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Isolation of Intestinal Mesenchymal Cells from Adult Mice
从成年小鼠分离肠道间充质细胞   

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参见作者原研究论文

本实验方案简略版
The Journal of Experimental Medicine
Dec 2015

 

Abstract

During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells from the adult mouse intestine.

Keywords: Mouse intestine (小鼠小肠), Mesenchyme (间充质), Fibroblasts (成纤维细胞), Tissue digestion (组织消化), Collagenase (胶原酶)

Materials and Reagents

  1. Petri dishes (100 mm)
    Note: Any Petri dish can be used, according to each researcher’s preference and availability.
  2. Falcon tube
  3. Transfer pipettes 3.5 ml (SARSTEDT, catalog number: 86.1172.001 )
  4. 50 ml conical tubes (SARSTEDT, catalog number: 62.547.004 )
  5. Syringe filters, 0.2 μm pore size (SARSTEDT, catalog number: 83.1826.001 )
  6. Cell strainers 70 μm (Corning, Falcon®, catalog number: 352350 )
  7. Cell culture treated flasks (25 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 136196 )
  8. Cell culture treated flasks (175 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
  9. Cell culture treated flasks (80 cm2) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178905 )
  10. Mice
  11. Ethanol
    Note: Any ethanol can be used, according to each researcher’s preference and availability.
  12. HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14170088 )
  13. Antibiotic-antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
  14. EDTA (Thermo Fisher Scientific, Fisher Scientific, catalog number: 327205000 )
  15. DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: D9779 )
  16. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
  17. Fetal bovine serum (FBS) (Biochrom, catalog number: S0115 )
  18. Penicillin-streptomycin 100x (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  19. L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  20. MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-068 )
  21. Phosphate-buffered saline (PBS) Dulbecco (10x) (Biochrom, catalog number: L1835 )
  22. Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
  23. Collagenase from Clostridium histolyticum, type XI (Sigma-Aldrich, catalog number: C7657 )
  24. Dispase II, neutral protease grade II, from Bacillus polymyxa (Roche Diagnostics, catalog number: 04942078001 )
  25. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  26. FACS staining antibodies and material
    1. Vimentin-A647 (Abcam, catalog number: ab194719 )
    2. CD45-PE-Cy5 (BD, catalog number: 553082 )
    3. Intracellular fixation & permeabilization buffer set (eBioscience, catalog number: 88-8824-00 )
  27. HBSS/antibiotics (see Recipes)
  28. HBSS/EDTA/DTT (see Recipes)
  29. Digestion solution (see Recipes)
  30. DMEM medium (see Recipes)

Equipment

  1. Dissection kit
    1. Two scissors (at least one medium size and one small)
    2. Two forceps
      Note: Any scissors and forceps can be used, according to each researcher’s preference and availability.
    3. A 10 ml syringe equipped with a 21-gauge needle for flushing the intestine (BD, PlastipakTM, catalog number: 308063 )
  2. Cell culture hood
  3. Cell culture incubator, 37 °C, 5% CO2
  4. Shaking water-bath
  5. Centrifuge compatible with 50 ml conical tubes
  6. Bright-field inverted microscope for cell culture
  7. Multicolor FACS analyser (BD, model: BD FACS Canto II )

Software

  1. FlowJo software (Tree Star Inc.)

Procedure

  1. At the bench
    1. Euthanize the mouse by CO2 asphyxiation, following guidelines approved by your Institutional Animal Care and Use Committee.
    2. Sterilize the skin of the abdomen with 70% ethanol and open it.
    3. Remove the colon and/or 7-8 cm of the small intestine (from the cecum to the stomach) and place separately in Petri dishes filled with ice-cold HBSS/antibiotics (Figure 1).
      Note: It is better to avoid cell isolation from the colon and small intestine together as they need different incubation times in the digestion solution.


      Figure 1. Dissection of the mouse intestine. A. The skin is removed and the abdomen is exposed. B. The peritoneum is carefully opened to expose the intestine. C. The intestine is removed from the cavity to expose the stomach and the anus (indicated by arrows). D. The intestine is carefully removed from the anus to the stomach. The colon and small intestine are removed and placed in separate Petri dishes. Intestinal parts are indicated.

