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Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells
小鼠小肠干细胞的分离、培养和维持   

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Abstract

In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

Keywords: Organoid(组织化), Lgr5(Lgr5), Intestine(肠), Colon(结肠), Stem cell(干细胞)

Materials and Reagents

  1. Cover glass (Corning, catalog number: 2998075X25 )
  2. 48-Well Tissue Culture Plate (Corning, Falcon®, catalog number: 351178 )
  3. 50 ml and 15 ml Conical centrifuge tubes (Corning, Falcon®, catalog number: 352098 and 352097 )
  4. 10 ml Syringe (BD, catalog number: 309604 )
  5. 21G Needle (BD, catalog number: 305165 )
  6. Pipette Tips
  7. 70 μM Cell Strainer (Corning, Falcon®, catalog number: 352350 )
  8. 100 μM Cell Strainer (Corning, Falcon®, catalog number: 352360 )
  9. Mice to be harvested for this protocol must be approved for use by the Institutional Animal Care and Use Committee (IACUC) at the institution, which sponsors the laboratory research
  10. Phosphate Buffered Saline (PBS) (Invitrogen, catalog number: 10010023 )
    Note: Currently, it is “ Thermo Fisher Scientific, GibcoTM, catalog number: 10010023”.
  11. Growth Factor Reduced Matrigel (BD, catalog number: 356230 )
    Note: Currently, it is “Corning, Matrigel®, catalog number: 356230”.
  12. Fetal Bovine Serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-044 )
  13. Collagenase Type IV (Worthington, catalog number: LS004188 )
  14. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A2058 )
  15. 1% BSA-PBS (sterile)
  16. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  17. 5 mM EDTA-PBS
  18. Recombinant DNase I, RNase Free (which is provided at 10 U/μl) (Sigma-Aldrich, catalog number: 4716728001 )
  19. Optional: 10 μM Rho Kinase Inhibitor Y-27632, provided as a 5 mM Solution (Merck Millipore Corporation, catalog number: 68801 )
  20. Advanced DMEM F/12 (Thermo Fisher Scientific, GibcoTM, catalog number: 12634-010 )
  21. Streptomycin (Gibco, catalog number: 15140-22 )
  22. N-Acetylcysteine (Sigma-Aldrich, catalog number: A9165 )
  23. HEPES (Sigma-Aldrich, catalog number: H3375 )
  24. EGF 50 ng/ml (Invitrogen, catalog number: PMG8043 )
    Note: Currently, it is “ Thermo Fisher Scientific, GibcoTM, catalog number: PMG8043”.
  25. Recombinant Murine EGF 50 ng/ml (Invitrogen, catalog number: PMG8043)
  26. Recombinant Murine Noggin 50 ng/ml (Peprotech, catalog number: 250-38 )
  27. Recombinant Human R-Spondin 1,500 ng/ml (R&D Systems, catalog number: 3474-RS-050 )
  28. Recombinant Murine Wnt-3A 100 ng/ml (Merck Millipore Corporation, catalog number: GF-160 )
  29. 10 mM Nicotinamide (Sigma-Aldrich, catalog number: N3376 )
  30. Intestinal basal medium (see Recipes)
  31. Small intestinal organoid growth media (see Recipes)
  32. Large intestinal organoid growth media (see Recipes)

Equipment

  1. Pipettes, Pipetaid and Micro-pipettes
  2. Dissection Forceps and Scissors (Fine Science Tools, catalog number: 11150-10 and 14058-09 )
  3. Centrifuge 5810R (Eppendorf, model: 5810R )
  4. Brightfield inverted Microscope
  5. Tabletop Roller in 4 °C Room (Bibby Scientific Limited, Stuart Equipment, model: SRT9D )
  6. 37 °C, 5% CO2 cell culture incubator
  7. Biosafety cabinet (Tissue culture hood)
  8. For mouse sacrifice by carbon dioxide asphyxiation, a carbon dioxide source, regulated dispenser, and euthanasia chamber, must be used in accordance with approved animal use protocols at the laboratory’s sponsoring institution.

