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Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells
成人脂肪源性基质/干细胞的分离和原代培养   

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Abstract

Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.

Keywords: Adipose-derived stem cells(脂肪源性干细胞), Adipose-derived stromal cells(脂肪源性基质细胞), Cell isolation(细胞分离), Primary cell culture(原代细胞培养), Cell expansion(细胞扩增), Cryopreservation(冷冻保存)

Background

Adipose-derived stromal/stem cells (ASCs) demonstrate vast potential for the field of stem cells. Following the therapeutic marvel of hematopoietic stem cells transplantation, ASCs represent the future for stem cells due to their more freely accessible source – adipose tissue. The ability of ASCs to self-renew and differentiate into various tissue lineages including adipocyte, chondrocyte, myocyte, and osteoblast lineages, allows restoration of damaged tissue. Additionally, it is speculated that ASCs have the potential to replicate tissue in vitro. In vitro organs will allow more readily available assessment of novel pharmaceuticals and thus reduce drug production costs significantly. However, inconsistencies in the processes of isolation, maintenance, and cryopreservation, prohibit collective analysis of results from different laboratories worldwide. A standard protocol for isolation and culture of ASCs is necessary to ensure consistent data analysis.

Materials and Reagents

  1. Stericup-GP 0.22 µm polyethersulfone 500 ml radio-sterilized vacuum filtration flask (EMD Millipore, catalog number: SCGPU05RE )
  2. Centrifuge tubes :
    15 ml (Corning, catalog number: 430790 )
    50 ml (Corning, catalog number: 430828 )
  3. Sterile disposable serologic pipets:
    2 ml (Corning, Falcon®, catalog number: 357558 )
    5 ml (Corning, Falcon®, catalog number: 357543 )
    10 ml (Corning, Falcon®, catalog number: 357551 )
    25 ml (Corning, Falcon®, catalog number: 357525 )
    50 ml (Corning, Falcon®, catalog number: 357550 )
  4. NuncTM 15 cm diameter, 145 cm2 culture area cell culture/Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 168381 )
  5. FisherbrandTM Premium microcentrifuge tubes: 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  6. Cryogenic vials, 1.2 ml (Corning, catalog number: 430487 )
  7. TipOne RPT 10 µl ultra-low retention filter pipet tips, sterile (USA Scientific, catalog number: 1181-3810 )
  8. ARTTM Barrier pipette 100 µl tips 100E low retention (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2065E-05 )
  9. Human adipose tissue obtained from liposuction
  10. 1x phosphate-buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30256 )
  11. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  12. Ethidium bromide
  13. Acridine orange
  14. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  15. Isopropanol 95% (v/v)
  16. Liquid nitrogen
  17. Type I collagenase (Worthington Biochemical, catalog number: LS004196 )
  18. Bovine serum albumin (BSA), fraction V (Sigma-Aldrich, catalog number: 10735078001 )
  19. Calcium chloride (CaCl2), ≥ 96.0% anhydrous (Sigma-Aldrich, catalog number: C4901 )
  20. Dulbecco’s modified Eagle medium (DMEM): Nutrient Mixture F-12 (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11330032 )
  21. α-minimal essential medium (α-MEM), no nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 12561056 )
  22. Fetal bovine serum (FBS), premium select (Atlanta Biologicals, catalog number: S11550 )
  23. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  24. L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  25. DMSO
  26. Digestion solution (see Recipes)
  27. Resuspension solution (see Recipes)
  28. Complete culture medium (CCM) (see Recipes)
  29. Freezing medium (see Recipes)

Equipment

  1. Precision scale
  2. IsotempTM digital-control water bath (Fisher Scientific, model: 215 )
  3. Sterile cell culture hood
  4. Motorized pipette aid
  5. Micropipettes: 10 µl, 200 µl, and 1,000 µl (Eppendorf, model: Research® plus )
  6. Centrifuge (Eppendorf, model: 5810 R )
  7. Cell culture incubator
  8. Inverted routine light microscope (Nikon Instruments, model: Eclipse TS100 )
  9. Hemocytometer
  10. Nalgene® freezing container (Sigma-Aldrich, catalog number: C1562 )
  11. Cryogenic storage dewar (Custom BioGenic Systems, model: 6001 Value Added Cryosystem Dewar )