    4. Wash the intestinal parts repeatedly by flushing with HBSS/antibiotics using a 10 ml syringe equipped with a 21-gauge needle (Figure 2A). Place the tissue on a pre-wet paper and remove fat and adherent connective tissue (Figure 2B). Cut the intestinal tissue longitudinally and place in 10 ml ice-cold HBSS/antibiotics in a falcon tube per mouse on ice (Figure 2C).
      Notes:
      1. Intestinal tissue is sensitive and the duration of this step should be as short as possible. Each mouse tissue should be prepared separately and the tissue should then be placed on ice. If you need to pause the protocol in this step, although not recommended, place all tissues on ice.
      2. For the small intestine, it is better to also remove Peyer’s patches (Figure 2B).
      3. Using blunt ended needles in this step might be more convenient for less experienced users.


        Figure 2. Representative images of the steps for the preparation of the intestine. A. Flushing of the intestine. B. Removal of fat and connective tissue with the help of forceps. Peyer’s Patches are indicated with arrows. C. Longitudinal opening of the intestinal tissue.

  2. In the cell culture hood
    1. Wash colon or small intestine with ice-cold HBSS/antibiotics (~10 ml) at least 3 times by vigorously shaking the tube, removing the supernatant and adding new HBSS/antibiotics each time.
      Notes:
      1. The supernatant should be clear before you move to the next step.
      2. Removal of the supernatant can be easier if you use the sterile transfer pipettes.
    2. Place the tissue in a Petri dish and cut the tissue at 0.5-1 cm pieces with the help of scissors and forceps.
      Note: Scissors and forceps can be sterilized before inserting in the cell culture hood. Alternatively, scissors and forceps can be sprayed with 70% ethanol before use. They can be kept in 70% ethanol and emerged in sterile water before manipulation of the intestine, or in-between samples.
    3. Wash the pieces once with ice-cold HBSS/antibiotics (~10 ml) with vigorous shaking and remove the supernatant.
    4. Add 10 ml pre-warmed HBSS/EDTA/DTT solution and incubate for 20 min, at 37 °C, in a shaking water-bath (~200 rpm) to remove intestinal epithelial cells (both for the colon and small intestine).
    5. Wash the intestinal pieces at least 3 times with HBSS/antibiotics (it can be at room temperature now) by vigorously shaking the tubes for ~15 sec, removing the supernatant and adding new HBSS/antibiotics each time. After the last wash the supernatant should no longer be cloudy. Decant through a cell strainer (70 μm) to remove all liquid and place in a new Falcon tube.
      Note: EDTA can inhibit collagenase, so it has to be removed completely.
    6. Add 10 ml digestion solution and incubate for 1 h (colon) or 30-40 min (small intestine), at 37 °C, in a shaking water-bath (~200 rpm).
      Note: You should see the supernatant become cloudy and the tissue more transparent but not completely dissolved (Figure 3).


      Figure 3. Intestinal tissue before and after enzymatic digestion. The arrow indicates the presence of some intact tissue after enzymatic digestion.

    7. Shake vigorously and place supernatant in a new Falcon tube.
      Optional: You can pass the supernatant through a cell strainer (70 μm).
    8. Centrifuge at 280 x g for 10 min at room temperature.
    9. Resuspend the cell pellet in 5-6 ml DMEM medium + amphotericin B (2.5 μg/ml) and place in a 25 cm2 flask per mouse.
      Important: After 2-3 h you can notice cells adhering to the bottom of the flasks (Figure 4A). Change the medium at this point or the next day by removing the supernatant and adding new medium to reduce chances of contamination.
    10. Watch out daily for signs of bacterial contamination and feed every 2-3 days. Usually, at 4-5 days cells are confluent and can be passaged to a 75 cm2 flask (Figure 4B).


      Figure 4. Representative images from IMCs in cell culture after plating (A) and at passage 2 (P2) (B). In (A) selected cells that have attached to the flask are indicated by arrows (Magnification lens 10x).

