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Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells
原代鼠小肠上皮细胞的分离和三维培养   

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Abstract

The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which are located at the bottom of the crypt. Here, we recapitulate a detailed protocol for the isolation and culture procedures of primary epithelial intestinal cells in a three - dimensional (3D) in vitro system, described for the first time by Hans Clevers group (Sato et al., 2009). This specific 3D culture preserves intestinal stem cells, which give rise to differentiated progeny for example goblet cells. The culture has many applications and represents a useful model to study stem cell biology, epithelial cell regeneration, and transplantation studies. Moreover, the presented 3D culture can be used to investigate the barrier function of intestinal epithelial cells, as well as heterotypic cell interactions between epithelial cells and stromal cells.

Keywords: Intestinal epithelium(肠上皮细胞), Intestinal stem cells(肠道干细胞), Organoid(组织化), Three-dimensional cell culture(三维细胞培养), Small intestine(小肠)

Materials and Reagents

  1. Mouse (4 weeks-16 weeks old)
  2. Advanced DMEM/F12 (Life Technologies, Gibco®, catalog number: 12634-010 )
  3. Heat Inactivated Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10500-064 )
  4. PBS without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14190-094 )
  5. Penicillin/Streptomycin (10,000 Units/ml Penicillin; 10,000 µg/ml Streptomycin) (Life Technologies, Gibco®, catalog number: 15140-22 )
  6. 70% ethanol
  7. 100x GlutaMAXTM-I (Life Technologies, Gibco®, catalog number: 35050-038 )
  8. 1 M HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
  9. 50x B-27 Supplement (Life Technologies, Gibco®, catalog number: 17504-044 )
  10. 100x N-2 Supplement (Life Technologies, Gibco®, catalog number: 17502-048 )
  11. BD Matrigel Basement Membrane Matrix (10 ml) (BD Biosciences, catalog number: 354234 )
  12. Gentamicin Reagent Solution (50 mg/ml) (Life Technologies, catalog number: 15750-060 )
  13. Sterile 0.1% BSA (in PBS)
  14. Sterile 2 mM EDTA (see Recipes)
  15. Washing solution (see Recipes)
  16. N-Acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165-5G ) (see Recipes)
  17. Recombinant Murine EGF (Pepro Tech, catalog number: AF-315-09 ) (see Recipes)
  18. Recombinant Murine Noggin (Pepro Tech, catalog number: 250-38 ) (see Recipes)
  19. Recombinant Human R-Spondin-1 (Pepro Tech, catalog number: 120-38 ) (see Recipes)

Equipment

  1. Forceps and short, sharp-point scissors (e.g. Hardened Fine Iris Scissors, Fine Science Tools, catalog number: 14090-09 )
  2. 70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
  3. Centrifuge 5702R (Eppendorf)
  4. Cover glass
  5. Microscope
  6. 24-well tissue culture plate (Sarstedt AG, catalog number: 83.1836 )
  7. Petri dishes
  8. Falcon tubes (50 ml, 15 ml)
  9. Pipettes (20 ml, 10 ml, 1 ml, 100 µl)
  10. Pipetboy
  11. 37 °C, 5% CO2 cell culture incubator

Procedure

Part I. Isolation
Before starting: put matrigel on ice in order to thaw it.
Note: For the matrigel handling, please follow the manufacturer’s instructions.

  1. Harvesting
    1. The small intestine is harvested from a mouse.
    2. The intestine is placed in a Petri dish containing ice-cold washing solution and then fat and the adjacent tissue are removed.
      Note: Both tissue and Washing solution should be stored on ice during the whole procedure.
    3. The intestine is opened lengthwise and washed out from the luminal contents using washing solution.
    4. The villi of an intestine are scraped off using a cover glass.
      Note: From this step the procedure should be continued under sterile conditions. To be on a safe side it is recommended to perform scraping on both sides of an intestine. Detached villi are seen as cloudy solution.
    5. The intestine is washed with washing solution and then cut with sharp-point scissors into 2-4 mm pieces.
    6. The material is transferred into a 50 ml falcon tube containing 10-20 ml of washing solution.
      Note: Tissue fragments might stick to the pipette. To avoid this, pre-wetting of the pipette is recommended.
    7. The material is pipetted up and down a few times with a 10 ml pipette.
    8. After sedimentation of tissue fragments, the supernatant is removed.

