搜索

3 users have reported that they have successfully carried out the experiment using this protocol.
Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments
来源于皮下和内脏白脂肪组织腔室的人脂肪源性干细胞的分离和培养   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Development
Mar 2016

Abstract

Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications.

This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.

Background

Human ASCs are an invaluable in vitro cell model to study molecular pathways important for the etiology of metabolic diseases, including obesity and type 2 diabetes. Human ASC cultures derived from different WAT sources, including subcutaneous and visceral compartments, can also help us to understand functional differences between different WAT compartments. Short protocols have been published previously to describe the isolation of human ASCs from a maximum of two different WAT compartments (Baglioni et al., 2009; 2012; van Harmelen et al., 2005). Here we describe a detailed protocol for both, isolation and characterization of human ASCs from human WAT biopsies collected from several WAT compartments. This protocol has been used to reliably derive human ASCs from four different WAT compartments, including superficial subcutaneous, deep subcutaneous, omental and mesenteric WAT. The isolated primary cultures display homogeneous morphology and are pure, with a high percentage of cells displaying typical MSC marker expression. The isolated human ASCs also have the ability to differentiate into mature adipocytes, with accumulation of intracellular triglyceride droplets. In summary, this protocol reliably results in the isolation of pure human primary ASCs that maintain robust adipogenic differentiation capacity in vitro.

Materials and Reagents

  1. 15 ml centrifugation tube (Corning, Falcon®, catalog number: 352099 )
  2. 50 ml centrifugation tube (Corning, Falcon®, catalog number: 352070 )
  3. 10 cm cell culture dish (Greiner Bio One, CellStar®, catalog number: 664160 )
  4. 0.2 μm 25 mm syringe filter (Pall, Acrodisc®, catalog number: 4612 )
  5. 30 ml syringe (BD, Luer-LokTM, catalog number: 302832 )
  6. 5 ml polystyrene fluorescence activated cell sorting (FACS) tubes (Corning, Falcon®, catalog number: 352054 )
  7. 100 μm nylon mesh cell strainer (Corning, Falcon®, catalog number: 352360 )
  8. 6-well-plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  9. Human WAT from patients
  10. OXOIDTM Phosphate buffered saline (PBS) tablets (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: BR0014G )
  11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  12. Fetal bovine serum (FBS), heat inactivated (Thermo Fisher Scientific, GibcoTM, catalog number: 16140071 )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  14. Antibodies (see Table 1)
  15. Isopropanol (EMD Millipore, catalog number: 109634 )
  16. Collagenase type IA (Sigma-Aldrich, catalog number: C9891-1G )
  17. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A0171 )
    Note: This product has been discontinued.
  18. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: P7682 )
    Note: This product has been discontinued.
  19. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  20. Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12) (1:1) (Thermo Fisher Scientific, GibcoTM, catalog number: 11330032 )
  21. Penicillin-streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  22. Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
  23. 10 mg/ml insulin solution from bovine pancreas (Sigma-Aldrich, catalog number: I0516 )
  24. 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 )
  25. Indomethacin (Sigma-Aldrich, catalog number: 17378 )
  26. 100% ethanol (EMD Millipore, catalog number: 100983 )

    Table 1. Antibody information
    Antigen

    Antibody
    Manufacturer
    Dilution
    Host/Isotype
    CD34
    hematopoietic cells
    Anti-Human CD34 APC
    Affymetrix, eBioscience,
    catalog number: 17-0349
    1:100
    Mouse IgG1, kappa
    CD31
    Endothelial cells
    Anti-Human CD31 APC
    Affymetrix, eBioscience,
    catalog number: 17-0319
    1:100
    Mouse IgG1, kappa
    CD14
    (Macrophages) hematopoietic cells
    Anti-Human CD14 APC
    Affymetrix, eBioscience,
    catalog number: 17-0149
    1:100
    Mouse IgG1, kappa
    CD11b
    (Leukocytes) Monocytes
    Anti-Human CD11b APC
    Affymetrix, eBioscience,
    catalog number: 17-0113
    1:100
    Mouse IgG1, kappa
    CD45
    (Nucleated cells of hematopoietic origin) lymphocytes
    Anti-Human CD45 APC
    Affymetrix, eBioscience,
    catalog number: 17-9459
    1:100
    Mouse IgG1, kappa
    CD106
    (Activated) endothelial cells
    Anti-Human CD106 PE
    Affymetrix, eBioscience,
    catalog number: 12-1069
    1:100
    Mouse IgG1, kappa
    CD90
    MSCs
    Anti-Human CD90 APC
    Affymetrix, eBioscience,
    catalog number: 17-0909
    1:100
    Mouse IgG1, kappa
    CD44
    MSCs
    Anti-Human CD44 APC
    Affymetrix, eBioscience,
    catalog number: 17-0441
    1:100
    Rat IgG2b, kappa
    CD29
    MSCs
    Anti-Human CD29 APC
    Affymetrix, eBioscience,
    catalog number: 17-0299
    1:100
    Mouse IgG1, kappa
    CD73
    MSCs
    Anti-Human CD73 APC
    Affymetrix, eBioscience,
    catalog number: 17-0739
    1:100
    Mouse IgG1, kappa
    CD105
    MSCs
    Anti-Human CD105 APC
    Affymetrix, eBioscience,
    catalog number: 17-1057
    1:100
    Mouse IgG1