    11. Passaging of cells can be done by washing the cells twice with 10 ml of 1x PBS (diluted 10x PBS to 1x with H2O) each time and adding trypsin/EDTA (1 ml for a 25 cm2 flask). Incubation with trypsin/EDTA for 5 min, at room temperature should be enough for cells to detach. DMEM medium is then added to inactivate trypsin and cells are split at a 1:2 ratio.
    12. After the first passage, cells are usually passaged every 3-6 days in a split ratio 1:2 (always split when the cells are almost 100% confluent). Feed every 2-3 days. At passages 1 and 2, amphotericin B is added at 0.25 μg/ml and at passage 3 it is omitted.
    13. At passage 3, check cells with FACS analysis for their purity. At this stage, cultures are normally CD45 (< 3-4%), therefore negative for hematopoietic cells, and can be used in subsequent experiments. Vimentin, an intracellular cytoskeleton protein, can be used as a positive marker and should stain > 90% of the cells (See example of flow cytometry analysis in Figure 5 and FACS staining details under “Notes” section).


      Figure 5. Representative FACS analysis of isolated IMCs at passage 3 (P3)

    14. Try to use isolated IMCs between passages 3-5 and don’t keep them more than one month or if you notice changes in their morphology. One 175 cm2 flask usually yields 3-6 x 106 cells.
    15. Cells can be used for several functional and biochemical assays. Plating cell concentration to reach ~80% confluence the next day is as follows:
      1. 96-well plate: 2 x 104 per well
      2. 24-well plate: 8 x 104 per well
      3. 6-well plate: 2.5 x 104 per well
      4. 100 mm plate: 1.6 x 106 per plate

Notes

  1. The above protocol refers to the use of 1 mouse/sample. You can group colon or small intestine from 3 mice and add 15 ml of HBSS/EDTA/DTT and 15 ml of digestion solution. In this case, more washes may be necessary before the supernatant is clear in steps B1 and B5. After the end of the protocol you can plate the isolated cells in a 75 cm2 flask.
  2. There is increased chance of bacterial contamination after isolation and before the first cell passage. For this reason try to change the medium 2-3 h or the next day after isolation, check cells daily and change the medium every 2-3 days. Make sure to always add amphotericin at the indicated concentration, preferably prepare the amphotericin-supplemented DMEM medium fresh and handle it appropriately, as it is light sensitive.
  3. FACS staining
    1. Cell staining for CD45 (0.2 μl per 1-2 x 106 cells) is performed first in 100 μl samples (1-2 x 106 cells) in PBS/5% FBS, for 30 min, at 4 °C, in the dark, followed by two washes with FACS buffer (PBS/2% FBS).
    2. Cells are then fixed and permeabilized, using the Fixation & Permeabilization kit for 40 min at room temperature, followed by two washes with 1x permeabilization buffer (Perm buffer).
    3. Cell staining with vimentin (0.2 μl per 1-2 x 106 cells) is then performed in 100 μl 1x Perm buffer, for 1 h, at room temperature, followed by one wash with Perm buffer and one wash with FACS buffer before analysis using a FACS BD Canto II cytometer and the FlowJo software.
  4. In case the intestine is inflamed you may notice a higher number of CD45+ cells. In this case and if this is a problem for your subsequent experiments, you may have to add an extra magnetic-bead depletion or cell-sorting step.
  5. Cryopreservation of primary intestinal mesenchymal cells is not recommended.

Recipes

  1. HBSS/antibiotics
    5 ml antibiotic-antimycotic in 500 ml HBSS
    Note: Ice cold before use.
  2. HBSS/EDTA/DTT
    5 mM EDTA
    1 mM DTT
    1x pen/strep
    Dissolve in HBSS
    Note: Pre-warmed at 37 °C before use.
  3. Digestion solution
    300 U/ml collagenase XI
    0.08 U/ml dispase II
    Dissolve in DMEM medium
    Note: The digestion solution should be made fresh. Weigh the appropriate amount of collagenase and dispase according to the number of mice you will use and dilute it in HBSS at 1/10 of the final volume. Sterile filter the solution through a syringe filter with 0.2 μm pore size and then add DMEM medium to the final volume.
  4. DMEM medium
    50 ml heat-inactivated FBS
    5 ml pen/strep
    5 ml L-glutamine
    5 ml non-essential amino acids
    Dissolve in 500 ml DMEM
    Notes:
    1. All mediums apart from the digestion solution can be made in advance and stored at 4 °C.
    2. Pre-warmed at 37 °C before use.

