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Isolation of Cells from Human Intestinal Tissue
从人小肠组织分离细胞   

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Abstract

The intestinal lamina propria contains a dense network of T cells, dendritic cells (DCs) and macrophages, which play an important role in local innate and adaptive immune responses. We have recently identified distinct subsets of DCs (Persson et al., 2013) and macrophages (Bain et al., 2013) in the human intestine. In addition, we have studied T cells in healthy and diseased intestine. Here, we describe two methods for isolating these cell populations: 1) enzymatic treatment and 2) migration based isolation. The enzymatic method can be used to isolate T cells, DC and macrophages, whereas the migration based ‘walk-out’ protocol is suitable for DC isolation, as these cells migrate out from the tissues.

Materials and Reagents

  1. Tissue specimens of small (terminal ileum) and large intestine
  2. RPMI 1640 (Life Technologies, catalog number: 21875-034 )
  3. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F7424 )
  4. HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
  5. Penicillin and Streptomycin (Life Technologies, catalog number: 15140-122 )
  6. HBSS (Life Technologies, catalog number: 14180-046 )
  7. EDTA (Life Technologies, catalog number: AM9261 )
  8. Liberase TM (Roche Diagnostics, catalog number: 05401127001 )
  9. DNase I (Sigma-Aldrich, catalog number: D4263 )
  10. Collagenase 1A (0.2 μm-filtered) (Sigma-Aldrich, catalog number: C9891 )
  11. FACS antibodies
    1. T cells
      CD3-PE-Cy7 (SK7) (BD biosciences)
      Pacific blue (PB)-CD8 (RPA-T8) (BD biosciences)
      Quantum dot (QD) 605-CD4 (S3.5) (Life Technologies, InvitrogenTM)
      LIVE/DEAD® Fixable Near IR Dead Cell Stain Kit (Life Technologies, InvitrogenTM)
    2. DC/macrophages
      CD3-PE-Cy5 (UCHT1) (eBioscience)
      CD19-PE-Cy5 (HIB19) (eBioscience)
      CD11c-PE-Cy7 (3.9) (eBioscience)
      CD103-PE (Ber-ACT8) or CD103-eFluor647 (B-Ly7) (eBioscience)
      CD14-eFluor450 (61D3) (eBioscience)
      HLA-DR-APCeFluor780 (L43) (eBioscience)
      CD20 PE-Cy5 (2H7) (BioLegend)
      TCRab-PE-Cy5 (IP26) (BioLegend)
      Biotin-or PE-Cy7 CD172a (SE5A5) (BioLegend)
      CD56-PE-Cy5 (Alpha Diagnostic Intl)
      Biotin- or FITC-CD11c (MJ4-27G12) (Miltenyi Biotec)
      CD141-PE (AD5-14H12) (Miltenyi Biotec)
      CD45 V500 (HI30) (BD Biosciences)
      Biotinylated antibodies were detected using streptavidin conjugated to PE-Cy7 (eBiosicence) or QDot605 (Life Technologies, InvitrogenTM).
      Dead cells were excluded from analysis using propidium iodide PI (Life Technologies, Molecular Probes®).
  12. R10 medium (see Recipes)

Equipment

  1. Cell strainer (100 μm) (Thermo Fisher Scientific, catalog number: 22363549 )
  2. 50 ml Falcon tube
  3. Polyester filters cut in 10 x 10 cm squares (mesh count 27 threads/cm, mesh opening 250 μm, thread diameter 120 μm) (Tekniska Precisionsfilter JR AB)
  4. Petri dish (SARSTEDT AG, catalog number: 82.1473 )
  5. Ultra low attachment 24-well culture plate (Sigma-Aldrich, catalog number: CLS3473 )
  6. Pipette
  7. Needle/forceps
  8. Surgical scissor/scalpel
  9. Beakers with lid (VWR International, catalog number: 216-2694 )
  10. 37 °C, 5% CO2 cell culture incubator
  11. Shaker
  12. Centrifuge
  13. Microscope
  14. Multicolour FACS analyser (BD Lsr II flow cytometer)

Software

  1. FlowJo software (Tree Star Inc)

Procedure

  1. Enzymatic isolation protocol
    1. Surgical specimens of human intestine are collected in R10 medium. The tissue is kept on ice and the isolation procedure started optimally within 1 h.
    2. Underlying muscular layers and fat are removed with scissors and the remaining tissue is cut into small (< 5 mm) pieces with surgical knives (Figure 1). The tissue is placed into a 50 ml Falcon tube. The maximum amount of tissue in one tube should be approximately 4 g.