  27.  Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  28. Oil Red O powder (Sigma-Aldrich, catalog number: O0625 )
  29. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  30. Collagenase solution (1 mg/ml) (see Recipes)
  31. Red blood cell (RBC) lysis buffer (10x) (see Recipes)
  32. Proliferation medium (see Recipes)
  33. Differentiation medium (see Recipes)
  34. Induction medium (see Recipes)
  35. Insulin medium (see Recipes)
  36. 10 mM dexamethasone stock solution (see Recipes)
  37. 1 mM dexamethasone working solution (see Recipes)
  38. 0.5 M IBMX stock solution (1,000x) (see Recipes)
  39. 200 mM indomethacin stock solution (1,600x) (see Recipes)
  40. 4% paraformaldehyde (PFA) (pH = 7.4) (see Recipes)
  41. Oil Red O working solution (see Recipes)

Equipment

  1. Cryogenic vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 366656 )
  2. Laminar flow tissue culture hood
  3. Sterilized surgical tools including forceps and scalpel or scissors
  4. Mr. FrostyTM freezing container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5100-0001 )
  5. Locator 6 Plus Rack and Box Systems, liquid nitrogen Dewar (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CY50985-70 )
  6. BD FACSCanto II flow cytometer (BD, model: BD FACSCanto II ) or similar equipment
  7. Analytical balance (Sartorius, model: CPA124S )
  8. MaxQTM 37 °C orbital shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: 4450 )
  9. Centrifuge with swinging bucket rotor (KUBOTA, model: 2800 )
  10. Automated cell counter (Thermo Fisher Scientific, Countess®, catalog number: AMQAF1000 )

Software

  1. FACSDiva software (BD)

Procedure

  1. Cell isolation
    1. Collect 0.2-1.0 g of human WAT from patients
      Note: We have utilized this protocol to isolate ASCs from superficial subcutaneous WAT isolated from morbidly obese individuals who underwent either gastrectomy or gastric bypass surgical procedures, as previously described (Leow et al., 2016). However, in unpublished studies from our lab we have also utilized this protocol to successfully isolate ASCs from deep subcutaneous, omental and mesenteric WAT from lean patients who underwent laparoscopic inguinal and ventral hernia surgery.
    2. Transfer human WAT biopsy into pre-weighed 50 ml tube with PBS.
      Note: A maximum of 1 g of WAT should be digested in one 50 ml centrifugation tube.
    3. Calculate WAT tissue weight.

    Note: For the following steps work in a laminar flow tissue culture hood.

    1. Transfer tissue into a 10 cm cell culture dish and mince the tissue into small pieces with sterilized scalpel or scissors.
      Note: For better tissue digestion, WAT should be minced thoroughly.
    2. Transfer the minced tissue into 50 ml tube. Add 3 ml collagenase solution/1 g of tissue. For tissue less than 0.3 g, use 1 ml of collagenase solution.
      Note: Collagenase solution should be prepared freshly with pre-warmed PBS (from 37 °C water bath).
    3. Digest the tissue in 37 °C incubator with shaking (120 rpm) for 1.5 h.
      Note: No chunks of WAT should be visible after digestion. If chunks of WAT are still visible then digestion time can be extended accordingly.
    4. Inactivate collagenase by adding 35 ml of proliferation medium into the 50 ml tube.
    5. Immediately centrifuge at 800 x g for 10 min at room temperature.
    6. After centrifugation the cell pellet should be visible at the bottom of the 50 ml tube.
    7. Carefully aspirate the supernatant (including fat layer) and re-suspend the cell pellet in RBC lysis buffer. For 1 ml of pellet, add 9 ml of RBC lysis buffer (1:9, v:v).
    8. Incubate for 10 min at room temperature.
    9. Immediately centrifuge at 800 x g for 10 min at room temperature.
    10. Re-suspend the cell pellet in 20 ml proliferation medium by pipetting up and down.
    11. Filter the cell suspension through a 100 μm nylon mesh cell strainer into a new 50 ml tube to remove debris.
    12. Immediately centrifuge at 800 x g for 10 min at room temperature.
    13. Carefully aspirate the supernatant and re-suspend the cell pellet containing ASCs in 10 ml proliferation medium.
    14. Transfer the cell suspension onto a cell culture treated 10 cm plate and incubate at 37 °C/5% CO2 for 24 h.
    15. After 24 h cell culturing, non-adherent cells are removed and adherent cells are washed with PBS. Adherent cells (ASCs) are cultured in fresh proliferation medium until 70% confluence.
    16. Passage ASCs 1-2 times to expand cell population. A representative image of growing ASCs is shown in Figure 1A. After several days of culture human ASCs show a typical fibroblastic-like morphology (Figure 1A).
    17. ASCs can then be either utilized for relevant in vitro investigations or cryopreserved. To cryopreserve ASCs, trypsinize and count 0.5-1 million cells and centrifuge at 800 x g for 10 min at room temperature. Aspirate the supernatant and re-suspend the cell pellet in 1 ml of pre-chilled (4 °C) proliferation medium containing 5% DMSO in cryogenic vials. Store vials in Mr. FrostyTM freezing container filled with isopropanol at -80 °C overnight, before transferring to a liquid nitrogen Dewar for long-term storage.