  2. Washing
    1. For the washing, 10-20 ml of washing solution is added. Then, tissue fragments are sedimented. After that, the supernatant is removed.
    2. Step B1 is being repeated until the supernatant is clear (approximately 10 times).
    3. 25 ml of 2 mM EDTA is added to the tissue fragments.
    4. The falcon tube that contains tissue fragments and EDTA is placed on a shaker and incubated for 15 min at 4 °C.
    5. Tissue fragments are sedimented followed by the removal of the supernatant.
    6. 10-20 ml of washing solution is added. Then, tissue fragments are sedimented. After that, the supernatant is removed.
    7. The Washing solution is added to the tissue fragments, pipetted up and down 3-5 times and passed through a 70 µm cell strainer.
    8. The flow-through (I fraction) is stored on ice and will be further proceeded in step C3 of Elution.
      Note: Crypts are present in the supernatant and go through the pores of the cell strainer, whereas the tissue fragments stay on the top of the cell strainer.
    9. Tissue fragments that remained on the cell strainer are proceeded further.
    10. On a pipette tip the tissue fragments are transferred into a 50 ml falcon tube containing 25 ml of 2 mM EDTA.
    11. The falcon tube is placed on a shaker and incubated for 30 min at 4 °C.

  3. Elution
    1. After tissue sedimentation, the supernatant is discarded.
    2. Washing solution is added to the tissue fragments, gently pipetted up and down 3-5 times and passed through a 70 µm cell strainer (II fraction).
      Note: This step is repeated; as a result fraction III will be collected (optional). Tissue fragments might stick to the pipette. To avoid that, pre-wetting of the pipette is recommended.
    3. Crypts from certain fractions are observed under the microscope. Presence of finger-like structures (= crypts, see Figure 1) indicates a successful isolation.


      Figure 1. A representative of a freshly isolated crypt. Objective 10x, scale bar 100 µm

    4. Crypts are spin down at 600 rpm for 5 min at 4 °C to remove single cells. A 24-well plate is pre-warmed by placing it into the cell culture incubator.
    5. After centrifugation the supernatant is discarded.
      Note: If the pellet is not visible, the centrifugation should be repeated.
    6. The pellet is resuspended in 10 ml of ice-cold Basal Medium 2 (BM2; see Table 1). Crypts are counted under the microscope.

      Table 1. Composition of BM2 (50 ml)
      Component   
      Volume
      50x B27
      100x N2
      N-Acetyl-L-cysteine (500 mM)
      1 ml
      0.5 ml
      125 µl
      BM1 (see Table 2)   
      To final volume of 50 ml
      Stored at 4 °C or -20 °C (avoid repeated freeze thaw cycles)

    7. The crypts are centrifuged at 800 rpm for 5 min at 4 °C.
    8. Crypt pellet is resuspended in matrigel:
      1. Use 100-500 crypts per 50 µl of matrigel
      2. Pipette gently, avoid bubbles
    9. 50 µl of matrigel is pipetted per well into the pre-warmed 24-well plate and then the plate is left for 2-3 min at room temperature.
      Note: Pipette matrigel exactly in the middle of the well, so that as a result matrigel drop is formed (see Figure 2).


      Figure 2. Scheme of the organoid culture

    10. The plate is transferred into the cell culture incubator and incubated at 37 °C for 5-10 min (until the matrigel solidifies completely).
    11. 0.5 ml of crypt culture medium (CCM; see Table 3) is added per well.
    12. The plate is transferred back to the cell culture incubator.

Part II. Culture

2-3 times per week the medium is removed and 0,5 ml/well of fresh medium (CCM, see Table 3) is added. When organoids are big (see Figure 3C), they should be passed. Usually the passage is performed once a week. Organoids should be splitted in 1:4 ratio.


Figure 3. Isolated small intestinal crypts in 3D culture. A, B, C - 48 h/5 days/12 days in culture, respectively. A and B, passage 0. C, passage 1. Phase contrast, objective 10x, scale bars 100 µm

Passage procedure:

  1. Medium is removed.
  2. 0.5 ml of ice-cold Basal medium 1 (see Table 1) well is added.
  3. Plate is incubated on ice for at least 5 min.
    Note: Starting from this step organoids will be kept on ice for the whole time.
  4. Organoids are pipetted up and down several times (1 ml pipette is used.).
  5. All organoids are transferred into one ice-cold 15 ml falcon tube and fill up with Basal Medium 1 (BM1; see Table 2) to final volume of 15 ml.