Procedure

  1. ASC isolation (Figure 1)


    Figure 1. Schematic representation of the ASC isolation process. The lipoaspirate is separated into 50 ml conical tubes, blood is aspirated, washed with PBS, incubated with digestion solution, the digestion solution is neutralized, and the tube is centrifuged. Once the tube is centrifuged, the supernatant is discarded and the SVF is washed until it is white. The SVF is then resuspended, cells are counted, then plated on cell culture plates.

    1. Warm water bath to 37 °C.
    2. Collect lipoaspirate from operating room in a sterile specimen collection container. Lipoaspiration samples commonly drain directly into vacuum containers in the operative field. These containers are sterile and can be safely transported to the laboratory where cell isolation can occur. Keep lipoaspirate at room temperature until ready to isolate cells and maintain sterile conditions when transferring to the tissue culture hood.
    3. Prepare digestion solution (see Recipe 1). Filter the solution under sterile conditions with a vacuum filter.
    4. Prepare resuspension solution (see Recipe 2). Filter the solution under sterile conditions with vacuum filter. Place resuspension solution in a 37 °C water bath.
    5. Warm digestion solution and PBS in the water bath to 37 °C.
    6. Place lipoaspirate and prepared solutions into a sterile cell culture hood and perform the following steps under sterile conditions.
    7. Separate lipoaspirate into sterile 50 ml centrifuge tubes by placing about 15 ml of lipoaspirate in each tube. Allow lipoaspirate to separate into blood and fat layers. The fat layer will float to the top of the tube, and the blood layer will be underneath it (see Figure 1).
    8. Pierce the fat layer with the aspiration pipette, and aspirate the blood layer using a 2 ml aspiration pipet. Avoid aspirating the fat layer, as this will clog the aspiration pipette (see Figure 1).
    9. Wash the fat layer with PBS until the lipoaspirate is a light reddish-yellow hue. The volume of PBS required will vary depending on how much blood is present in the lipoaspirate sample. The volume of PBS needed will range from 5 to 20 ml, and multiple washes will be required until the wash solution is clear. Centrifuge the solution at 300 x g at room temperature for 5 min to separate the wash from the fat layer.
    10. Aspirate the PBS wash below the fat layer, and avoid aspirating the fat layer and the cell pellet at the bottom of the centrifuge tube (see Figure 1).
    11. Incubate lipoaspirate in a volume of digestion solution equivalent to the original amount of lipoaspirate (15 ml) for 60 min at 37 °C. For example, 15 ml of lipoaspirate should be incubated in 15 ml of digestion solution. Mix the tube(s) by vigorously shaking the tube for 1 min and mix the tubes intermittently every 5 to10 min throughout the digestion time.
    12. Neutralize collagenase by adding an equal amount of resuspension solution to the digestion solution used. For example, 15 ml of lipoaspirate incubated in 15 ml of digestion solution should be neutralized by 15 ml of resuspension solution.
    13. Centrifuge the solution at 300 x g at room temperature for 5 min. This step separates floating adipocytes, oil, fat, collagenase solution and the SVF.
    14. Discard supernatant, leaving about 5 ml of solution and the SVF cell pellet. Resuspend the SVF cell pellet in 10 ml PBS.
    15. Repeat steps A12 and A13 two times or until pellet appears white. These washes remove red blood cells and other non-adherent cells without compromising the yield of ASCs.
    16. Resuspend the SVF in 5 ml of resuspension solution. Pipet 10 µl of cells into a 1.5 ml microcentrifuge tube.
    17. Add 10 µl of trypan blue. Mix the solution with the cells by pipetting up and down 3-5 times.
    18. Add 10 µl of the mixed trypan blue-cell solution to the hemocytometer. Count cells in the four outer quadrants.
    19. As an alternative to trypan blue, mix 1 µl of 1:1 solution of 100 µg/ml ethidium bromide and 100 µg/ml acridine orange with 25 µl of cells. Add 10 µl of the mixture to the hemocytometer. Count the cells under fluorescent microscopy.
    20. Use the following equations to determine the total number of cells per milliliter and the total number of cells:

      Where,
      ‘2’ represents the dilution factor,
      ‘10,000’ represents the hemocytometer constant.
    21. Plate the cells on 145 cm2 culture dishes at a density of 100 cells per cm2 in 20 ml of CCM (approximately 14,500 cells per 145 cm2 culture dish). The expected cell yield ranges from 1.6 x 105 to 1.1 x 106 cells per ml of liposuction tissue.
    22. Maintain cells in a humidified 5% CO2 incubator at 37 °C for 24-72 h. Since donor variability is expected with human samples, the variability in initial incubation duration is associated with differences in the rate at which different donors’ cells will adhere to the plate.
    23. After 24-72 h, gently wash cell layer with PBS to remove non-adherent cells, leaving behind the adherent ASCs on the plate.
    24. Aspirate and discard PBS wash.
    25. Replace medium with CCM (15 to 20 ml for 145 cm2 plate) following the aspiration of PBS. Replace the medium with CCM every 2 to 3 days until cells have reached 70-80% confluence. Time to confluency will depend on the proliferative rate of the cells. Confluency of 80% can be expected within 4 to 10 days with medium replacements every 2-3 days. The cells should be passaged (see Procedure B) or cryopreserved (see Procedure C) once sufficient proliferation has occurred.
    26. Visualize cells under light microscopy to check for the absence of floating cells. The presence of floating cells indicates poor adherence and cell death.
    27. Continue to maintain cells in a humidified 5% CO2 incubator at 37 °C for 24-72 h.

  2. Expansion of ASCs
    1. Visualize the cells under light microscopy (see Figure 2). The cells should be approximately 80% confluent at the time of expansion. This confluence level is optimal as it prevents changes in cytokine expression that occurs when ASCs overgrow.


      Figure 2. Light microscopy of passage (p) 0 and p4 ASCs under 4x and 10x magnification. The ASCs are identified by their spindle-shaped morphology. Scale bars = 100 μm.

    2. Warm 0.25% trypsin-EDTA, media, and PBS in 37 °C water bath.
    3. Aspirate off CCM from the cell culture plate.
    4. Wash cells with 10 ml PBS and aspirate the wash.
    5. Add 2-5 ml of 0.25% trypsin to a 145 cm2 plate to adequately cover the entire area.
    6. Place 145 cm2 plates in the incubator set to 37 °C for a minimum of 5 min and no more than 20 min as cells will begin to lift into solution.
    7. Visualize the cells under light microscopy to determine whether the enzymatic reaction is complete and the cells have lifted. Neutralize the trypsin by adding an equal volume of CCM. The reaction should not take longer than 10 min and should be neutralized if 10 min have passed to prevent degradation of the cell membrane.
    8. Pipet the cell solution into a sterile 50 ml conical centrifuge tube and spin at 300 x g for 5 min at room temperature.
    9. Aspirate the supernatant, leaving behind the cell pellet.
    10. Resuspend the cell pellet in CCM.
    11. Plate cells at a density of 100 cells per cm2.
       