      Figure 1. Human ASCs and ASCs-derived mature adipocytes. A. Representative phase contrast image of proliferating human ASCs cultures isolated from superficial subcutaneous WAT (Scale bar = 100 μm). B. Representative phase contrast image of human ASCs-derived mature adipocytes (Scale bar = 200 μm). C. Representative phase contrast image of Oil Red O stained lipid droplets within ASCs-derived mature adipocytes (Scale bar = 200 μm). D. Representative high magnification phase contrast image of Oil Red O stained lipid droplets within ASCs-derived mature adipocytes (Scale bar = 50 μm).

  2. Cell surface markers staining and flow cytometry
    1. Trypsinize ASCs and count cell number.
    2. Collect ~0.2 million cells in each 5 ml FACS tube.
    3. Immediately centrifuge at 200 x g for 5 min at 4 °C and aspirate the supernatant.
    4. Wash the cell pellet with 3 ml PBS twice.
    5. Add 2 ml of ice-cold 4% PFA to each cell pellet and re-suspend the cell pellet by vortexing gently.
    6. Incubate at 4 °C for 30 min or keep the fixed cells at 4 °C for subsequent immunofluorescent staining (up to 6 months).
    7. Wash the fixed cells with 3 ml of PBS twice.
    8. Re-suspend the cell pellet with 1 ml of 15% FBS in PBS and incubate for 20 min for blocking.
    9. Immediately centrifuge at 200 x g for 5 min and aspirate the supernatant.
    10. Re-suspend the cell pellet with 50 μl of primary antibody diluted in ice-cold 15% FBS in PBS (For antibody information please see Table 1).
    11. Incubate on ice for 45 min.


      Figure 2. Surface marker expression profile analysis of human ASCs by flow cytometry. Isolated human ASCs (from superficial subcutaneous WAT) were analyzed for MSC markers (CD90, CD44, CD29, CD73 and CD105), hematopoietic lineage markers (CD34, CD14, CD11b, and CD45) and endothelial markers (CD31 and CD106) by flow cytometry. Histograms show results of staining of ASCs for indicated surface markers from one representative experiment. Cell counts are indicated on the y-axis and fluorescence intensity on the x-axis. Gate P2 was set according to staining with appropriate isotype-matched antibody controls. The percentages of ASCs stained positively are indicated in each panel.

    12. Add 5 ml of ice-cold 0.5% BSA in PBS.
    13. Immediately centrifuge at 200 x g for 5 min at 4 °C and aspirate the supernatant.
    14. Re-suspend the cell pellet with 200 μl of ice-cold 0.5% BSA in PBS.
    15. Analyze samples using the BD FACSCanto II machine (BD) and FACSDiva software (BD) software for data acquisition.
      Note: Alternative FACS hardware and software packages can be utilized.
    16. Typically we find that more than 98% of the isolated ASCs express MSC markers, including CD90, CD44, CD29, CD73 and CD105. However, less than 1% of the isolated ASCs express cell surface markers for hematopoietic cells (CD34, CD14, CD11b, and CD45) and markers for endothelial cells (CD31 and CD106) (Figure 2).

  3. Adipogenic differentiation
    1. Trypsinize ASCs and count cell number.
    2. Plate cells on cell culture treated 6-well-plates in proliferation medium (see Recipes) at the density of 10,000 cells/cm2.
    3. Let cells grow to confluency and wait for another 48 h to arrest cell division.
    4. Treat cells with induction medium (Differentiation medium, containing 1 μM dexamethasone [diluted from 10 mM dexamethasone stock solution], 58 μg/ml insulin, 0.5 mM IBMX, and 200 μM indomethacin, see Recipes) for 2 weeks (We have observed that for longer term and more complex experimental studies [Leow et al., 2016], the treatment with induction medium can be decreased to 1 week). Change medium with fresh induction medium every 3 days. Treat cells with insulin medium (Differentiation medium, containing 10 μg/ml insulin, see Recipes). Change medium with fresh insulin medium every 2 days. Cells can be assayed from day-4 to day-7 post treatment with insulin medium. A representative image of differentiated ASC cultures is shown in Figure 1B, clusters of mature adipocytes filled with lipid droplets are visible.
    5. To assess lipid accumulation, Oil Red O staining is performed on differentiated mature adipocytes (Ramirez-Zacarias et al., 1992). For Oil Red O staining, remove medium and wash the cells with 2 ml of PBS/well. Fix the cells with 2 ml of 4% paraformaldehyde (PFA)/well for 30 min at room temperature before washing the cells with 4 ml of H2O/well. Incubate the cells with 2 ml of 60% isopropanol/well for 5 min. Aspirate the isopropanol and incubate the cells with 2 ml of Oil Red O working solution/well for 30 min at room temperature. Aspirate Oil Red O working solution and wash the cells with H2O before taking images. Representative images of Oil Red O stained mature adipocytes are shown in Figures 1C (4x objective) and 1D (20x objective). Oil Red O stained lipid droplets are clearly visible.

Data analysis

For characterization of surface marker expression of isolated human ASCs, flow cytometry data were collected with acquisition of 10,000 events per sample using the BD FACSCanto II machine (BD). Gate P2 was set according to staining intensity of appropriate isotype-matched control antibodies. Data analysis was performed using FACSDiva software (BD). Only representative data from one ASC culture are shown in this protocol. However, this protocol has been used to assess purity of several independent ASC cultures, with similar results obtained. No statistical analysis was performed in this protocol.

