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Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants
人体皮下脂肪组织外植体脂解作用的离体分析   

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Stem Cells
Jul 2017

Abstract

Most studies of human adipose tissue (AT) metabolism and functionality have been performed in vitro on isolated mature adipocyte or in situ using the microdialysis technique (Lafontan, 2012). However, these approaches have several limitations. The use of mature isolated adipocytes is limiting as adipocytes are not in their physiological environment and the collagenase digestion process could affect both adipocyte survival and functionality. While metabolic studies using microdialysis have brought the advantage of studying the lipolytic response of the adipose tissue in situ, it provides only qualitative measures but does not give any information on the contribution of different adipose tissue cell components. Moreover, the number of microdialysis probes that can be used concomitantly in one subject is limited and can be influenced by local blood flow changes and by the molecular size cut-off of the microdialysis probe. Here we present a protocol to assess adipose tissue functionality ex vivo in AT explants allowing the studies of adipose tissue in its whole context, for several hours. In addition, the isolation of the different cell components to evaluate the cell-specific impact of lipolysis can be performed. We recently used the present protocol and demonstrated that fatty acid release during lipolysis impacts directly on a specific cell subset present in the adipose tissue stroma-vascular compartment. This assay can be adapted to address other research questions such as the effects of hormones or drugs treatment on the phenotype of the various cell types present in adipose tissue (Gao et al., 2016).

Keywords: Human adipocyte biology (人类脂肪细胞生物学), Lipolysis (脂解作用), ex vivo (离体), Explant (外植体)

Background

Human white adipose tissue (WAT) plays a major role in body energy homeostasis. Adipocytes, specialized cells expressing specific lipid handling metabolic activities, constitute more than 90% of the volume of WAT (Lafontan, 2012). In addition to adipocytes, other cell types are present within human WAT e.g., vascular cells, immune cells (lymphocytes and macrophages) and progenitor cells involved in WAT remodeling and renewal. The metabolic activity of adipocytes is tightly controlled by the integration of both local and systemic pathways. Neurohumoral signals modulated in anabolic or catabolic conditions impact on the net adipocyte metabolic activity, i.e., energy storage or release. In post-prandial conditions, non-esterified fatty acids (NEFAs) originating from the hydrolysis of VLDL and chylomicron particles are taken up by the adipocytes and esterified to glycerol phosphate to form triglycerides packaged into a single lipid vacuole (Large et al., 2004). This process called lipogenesis, is mainly under the control of insulin. In conditions of energy demand such as exercise or fasting, hydrolysis of triacylglycerol through a process called lipolysis results in the release of glycerol and NEFAs into the circulation thereby providing energy to other tissues and organs. Lipolysis involves the sequential hydrolysis of triacylglycerol through the successive action of lipolytic enzymes, i.e., adipose triglyceride lipase, hormone sensitive lipase and monoacylglycerol lipase. In human adipocytes, lipolysis is mainly stimulated by catecholamines and atrial natriuretic peptide (ANP). Catecholamines mediate their pro-lipolytic effects through β-adrenoceptors (β1-AR and β2-AR), while alpha2-adrenoceptors are anti-lipolytic; ANP acts via NPR-A to activate lipolysis (Lafontan, 2012). It should be noted that in human AT the beta3-adrenergic receptor is almost not expressed and is not functional. The classical approach to evaluate adipocyte lipolytic responses was developed by Robdell (Robdell, 1964) who first described mature adipocyte isolation based on flotation after collagenase digestion. Although this technique is used worldwide, it presents several limitations. Firstly, the buoyancy of isolated mature adipocytes will prevent, with increasing time in vitro, their immersion into media and promote direct toxic effects through air contact, ultimately leading to cell damage and disintegration. Secondly, the isolation process per se alters adipocyte phenotype (Ruan et al., 2003). Thirdly, the isolated mature adipocytes are disconnected from their natural microenvironment including extracellular matrix and from other cell types (vascular cells, immune cells (lymphocytes and macrophages) and progenitor cells). Moreover, cytokines such as TNF-alpha and IL6, present in the AT microenvironment, are well described to impact adipocyte lipolysis. Finally, NEFAs originating from in situ lipolysis may have different fates: 1) release into the circulation, 2) re-esterification within the mature adipocytes, 3) potentially taken up by other cell types in the near vicinity of mature adipocytes including progenitor cells. Thus studies on isolated adipocytes do not take into account these different factors.