  3. Cryopreservation of ASCs
    1. Remove the vial compartment of the freezing container and ensure that the freezing container is filled with isopropanol to the appropriate volume.
    2. Prepare freezing medium (see Recipe 4).
    3. Aspirate the media, wash with 10 ml PBS, and aspirate the PBS. The PBS washes away residual sera which inhibits trypsin.
    4. Add 2-5 ml of 0.25% trypsin to a 145 cm2 plate to adequately cover the entire area.
    5. Place cells in a 37 °C cell culture incubator for 5 min.
    6. Visualize the cells under light microscopy to determine whether the enzymatic reaction is complete and the cells have lifted. Neutralize the trypsin by adding an equal volume of CCM. The reaction should not take longer than 10 min and should be neutralized if 10 min has passed.
    7. Centrifuge 300 x g for 5 min at room temperature.
    8. Resuspend in 1 ml PBS. Count cells using the hemocytometer method as above.
    9. Centrifuge 300 x g for 5 min at room temperature.
    10. Aspirate supernatant and resuspend cells in freezing media to approximately 1 x 106 cells/ml.
    11. Pipet 1 ml of cell suspension into clean 1.2 ml cryogenic vials. Using this volume of cell suspension, each cryogenic vial will have approximately 1 x 106 frozen cells. Label vials with appropriate sample titles and date of freezing. Once the cells are in the freezing media, move vials to -80 °C freezer within 5 min. Extended exposure to DMSO at room temperature can lead to decreased cell viability.
    12. Place vials into an alcohol jacketed freezing container. The alcohol jacketed container slows the rate of cooling to about -1 °C/min, which is the ideal rate of cooling for cell preservation.
    13. Place alcohol jacketed freezing container into -80 °C freezer.
    14. Once the cells have been in the freezer for 24 h, they may be moved into liquid nitrogen.

Data analysis

For representative photos and results, please refer to Estes et al. (2010).

Notes

  1. Using cells from lower passages leads to better reproducibility. Therefore, we recommend using ASC between passages 2-6 for all experiments. Passage 1 cells represent the initial population of ASCs frozen down after isolation.
  2. Prepare all solutions the day of or the day prior to use for best results.
  3. Cell isolation should be performed the same day as tissue harvest from the patient for the highest viability, though our group has also determined that > 90% of adherent ASCs can be recovered from tissue as long as 24 h after collection.
  4. All lipoaspirate samples must be obtained according to Institution Review Board policies regarding human tissue collection. These protocols include informed consent from patients and tissue manipulation only by those who have been trained to work with blood borne pathogens.

Recipes

  1. Digestion solution
    100 ml PBS
    0.1 g collagenase I
    1 g BSA stock
    200 µl of 200 mM CaCl2
    Gently rock tube until solutions are homogenous and CaCl2 has dissolved completely 
    Sterile filter immediately prior to use
  2. Resuspension solution
    900 ml DMEM/F-12
    100 ml FBS
  3. Complete culture medium (CCM) (1,000 ml)
    780 ml α-MEM
    200 ml FBS
    10 ml 100x L-glutamine
    10 ml penicillin-streptomycin 10,000 U/ml
  4. Freezing medium (100 ml)
    65 ml of α-MEM
    30 ml of FBS
    5 ml of DMSO
    Filter in sterile filter unit
    Note: DMSO should be added to the medium and allowed to cool before adding the sera. Be sure to use a DMSO safe filter unit. Once thawed, discard any unused medium.

Acknowledgments

This protocol is an expansion of the protocol listed in Strong et al. (2016). The authors would like to thank Marjorie McCants for assisting with organization of materials and equipment, and Stephen Dickinson, Annie Bowles, and Rachel Sabol for helping with creation of the figures. J.M.G. is co-founder and co-owner of LaCell, LLC. All other authors indicate no conflicts of interest.

References

  1. Aust, L., Devlin, B., Foster, S. J., Halvorsen, Y. D., Hicok, K., du Laney, T., Sen, A., Willingmyre, G. D. and Gimble, J. M. (2004). Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy 6(1): 7-14.
  2. Estes, B. T., Diekman, B. O., Gimble, J. M. and Guilak, F. (2010). Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 5(7): 1294-1311.
  3. Gimble, J. and Guilak, F. (2003). Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5(5): 362-369.
  4. Strong, A. L., Bowles, A. C., Wise, R. M., Morand, J. P., Dutreil, M. F., Gimble, J. M. and Bunnell, B. A. (2016). Human adipose stromal/stem cells from obese donors show reduced efficacy in halting disease progression in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Stem Cells 34(3): 614-626.