Notes

  1. This protocol has been used to isolate human ASCs from several WAT biopsies and the protocol has proved to be reproducible.
  2. The highest passage of ASCs we have used to study adipogenic differentiation potential is passage 10. However, a previous study has stated that ASCs maintain adipogenic differentiation capacity through multiple passages (up to at least passage 15) (Dicker et al., 2005).

Recipes

  1. Collagenase solution (1 mg/ml) (prepare freshly)
    10 mg collagenase type IA
    200 mg BSA
    Top up with pre-warmed (37 °C) PBS to final volume of 10 ml
    Filter sterilize using a 0.22 μm syringe filter before use
  2. Red blood cell (RBC) lysis buffer (10x)
    824 mg NH4Cl
    100 mg KHCO3
    4 mg EDTA
    Top up to 100 ml with PBS
    Filter sterilize using a 0.22 μm filter unit before use
  3. Proliferation medium
    DMEM/F-12
    20% FBS
    1% P/S
    Filter sterilize using a 0.22 μm filter unit before use
  4. Differentiation medium
    DMEM/F-12
    10% FBS
    1% P/S
    Filter sterilize using a 0.22 μm filter unit before use.
  5. Induction medium (prepare freshly)
    Differentiation medium
    1 μM dexamethasone
    58 μg/ml insulin
    0.5 mM IBMX
    200 μM indomethacin
  6. Insulin medium (prepare freshly)
    Differentiation medium
    10 μg/ml insulin
  7. 10 mM dexamethasone stock solution (2,000x)
    Reconstitute 78.5 mg dexamethasone in 20 ml of 100% ethanol
    Filter sterilize using a 0.22 μm syringe filter and store at 4 °C
  8. 1 mM dexamethasone working solution
    Dilute dexamethasone stock solution into 1 mM of dexamethasone working solution with PBS before using.
  9. 0.5 M IBMX stock solution (1,000x)
    Reconstitute 1 g of IBMX in 9 ml of DMSO
    Filter sterilize using a 0.22 μm syringe filter
    Aliquot into 1 ml fractions and store at -20 °C
  10. 200 mM indomethacin stock solution (1,600x)
    Reconstitute 71.567 mg of indomethacin in 1 ml of DMSO
    Filter sterilize using a 0.22 μm syringe filter and store at -20 °C
  11. 4% paraformaldehyde (PFA) (pH = 7.4)
    Add 4 g of paraformaldehyde to 48 ml of H2O
    Heat to dissolve
    Add 2 N NaOH dropwise until solution becomes clear
    Add 50 ml of 2x PBS and mix
    Remove from heat and cool down the solution
    Adjust pH to 7.4
    Aliquot into 10 ml fractions and store at -20 °C
  12. Oil Red O working solution (freshly prepared)
    Dissolve 300 mg of Oil Red O powder into 100 ml of 99% isopropanol as Oil Red O stock solution (store at room temperature)
    To prepare Oil Red O working solution, mix 3 parts of Oil Red O stock solution and 2 parts of H2O and incubate at room temperature for 10 min
    Filter the Oil Red O working solution through filter paper
    Use the filtered working solution for staining within 2 h

Acknowledgments

We would like to thank Khin Thida Soe for patient recruitment and coordinating human WAT biopsy collection at National University Hospital (NUH), Singapore. This work was supported by Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*STAR) core funding. We also acknowledge the previously published protocols from Baglioni et al. (2009), Baglioni et al. (2012) and van Harmelen et al. (2005), based on which this current protocol was modified.

References

  1. Baglioni, S., Cantini, G., Poli, G., Francalanci, M., Squecco, R., Di Franco, A., Borgogni, E., Frontera, S., Nesi, G., Liotta, F., Lucchese, M., Perigli, G., Francini, F., Forti, G., Serio, M. and Luconi, M. (2012). Functional differences in visceral and subcutaneous fat pads originate from differences in the adipose stem cell. PLoS One 7(5): e36569.
  2. Baglioni, S., Francalanci, M., Squecco, R., Lombardi, A., Cantini, G., Angeli, R., Gelmini, S., Guasti, D., Benvenuti, S., Annunziato, F., Bani, D., Liotta, F., Francini, F., Perigli, G., Serio, M. and Luconi, M. (2009). Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J 23(10): 3494-3505.
  3. Dicker, A., Le Blanc, K., Astrom, G., van Harmelen, V., Gotherstrom, C., Blomqvist, L., Arner, P. and Ryden, M. (2005). Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308(2): 283-290.
  4. Leow, S. C., Poschmann, J., Too, P. G., Yin, J., Joseph, R., McFarlane, C., Dogra, S., Shabbir, A., Ingham, P. W., Prabhakar, S., Leow, M. K., Lee, Y. S., Ng, K. L., Chong, Y. S., Gluckman, P. D. and Stunkel, W. (2016). The transcription factor SOX6 contributes to the developmental origins of obesity by promoting adipogenesis. Development 143(6): 950-961.
  5. Ramirez-Zacarias, J. L., Castro-Munozledo, F. and Kuri-Harcuch, W. (1992). Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97(6): 493-497.
  6. van Harmelen, V., Skurk, T. and Hauner, H. (2005). Primary culture and differentiation of human adipocyte precursor cells. Methods Mol Med 107: 125-135.