    Table 2. Composition of BM1 (500 ml)
    Component   
    Volume
    Advanced DMEM/F12
    100x GlutaMax
    100x Pen/Strep
    Hepes (1 M)
    485 ml
    5 ml
    5 ml
    5 ml
    Stored at 4 °C

  6. Organoids are centrifuged for 600 rpm for 5 min at 4 °C. During centrifugation a 24-well plate is pre-warmed at 37 °C.
  7. The supernatant is carefully removed.
    Note: Remove as much supernatant as possible. Be careful not to remove organoids.
  8. 10 ml of ice-cold BM1 is added to the organoid pellet.
  9. The organoids are centrifuged for 800 rpm for 5 min at 4 °C.
  10. The supernatant is carefully removed.
    Note: During this step nice pellet should be visible and the supernatant should be removed completely.
  11. The organoids are mixed with matrigel and pipetted up and down.
    Note: Avoid bubbles during pipetting. Use 4x volume of matrigel used for the first time.
  12. 50 µl matrigel/well is pipetted into the pre-warmed 24-well plate.
    Note: Pipette matrigel exactly in the middle of the well, so that as a result matrigel drop is formed.
  13. The plate is left for 2-3 min at room temperature.
  14. The plate is transferred into the cell culture incubator and incubated at 37 °C for 5-10 min (until the matrigel solidifies completely).
  15. 0.5 ml of CCM (see Table 3) is added per well.
  16. The plate is transferred back to the cell culture incubator.

    Table 3. Composition of CCM (20 ml)
    Component   
    Volume
    BM2
    EGF (50 ng/µl)
    Noggin (100 ng/µl)
    R-Spondin-1 (1 µg/µl)
    20 ml
    10 µl
    20 µl
    10 µl
    Stored short-term at 4 °C or -20 °C (avoid repeated freeze thaw cycles)

Notes

  1. The percentage of viable crypts in the culture is approximately 90-95%. Crypt viability in vitro strictly depends on the quality of the growth factors used for preparation of the culture medium. Moreover, medium should be either freshly prepared (and stored at 4 °C up to 5 days) or single-use aliquots should be made and stored at -20 °C for one month.
  2. Viable and healthy organoid culture is characterized by the presence of buds and increase in organoid size over the time. Just after the passage, organoids are small, but then they grow again into structures as seen in the Figure 3C. Crypt culture contains intestinal stem cells, which represent a multipotent stem cell population, as they have potential to differentatiate only into intestinal epithelial cells. There are certain types of intestinal epithelial cells and they can be identified by stainings: stem cells (e.g. Lgr5 staining), Goblet cells (e.g. periodic acid-Schiff staining), Paneth cells (e.g. lyzozyme staining), enterocytes (e.g. intestinal alkaline phosphatase staining), enteroendocrine cells (e.g. chromogranin A staining) (Sato et al., 2009). Some other possible stainings are: b-catenin (active Wnt signaling) and Ki-67 (proliferation marker).

Recipes

  1. 2 mM EDTA
    500 ml PBS
    2.5 ml 0.5 M EDTA (pH 8) in distilled water (sterile filtered)
    Stored at 4 °C
  2. Washing solution
    50 ml FBS
    450 ml PBS
    0.5 ml gentamicin, stock 50 mg/ml (optional)
    Stored at 4 °C
  3. 500 mM N-Acetyl-L-cysteine
    Dissolve 407.5 mg of N-Acetyl-L-cysteine in 5 ml of distilled water
    Sterile filter
    Make qliquots
    Stored at 4 °C
  4. EGF, Noggin, R-Spondin-1
    Prepare stock solutions (EGF 50 ng/µl; Noggin 100 ng/µl; R-Spondin 1 µg/µl) according to the manufacter´s instructions
    Example:
    Noggin 100 ng/µl
    Add 1 ml of 0.1% BSA (in PBS) into a vial with 100 µg Noggin
    Mix by pipetting
    Make aliquots
    Stored at -20 °C

Acknowledgments

This protocol was adapted from Sato et al. (2009).

References

  1. Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-265.