简介

脂肪来源的基质/干细胞(ASCs)是可以从脂肪组织分离的多能细胞。研究表明,细胞具有自我更新和分化成脂肪细胞,软骨细胞,肌细胞和成骨细胞谱系的能力。因此,近几十年来,对再生用途恢复老化或损伤组织的兴趣越来越大。这些细胞也被证明可以免疫微环境并分泌丰富的生长因子,从而使炎症最小化并辅助修复和再生。 ASCs可以容易地从脂质体的基质血管分数(SVF)中分离出来。鉴于其易于获取,丰富的来源和在再生医学和组织工程中的潜在应用,对于ASC的表征和利用越来越感兴趣。该方案描述了从成人人类脂肪组织中分离的ASC以及用于培养维持的方法,包括扩增和低温保存。

背景 脂肪来源的基质/干细胞(ASCs)表现出干细胞领域的巨大潜力。根据造血干细胞移植的治疗奇迹,ASCs代表干细胞的未来,因为它们更容易获得源 - 脂肪组织。 ASCs自我更新和分化成各种组织谱系(包括脂肪细胞,软骨细胞,肌细胞和成骨细胞谱系)的能力允许恢复损伤的组织。另外,推测ASCs有可能在体外复制组织。器官将允许更容易获得新颖药物的评估,从而显着降低药物生产成本。然而,隔离,维护和冷冻保存过程中的不一致,禁止集体分析世界各地不同实验室的结果。用于分离和培养ASC的标准方案对于确保一致的数据分析是必要的。

关键字:脂肪源性干细胞, 脂肪源性基质细胞, 细胞分离, 原代细胞培养, 细胞扩增, 冷冻保存

材料和试剂

  1. Stericup-GP0.22μm聚醚砜500ml无菌灭菌真空过滤瓶(EMD Millipore,目录号:SCGPU05RE)
  2. 离心管:
    15毫升(康宁,目录号:430790)
    50毫升(康宁,目录号:430828)
  3. 无菌一次性血清学移液管:
    2 ml(Corning,Falcon ®,目录号:357558)
    5 ml(Corning,Falcon ®,目录号:357543)
    10 ml(Corning,Falcon ®,目录号:357551)
    25 ml(Corning,Falcon ®,目录号:357525)
    50ml(Corning,Falcon ®,目录号:357550)
  4. Nunc TM直径15cm,145cm 2培养区细胞培养/培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:168381 )
  5. Fisherbrand TM 高级微量离心管:1.5ml(Fisher Scientific,目录号:05-408-129)
  6. 低温小瓶1.2ml(Corning,目录号:430487)
  7. TipOne RPT 10μl超低保留过滤器吸头,无菌(USA Scientific,目录号:1181-3810)
  8. ART TM 阻隔移液管100μl提示100E低保留率(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2065E-05)
  9. 从脂肪抽吸获得的人体脂肪组织
  10. 1x磷酸缓冲盐水(PBS)(GE Healthcare,HyClone TM,目录号:SH30256)
  11. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  12. 溴化乙锭
  13. 吖啶橙
  14. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200072)
  15. 异丙醇95%(v/v)
  16. 液氮
  17. I型胶原酶(Worthington Biochemical,目录号:LS004196)
  18. 牛血清白蛋白(BSA),级分V(Sigma-Aldrich,目录号:10735078001)
  19. 氯化钙(CaCl 2),≥96.0%无水(Sigma-Aldrich,目录号:C4901)
  20. Dulbecco改性Eagle培养基(DMEM):营养混合物F-12(DMEM/F-12)(Thermo Fisher Scientific,Gibco TM,目录号:11330032)
  21. α-最小必需培养基(α-MEM),无核苷(Thermo Fisher Scientific,Gibco TM,目录号:12561056)
  22. 胎牛血清(FBS),高级选择(Atlanta Biologicals,目录号:S11550)
  23. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  24. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  25. DMSO
  26. 消化溶液(参见食谱)
  27. 再悬浮液(参见食谱)
  28. 完全培养基(CCM)(见食谱)
  29. 冷冻介质(参见食谱)