The present technique allows the study of the lipolytic responsiveness of mature adipocytes for a longer time in their natural context 1) in a closed culture chamber avoiding direct contact with air but with adequate and modulable gas exchange, 2) without the necessity of a collagenase digestion step and 3) in a maintained viable microenvironment. This approach was recently published by our groups and clearly demonstrate that lipolytic stimulation is associated with increased fatty acid uptake by the progenitor cells leading to enhanced adipogenic capacity (Gao et al., 2016).

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 ScienticTM, catalog number: 168381 )
  6. 10 ml syringe (Terumo, catalog number: SS-10ES1 )
  7. Sterile individually needle 21 G x 11/2” (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 ScienticTM, 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 ScienticTM, model: HeraCellTM 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

  1. Gao, H., Volat, F., Sandhow, L., Galitzky, J., Nguyen, T., Esteve, D., Åström, G., Mejhert, N., Ledoux, S., Thalamas, C., Arner, P., Guillemot, J. C., Qian, H., Rydén, M. and Bouloumié, A. (2016). CD36 is a marker of human adipocyte progenitors with pronounced adipogenic and triglyceride accumulation potential. Stem Cells 35(7): 1799-1814.
  2. Lafontan, M. (2012). Historical perspectives in fat cell biology: the fat cell as a model for the investigation of hormonal and metabolic pathways. Am J Physiol Cell Physiol 302(2): C327-359.
  3. Large, V., Peroni, O., Letexier, D., Ray, H. and Beylot, M. (2004). Metabolism of lipids in human white adipocyte. Diabetes Metab 30(4): 294-309.
  4. Robdel, M. (1964). Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239: 375-380.
  5. Ruan, H., Zarnowski, M. J., Cushman, S. W. and Lodish, H. F. (2003). Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes. J Biol Chem 278(48): 47585-93.

简介

人脂肪组织(AT)代谢的上havebeen执行的大多数研究和功能的体外上分离的脂肪细胞成熟或原位使用微透析技术(Lafontan,2012)。但是,这些方法有几个限制。成熟的分离的脂肪细胞的使用是有限的,因为脂肪细胞不在其生理环境中,胶原酶消化过程可能影响脂肪细胞的存活和功能。虽然微透析代谢研究带来了学习原位脂肪组织中的的脂肪分解反应的优点的它仅提供定性的措施,但并没有给出不同的脂肪组织细胞成分的贡献的任何信息。此外,可以在受试者中同时使用的微透析探针的数量是有限的,并且可以受到局部血流量变化和微透析样品的分子大小截断的影响。在这里,我们提出了一个协议,以评估对脂肪组织的功能的体外在AT植允许脂肪组织的研究中其整个范围内,几个小时。此外,可以评估脂肪分解的细胞特异性影响。直接在脂肪组织基质血管室。在脂肪组织中(Gao等人,2016)。