      Figure 1. Preparation of terminal ileum for enzymatic or migration based isolation protocols. A) Tissue is placed on a Petri dish and cut longitudinally to expose the mucosa (now facing upwards). B) The soft mucosal layer (containing the immune cells) is held by forceps and the underlying rigid muscular layer together with fat is then removed by cutting with scissors.
 C) Prior to EDTA treatment, the mucosal layer obtained in B) is cut into approximately 5 mm sized pieces.
 D) After the EDTA treatment, the tissue is cut into even smaller (1 mm) pieces. These can be then added
directly in the bottom of a cell culture well (migration protocol) or in a beaker with enzymes (enzymatic protocol).

    3. Epithelial cells are removed by incubating tissue fragments in 15 ml of HBSS supplemented with 5% FCS, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and EDTA (2 mM) for 15 min at 37 °C. After the incubation, the tissue is shaken vigorously by hand for 15 sec (foam will appear) and the epithelial cells in suspension are removed by filtering through a nylon filter. The tissue pieces are collected from the filter and placed again in 50 ml Falcon tubes.
    4. Repeat step A3 for a total of three times.
    5. Confirm the denuded appearance of the tissue by light microscopy (Figure 2).


      Figure 2. Light microscope images of human terminal ileum A) before and B) after treatment with EDTA
 to remove epithelial cells. Arrowheads in A) point to numerous villi with the clearly visible epithelial cell layers
still intact. In B) the arrow heads point to the missing epithelium. Tiny pieces (1 mm) of intestine were placed directly on glass objective slides together with a drop of R10 medium and a glass coverslip. Images were acquired
with a 5x objective and a digital camera attached to the microscope.

    6. Transfer remaining tissue pieces to a Petri dish. Cut in very fine pieces (< 1 mm) using a needle/forceps and a surgical scissor/scalpel. Look carefully if there are any big pieces left, cut more (Figure 1).
    7. Transfer the fragments into plastic beakers making sure that all pieces are transferred from the Petri dish.
    8. Wash the tissue fragments with 20 ml of R10 to remove any remaining EDTA, which may otherwise interfere with enzyme activity in the subsequent step. The pieces will sediment to the bottom of the beaker, remove the R10 with a pipette.
    9. For isolation of macrophages and DC, the tissue fragments are digested for 2 x 60 min at 37 °C in 10 ml of R10 containing Liberase TM (0.13 Wünsch units/ml) and DNase I (20 IU/ml) with magnetic stirring in plastic beakers with lid. Change the medium and enzymes after the first 60 min and then incubate another 60 min. The cell suspensions are pooled after filtering through 100 µM cell strainers. For T cells, incubate in 20 ml of R10 containing collagenase 1A (1 mg/ml) and DNase I (10 U/ml) with agitation for 60 min. The obtained cell suspension is filtered through 70 µM cell strainer.
    10. The single cell suspension is washed in 20 ml of R10 and pelleted at 177 x g for 10 min.
    11. Approximately 4 x 106 T cells, 5,000 CD11c+CD103+ DC and 50,000 CD14+ cells can be isolated from each gram of tissue as calculated by cell sorting (weighed after removal of muscular layer). These numbers are however only an estimate as the donor/preparation variation is high.
    12. Example of flow cytometry analysis from retrieved cell populations is shown in Figure 3.