设备

  1. 精准量表
  2. Isotemp TM数字控制水浴(Fisher Scientific,型号:215)
  3. 无菌细胞培养罩
  4. 电动吸管辅助装置
  5. 微量移液器:10μl,200μl和1,000μl(Eppendorf,型号:Research plus)
  6. 离心机(Eppendorf,型号:5810 R)
  7. 细胞培养箱
  8. 倒置常规光学显微镜(Nikon Instruments,型号:Eclipse TS100)
  9. 血细胞计数器
  10. 冷藏容器(Sigma-Aldrich,目录号:C1562)
    Nalgene
  11. 低温储存杜瓦瓶(Custom BioGenic Systems,型号:6001加值冷冻系统杜瓦)

程序

  1. ASC隔离(图1)


    图1. ASC分离过程的示意图。将Lipoaspirate分离成50ml锥形管,抽吸血液,用PBS洗涤,与消化液一起温育,消化溶液中和,离心。将管离心后,弃去上清液,洗涤SVF至白色。然后重新悬浮SVF,计数细胞,然后铺板在细胞培养板上
    1. 温水浴至37°C
    2. 从无菌样品收集容器中的手术室收集脂肪抽吸物。吸脂样品通常在操作区域直接排入真空容器。这些容器是无菌的,可以安全地运送到可以发生细胞隔离的实验室。将Lipoaspirate保持在室温,直至准备好分离细胞,并在转移到组织培养罩时保持无菌条件。
    3. 准备消化液(参见食谱1)。用无菌条件下用真空过滤器过滤溶液。
    4. 准备再悬浮液(参见配方2)。使用真空过滤器在无菌条件下过滤溶液。将再悬浮溶液置于37°C水浴中。
    5. 温热消解溶液和PBS中的水浴至37°C
    6. 将lipoaspirate和制备的溶液置于无菌细胞培养罩中,并在无菌条件下进行以下步骤
    7. 通过在每个管中放置约15ml的lipoaspirate将lipoaspirate分离成无菌的50ml离心管。允许lipoaspirate分离成血液和脂肪层。脂肪层将浮在管的顶部,血液层将在其下面(见图1)。
    8. 用吸液管穿透脂肪层,并使用2 ml吸液管抽吸血液层。避免吸入脂肪层,因为这会堵塞吸液管(见图1)。
    9. 用PBS清洗脂肪层,直到lipoaspirate为浅黄红色。所需的PBS体积将根据Lipoaspirate样品中存在多少血液而变化。所需的PBS体积范围为5至20ml,并且需要多次洗涤,直到洗涤液清洁。在室温下将溶液以300g离心5分钟,以从脂肪层分离洗涤液。
    10. 吸出脂肪层下方的PBS洗液,并避免吸出离心管底部的脂肪层和细胞团块(见图1)。
    11. 将Lipoaspirate在相当于初始量的Lipoaspirate(15ml)的消化液体积中孵育60分钟,37℃。例如,15ml的脂肪酸酯应该在15ml消化溶液中孵育。通过大量摇动管子将管混合1分钟,并在整个消化时间内每隔5至10分钟间歇地混合管子。
    12. 通过向所使用的消化溶液中加入等量的再悬浮溶液中和胶原酶。例如,在15ml消化溶液中孵育的15ml Lipoaspirate应该被15ml的再悬浮溶液中和。
    13. 将溶液在室温下以300×g离心5分钟。该步骤分离浮游脂肪细胞,油脂,脂肪,胶原酶溶液和SVF
    14. 弃去上清液,留下约5ml溶液和SVF细胞沉淀。将SVF细胞沉淀重悬于10ml PBS中
    15. 重复步骤A12和A13两次,直到颗粒显示为白色。这些洗涤液可以清除红细胞和其他非粘附细胞,而不会影响到ASCs的产量
    16. 将SVF重悬于5ml的悬浮液中。将10μl细胞吸入1.5 ml微量离心管中
    17. 加入10μl台盼蓝。通过上下移动3-5次将溶液与细胞混合。
    18. 将10μl混合的台盼蓝细胞溶液加入血细胞计数器。计数四个外象限的细胞。
    19. 作为台盼蓝的替代品,将1μl的100μg/ml溴化乙锭和100μg/ml吖啶橙的1:1溶液与25μl细胞混合。加入10μl混合物到血细胞计数器。在荧光显微镜下计数细胞。
    20. 使用以下等式确定每毫升的细胞总数和总细胞数:

      哪里,
      '2'表示稀释因子,
      '10 000'代表血细胞计数器常数。
    21. 将细胞在145cm 2培养皿上以100个细胞/cm 2的密度在20ml CCM(每145cm 2约14,500个细胞中) sup>培养皿)。预期的细胞产量范围为每毫升吸脂组织的1.6×10 5至1.1×10 6细胞。
    22. 将细胞在37℃的湿润5%CO 2培养箱中保持24-72小时。由于供体的变异性预期与人类样品一起,初始孵化时间的变化与不同供体细胞粘附在板上的速率差异相关。
    23. 24-72小时后,用PBS轻轻洗涤细胞层以除去非贴壁细胞,留下板上贴壁的ASC。
    24. 吸出并丢弃PBS洗涤。
    25. 在PBS吸入后,用CCM替换培养基(145至20ml,145cm 2 板)。每2至3天更换一次CCM培养基,直至细胞达到70-80%汇合。融合时间取决于细胞的增殖率。可以预期在4至10天内达到80%的汇合,每2-3天更换一次。一旦发生了足够的增殖,细胞应该被传代(见程序B)或冷冻保存(见程序C)。
    26. 在光学显微镜下观察细胞以检查是否存在浮游细胞。浮游细胞的存在表明不良依从性和细胞死亡。
    27. 继续在37℃的潮湿5%CO 2培养箱中将细胞维持24-72小时。

  2. 扩展ASCs
    1. 在光学显微镜下观察细胞(见图2)。细胞在扩张时约为80%汇合。这种汇合水平是最佳的,因为它可以防止当ASC过度生长时发生的细胞因子表达的变化

      图2.在4x和10x放大倍数下通过(p)0和p4ASCs的光学显微镜。
      通过其纺锤形形态鉴定ASCs。比例尺=100μm
    2. 在37℃水浴中加热0.25%胰蛋白酶-EDTA,培养基和PBS。
    3. 从细胞培养板吸出CCM。
    4. 用10ml PBS洗涤细胞并吸出洗液。
    5. 加入2-5毫升0.25%胰蛋白酶至145厘米2平板,以充分覆盖整个区域。
    6. 将145厘米2厘米的板放置在37℃的培养箱中至少5分钟,不超过20分钟,因为细胞将开始提升到溶液中。
    7. 在光学显微镜下可视化细胞以确定酶反应是否完整并且细胞已经提升。通过加入等量的CCM中和胰蛋白酶。反应不应超过10分钟,如果10分钟过去,应中和,以防止细胞膜的降解。
    8. 将细胞溶液吸入无菌的50ml锥形离心管中,并在室温下以300×g旋转5分钟。
    9. 吸出上清液,留下细胞沉淀。
    10. 将细胞沉淀重悬于CCM。
    11. 密度为100个细胞/cm 2的平板细胞  
  3. ASC的冷冻保存
    1. 取出冷冻容器的小瓶隔间,并确保冷冻容器中装满异丙醇至适当的体积
    2. 准备冷冻介质(参见配方4)。
    3. 吸出培养基,用10ml PBS洗涤,并吸出PBS。 PBS洗涤残留的血清,抑制胰蛋白酶。
    4. 加入2-5毫升0.25%胰蛋白酶至145厘米2平板,以充分覆盖整个区域。
    5. 将细胞置于37℃的细胞培养箱中5分钟
    6. 在光学显微镜下可视化细胞以确定酶反应是否完整并且细胞已经升高。通过加入等量的CCM中和胰蛋白酶。反应不应超过10分钟,如果10分钟过去,应该中和。
    7. 在室温下离心300×g 5分钟。
    8. 重悬于1ml PBS中。使用如上所述的血细胞计数器计数细胞。
    9. 在室温下离心300×g 5分钟。
    10. 吸出上清液并将细胞在冷冻介质中重新悬浮至约1×10 6细胞/ml。
    11. 将1ml细胞悬液吸入干净的1.2ml低温小瓶中。使用该体积的细胞悬浮液,每个低温小瓶将具有约1×10 6个冷冻细胞。标签样品瓶,具有适当的样品标题和冻结日期。一旦细胞处于冷冻介质中,在5分钟内将小瓶移至-80°C冰箱。在室温下延长暴露于DMSO可导致细胞活力降低。
    12. 将小瓶放入酒精夹套的冷冻容器中。酒精夹套容器将冷却速度降低至约-1℃/min,这是细胞保存的理想冷却速度。
    13. 将酒精夹套冷冻容器放入-80°C冰箱。
    14. 一旦细胞进入冷冻箱24小时,就可以将其移入液氮