简介

肠道与皮肤和血液一起属于具有最高细胞更新的器官,这使得它成为研究细胞过程如增殖和分化的完美模型。由于存在位于隐窝底部的肠干细胞,肠上皮细胞更新是可能的。在这里,我们重述了在三维(3D)体外系统中原发性上皮肠细胞的分离和培养程序的详细方案,首次由Hans Clevers组(Sato > et al。,2009)。这种特定的3D培养物保留肠干细胞,其产生分化的子代,例如杯状细胞。该培养物具有许多应用并且代表研究干细胞生物学,上皮细胞再生和移植研究的有用模型。此外,呈现的3D文化可用于调查肠上皮细胞的屏障功能,以及上皮细胞和基质细胞之间的异型细胞相互作用。

关键字:肠上皮细胞, 肠道干细胞, 组织化, 三维细胞培养, 小肠

材料和试剂

  1. 小鼠(4周-16周龄)
  2. 高级DMEM/F12(Life Technologies,Gibco ,目录号:12634-010)
  3. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10500-064)
  4. PBS(不含Ca 2+和Mg 2+)(Life Technologies,Gibco ,目录号:14190-094)。
  5. 青霉素/链霉素(10,000单位/ml青霉素;10,000μg/ml链霉素)(Life Technologies,Gibco ,目录号:15140-22)
  6. 70%乙醇
  7. 100x GlutaMAX TM-I(Life Technologies,Gibco ,目录号:35050-038)
  8. 1 M HEPES(Life Technologies,Gibco ,目录号:15630-080)
  9. 50x B-27补充剂(Life Technologies,Gibco ,目录号:17504-044)
  10. 100x N-2补充剂(Life Technologies,Gibco ,目录号:17502-048)
  11. BD Matrigel基底膜基质(10ml)(BD Biosciences,目录号:354234)
  12. 庆大霉素试剂溶液(50mg/ml)(Life Technologies,目录号:15750-060)
  13. 无菌0.1%BSA(在PBS中)
  14. 无菌2mM EDTA(见配方)
  15. 洗涤液(见配方)
  16. N-乙酰基-L-半胱氨酸(Sigma-Aldrich,目录号:A9165-5G)(参见配方)
  17. 重组鼠EGF(Pepro Tech,目录号:AF-315-09)(参见Recipes)
  18. 重组鼠头蛋白(Pepro Tech,目录号:250-38)(参见Recipes)
  19. 重组人类R-Spondin-1(Pepro Tech,目录号:120-38)(参见Recipes)

设备

  1. 镊子和短尖尖剪刀(例如 Hardened Fine Iris Scissors,Fine Science Tools,目录号:14090-09)
  2. 70μm细胞滤器(BD Biosciences,Falcon ,目录号:352350)
  3. 离心机5702R(Eppendorf)
  4. 盖玻璃
  5. 显微镜
  6. 24-孔组织培养板(Sarstedt AG,目录号:83.1836)
  7. 培养皿
  8. Falcon管(50ml,15ml)
  9. 移液管(20ml,10ml,1ml,100μl)
  10. Pipetboy
  11. 37℃,5%CO 2细胞培养箱中培养