简介

Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications.
  This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.

[Background] Human ASCs are an invaluable in vitro cell model to study molecular pathways important for the etiology of metabolic diseases, including obesity and type 2 diabetes. Human ASC cultures derived from different WAT sources, including subcutaneous and visceral compartments, can also help us to understand functional differences between different WAT compartments. Short protocols have been published previously to describe the isolation of human ASCs from a maximum of two different WAT compartments (Baglioni et al., 2009; 2012; van Harmelen et al., 2005). Here we describe a detailed protocol for both, isolation and characterization of human ASCs from human WAT biopsies collected from several WAT compartments. This protocol has been used to reliably derive human ASCs from four different WAT compartments, including superficial subcutaneous, deep subcutaneous, omental and mesenteric WAT. The isolated primary cultures display homogeneous morphology and are pure, with a high percentage of cells displaying typical MSC marker expression. The isolated human ASCs also have the ability to differentiate into mature adipocytes, with accumulation of intracellular triglyceride droplets. In summary, this protocol reliably results in the isolation of pure human primary ASCs that maintain robust adipogenic differentiation capacity in vitro.

材料和试剂

  1. 15ml离心管(Corning,Falcon ,目录号:352099)
  2. 50ml离心管(Corning,Falcon ,目录号:352070)
  3. 10cm细胞培养皿(Greiner Bio One,CellStar ,目录号:664160)。
  4. 0.2μm25mm注射器过滤器(Pall,Acrodisc ,目录号:4612)
  5. 30ml注射器(BD,Luer-Lok TM ,目录号:302832)
  6. 5ml聚苯乙烯荧光激活细胞分选(FACS)管(Corning,Falcon ,目录号:352054)。
  7. 100μm的尼龙网孔过滤器(Corning,Falcon ,目录号:352360)
  8. 6孔板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:140675)
  9. 来自患者的人类WATRW />
  10. OXOID TM磷酸盐缓冲盐水(PBS)片剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:BR0014G)
  11. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  12. 胎牛血清(FBS),热灭活(Thermo Fisher Scientific,Gibco TM ,目录号:16140071)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  14. 抗体(见表1)
  15. 异丙醇(EMD Millipore,目录号:109634)
  16. 胶原酶IA型(Sigma-Aldrich,目录号:C9891-1G)
  17. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A0171)
    注意:此产品已停产。
  18. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:P7682)
    注意:此产品已停产。
  19. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25200056)
  20. Dulbecco改良的Eagle培养基:营养混合物F-12(DMEM/F-12)(1:1)(Thermo Fisher Scientific,Gibco TM,目录号:11330032)
  21. 青霉素 - 链霉素(P/S)(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  22. 地塞米松(Sigma-Aldrich,目录号:D4902)
  23. 10mg/ml来自牛胰腺的胰岛素溶液(Sigma-Aldrich,目录号:I0516)
  24. 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich,目录号:I5879)
  25. 吲哚美辛(Sigma-Aldrich,目录号:17378)
  26. 100%乙醇(EMD Millipore,目录号:100983)
    表1.抗体信息
    抗原

    抗体
    制造商
    稀释
    主机 /同种型
    CD34
    造血细胞
    抗人CD34 APC
    Affymetrix,eBioscience,
    目录号:17-0349
    1:100
    小鼠IgG1,κ
    CD31
    内皮细胞
    抗人CD31 APC
    Affymetrix,eBioscience,
    目录号:17-0319
    1:100
    小鼠IgG1,κ
    CD14
    (巨噬细胞)造血细胞
    抗人CD14 APC
    Affymetrix,eBioscience,
    目录号:17-0149
    1:100
    小鼠IgG1,κ
    CD11b
    (白细胞)单核细胞
    抗人CD11b APC
    Affymetrix,eBioscience,
    目录号:17-0113
    1:100
    小鼠IgG1,κ
    CD45
    (造血起源的成核细胞)淋巴细胞
    抗人CD45 APC
    Affymetrix,eBioscience,
    目录号:17-9459
    1:100
    小鼠IgG1,κ
    CD106
    (活化的)内皮细胞
    抗人类CD106 PE
    Affymetrix,eBioscience,
    目录号:12-1069
    1:100
    小鼠IgG1,κ
    CD90
    MSC
    抗人CD90 APC
    Affymetrix,eBioscience,
    目录号:17-0909
    1:100
    小鼠IgG1,κ
    CD44
    MSC
    抗人CD44 APC
    Affymetrix,eBioscience,
    目录号:17-0441
    1:100
    大鼠IgG2b,κ
    CD29
    MSC
    抗人CD29 APC
    Affymetrix,eBioscience,
    目录号:17-0299
    1:100
    小鼠IgG1,κ
    CD73
    MSC
    抗人CD73 APC
    Affymetrix,eBioscience,
    目录号:17-0739
    1:100
    小鼠IgG1,κ
    CD105
    MSC
    抗人CD105 APC
    Affymetrix,eBioscience,
    目录号:17-1057
    1:100
    小鼠IgG1