【背景】人类白色脂肪组织(WAT)在体内能量平衡中起主要作用。脂肪细胞是表达特异性脂质处理代谢活动的特化细胞,构成超过90%的WAT体积(Lafontan,2012)。除了脂肪细胞,其他细胞类型是人类WAT中存在的例如,血管细胞,免疫细胞(淋巴细胞和巨噬细胞)和祖细胞在WAT重构和更新参与。脂肪细胞的代谢活动受到局部和全身途径整合的严格控制。在合成代谢或分解代谢条件影响调制的净脂肪细胞的代谢活性神经体液信号, I.E. ,储能或释放。在餐后的条件下,从VLDL和乳糜微粒的粒子的水解的非酯化脂肪酸(NEFA)始发采取了由脂肪细胞和酯化甘油磷酸,以形成封装到单个脂质液泡(大等人的甘油三酯,2004)。这个过程称为脂肪生成,主要是在胰岛素的控制下。能源需求的条件:例如,通过这个过程被称为脂肪分解导致甘油和NEFA释放到循环中,从而其它组织和器官提供能量锻炼或禁食,三酰基甘油的水解。脂解作用涉及到甘油三酯通过脂解酶的连续作用顺序水解, I.E. ,脂肪甘油三酯的脂肪酶,激素敏感脂肪酶和单酰甘油脂肪酶。在人类脂肪细胞中,脂肪分解主要由儿茶酚胺和心房利钠肽(ANP)刺激。儿茶酚胺介导其通过β肾上腺素受体(β1-AR和β2-AR)亲脂解作用,而α-2肾上腺素受体是抗脂肪分解; ANP通过NPR-A起作用来激活脂肪分解(Lafontan,2012)。应该指出的是,在人AT中,β3-肾上腺素能受体不表达且不起作用。最经典的方法来评估其由Robdell(Robdell,1964年),谁首先描述了基于胶原酶消化后浮选成熟脂肪细胞分离脂肪细胞开发的脂肪分解反应。尽管这种技术在全世界范围内使用,但是它有一些局限首先,分离的成熟脂肪细胞的浮力将防止,随着时间的增加的体外它们浸入到介质,并促进通过空气接触直接的毒性作用,最终导致细胞损伤和崩解。其次,分离过程本身是脂肪细胞表型(Ruan et al。,2003)。第三,分离的成熟脂肪细胞从它们的天然微环境包括细胞外基质,并从其他细胞类型(血管细胞,免疫细胞(淋巴细胞和巨噬细胞)和祖细胞)断开。更以上,细胞因子:例如在AT TNF-α和IL-6,本微环境,有很好的描述来影响脂肪细胞脂解作用。最后,从NEFA始发原位脂解可以具有不同的命运:1)释放到循环中的成熟脂肪细胞内,2)再酯化,3)有可能在不久的附近采取由其他细胞类型成熟的脂肪细胞包括祖细胞。因此对分离的脂肪细胞的研究没有考虑到这些不同的因素。

本技术允许在其天然环境中1)成熟的脂肪细胞在较长时间的脂解反应性的研究在一个封闭的培养室避免与空气直接接触,而是与适当和可调制的气体交换,2)不具有胶原酶消化的必要性步骤和3)维持可行的微环境。通过祖细胞导致增强的脂肪生成能力(Gao等人,2016)。

关键字:人类脂肪细胞生物学, 脂解作用, 离体, 外植体

材料和试剂

  1. 移液器吸头(Dutscher,ClearLine,目录号:037660CL(10μl); 032260CL(200μl); 027120CL(1.000μl))。
  2. 50毫升锥形管(Coming,Falcon ,目录号:352070)
  3. 20毫升注射器(Terumo,目录号:SS-20ES1)
  4. 无菌独立包装的5毫升刻度移液器(Corning,Falcon ,目录号:357543)
  5. 无菌的150×20mm细胞培养聚苯乙烯培养皿(Thermo Fisher Scientific,Thermo Scientic TM,产品目录号:168381)
  6. 10毫升注射器(Terumo,产品目录号:SS-10ES1)
  7. 无菌个体针21 G x 1 1/2“(Terumo,目录号:NN-2138R)