程序

第I部分。隔离
开始之前:把matrigel放在冰上,以解冻它 注意:对于基质胶处理,请按照制造商的说明进行操作。

  1. 收获
    1. 小肠是从小鼠收获的。
    2. 将肠放置在含有冰冷的洗涤溶液的培养皿中,然后除去脂肪和相邻的组织。
      注意:在整个过程中,组织和洗涤液都应存放在冰上。
    3. 肠纵向打开并用洗涤液从腔内容物中洗出
    4. 使用盖玻璃刮除肠的绒毛。
      注意:从这一步,程序应在无菌条件下继续。为了安全起见,建议在肠道两侧进行刮擦。分离的绒毛被视为多云的解决方案。
    5. 用洗涤液洗涤肠,然后用尖点剪刀切成2-4mm片
    6. 将材料转移到含有10-20ml洗涤溶液的50ml falcon管中 注意:组织碎片可能粘在移液器上。 为避免这种情况,建议预先润湿移液器。
    7. 将材料用10ml移液管上下移动几次。
    8. 在组织碎片沉降后,除去上清液
  2. 洗涤
    1. 对于洗涤,加入10-20ml洗涤溶液。 然后,沉淀组织碎片。 之后,除去上清液。
    2. 重复步骤B1直到上清液澄清(约10次)
    3. 向组织碎片中加入25ml 2mM EDTA
    4. 将含有组织碎片和EDTA的猎鹰管置于振荡器上并在4℃下孵育15分钟。
    5. 将组织碎片沉淀,然后除去上清液
    6. 加入10-20ml洗涤溶液。 然后,沉淀组织碎片。 之后,除去上清液。
    7. 将洗涤溶液加入到组织碎片中,向上和向下吸移3-5次,并通过70μm细胞过滤器。
    8. 流出物(I馏分)储存在冰上,并在洗脱的步骤C3中进一步进行 注意:隐窝存在于上清液中,通过细胞滤网的孔,而组织碎片留在细胞滤网的顶部。
    9. 向组织碎片中加入25ml 2mM EDTA
    10. 将含有组织碎片和EDTA的猎鹰管置于振荡器上并在4℃下孵育15分钟。
    11. 将组织碎片沉淀,然后除去上清液
    12. 加入10-20ml洗涤溶液。 然后,沉淀组织碎片。 之后,除去上清液。
    13. 将洗涤溶液加入到组织碎片中,向上和向下吸移3-5次,并通过70μm细胞过滤器。
    14. 流出物(I馏分)储存在冰上,并在洗脱的步骤C3中进一步进行 注意:隐窝存在于上清液中,通过细胞滤网的孔,而组织碎片留在细胞滤网的顶部。
      ...
    15. Washing solution is added to the tissue fragments, gently pipetted up and down 3-5 times and passed through a 70 µm cell strainer (II fraction).
      Note: This step is repeated; as a result fraction III will be collected (optional). Tissue fragments might stick to the pipette. To avoid that, pre-wetting of the pipette is recommended.
    16. Crypts from certain fractions are observed under the microscope. Presence of finger-like structures (= crypts, see Figure 1) indicates a successful isolation.


      Figure 1. A representative of a freshly isolated crypt. Objective 10x, scale bar 100 µm

    17. Crypts are spin down at 600 rpm for 5 min at 4 °C to remove single cells. A 24-well plate is pre-warmed by placing it into the cell culture incubator.
    18. After centrifugation the supernatant is discarded.
      Note: If the pellet is not visible, the centrifugation should be repeated.
    19. The pellet is resuspended in 10 ml of ice-cold Basal Medium 2 (BM2; see Table 1). Crypts are counted under the microscope.

      Table 1. Composition of BM2 (50 ml)
      Component   
      Volume
      50x B27
      100x N2
      N-乙酰基-L-半胱氨酸(500mM)
      1ml
      0.5ml
      125μl
      BM1(见表2)   
      至最终体积为50ml
      储存在4°C或-20°C(避免反复冻融循环)

    20. 将隐窝在4℃下以800rpm离心5分钟。
    21. 将冻结颗粒重悬在基质胶中:
      1. 每50μl基质胶使用100-500个隐窝
      2. 轻轻吸取,避免气泡
    22. 将50μl基质胶每孔移入预热的24孔板中,然后将板在室温下放置2-3分钟。
      注意:移取基质胶恰好在孔中间,因此形成基质胶滴(见图2)。


      图2.器官培养方案

    23. 将板转移到细胞培养孵育器中并在37℃孵育5-10分钟(直到基质胶完全凝固)。
    24. 每孔加入0.5ml隐窝培养基(CCM;参见表3)
    25. 将板转移回细胞培养孵化器

第二部分。 文化

每周2-3次除去培养基,加入0.5ml /孔的新鲜培养基(CCM,参见表3)。 当类器官大(见图3C),他们应该通过。 通常每周进行一次。 机体应以1:4的比例分裂

图3.分别在3D培养物中的分离的小肠隐窝,A,B,C-48h/5天/12天。 A和B,通道O.C,通道1.相差,物镜10x,比例尺100μm
通行程序:

  1. 中等已删除。
  2. 加入0.5ml冰冷的Basal培养基1(见表1)
  3. 将板在冰上孵育至少5分钟 注意:从这个步骤开始,类固醇将一直保持在冰上。
  4. 用移液管吸取上清液和下清液几次(使用1ml移液管)。
  5. 将所有类器官转移至一个冰冷的15ml falcon管中,并用基础培养基1(BM1;参见表2)填充至最终体积为15ml。