      Figure 3. Isolation of human T cells, DC and macrophages. Human intestine was treated with either Collagenase 1A to isolate T cells or Liberase TM to isolate DC macrophages, as described above.
The resulting cell suspensions were stained for multicolour FACS analysis. After gating for size and granularity on the FSC/SSC profile, single, live CD3+ cells were gated and analysed for the expression of CD4 and CD8. For DC/macrophage analysis, live CD45+Lin-HLADR+ events were analysed for the expression of CD103 and CD14 showing both CD103+ DC and CD14hi/CD14lo macrophages. Comparison between liberase and migration based isolation method shows less CD14+ macrophages when the cells were allowed to migate out from the tissue. (Lin= CD3, CD19, CD20, CD56, TCRab)

  2.  Migration based isolation protocol
    1. The intestine is collected, cleaned and the epithelial cells removed as in the previous section resulting in tiny pieces (< 1 mm) of denuded tissue.
    2. Approximately 5 pieces of tissue/well are placed in 2 ml of R10 medium in ultra low attachment 24-well culture plates.
    3. The tissues are incubated for 18 h at 37 °C in 5% CO2 to allow cells to migrate out of the tissue.
    4. Place the plate on ice for 30 min in order to detach any plate bound cells. The tissue pieces are discarded and the medium containing migrated cells, is collected from the wells and cells pelleted by centrifugation at 177 x g for 10 min at 4 °C.
    5. Example of staining of the DC subsets is shown in Figure 4.


      Figure 4. Characterisation of CD103+ DC subsets in the human intestine. DC were allowed to migrate out of human small intestinal preparations as described above. The cells were stained for multicolour FACS analysis with antibodies to CD3, CD19, CD20, CD56, TCRαβ(Lin), CD45, HLA-DR,
CD11c, CD14 and CD103. The contour plots show Live CD45+Lin-HLADR+CD11c+CD103+CD14- events. DCs were assessed for expression of SIRPα and CD141.

Notes

  1. Both the enzymatic and migration based protocols can also be applied to small biopsy specimens. In this case, tissue does not need to be further trimmed for removal of fat or size reduction. In addition, the epithelial cell removal step should not include shaking and filtering as the amount of starting material can be very low. Instead, the biopsies are kept in the HBSS/EDTA solution for 45 min at 37 °C with low speed magnetic stirring. After the incubation, discard the medium (and the epithelial cells) and proceed as in step A6. We recommend needles instead of pipette tips for the handling of the tiniest biopsies.
  2. Collagenase 1A preserves the CD4 epitope on T cells, whereas Liberase cleaves CD4.
  3. FACS Staining
    1. Cells were preincubated on ice for 10 min in FACS buffer (PBS/2% FBS) containing 5% mouse serum to prevent unspecific antibody binding.
    2. Cell staining was performed in 100 μl aliquots for 30 min at 4 °C in the dark, followed by a wash in FACS buffer before being analyzed using an LSR II or FACSAria I and FlowJo software.

Recipes

  1. R10 medium
    RPMI 1640 supplemented with:
    10% FBS
    10 mM HEPES
    100 U/ml penicillin
    100 µg/ml streptomycin
    50 μg/ml gentamicin

Acknowledgments

This work was supported by grants from the Swedish Medical Research Council, the Göran Gustafsson, Crafoordska, Torsten and Ragnar Söderbergs, Kocks, Österlund, Swartz, Richard and Ruth Julins, and the IngaBritt and Arne Lundbergs Foundations, the Royal Physiographic Society, a clinical grant from the Swedish National Health Service, and the Swedish Foundation for Strategic Research FFL-2 program.

References

  1. Bain, C. C., Scott, C. L., Uronen-Hansson, H., Gudjonsson, S., Jansson, O., Grip, O., Guilliams, M., Malissen, B., Agace, W. W. and Mowat, A. M. (2013). Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 6(3): 498-510. 
  2. Persson, E. K., Uronen-Hansson, H., Semmrich, M., Rivollier, A., Hagerbrand, K., Marsal, J., Gudjonsson, S., Hakansson, U., Reizis, B., Kotarsky, K. and Agace, W. W. (2013). IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 38(5): 958-969. 