  27. <多聚甲醛(Sigma-Aldrich,目录号:P6148)
  28. 油红O粉末(Sigma-Aldrich,目录号:O0625)
  29. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  30. 胶原酶溶液(1mg/ml)(参见配方)
  31. 红细胞(RBC)裂解缓冲液(10x)(参见配方)
  32. 增殖培养基(参见配方)
  33. 分化介质(参见配方)
  34. 感应介质(见配方)
  35. 胰岛素介质(参见配方)
  36. 10 mM地塞米松储备溶液(见配方)
  37. 1 mM地塞米松工作溶液(见配方)
  38. 0.5 M IBMX储备溶液(1,000x)(参见配方)
  39. 200 mM吲哚美辛储备液(1,600x)(参见配方)
  40. 4%多聚甲醛(PFA)(pH = 7.4)(参见配方)
  41. 油红O工作溶液(见配方)

设备

  1. 低温小瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:366656)
  2. 层流组织培养罩
  3. 灭菌手术工具,包括镊子和手术刀或剪刀
  4. Frosty TM 冷冻容器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:5100-0001)
  5. Locator 6 Plus Rack and Box Systems,liquid nitrogen Dewar(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:CY50985-70)
  6. BD FACSCanto II流式细胞仪(BD,型号:BD FACSCanto II)或类似设备
  7. 分析天平(Sartorius,型号:CPA124S)
  8. 37℃轨道摇床(Thermo Fisher Scientific,Thermo Scientific TM ,型号:4450)。
  9. 用摆动式转子离心机(KUBOTA,型号:2800)
  10. 自动细胞计数器(Thermo Fisher Scientific,Countess ®,目录号:AMQAF1000)

软件

  1. FACSDiva软件(BD)

程序

  1. 细胞分离
    1. 从患者收集0.2-1.0 g的人类WAT 注意:我们已经利用这个协议从孤立从经历胃切除术或胃旁路手术过程的病态肥胖个体的表面皮下WAT分离ASCs,如前所述(Leow et al。,2016)。然而,在我们实验室的未发表的研究中,我们还利用该方案成功地从经历腹腔镜腹股沟和腹侧疝手术的瘦患者的深部皮下,网膜和肠系膜WAT中分离出ASC。
    2. 将人类WAT活检转移到预先称重的50ml PBS管中 注意:最多1 g的WAT应该在一个50 ml离心管中消化。
    3. 计算WAT组织重量

    注意:对于以下步骤,在层流组织培养罩中工作。

    1. 将组织转移到10厘米的细胞培养皿,并用灭菌的手术刀或剪刀将组织切成小块。
      注意:为了更好的组织消化,WAT应彻底切碎。
    2. 将切碎的组织转移到50ml管中。加入3毫升胶原酶溶液/1克组织。对于小于0.3 g的组织,使用1毫升胶原酶溶液。
      注意:胶原酶溶液应该用预热的PBS(从37℃水浴)新鲜配制。
    3. 在37℃培养箱中振荡(120 rpm)1.5小时,消化组织。
      注意:消化后不应显示WAT的大块。如果WAT的块仍然可见,则消化时间可以相应地延长。
    4. 通过加入35毫升增殖培养基到50毫升管中的灭活胶原酶。
    5. 立即在室温下以800×g离心10分钟
    6. 离心后,细胞沉淀应在50ml管的底部可见。
    7. 小心吸出上清液(包括脂肪层),并重悬细胞沉淀在RBC裂解缓冲液中。对于1ml沉淀,加入9ml RBC裂解缓冲液(1:9,v:v)。
    8. 在室温下孵育10分钟。
    9. 立即在室温下以800×g离心10分钟
    10. 通过上下吹吸将细胞沉淀重悬在20ml增殖培养基中。
    11. 通过一个100微米尼龙网格细胞过滤器过滤细胞悬浮液到一个新的50ml管,以清除碎片
    12. 立即在室温下以800×g离心10分钟
    13. 小心吸出上清液并将含有ASCs的细胞沉淀重悬于10ml增殖培养基中
    14. 将细胞悬液转移到细胞培养处理的10cm板上,并在37℃/5%CO 2下孵育24小时。
    15. 在24小时细胞培养后,除去非贴壁细胞,并用PBS洗涤贴壁细胞。将贴壁细胞(ASCs)在新鲜的增殖培养基中培养直到70%汇合
    16. 通过ASCs 1-2次以扩大细胞群。生长的ASC的代表性图像显示在图1A中。在培养几天后,人类ASCs显示典型的成纤维细胞样形态(图1A)。
    17. 然后可以将ASC用于相关的体外研究或冷冻保存。为了冷冻保存ASC,胰蛋白酶消化并计数0.5-1百万个细胞,并在室温下以800×g离心10分钟。吸出上清液并将细胞沉淀物重悬在1ml含有5%DMSO的低温小瓶中的预冷冻(4℃)增殖培养基中。将装有异丙醇的Frosty TM 冷冻容器在-80℃下储存过夜,然后转移到液氮杜瓦瓶中长期储存。