  8. 15 ml锥形管(Greiner Bio One International,目录号:188271)
  9. 0.22μm注射器过滤器(Dutscher,目录号:051732)
  10. 96孔微量平板清洁平底(Thermo Fisher Scientific,Thermo Scientic TM,产品目录号:269620)
  11. 50毫升注射器(Terumo,目录号:SS-50L1)
  12. Stericup 500 ml(Merck,目录号:SCGPU05RE)
  13. Clinicell®25盒(Mabio International,目录号:00109)
  14. RNA裂解缓冲液(Quiazole,QIAGEN,目录号:79306)
  15. 游离甘油试剂(Sigma-Aldrich,目录号:F6428)。
  16. Wako NEFA(SOBIODA,产品目录号:W1W434-91795和W1W436-91995)。
  17. 乳酸FS(Diasys诊断,目录号:1400199109)。
  18. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537)。
  19. 葡萄糖GOD FS(Diasys Diagnostics,目录号:1250099100)
  20. 庆大霉素(Sigma-Aldrich,目录号:G1272)。
  21. ECBM缓冲液(PromoCell,目录号:C-22210)
  22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)。
  23. HEPES 1M(PAA,目录号:S11-001)
  24. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  25. 克雷布斯林格粉(西格玛奥德里奇,目录号:K4002)
  26. ( - )异丙肾上腺素盐酸盐(Sigma-Aldrich,目录号:I6504)
  27. 人α-ANF(1-28)(R&D Systems,目录号:1906/1)。
  28. ECBM中等(见食谱)
  29. BSA游离脂肪酸20%(见食谱)
  30. 脂肪组织外植体介质(ATEM)(见食谱)
  31. 癌症碳酸氢盐碳酸氢盐HEPES 0.1%BSA(KRBHA)(见食谱)
  32. 异丙肾上腺素储备液(见食谱)
  33. ANP储备液200μM(见食谱)

设备

  1. P20 pipetman(Gilson,目录号:F123600)
  2. P200移液器(Gilson,目录号:F123601)
  3. P1000移液器(吉尔森,目录号:F123602)
  4. 层流罩(更快,型号:BHA36)
  5. 无菌钢不锈钢剪刀,直径16厘米(杜卡迪,目录号:005055)

  6. 无菌不锈钢修整镊子直径11.5厘米(Dutscher,目录号:711202)
  7. 不锈钢圆形针14 G x 6“(Dutscher,目录号:075515)
  8. 用于15和50毫升锥形管的冷冻台式离心机(Eppendorf,型号:5810 R)
  9. 37℃,5%CO <子> 2 水夹套培养箱(赛默飞世尔科技,热Scientic TM ,型号:Heracell TM 150 1)
  10. Pipettor PipetGirl(Integra Biosciences,目录号:155 021)。
  11. 台式干浴(Thermo Electron LED,目录号:D-63505)
  12. 高压灭菌器
  13. -20°C冷冻机
  14. 100毫升烧杯(康宁公司,PYREX®,目录:1000-100)
  15. 1个烧杯中(Corning,PYREX ®,目录:1000-1L)
  16. 微孔板分光光度计(Labsystem,型号:iEMS Reader MF)
  17. pH计(梅特勒 - 托利多,型号:EL20)

软件

  1. Excel 2013
  2. GraphPad Prism6软件

程序

注意:


  1. 预热KRBHA缓冲液和ATEM(见食谱)到37°C
  2. 该协议在无菌条件下在层流罩下进行。


  1. 脂肪组织外植体的制备(图1)
    1. 1-2厘米3并将其放入50毫升锥形管中(图1A)。每50毫升锥形管不要超过25毫升的AT外植体(这个数量足以完成4-5 x Clinicell AT外植体)。

    2. 剪刀剪2分钟(图1B)
    3. 加入20毫升的KRBHA,关闭试管,轻轻混合,倒置试管3-4次。

    4. 100×g离心管2分钟(图1C)。
    5. 通过使用14g不锈钢套管和20ml注射器去除infranatant(图H-1)。
    6. 重复步骤A2-A6,直到可以用5毫升塑料移液管移取移液器。
    7. 当AT外植体充分切割时,用20ml ATEM替代KRBHA(见食谱)。
    8. 关闭试管,轻轻混匀,颠倒试管,在37℃的台式干浴中放置15分钟。
    9. 通过使用14 G不锈钢套管和20毫升注射器(图G-1)去除infranatant。


      图1.制备脂肪组织外植体A.切碎前的AT片段; B.开始2分钟切碎; C.在第一次切碎,洗涤和离心后的外植体中; d-F。每张照片显示2分钟后用剪刀和添加ATEM和100×g离心后的脂肪组织外植体的方面。 G-我。在最后一次ATEM洗涤后的外植体中,休息15分钟并使用14g不锈钢套管和20ml注射器除去infranatant。