简介

肠固有层含有T细胞,树突细胞(DC)和巨噬细胞的密集网络,其在局部先天和适应性免疫应答中起重要作用。 我们最近在人肠中鉴定了不同的DC亚群(Persson等人,2013)和巨噬细胞亚群(Bain等人,2013)。 此外,我们研究了健康和患病肠中的T细胞。 在这里,我们描述了两种方法隔离这些细胞群体:1)酶处理和2)基于迁移的隔离。 酶法可用于分离T细胞,DC和巨噬细胞,而基于迁移的"走出"方案适合于DC分离,因为这些细胞从组织迁出。

材料和试剂

  1. 小(末回肠)和大肠的组织标本
  2. RPMI 1640(Life Technologies,目录号:21875-034)
  3. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7424)
  4. HEPES(Life Technologies,Gibco ,目录号:15630-080)
  5. 青霉素和链霉素(Life Technologies,目录号:15140-122)
  6. HBSS(Life Technologies,目录号:14180-046)
  7. EDTA(Life Technologies,目录号:AM9261)
  8. Liberase TM(Roche Diagnostics,目录号:05401127001)
  9. DNase I(Sigma-Aldrich,目录号:D4263)
  10. 胶原酶1A(0.2μm过滤)(Sigma-Aldrich,目录号:C9891)
  11. FACS抗体
    1. T细胞
      CD3-PE-Cy7(SK7)(BD biosciences)
      太平洋蓝(PB)-CD8(RPA-T8)(BD biosciences)
      量子点(QD)605-CD4(S3.5)(Life Technologies,Invitrogen TM
      LIVE/DEAD 可固定近红外死细胞染色试剂盒(Life Technologies,Invitrogen TM
    2. DC /巨噬细胞
      CD3-PE-Cy5(UCHT1)(eBioscience)
      CD19-PE-Cy5(HIB19)(eBioscience)
      CD11c-PE-Cy7(3.9)(eBioscience)
      CD103-PE(Ber-ACT8)或CD103-eFluor647(B-Ly7)(eBioscience)
      CD14-eFluor450(61D3)(eBioscience)
      HLA-DR-APCeFluor780(L43)(eBioscience)
      CD20 PE-Cy5(2H7)(BioLegend)
      TCRab-PE-Cy5(IP26)(BioLegend)
      生物素或PE-Cy7 CD172a(SE5A5)(BioLegend)
      CD56-PE-Cy5(Alpha诊断国家)
      生物素 - 或FITC-CD11c(MJ4-27G12)(Miltenyi Biotec) CD141-PE(AD5-14H12)(Miltenyi Biotec)
      CD45 V500(HI30)(BD Biosciences) 使用与PE-Cy7(eBiosicence)或QDot605(Life Technologies,Invitrogen)连接的链霉抗生物素检测生物素化的抗体。
      使用碘化丙锭PI(Life Technologies,Molecular Probes )从分析中排除死细胞。
  12. R10介质(见配方)

设备

  1. 细胞过滤器(100μm)(Thermo Fisher Scientific,目录号:22363549)
  2. 50ml Falcon管
  3. 以10×10cm正方形(筛目27线/cm,筛孔250μm,线径120μm)(Tekniska Precisionsfilter JR AB)切割的聚酯过滤器
  4. 培养皿(SARSTEDT AG,目录号:82.1473)
  5. 超低附着24孔培养板(Sigma-Aldrich,目录号:CLS3473)
  6. 移液器
  7. 针/镊子
  8. 外科剪刀/手术刀
  9. 带盖的烧杯(VWR International,目录号:216-2694)
  10. 37℃,5%CO 2细胞培养箱中培养
  11. 振动器
  12. 离心机
  13. 显微镜
  14. 多色FACS分析仪(BD Lsr II流式细胞仪)