      图1.人类ASCs和来自ASCs的成熟脂肪细胞 A.从表面皮下WAT(比例尺=100μm)分离的增殖的人ASCs培养物的代表性相差图像。 B.人类ASCs衍生的成熟脂肪细胞的代表性相差图像(比例尺=200μm)。 C.来源于ASCs的成熟脂肪细胞中油红O染色的脂滴的代表性相差图像(比例尺=200μm)。 D.在来源于ASCs的成熟脂肪细胞(比例尺=50μm)内油红O染色的脂滴的代表性高放大率相位对比图像。
  2. 细胞表面标记染色和流式细胞术
    1. 胰酶消化ASC和计数细胞数。
    2. 在每个5ml FACS管中收集约20万个细胞
    3. 立即在4℃下以200×g离心5分钟,并抽吸上清液。
    4. 用3ml PBS洗涤细胞沉淀两次。
    5. 加入2毫升冰冷的4%PFA到每个细胞沉淀,并通过轻轻涡旋重新悬浮细胞沉淀。
    6. 在4°C孵育30分钟或保持固定细胞在4°C进行随后的免疫荧光染色(长达6个月)。
    7. 用3ml PBS洗涤固定的细胞两次。
    8. 用1ml 15%FBS的PBS悬浮细胞沉淀,孵育20分钟阻断
    9. 立即在200×g离心5分钟,并抽吸上清液
    10. 用在PBS中的冰冷的15%FBS中稀释的50μl一级抗体重悬细胞沉淀(关于抗体信息,请参见表1)。
    11. 在冰上孵育45分钟。


      图2.通过流式细胞术分析人ASCs的表面标志物表达谱。分析分离的人ASCs(来自浅表皮下WAT)的MSC标志物(CD90,CD44,CD29,CD73和CD105),造血谱系标记(CD34,CD14,CD11b和CD45)和内皮标记(CD31和CD106)。直方图显示来自一个代表性实验的指示表面标记物的ASC的染色结果。细胞计数在y轴上指示,荧光强度在x轴上指示。根据用合适的同种型匹配的抗体对照的染色设置门P2。每个面板中显示了阳性染色的ASC的百分比
    12. 加入5ml冰冷的0.5%BSA的PBS溶液。
    13. 立即在4℃下以200×g离心5分钟,并抽吸上清液。
    14. 用200μl冰冷的0.5%BSA的PBS悬浮细胞沉淀
    15. 使用BD FACSCanto II机器(BD)和FACSDiva软件(BD)软件分析样品,用于数据采集。
      注意:可以使用其他FACS硬件和软件包。
    16. 通常,我们发现超过98%的分离的ASCs表达MSC标志物,包括CD90,CD44,CD29,CD73和CD105。然而,少于1%的分离的ASCs表达造血细胞(CD34,CD14,CD11b和CD45)和内皮细胞(CD31和CD106)的标记的细胞表面标记(图2)。

  3. 脂肪分化
    1. 胰蛋白酶消化ASC和计数细胞数量
    2. 平板细胞在细胞培养物处理的增殖培养基中的6孔板(参见Recipes)中以10,000细胞/cm 2的密度处理。
    3. 让细胞生长至融合,并等待另外48小时阻止细胞分裂
    4. 用诱导培养基(分化培养基,含有1μM地塞米松[从10mM地塞米松储备溶液稀释],58μg/ml胰岛素,0.5mM IBMX和200μM吲哚美辛,参见Recipes)处理细胞2周(我们观察到长期和更复杂的实验研究[Leow等人,2016],用诱导培养基的处理可以减少到1周)。用新鲜诱导培养基每3天更换培养基。用胰岛素培养基(分化培养基,含有10μg/ml胰岛素,参见Recipes)处理细胞。每2天更换培养基与新鲜胰岛素培养基。可以在用胰岛素介质治疗后第4天至第7天测定细胞。分化的ASC培养物的代表性图像显示在图1B中,填充有脂滴的成熟脂肪细胞的簇是可见的
    5. 为了评估脂质积累,在分化的成熟脂肪细胞上进行油红O染色(Ramirez-Zacarias等人,1992)。对于油红O染色,除去培养基并用2ml PBS /孔洗涤细胞。用2ml的4%多聚甲醛(PFA)/孔在室温下固定细胞30分钟,然后用4ml H 2 O /孔洗涤细胞。用2ml的60%异丙醇/孔孵育细胞5分钟。吸出异丙醇,细胞与2毫升油红O工作溶液/孔在室温下孵育30分钟。吸取油红O工作溶液,并在拍摄图像之前用H 2 O洗涤细胞。油红O染色的成熟脂肪细胞的代表性图像显示在图1C(4x物镜)和1D(20x物镜)中。油红O染色的脂滴是清晰可见的

数据分析

为了表征分离的人ASCs的表面标志物表达,使用BD FACSCanto II仪器(BD)收集流式细胞术数据,每个样品获得10,000个事件。根据合适的同种型匹配的对照抗体的染色强度设定门P2。使用FACSDiva软件(BD)进行数据分析。在该方案中仅显示来自一个ASC培养物的代表性数据。然而,该方案已经用于评估几种独立ASC培养物的纯度,获得相似的结果。在该方案中没有进行统计分析。

笔记

  1. 该协议已被用于从几个WAT活检中分离人类ASCs,并且该方案被证明是可重复的。
  2. 我们已经用于研究脂肪形成分化潜能的ASCs的最高传代是第10代。然而,先前的研究已经表明,ASCs通过多次传代维持成脂分化能力(直到至少第15代)(Dicker等, ,2005)。

