect a 0.22 µm filter to the syringe and filtrate the conditioned media Figure 3G).
      
   5. Make aliquots of the conditioned media and freeze it at -20 °C or -80 °C for long-term storage. Keep an aliquot of 300 to 500 µl at -20 °C to perform metabolite determination subsequently.
      
   6. The AT explants could either be dissociated using collagenase digestion to isolate the stroma-vascular fraction and the mature adipocytes or aliquoted by 0.5 ml and frozen and stored at -80 °C in 0.5 ml RNA lysis buffer.
      
      
      Figure 3. Recovery of AT explant and conditioned medium. A. Aspect of the explant 24 h after incubation; B-E. Recovery of AT explant and medium; F. Recovery of the conditioned medium after 100 x g centrifugation; G. Representative photography of the explants and the medium after filtration through a 0.22 µm filter.
      
   
   
4. Lipolysis and metabolites determinations (Figure 4)
   Glycerol and free fatty acid determination could be performed using the commercial kits described in the materials and reagents in 96-well microplates and a microplate spectrophotometer. Typically 20 µl of media is sufficient for glycerol and NEFA determination and 5 to 10 µl for glucose and lactate. The standard curves are performed in ATEM medium except for the glucose one which is done in PBS (the glucose concentration in the ATEM medium, which is around 5.5 mM, should be measured in order to be able to calculate the glucose consumption [difference between glucose concentration in ATEM and in AT explants conditioned medium]). Examples of data obtained are presented in Figure 4.
   
   
   Figure 4. Typical metabolites determination in conditioned media of human AT explants. Data represent the mean ± SEM of 4 to 8 experiments. Lipolysis was induced by either the β-agonist isoprenaline at 1 µM or ANP at 500 nM for the time indicated. Cont represents the basal condition without drugs.
   

Data analysis

Data calculation and analysis were performed using Excel 2013 and GraphPad Prism6 software.

Recipes

1. ECBM medium
   Add 2.5 ml of gentamycin solution for one 500 ml bottle of ECBM
   
2. BSA free fatty acid 20% (50 ml)
   1. Prewarm at 37 °C 40 ml of ECBM medium in a 100 ml beaker
      
   2. Weight 10 g of BSA in a 50 ml conical tube
      
   3. Add BSA on the top of ECBM medium and let it dissolve at 37 °C
      
   4. Adjust pH to 7.4 and complete to 50 ml with ECBM
      
   5. Sterilize with a 0.22 µm filter connected to a 50 ml syringe
      
   6. Store at 4 °C for several weeks
      
3. Adipose tissue explant medium (ATEM) (100 ml)
   1. Add 1 ml 1 M HEPES and 2.5 ml of 20% BSA free fatty acid to 97.5 ml ECBM
      
   2. Adjust to pH = 7.4
      
   3. Sterilize by 0.22 µm filtration on a 500 ml Stericup connected to vacuum
      
   4. Store at 4 °C
      
4. Krebs-Ringer Bicarbonate HEPES 0.1% BSA (KRBHA) (1 L)
   1. Weight 1,260 g of sodium bicarbonate in a 15 ml conical tube
      
   2. Weight 2.38 g of HEPES in a 15 ml conical tube
      
   3. In a 1 L beaker put the HEPES, the sodium bicarbonate and one vial of Krebs Ringer powder
      
   4. Add 950 ml of ddH₂O
      
   5. Add 5 ml of 20% BSA , FFA free (Recipe 2)
      
   6. Adjust pH to 7.4
      
   7. Complete the volume to 1 L with ddH₂O
      
   8. Sterilize by 0.22 µm filtration on a 500 ml Stericup connected to vacuum
      
   9. Store at 4 °C
      
5. Isoproterenol stock solution (10 mM)
   1. Weight 5 mg of isoproterenol and dilute in 2,018 µl of double distilled water
      
   2. Sterilize by 0.22 µm filtration
      
   3. Make 50 µl aliquots and store at -20 °C
      
6. ANP stock solution at 200 µM
   1. Dilute 0.5 mg of human ANP in 812 µl of sterile water
      
   2. Make 10 µl aliquots and store at -20 °C
      

Acknowledgments

This work was financially supported by the INSERM and Clarins Dermocosmetique. We acknowledge Grolleau-Raoux from aesthetic surgery department, Rangueil Hospital for the collection of AT samples. This approach was recently published by our groups (Gao et al., 2016). The authors have no conflict or competing interests to disclose.

References

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