软件

  1. FlowJo软件(Tree Star Inc)

程序

  1. 酶分离方案
    1. 将人肠的外科手术标本收集在R10培养基中。 将组织保持在冰上,并在1小时内最佳地开始分离程序
    2. 用剪刀除去下面的肌肉层和脂肪,并用手术刀(图1)将剩余的组织切成小(<5mm)片。将组织置于50ml Falcon管中。一个试管中的最大组织量应为约4g

      图1.制备用于酶促或基于迁移的分离方案的末端回肠。 A)将组织置于培养皿上并纵向切开以暴露粘膜(现面向上)。 B)软粘膜层(含有免疫细胞)由镊子保持,并且随后用剪刀通过切割除去与脂肪一起的下面的刚性肌层。 C)在EDTA处理之前,将B)中获得的粘膜层切成约5mm大小的片。 D)EDTA处理后,将组织切成甚至更小(1mm)的片。然后可以将它们直接加入细胞培养孔(迁移方案)的底部或具有酶的烧杯中(酶方案)。

    3. 通过将组织片段在补充有5%FCS,10mM HEPES,100U/ml青霉素,100μg/ml链霉素和EDTA(2mM)的15ml HBSS中在37℃下孵育15分钟来除去上皮细胞。孵育后,用手剧烈摇动组织15秒(出现泡沫),并通过尼龙过滤器过滤除去悬浮液中的上皮细胞。从过滤器收集组织片,并再次放置在50ml Falcon管中。
    4. 重复步骤A3,总共三次。
    5. 通过光学显微镜检查确认组织的裸露外观(图2)

      图2.人类末端回肠的光学显微镜图像A)在用EDTA处理以去除上皮细胞之前和B)。 A)中的箭头指向具有清晰可见的上皮细胞层的多个绒毛保持完整。在B)中,箭头指向缺失的上皮。将小块(1mm)的肠直接放置在玻璃目标载玻片上,一滴R10培养基和玻璃盖玻片。使用5x物镜和连接到显微镜的数码相机获取图像
    6. 将剩余的组织片转移到培养皿中。使用针/镊子和外科手术剪刀/手术刀切成非常细的碎片(<1mm)。仔细检查是否有大块,留下更多(图1)。
    7. 将碎片转移到塑料烧杯中,确保所有的碎片都从培养皿中转移出来
    8. 用20ml R10洗涤组织片段以除去任何剩余的EDTA,否则可能干扰后续步骤中的酶活性。碎片将沉淀到烧杯底部,用移液管取出R10
    9. 为了分离巨噬细胞和DC,将组织片段在37℃下在10ml含有Liberase TM(0.13Wünsch单位/ml)和DNase I(20IU/ml)的R10中在塑料中磁力搅拌下消化2×60分钟烧杯带盖。在第一次60分钟后更换培养基和酶,然后再孵育60分钟。在通过100μM细胞过滤器过滤后合并细胞悬浮液。对于T细胞,在20ml含有胶原酶1A(1mg/ml)和DNase I(10U/ml)的R10中孵育60分钟。将获得的细胞悬浮液通过70μM细胞过滤器过滤。
    10. 将单细胞悬浮液在20ml R10中洗涤并在177×g下沉淀10分钟。
    11. 可以分离约4×10 6个T细胞,5,000个CD11c + CD103 + sup DC和50,000个CD14 + sup +细胞通过细胞分选(除去肌肉层后称重)计算的每克组织。然而,这些数字仅仅是供体/制剂变化高的估计值
    12. 从检索的细胞群体的流式细胞术分析的实例显示于图3中

      图3.人T细胞,DC和巨噬细胞的分离。如上所述,用胶原酶1A处理人肠以分离T细胞或用Liberase TM处理以分离DC巨噬细胞。将所得细胞悬浮液染色以进行多色FACS分析。在FSC/SSC图谱上对大小和粒度进行门控后,门控单个活的CD3 +细胞并分析CD4和CD8的表达。对于DC /巨噬细胞 分析,分析活CD45 +/- Lin-HLADR + 事件的CD103和CD14的表达,显示CD103 + DC和CD14 - hi/CD14 sup/lo巨噬细胞。 当允许细胞从组织迁移出来时,基于释放和基于迁移的分离方法之间的比较显示较少的CD14 + sup/+巨噬细胞。 (Lin = CD3,CD19,CD20,CD56,TCRab)