    表2. BM1(500ml)的组成
    组件   

    高级DMEM/F12
    100x GlutaMax
    100x Pen/Strep
    Hepes(1M)
    485ml
    5毫升
    5毫升
    5毫升
    储存在4°C

  6. 将组织在4℃下以600rpm离心5分钟。 在离心过程中,24孔板在37℃预热
  7. 小心除去上清液。
    注意:去除尽可能多的上清液。 小心不要去除类器官。
  8. 将10ml冰冷的BM1加入到有机物小丸中
  9. 将类器官在4℃下以800rpm离心5分钟
  10. 小心地除去上清液。
    注意:在这个步骤中,应该可以看到好的颗粒,上清液应该完全去除。
  11. 将类固醇与基质胶混合并用移液管上下移动 注意:在移液期间避免气泡。 使用4倍体积的第一次使用的基质胶。
  12. 将50μl基质胶/孔移入预热的24孔板中 注意:移液器正好在孔中间,这样就形成了基质胶滴。
  13. 将板在室温下放置2-3分钟。
  14. 将板转移到细胞培养孵育器中并在37℃孵育5-10分钟(直到基质胶完全凝固)。
  15. 每孔加入0.5ml CCM(参见表3)
  16. 将板转移回细胞培养孵化器
    表3.CCM(20ml)的组成
    组件   

    BM2
    EGF(50ng /μl)
    头蛋白(100ng /μl)
    R-Spondin-1 (1 μ g /μl)
    20毫升
    10μl
    20μl
    10μl
    短期储存在4°C或-20°C(避免反复冻融循环)

笔记

  1. 培养物中活隐窝的百分比为约90-95%。隐窝生存力在体外严重依赖于用于制备培养基的生长因子的质量。此外,培养基应该是新鲜制备的(并且在4℃下储存至多5天),或者应当制备单次使用的等分试样并在-20℃下储存一个月。
  2. 活的和健康的器官培养的特征在于芽的存在和随时间的器官大小的增加。刚刚通过后,类器官很小,但是然后他们再次成长为如图3C所示的结构。地穴培养物包含肠干细胞,其代表多能干细胞群体,因为它们具有仅分化成肠上皮细胞的潜力。存在某些类型的肠上皮细胞,并且它们可以通过染色鉴定:干细胞(例如,Lgr5染色),杯状细胞(例如,高碘酸 - 希夫染色),Paneth细胞(例如溶菌酶染色),肠细胞(例如肠碱性磷酸酶染色),肠内分泌细胞(例如嗜铬粒蛋白A染色)(Sato et al。,2009)。一些其它可能的染色是:β-连环蛋白(活性Wnt信号传导)和Ki-67(增殖标记)。

食谱

  1. 2mM EDTA 500 ml PBS
    2.5ml 0.5M EDTA(pH 8)的蒸馏水(无菌过滤) 储存在4°C
  2. 洗涤溶液
    50ml FBS
    450 ml PBS
    0.5 ml庆大霉素,原液50 mg/ml(可选)
    储存在4°C
  3. 500mM N-乙酰基-L-半胱氨酸 将407.5mg N-乙酰基-L-半胱氨酸溶于5ml蒸馏水中
    无菌过滤器
    制作报表
    储存在4°C
  4. EGF,头蛋白,R- Spondin-1 根据制造商说明书制备储备溶液(EGF 50 ng /μl;头蛋白100 ng /μl; R-Spondin 1μg/μl)
    示例:
    头蛋白100ng /μl
    加入1ml的0.1%BSA(在PBS中)到具有100μgNoggin的小瓶中 通过吹打混合
    制作等份
    储存于-20°C

致谢

该方案从Sato et al。 (2009)。

参考文献

  1. Sato,T.,Vries,RG,Snippert,HJ,van de Wetering,M.,Barker,N.,Stange,DE,van Es,JH,Abo,A.,Kujala,P.,Peters,PJand Clevers, H.(2009)。 单个Lgr5干细胞体外构建隐窝 - 绒毛结构,无间质 利基。 自然 459(7244):262-265。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pastuła, A. and Quante, M. (2014). Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells. Bio-protocol 4(10): e1125. DOI: 10.21769/BioProtoc.1125.
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Sau-Yee Kok
Kanazawa University
Hi, I failed to isolate primary murine intestinal epithelial crypts and cultured in 3D culture since last month. I did change the mEGF and R-spondin but the crypts failed to budding. They formed enterosphere nicely on day 1 but could not turn into enteroid and started to dying. Do you have any ideas what the cause?
8/13/2017 8:24:45 PM Reply
Agnieszka Pastula
Columbia University in the City of New York

Hi Sau-Yee Kok, thank for your interest in the protocol that we published and for your question. Did you isolate crypts from small intestine or colon? Did you check if cultures are not contaminated with bacteria? Was the medium pink or yellow when crypts started to die? What was the source of R-Spondin? Did you use recombinant R-Spondin?