  2. 诊所填充(图2)
    1. 在引擎盖下,取一个150毫米的无菌培养皿,并使用下部。
    2. 每个Clinicell准备10毫升的预热ATEM在一个50毫升的锥形管。对于异丙肾上腺素和ANP的脂解,终浓度通常分别为1和0.5μM。考虑到ATEM中盒的总体积分别为异丙肾上腺素和ANP的1.33和0.667μM。
    3. 填充与21 G针连接的10毫升注射器。

    4. 打开诊所的一侧和另一侧,以填补临床与AT外植体(图2B)
    5. 取下外罩下的AT外植体,用14G不锈钢插管和20毫升注射器抽吸ATEM。
    6. 移取3.5 ml的AT外植体和5 ml移液管连接移液器(图2B)。
    7. 使用连接到21G针的10ml注射器一侧完全填充诊所,并轻轻倾斜(图2E-2F)。当介质水平靠近另一侧的顶部(图2G)时,关闭此侧并完全填满盒子并关闭第二侧(图2H)。
    8. 通过旋转运动将诊所倾斜并将其水平放置在150mm培养皿中(图2I),轻轻混合AT外植体和培养基。
    9. 将所有系统放入培养箱(37°C,5%CO 2)中,并保留一段时间。通常4小时的孵育时间足以研究短期的脂肪分解。
      。为了研究脂肪组织基质细胞的长期效应,孵育时间可以延长至48小时
      “”
      图2.诊所盒带的填充。 A.在填充前处理诊所B-d。用5ml移液管用AT外植体填充诊所; E-H。使用连接到21G针的10ml注射器用ATEM完成诊所的填充; I.在培养箱中培养之前,将培养皿置于培养皿中。

  3. 外植体和培养基回收(图3)
    1. 准备15毫升锥形管,10毫升注射器和不锈钢插管。
    2. 将诊所从培养箱中取出,在层流罩下转移。大力倾斜(图3B)打开一侧后,把它竖直地15ml锥形管上方,然后打开第二侧,并让外植体和所述媒体倒出来(图3C-3E)。

    3. 100 g离心管2分钟。
    4. 使用连接到不锈钢插管的10毫升注射器回收条件培养基(图3F)。完成后,注射器和套管垂直返回,吸入1毫升空气,丢弃套管,将0.22微米的过滤器连接到注射器,并过滤条件培养基(图3G)。
    5. 在-20°C或-80°C下将等分的条件培养基进行长期储存。
      在-20°C保持300至500μl的等分试样进行代谢物测定。
    6. 所述AT外植体可以或者使用胶原酶消化以分离基质血管级分和成熟脂肪细胞被离解或在0.5ml RNA裂解缓冲液的加入0.5mL等分并冷冻并储存在-80℃。

      “”
      图3. AT外植体和条件培养基的回收A.孵育后24小时外植体的外观; B至E。 AT外植体和培养基的回收; F.在100×g离心后回收条件培养基; G.代表性摄影的外植体和介质经过0.22微米的过滤器过滤后。

  4. 脂解和代谢物测定(图4)。
    甘油和游离脂肪酸的测定可以使用96孔微量培养板和微孔板分光光度计进行。通常,20μl的培养基足以用于甘油和NEFA的测定,而对于葡萄糖和乳酸盐则为5至10μl。标准曲线进行在ATEM介质除了葡萄糖酮所有其在PBS(在ATEM培养基中的葡萄糖浓度,全部完成这大约是5.5毫米,如果为了进行测量,以便能够计算葡萄糖之间的葡萄糖消耗[差在ATEM和AT外植体条件培养基中的浓度])。所获得的数据的例子如图4所示。

    “”
    图4.人类AT外植体的条件培养基中的典型代谢物测定。数据表示4至8次实验的平均值±SEM。在1μM的β-激动剂异丙肾上腺素或在500nM的ANP诱导的脂解可持续指定的时间。 Cont代表没有药物的基础状况。