  2.  基于迁移的隔离协议
    1. 收集,清洁肠,并如上一节所述除去上皮细胞,得到微小碎片(<1mm)的剥离组织。
    2. 将约5片组织/孔置于超低附着24孔培养板中的2ml R10培养基中。
    3. 将组织在37℃下在5%CO 2中孵育18小时以允许细胞迁移出组织。
    4. 将板置于冰上30分钟,以分离任何板结合的细胞。弃去组织片,从孔中收集含有迁移细胞的培养基,并通过在177℃下以4℃离心10分钟使细胞沉淀。
    5. DC子集的染色实例如图4所示。


      图4.人类肠中CD103 + DC亚型的表征。 DC如上所述从人类小肠制剂中迁移出来。使用针对CD3,CD19,CD20,CD56,TCRαβ(Lin),CD45,HLA-DR,CD11c,CD14和CD103的抗体对细胞进行染色以进行多色FACS分析。轮廓图显示活CD45 + Lin-HLADR + CD11c + CD103 + CD14 sup>事件。评估DCs的SIRPα和CD141的表达

笔记

  1. 酶和基于迁移的协议也可以应用于小活检标本。在这种情况下,组织不需要进一步修整以去除脂肪或减小尺寸。此外,上皮细胞去除步骤不应包括摇动和过滤,因为起始材料的量可以非常低。相反,活检在HBSS/EDTA溶液中在37℃下用低速磁力搅拌保持45分钟。孵育后,丢弃培养基(和上皮细胞),并如步骤A6进行。我们建议使用针头而不是移液管尖端来处理最小活检
  2. 胶原酶1A保留T细胞上的CD4表位,而Liberase切割CD4。
  3. FACS染色
    1. 将细胞在含有5%小鼠血清的FACS缓冲液(PBS/2%FBS)中在冰上预温育10分钟,以防止非特异性抗体结合。
    2. 细胞染色在100μl等分试样中在4℃黑暗中进行30分钟,然后在FACS缓冲液中洗涤,然后使用LSR II或FACSAria I和FlowJo软件分析。

食谱

  1. R10介质
    RPMI 1640补充:
    10%FBS
    10 mM HEPES
    100 U/ml青霉素
    100μg/ml链霉素 50μg/ml庆大霉素

致谢

这项工作得到瑞典医学研究委员会,GöranGustafsson,Crafoordska,Torsten和RagnarSöderbergs,Kocks,Österlund,Swartz,Richard和Ruth Julins以及IngaBritt和Arne Lundbergs基金会,皇家生理协会,瑞典国家卫生服务临床拨款和瑞典战略研究基金FFL-2计划。

参考文献

  1. B,CC,Scott,CL,Uronen-Hansson,H.,Gudjonsson,S.,Jansson,O.,Grip,O.,Guilliams,M.,Malissen,B.,Agace,WW和Mowat,AM 。 结肠中的常驻和促炎性巨噬细胞代表相同Ly6Chi单核细胞前体的替代性上下文相关命运。 Mucosal Immunol 6(3):498-510。 
  2. Persson,EK,Uronen-Hansson,H.,Semmrich,M.,Rivollier,A.,Hagerbrand,K.,Marsal,J.,Gudjonsson,S.,Hakansson,U.,Reizis,B.,Kotarsky,和Agace,WW(2013)。 IRF4转录因子依赖性CD103(+)CD11b(+)树突细胞驱动粘膜T辅助细胞17细胞分化。 免疫 38(5):958-969。
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引用:Uronen-Hansson, H., Persson, E., Nilsson, P. and Agace, W. (2014). Isolation of Cells from Human Intestinal Tissue. Bio-protocol 4(7): e1092. DOI: 10.21769/BioProtoc.1092.
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