Acknowledgments

This protocol was based on a previous protocol used in our lab (Armaka et al., 2008). This work was supported by FP7 Advanced ERC grant MCs-inTEST (Grant Agreement No. 340217) and Innovative Medicines Initiative Joint Undertaking (IMI JU) project BTCure (Grant Agreement No.115142) to GK.

References

  1. Armaka, M., Apostolaki, M., Jacques, P., Kontoyiannis, D. L., Elewaut, D. and Kollias, G. (2008). Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J Exp Med 205(2): 331-337.
  2. Koliaraki, V., Roulis, M. and Kollias, G. (2012). Tpl2 regulates intestinal myofibroblast HGF release to suppress colitis-associated tumorigenesis. J Clin Invest 122(11): 4231-4242.
  3. Koliaraki, V., Pasparakis, M. and Kollias, G. (2015). IKKβ in intestinal mesenchymal cells promotes initiation of colitis-associated cancer. J Exp Med 212(13): 2235-2251.
  4. Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X. and Mifflin, R. C. (2011). Mesenckhymal cells of the intestinal lamina propria. Annu Rev Physiol 73: 213-237.
  5. Roulis, M., Nikolaou, C., Kotsaki, E., Kaffe, E., Karagianni, N., Koliaraki, V., Salpea, K., Ragoussis, J., Aidinis, V., Martini, E., Becker, C., Herschman, H. R., Vetrano, S., Danese, S. and Kollias, G. (2014). Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis. Proc Natl Acad Sci U S A 111(43): E4658-4667.

简介

在过去20年间,肠间质细胞(IMC)已经作为重要的细胞类型出现,通过提供结构支持和生长调节元件在肠发育和体内平衡中起着中心作用。 IMC还积极参与伤口愈合反应,从而调节病理状况,例如组织修复,炎症,纤维化和癌发生(Powell等人,2011)。 我们最近已经证明肠间充质特异性信号在肠炎症和癌发生中在体内起重要的生理作用(Koliaraki等人,2012; Roulis等人,/em>。,2014; Koliaraki 。。,2015)。 在这里我们描述了用于从成年小鼠肠道分离和培养间充质细胞的酶法。

关键字:小鼠小肠, 间充质, 成纤维细胞, 组织消化, 胶原酶

材料和试剂

  1. 培养皿(100mm)
    注意:根据每个研究者的偏好和可用性,可以使用任何培养皿。
  2. 猎鹰管
  3. 移液管3.5ml(SARSTEDT,目录号:86.1172.001)
  4. 50ml锥形管(SARSTEDT,目录号:62.547.004)
  5. 注射过滤器,0.2μm孔径(SARSTEDT,目录号:83.1826.001)
  6. 细胞过滤器70μm(Corning,Falcon ,目录号:352350)
  7. 细胞培养处理的烧瓶(25cm 2)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:136196)
  8. 将细胞培养处理的烧瓶(175cm 2)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:178883)
  9. 细胞培养处理的烧瓶(80cm 2)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:178905)
  10. 小鼠
  11. 乙醇
    注意:根据每个研究者的偏好和可用性,可以使用任何乙醇。
  12. HBSS(Thermo Fisher Scientific,Gibco TM ,目录号:14170088)
  13. 抗生素 - 抗真菌的(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:15240096)
  14. EDTA(Thermo Fisher Scientific,Fisher Scientific,目录号:327205000)
  15. DTT(DL-二硫苏糖醇)(Sigma-Aldrich,目录号:D9779)
  16. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11960044)
  17. 胎牛血清(FBS)(Biochrom,目录号:S0115)
  18. 青霉素 - 链霉素100x(10,000U/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  19. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  20. MEM非必需氨基酸溶液(100x)(Thermo Fisher Scientific,Gibco< sup>,目录号:11140-068)
  21. 磷酸盐缓冲盐水(PBS)Dulbecco(10x)(Biochrom,目录号:L1835)
  22. 两性霉素B(Sigma-Aldrich,目录号:A2942)
  23. 来自溶组织梭菌(Clostridium histolyticum)III型(Sigma-Aldrich,目录号:C7657)的胶原酶
  24. Dispase II,中性蛋白酶II级,来自多粘芽孢杆菌(Roche Diagnostics,目录号:04942078001)
  25. 胰蛋白酶-EDTA(0.05%)(Thermo Fisher Scientific,Gibco TM ,目录号:25300054)
  26. FACS染色抗体和物质
    1. Vimentin-A647(Abcam,目录号:ab194719)
    2. CD45-PE-Cy5(BD,目录号:553082)
    3. 细胞内固定和 渗透缓冲液(eBioscience,目录号:88-8824-00)
  27. HBSS /抗生素(参见配方)
  28. HBSS/EDTA/DTT(参见配方)
  29. 消解解决方案(参见配方)
  30. DMEM介质(参见配方)