8/14/2017 6:32:02 PM


Sau-Yee Kok
Kanazawa University

The crypts were isolated from small intestine and no contamination observed. For troubleshooting purpose, we used 20% R-spondin condition medium and it was in yellow color when the crypts started dying. We used recombinant R-spondin 1 and Wnt signaling checked using TOP flash assay which met the criteria.

8/14/2017 7:04:41 PM


Agnieszka Pastula
Columbia University in the City of New York

This was murine small intestine (SI), right? Because cultures derived from human SI require additional growth factors. Generally, your crypt isolation seems to me very successful, because you observed enterospheres on day 1. However, it seems that there are inconsistencies with the culture conditions. At what day crypts started to die? Were the crypts overcrowded? (how many crypts did you observe in one well on day 1?) Did you exchange medium when the medium turned yellow, and observed if at least some crypts can survive? If we exclude bacterial/fungal contamination, then probably one of the growth factors was not working optimally. Did you add Noggin to the culture media? How do you store R-Spondin conditioned medium and recombinant proteins after reconstitution?

8/14/2017 7:43:35 PM


Sau-Yee Kok
Kanazawa University

Yes, it was murine small intestine. The crypts started to die after day 4 and they were not overcrowded. We were abled to observe few budding point when we used 20% R-spondin condition medium. We did exchange the medium but the crypts could not survive. We did check the Noggin and it met the criteria as well. After reconstitution, we kept in small volume and stored at -80 degree celsius meanwhile thawed solution kept at 4 deg celsius. Polyps culture able to growth with the same batch of Noggin and mEGF. We will check R-spondin 1 activity again for double confirmation.

8/14/2017 8:55:42 PM


Bingwei Wang
Immunology Institute of Maynooth University
Sorry, forget to say thank you.
Bingwei
2/24/2015 3:04:40 AM Reply
Bingwei Wang
Immunology Institute of Maynooth University
Hi, why Crypts need to be spin down at 4 °C in C Elution step 4 the and also in the passage procedure it is using cold medium?
Also I notice in the CCM there is no FBS, do B27 and N2 the job of nourish the cells?
2/24/2015 3:04:14 AM Reply
Agnieszka Pastula
Columbia University in the City of New York

Dear Bingwei, it is mainly about matrigel. At room temperature and 37°C matrigel is solid. At 4°C matrigel is in liquid form. During passage procedure crypts need to get out of matrigel, so cold medium is used + incubation on ice. For the centrifugation, temperature 4°C is used to limit temperature fluctuactions and to prepare crypts for seeding in ice-cold matrigel.

FBS may induce differentiation of stem cells.
Yes, supplements B27 and N2 are to nourish the crypts.

2/24/2015 4:39:39 PM


Bingwei Wang
Immunology Institute of Maynooth University

Thank you, Agnieszka.
I tried your protocol on colon crypts culture. The crypts looked great at the beginning 4 days, but on day 5 they started to die. I don't understand what's wrong with them, do you think I used the CCM frozen in -20 °C for changing cause the trouble? But for small culture at beginning it's impossible to prepare fresh and the supplements like Noggin, R-Spondin are so expensive.

3/9/2015 4:29:01 AM


Agnieszka Pastula
Columbia University in the City of New York

Dear Bingwei, the protocol is optimized for the crypts from small intestine. Murine colon crypts require more growth factors for the culture. I would advise you to add to the medium Wnt3a and BSA, as it was previously published (*). Good luck!

*Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell.
Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M. Nat Med. 2012.

3/10/2015 2:30:04 AM


Bingwei Wang
Immunology Institute of Maynooth University

Thank you so much Agnieszka. I thought the crypt from colon and small intestine were same. Really appreciate for your advice and paper. I will follow this paper and try again.

3/10/2015 3:36:09 AM