Procedure


Figure 1. An overview of the key steps for the isolation of intestinal crypt containing stem cells from mouse colon or small intestine

Part I. Isolation, culture, maintenance, passage and preservation of small intestinal organoids


  1. Procedure to isolate and culture small intestinal organoids (expected time to completion: 2-3 h)
    1. Mice are euthanized by carbon dioxide asphyxiation as recommended by the 2000 Report of the AVMA Panel on Euthanasia (Association, 2001).
    2. Dissect and remove 15 cm of the proximal small intestine. Be careful to avoid touching any of the mouse skin or hair to your tools to prevent microbial contamination. After dissected, flush the lumen of the intestine with ice-cold PBS using a 10 ml Syringe and 21G needle.
    3. Use scissors to open the intestine longitudinally and place in a 50 ml conical tube containing 25 ml ice-cold PBS. Invert 10-15 times, remove the PBS and replace with 25 ml of ice-cold PBS. Repeat this process until the supernatant no longer contains any visible debris.
    4. Cut the intestine into 5 millimeter pieces and place into 10 ml ice-cold 5 mM EDTA-PBS. Vigorously triturate the fragments by pipetting up and down into a 10 ml pipette 15 times. Let the fragments settle by gravity for 30 sec.
    5. Aspirate the supernatant, being careful to avoid the intestinal fragments, and replace with 10 ml, 5 mM EDTA-PBS and place at 4 °C on a benchtop roller for 10 min.
    6. Repeat step A5 by aspirating the supernatant, replacing with 10 ml of 5 mM EDTA-PBS, but now place at 4 °C on a benchtop roller for 30 min.
    7. Aspirate the supernatant, gently add 10 ml of cold PBS to wash the crypts and aspirate. Then, add 10 ml of cold PBS and then vigorously triturate with a 10 ml pipette 10 times.
    8. Collect this 10 ml supernatant fraction in a separate tube. Add 10 ml of cold PBS to the crypts, vigorously triturate 10 times, and then collect this fraction in a separate tube. Repeat step A7 once more to collect a total of three fractions.
    9. Examine 10 μl of each fraction under a microscope, looking for the presence of intact intestinal crypts and lack of larger, fragmented villi. An example of intact intestinal crypts isolated from a mouse that harbors a GFP knock-in allele at the Lgr5 locus (Barker et al., 2007) is shown in Figure 2.


      Figure 2. A brightfield/epifluorescent image overlay of freshly isolated small intestinal crypts that harbor the Lgr5-GFP knock in allele (Barker et al., 2007). Intact crypts are marked by white arrows, and contain Lgr5+ stem cells marked by the presence of GFP (green). Debris (non-viable cells) are marked by the white arrowhead. Scale bar is 50 μm.

    10. Using the 10 ml fraction that contains the most crypts, mix with 10 ml Basal Media + 15 U/ml DNase I (add 30 μl to 10 ml Basal Media) and filter through a 100 μm filter into a BSA (1%) coated 50 ml conical tube; to coat the tube, add 1 ml of 1% BSA, shake vigorously and discard excess. Coating the tube with BSA prevents adsorption of the crypts to the sides of the tube, therefore increasing yield.
    11. Filter the solution again, this time through a 70 μm filter into a BSA (1%) coated tube and then spin the filtrate at 300 x g in a tabletop centrifuge for 5 min.
    12. Aspirate the supernatant. Depending on multiple factors (animal diet, time of day of harvest, how vigorous each washing step was performed) there can be a layer of mucous that accumulates in the supernatant, sometimes near the pellet of cells. Aspirating this will increase the purity of your yield, however it can also pull the cell pellet with it during vacuum suction. Be careful to avoid losing your pellet at this step, it is possible to use a 10 ml pipette to gently aspirate the supernatant at this step.
    13. Resuspend the cell pellet with 5 ml basal media containing 5% FBS and centrifuge at 100 x g for 5 min.
    14. Resuspend the cell pellet in 200 μl of basal media and count crypts from a 10 μl aliquot. You may have to first dilute the aliquot that you count in order to get an accurate number. Keep the volume of media that you resuspend the crypts in as low as possible because you will aim to mix 100-1,000 crypts at a 1:10 mixture with Matrigel. In a typical isolation, we recover 250 crypts/μl (in basal media) and mix this 1:10 with Matrigel.
    15. Plate 40 μl of the Matrigel:crypt mixture as a bubble on one well of a 48 well plate, being careful to avoid letting the Matrigel touch the edges of the well (for a schematic see Figure 3). Although organoids will still grow in Matrigel that has ‘collapsed’ to the sides of the wells, it is easier to change media if the Matrigel remains as a single bubble in the center of the well. Incubate the plate in a 37 °C incubator to allow the Matrigel to polymerize for 10-15 min.
    16. Add 250 μl of Small Intestinal Organoid Growth Media to each well. Over the course of 7-10 d, viable intestinal stem cells should form organoids with a crypt-villus structure (for example, see Figure 4).