数据分析

有关代表性照片和结果,请参阅Estes 等人。 (2010)。

笔记

  1. 使用下部通道的细胞导致更好的再现性。因此,我们建议在所有实验的2-6段之间使用ASC。通道1细胞代表分离后冷冻的ASCs的初始种群。
  2. 在使用前一天或之前准备所有解决方案以获得最佳效果。
  3. 细胞分离应在同一天进行来自患者的组织收获以获得最高的生存能力,尽管我们组还确定了>收集后长达24小时,可以从组织中回收90%的贴壁ASC
  4. 所有lipoaspirate样品必须根据机构审查委员会关于人体组织收集的政策获得。这些方案包括患者的知情同意和组织操作,只有那些受过培训才能与血液传播的病原体合作的人。

食谱

  1. 消化液
    100毫升PBS
    0.1 g胶原酶I
    1克BSA原料
    200μl200mM CaCl 2
    轻轻摇动管,直至溶液均匀,CaCl 2完全溶解
    无菌过滤器即将使用前
  2. 重悬液
    900毫升DMEM/F-12
    100毫升FBS
  3. 完全培养基(CCM)(1000ml)
    780 mlα-MEM
    200毫升FBS
    10 ml 100x L-谷氨酰胺
    10 ml青霉素 - 链霉素10,000 U/ml
  4. 冷冻培养基(100 ml)
    65mlα-MEM
    30毫升FBS
    5毫升DMSO
    在无菌过滤器单元中过滤
    注意:将DMSO加入到培养基中,然后加入血清进行冷却。确保使用DMSO安全过滤器。一旦解冻,请丢弃任何未使用的介质。

致谢

该协议是Strong 等人中列出的协议的扩展。 (2016)。作者要感谢Marjorie McCants协助组织材料和设备,Stephen Dickinson,Annie Bowles和Rachel Sabol帮助创建了这些数字。马克思是LaCell,LLC的联合创始人和共同所有者。所有其他作者都表示没有利益冲突。

参考文献

  1. Aust,L.,Devlin,B.,Foster,SJ,Halvorsen,YD,Hicok,K.,du Laney,T.,Sen,A.,Willingmyre,GD and Gimble,JM(2004)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/14985162"target ="_ blank">来自吸脂抽吸物的人类脂肪来源的成体干细胞的产量。细胞疗法 6(1):7-14。
  2. Estes,BT,Diekman,BO,Gimble,JM和Guilak,F。(2010)。  分离脂肪来源的干细胞及其对软骨形成表型的诱导。 Nat Protoc 5(7):1294-1311。
  3. Gimble,J。和Guilak,F。(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/14578098"target ="_ blank" >脂肪来源的成体干细胞:分离,表征和分化潜能。细胞疗法 5(5):362-369。
  4. Strong,AL,Bowles,AC,Wise,RM,Morand,JP,Dutreil,MF,Gimble,JM and Bunnell,BA(2016)。 
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引用:Jones, R. B., Strong, A. L., Gimble, J. M. and Bunnell, B. A. (2017). Isolation and Primary Culture of Adult Human Adipose-derived Stromal/Stem Cells. Bio-protocol 7(5): e2161. DOI: 10.21769/BioProtoc.2161.
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