设备

  1. 解剖工具包
    1. 两把剪刀(至少一个中号和一个小号)
    2. 两只镊子
      注意:根据每个研究者的偏好和可用性,可以使用任何剪刀和镊子。
    3. 装配有用于冲洗肠的21号针(BD,Plastipak TM ,目录号:308063)的10ml注射器
  2. 细胞培养罩
  3. 细胞培养温箱,37℃,5%CO 2/s
  4. 摇水浴
  5. 离心机与50ml锥形管相容
  6. 用于细胞培养的明场倒置显微镜
  7. 多色FACS分析仪(BD,型号:BD FACS Canto II)

软件

  1. FlowJo软件(Tree Star Inc.)

程序

  1. 在长凳
    1. 按照机构动物护理和使用委员会批准的指南,通过CO 2窒息将小鼠安乐死。
    2. 用70%乙醇消毒腹部皮肤并打开它
    3. 取出结肠和/或7-8厘米的小肠(从盲肠到胃),并分别放置在装有冰冷的HBSS /抗生素的培养皿中(图1)。
      注意:最好避免细胞与结肠和小肠分离,因为它们在消化溶液中需要不同的孵育时间。

      span>
      图1.小鼠肠道的解剖。 A.去除皮肤,暴露腹部。 B.小心打开腹膜以暴露肠。 C.从腔中取出肠以暴露胃和肛门(由箭头指示)。 D.小肠从肛门小心地移至胃。取出结肠和小肠,并置于单独的培养皿中。指示肠部分。

    4. 使用装有21号针的10ml注射器(图2A)通过用HBSS /抗生素冲洗来重复洗涤肠部分。将组织放在预湿纸上,去除脂肪和粘附结缔组织(图2B)。纵向切开肠组织,并在冰上每只小鼠放在冰冷管中的10ml冰冷的HBSS /抗生素中(图2C)。
      注意:
      1. 肠组织是敏感的,并且该步骤的持续时间应该尽可能短。每个小鼠组织应单独制备,然后将组织置于冰上。如果您需要暂停此步骤中的协议,但 不推荐,请将所有组织放在冰上。
      2. 对于小肠,最好也除去Peyer的补丁(图2B)。
      3. 在此步骤中使用钝针可能会使经验较少的用户更方便。