数据分析

使用Excel 2013和GraphPad Prism6软件进行数据计算和分析。

食谱

  1. ECBM介质
    加入2.5 ml庆大霉素溶液,每500 ml一瓶ECBM
  2. BSA游离脂肪酸20%(50毫升)
    1. 在37℃预热在100ml烧杯中的40ml ECBM培养基
    2. 重量10克BSA在50毫升锥形管

    3. 在ECBM培养基的顶部添加BSA,让其在37°C溶解
    4. 使用ECBM
      将pH值调节至7.4,完成至50 ml
    5. 使用连接到50毫升注射器的0.22微米过滤器消毒

    6. 在4°C储存几个星期
  3. 脂肪组织外植体培养基(ATEM)(100ml)
    1. 添加1毫升1M HEPES和2.5毫升的20%BSA游离脂肪酸到97.5毫升ECBM
    2. 调整到pH = 7.4
    3. 在与真空连接的500ml Stericup上通过0.22μm过滤消毒
    4. 在4°C储存
  4. 癌症碳酸氢盐碳酸氢盐HEPES 0.1%BSA(KRBHA)(1 L)
    1. 在一个15毫升的锥形管中加入1,260克碳酸氢钠。
    2. 重量2.38克的HEPES在一个15毫升的锥形管
    3. 在1升的烧杯中放入HEPES,碳酸氢钠和一瓶Krebs林格粉。
    4. 加入950ml的ddH 2 O
    5. 添加5毫升的20%BSA,无FFA(方案2)。
    6. 调整pH值到7.4
    7. 用ddH <2> O完成体积至1L
    8. 在与真空连接的500ml Stericup上通过0.22μm过滤消毒
    9. 在4°C储存
  5. 异丙肾上腺素储备液(10mM)
    1. 称重5毫克的异丙肾上腺素,并在2.018微升的双蒸水稀释。
    2. 通过0.22μm过滤消毒
    3. 制成50μl等分试样并储存在-20°C
  6. ANP原液200μM
    1. 在812μl无菌水中稀释0.5 mg人ANP。
    2. 制作10μl等分试样并储存在-20°C

确认

这项工作得到了INSERM和Clarins Dermocosmetique的财务支持。我们感谢Rangueil医院美容外科的Grolleau-Raoux收集AT样本。这个方法最近由我们的小组发布(Gao等人,2016)。作者没有冲突或利益冲突的披露。

参考

  1. 高,H.,长凳,F.,Sandhow,L.,Galitzky,J.,阮,T.,埃斯泰夫,D.,乻tr歮-H,G.,Mejhert,N.,勒杜,S.,Thalamas,C., Arner,P.,Guillemot,JC,Qian,H.,Rydén,M.和Bouloumié,A。(2016)。 CD36是具有显着的生脂和甘油三酯积累潜在的人类脂肪细胞祖细胞的标志物。 干细胞 35(7):1799-1814。
  2. Lafontan,M.(2012)。 脂肪细胞生物学历史观脂肪细胞作为激素和代谢途径 上午生理学杂志细胞生理学 302(2)的研究的模型:C327 -359。
  3. Large,V.,Peroni,O.,Letexier,D.,Ray,H.and Beylot,M。(2004)。在人类脂肪细胞白脂质的代谢的影响。 糖尿病代谢 30(4):294-309。
  4. Robdel,M.(1964)。分离的脂肪细胞的代谢的影响。 I.对葡萄糖代谢和脂肪分解代码激素的影响的生物化学杂志 239:375-380 ..
  5. 阮,H.,Zarnowski,M. J.,库什曼,S.W。和洛迪什,H. F.(2003)。 从小鼠的附睾脂肪垫初级脂肪细胞的标准隔离诱导的炎症介质和下调基因脂肪细胞代码生物化学杂志 278(48):。47585-93。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Decaunes, P., Bouloumié, A., Ryden, M. and Galitzky, J. (2018). Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants. Bio-protocol 8(3): e2711. DOI: 10.21769/BioProtoc.2711.
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