食谱

  1. 胶原酶溶液(1mg/ml)(新鲜制备)
    10mg IA型胶原酶
    200mg BSA
    用预热的(37℃)PBS补充至最终体积为10ml 使用0.22μm注射器过滤器过滤灭菌
  2. 红细胞(RBC)裂解缓冲液(10x)
    824mg NH 4 Cl 100 mg KHCO 3
    4mg EDTA
    用PBS
    补充至100 ml 使用0.22μm过滤器过滤灭菌
  3. 增殖培养基
    DMEM/F-12
    20%FBS
    1%P/S
    使用0.22μm过滤器过滤灭菌
  4. 分化媒介
    DMEM/F-12 10%FBS
    1%P/S
    使用0.22μm过滤器过滤灭菌。
  5. 感应介质(新鲜制备)
    分化媒介
    1μM地塞米松
    58微克/毫升胰岛素
    0.5 mM IBMX
    200μM吲哚美辛
  6. 胰岛素培养基(新鲜制备)
    分化媒介
    10μg/ml胰岛素
  7. 10mM地塞米松储备溶液(2000x)
    在20ml的100%乙醇中重建78.5mg地塞米松 使用0.22μm注射器过滤器进行过滤灭菌并在4℃下保存
  8. 1mM地塞米松工作溶液 稀释地塞米松储备溶液到1mM的地塞米松工作溶液与PBS使用前。
  9. 0.5 M IBMX储存解决方案(1,000x)
    在9ml DMSO中重构1g的IBMX 使用0.22μm注射器过滤器过滤灭菌
    分成1ml级分,并储存在-20℃下
  10. 200 mM吲哚美辛储备液(1,600x)
    在1ml DMSO中重建71.567mg吲哚美辛
    使用0.22μm注射器过滤器进行过滤灭菌,并在-20℃下保存
  11. 4%多聚甲醛(PFA)(pH = 7.4) 向48ml H 2 O中加入4g多聚甲醛 加热溶解
    滴加2N NaOH,直到溶液变得澄清
    加入50ml的2x PBS并混合
    从热中取出并冷却溶液
    将pH调节至7.4
    分成10毫升级分,并储存在-20℃下
  12. 油红O工作溶液(新鲜制备)
    将300mg油红O粉末溶解在100ml的99%异丙醇中作为油红O储备溶液(在室温下储存)
    为了制备油红O工作溶液,将3份油红O储备溶液和2份H 2 O混合,并在室温下孵育10分钟
    通过滤纸
    过滤油红O工作溶液 使用过滤的工作溶液在2小时内染色

致谢

我们要感谢Khin Thida Soe在新加坡国立大学医院(NUH)的患者招聘和协调人类WAT活检收集。这项工作得到新加坡临床科学研究所,科学技术和研究机构(A * STAR)核心资助。我们还承认以前从Baglioni等人发表的协议 。 (2009),Baglioni等人。 (2012)和van Harmelen等人。 (2005),基于此修改了当前协议。

参考文献

  1. Baglioni,S.,Cantini,G.,Poli,G.,Francalanci,M.,Squecco,R.,Di Franco,A.,Borgogni,E.,Frontera,S.,Nesi,G.,Liotta, ,Lucchese,M.,Perigli,G.,Francini,F.,Forti,G.,Serio,M.和Luconi,M.(2012)。  内脏和皮下脂肪垫的功能差异起源于脂肪干细胞的差异。 em> 7(5):e36569。
  2. Baglioni,S.,Francalanci,M.,Squecco,R.,Lombardi,A.,Cantini,G.,Angeli,R.,Gelmini,S.,Guasti,D.,Benvenuti,S.,Annunziato, Bani,D.,Liotta,F.,Francini,F.,Perigli,G.,Serio,M.和Luconi,M.(2009)。  分离自内脏和皮下脂肪组织的人成体干细胞群的表征。 FASEB J 23(10):3494-3505。
  3. Dicker,A.,Le Blanc,K.,Astrom,G.,van Harmelen,V.,Gotherstrom,C.,Blomqvist,L.,Arner,P.and Ryden,M。(2005) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15925364"target ="_ blank">来自成人脂肪组织的间充质干细胞的功能研究。 Exp Cell Res 308(2):283-290。
  4. Leow,SC,Poschmann,J.,Too,PG,Yin,J.,Joseph,R.,McFarlane,C.,Dogra,S.,Shabbir,A.,Ingham,PW,Prabhakar,S.,Leow,MK ,Lee,YS,Ng,KL,Chong,YS,Gluckman,PD和Stunkel,W。(2016)。  转录因子SOX6通过促进脂肪形成促进肥胖的发育起源。 143(6):950-961。 br />
  5. Ramirez-Zacarias,JL,Castro-Munozledo,F.和Kuri-Harcuch,W。(1992)。  通过用油红O染色胞质内脂质来定量脂肪转化和甘油三酯。组织化学 97(6):493-497。 >
  6. van Harmelen,V.,Skurk,T。和Hauner,H。(2005)。  人类脂肪细胞前体细胞的原代培养和分化。 Methods Mol Med 107:125-135。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ge, X., Leow, S. C., Sathiakumar, D., Stünkel, W., Shabbir, A., So, J. B., Lomanto, D. and McFarlane, C. (2016). Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments. Bio-protocol 6(22): e2027. DOI: 10.21769/BioProtoc.2027.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。