        图2.用于制备肠的步骤的代表性图像。 A.冲洗肠。 B.利用镊子去除脂肪和结缔组织。 Peyer的补丁用箭头指示。 C.肠组织的纵向打开。

  2. 在细胞培养箱
    1. 用冰冷的HBSS /抗生素(约10ml)冲洗结肠或小肠至少3次,通过剧烈摇动试管,除去上清液,每次加入新的HBSS /抗生素。
      1. 在进入下一步之前,上清液应该是澄清的。
      2. 如果使用无菌转移移液器,去除上清液可能更容易。
    2. 将组织放置在培养皿中,并用剪刀和镊子帮助下切割0.5-1厘米的组织。
      注意:剪刀和镊子可以在插入细胞培养罩之前进行灭菌。或者,剪刀和镊子可在使用前用70%乙醇喷雾。它们可以保存在70%乙醇中,并在肠道操作之前或在样品之间出现在无菌水中。
    3. 用冰冷的HBSS /抗生素(约10ml)洗涤一次,并剧烈摇动,并除去上清液。
    4. 加入10ml预热的HBSS/EDTA/DTT溶液,并在37℃下在振荡水浴(〜200rpm)中孵育20分钟以除去肠上皮细胞(结肠和小肠两者)。 br />
    5. 通过剧烈摇动试管〜15秒,用HBSS /抗生素(可以在室温下)洗肠段至少3次,除去上清液,每次加入新的HBSS /抗生素。最后一次洗涤后,上清液应不再浑浊。倾析细胞过滤器(70μm),以去除所有液体,并放置在新的猎鹰管中。
      注意:EDTA可以抑制胶原酶,因此必须完全去除。
    6. 加入10ml消化溶液并在37℃下在振荡水浴(〜200rpm)中孵育1小时(结肠)或30-40分钟(小肠)。
      注意:您应该会看到上层变得浑浊,组织更透明,但不完全溶解(图3)。


      图3. 酶消化前后的肠组织。箭头表示酶消化后存在一些完整组织。

    7. 剧烈摇动并将上清液放置在新的falcon管中。
      可选:您可以通过细胞过滤器(70μm)传递su 上下文。
    8. 在室温下280℃离心10分钟。
    9. 将细胞沉淀重悬于5-6ml DMEM培养基+两性霉素B(2.5μg/ml)中,并置于每只小鼠25cm 2的培养瓶中。
      重要:2-3小时后,您可以注意到细胞粘附在烧瓶底部(图4A)。 第二天更换培养基。
    10. 每天观察细菌污染的迹象,每2-3天喂食一次。通常在4-5天,细胞汇合,并可以传代至75cm 2烧瓶(图4B)。


      图4.在铺板(A)和第2代(P2)(B)后细胞培养物中来自IMC的代表性图像。在(A)中,连接到烧瓶的所选细胞由箭头指示放大镜10x)。

    11. 细胞的传代可通过每次用10ml的1x PBS洗涤细胞两次(用10x PBS稀释至1x H 2 O)两次,并加入胰蛋白酶/EDTA(1ml,对于25cm 3) 2烧瓶)。用胰蛋白酶/EDTA孵育5分钟,在室温下应足以使细胞分离。然后加入DMEM培养基以灭活胰蛋白酶,并以1:2的比例分裂细胞。
    12. 在第一次传代后,细胞通常每3-6天以1:2的分流比传代(当细胞几乎100%汇合时总是分裂)。每2-3天饲喂一次。在第1和2代,两性霉素B以0.25μg/ml加入,第3代省略。
    13. 在第3代,用FACS分析检查细胞的纯度。在这一阶段,培养物通常是CD45(<3-4%),因此对于造血细胞是阴性的,并且可以用于随后的实验中。波形蛋白,细胞内细胞骨架蛋白,可以用作阳性标记,并且应该染色> 90%的细胞(参见图5中的流式细胞术分析的实例和"注释"部分下的FACS染色细节)。


      图5.在第3代(P3)
      的分离的IMC的代表性FACS分析
    14. 尝试在3-5代之间使用隔离的IMC,并且不要保持它们超过一个月,或者如果注意到其形态的变化。一个175cm 2烧瓶通常产生3-6×10 6个细胞。
    15. 细胞可用于几种功能和生化测定。第二天电镀细胞浓度达到〜80%汇合如下:
      1. 96孔板:每孔2×10 4个
      2. 24孔板:每孔8×10 4个
      3. 6孔板:每孔2.5×10 4个
      4. 100mm板:每个板1.6×10 6个

笔记

  1. 上述方案涉及使用1只小鼠/样品。您可以从3只小鼠分组结肠或小肠,加入15毫升HBSS/EDTA/DTT和15毫升消化溶液。在这种情况下,在步骤B1和B5中上清液澄清之前可能需要更多的洗涤。在方案结束后,您可以将分离的细胞在75cm 2烧瓶中铺板。
  2. 在分离后和第一次细胞传代之前,细菌污染的可能性增加。为此,尝试改变培养基2-3小时或隔离后的第二天,每天检查细胞,每2-3天更换培养基。确保始终添加指定浓度的两性霉素,优选制备新鲜的两性霉素补充的DMEM培养基并适当处理,因为它是光敏感的。
  3. FACS染色
    1. 首先在PBS/5中的100μl样品(1-2×10 6个细胞)中进行CD45的细胞染色(0.2μl每1-2×10 6个细胞) %FBS,在4℃在黑暗中孵育30分钟,然后用FACS缓冲液(PBS/2%FBS)洗涤两次。
    2. 然后将细胞固定并透化,使用Fixation&渗透试剂盒在室温下40分钟,然后用1x渗透缓冲液(Perm缓冲液)洗涤两次。
    3. 然后在室温下在100μl1×Perm缓冲液中用波形蛋白(0.2μm每1-2×10 6个细胞)进行细胞染色1小时,随后用Perm缓冲液洗涤一次使用FACS BD Canto II细胞仪和FlowJo软件在分析前用FACS缓冲液洗涤
  4. 在肠发炎的情况下,可以注意到更多数量的CD45 + 细胞。在这种情况下,如果这是您的后续实验的问题,您可能必须添加额外的磁珠消耗或细胞分选步骤。
  5. 不推荐对原代肠间充质细胞进行冷冻保存。

食谱

  1. HBSS /抗生素
    5ml在500ml HBSS中的抗生素 - 抗生素 注意:使用前应冷。
  2. HBSS/EDTA/DTT
    5 mM EDTA
    1 mM DTT
    1x pen/strep
    溶解在HBSS中
    注意:使用前在37°C预热。
  3. 消化解决方案
    300U/ml胶原酶XI 0.08 U/ml分散酶II
    溶于DMEM培养基中
    注意:消解溶液应该是新鲜的 。根据您将使用的小鼠的数量称重适量的胶原酶和分散酶,并在HBSS中以1/10的最终体积稀释。通过0.2μm孔径的注射器过滤器对溶液进行无菌过滤,然后加入DMEM培养基至最终体积。
  4. DMEM培养基
    50 ml热灭活的FBS
    5ml pen/strep
    5ml L-谷氨酰胺 5ml非必需氨基酸
    溶于500ml DMEM中
    注意:
    1. 除了消解溶液之外的所有介质都可以提前制备,并储存在4℃。
    2. 在37°C预热,然后使用。

致谢

该协议基于我们实验室中使用的先前协议(Armaka 。这项工作得到FP7高级ERC拨款MCs-inTEST(授予协议号340217)和创新药物倡议联合承诺(IMI JU)项目BTCure(授予协议No.115142)GK支持。

参考文献

  1. Armaka,M.,Apostolaki,M.,Jacques,P.,Kontoyiannis,DL,Elewaut,D.and Kollias,G。(2008)。  间充质细胞靶向TNF作为慢性炎症性关节和肠疾病中的常见致病原理。 205(2):331-337。
  2. Koliaraki,V.,Roulis,M.和Kollias,G。(2012)。  Tpl2调节肠肌纤维母细胞HGF释放以抑制结肠炎相关的肿瘤发生。 122(11):4231-4242。 br />
  3. Koliaraki,V.,Pasparakis,M.和Kollias,G.(2015)。  IKKβ在肠间质细胞中促进结肠炎相关癌的发生。 212(13):2235-2251。
  4. Powell,DW,Pinchuk,IV,Saada,JI,Chen,X.and Mifflin,RC(2011)。  肠粘膜固有层的间质细胞。 :213-237。
  5. Roigan,M.,Nikolaou,C.,Kotsaki,E.,Kaffe,E.,Karagianni,N.,Koliaraki,V.,Salpea,K.,Ragoussis,J.,Aidinis,V.,Martini, Becker,C.,Herschman,HR,Vetrano,S.,Danese,S.和Kollias,G。(2014)。  肠道肌成纤维细胞特异性Tpl2-Cox-2-PGE2通路将先天感知与上皮内环境稳定联系起来。 111(43):E4658-4667。
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引用:Koliaraki, V. and Kollias, G. (2016). Isolation of Intestinal Mesenchymal Cells from Adult Mice. Bio-protocol 6(18): e1940. DOI: 10.21769/BioProtoc.1940.
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