      Figure 3. A schematic illustrating a properly plated 40 μl ‘Matrigel bubble’ at the bottom of one well of a 48-well plate. 250 μl of growth media is added on top of the bubble after polymerization. Ensuring that the Matrigel does not contact the well edges allows for a pipette to be placed down the side of the well to remove media during media changes.


      Figure 4. A high-resolution bright field microscopic picture of a small intestinal organoid, accompanied by a schematic depiction of each of the individual cell types observed in the structure. Scale bar is 50 μm.

  2. Procedure to maintain, passage, and cryogenically preserve small intestinal organoids
    1. After isolation, small intestinal growth media is changed every two days.
    2. After 5-7 days of growth, or when the organoids show dark, necrotic cores, they are passaged. To do this, the growth media is removed and 500 μl of cold PBS is added to the well and Matrigel is broken up by pipette, being careful to avoid bubble formation in the resuspension mixture.
    3. Transfer the resuspension to a 15 ml conical tube. Using a p200 pipette, pipette up and down 50-100 times to mechanically disassociate the organoids into smaller fragments. When finished, another 7 ml of cold PBS is added to the mixture and pipetted up and down 20 times.
    4. Centrifuge the cells at 200 x g for 5 min in a tabletop centrifuge.
    5. Carefully aspirate the supernatant, being careful not to remove the pellet. The pellet may not be visible to the naked eye; this is especially true if only passaging 1 well. Resuspend the pellet in Matrigel, and replate 40 μl per well into a 48 well plate. For non-transformed organoid cultures, we typically split at a 1:4 ratio.
    6. If preparing cells for storage in liquid nitrogen, resuspend the pellet in Basal Media containing 10% FBS and 10% DMSO. Allow to freeze slowly in a -80 °C freezer and then move to liquid nitrogen for indefinite storage.

Part II. The isolation, culture, maintenance, passage and cryogenic preservation of large intestinal organoids


  1. Procedure to isolate and culture large intestinal organoids (Video 1)

    Video 1. Procedure to isolate and culture large intestinal organoids

    1. Mice are euthanized as recommended by the 2000 Report of the AVMA Panel on Euthanasia (Association, 2001).
    2. Dissect and remove 5-7 cm of the proximal large intestine. Be careful to avoid touching any of the mouse skin or hair to your tools to prevent microbial contamination. After dissected, flush the lumen of the intestine with cold PBS.
    3. Using scissors, open the colon longitudinally. Using a glass slide, gently scrape the lumen of the intestine to remove fecal matter and mucous. Place in a 50 ml conical tube with 25 ml ice-cold PBS. Invert 10-15 times, remove the PBS and replace with 25 ml of ice-cold PBS. Repeat this process until the supernatant no longer contains any visible debris.
    4. Cut the intestine into 5 millimeter pieces and place into 10 ml cold 5mM EDTA-PBS. Vigorously triturate the fragments by pipetting up and down into a 10 ml pipette 15 times. Let the fragments settle by gravity for 30 sec.
    5. Aspirate the supernatant, being careful to avoid the intestinal fragments, and replace with 10 ml, 5 mM EDTA-PBS and place at 4 °C on a benchtop roller for 15 min.
    6. Remove the EDTA from the tube by aspiration. Wash 2 x with PBS. Then add 3 ml of Basal Medium containing 500 U/ml Collagenase Type IV. Pipette up and down 5 times using a 5 ml pipette. Place in a 37 °C water bath for 30 min.
    7. Add 10 ml of ice-cold PBS and then vigorously triturate with a 10 ml pipette 10 times.
    8. Collect this 10 ml supernatant fraction in a separate tube. Add 10ml of cold PBS to the crypts, vigorously triturate 10 times, and then collect this fraction in a separate tube. Repeat step B7 once more to collect a total of three fractions.
    9. Pipette 10 μl of each fraction on to a glass slide and examine underneath a microscope, looking for the presence of intact colonic crypts.
    10. To the 10 ml fraction that contains the most crypts, add 10 ml Basal Media containing 15 U/ml DNase I (add 30 μl to 10 ml Basal Media) and then filter through a 100 μm filter into a BSA (1%) coated 50 ml Falcon tube. To coat a tube, pipette 1 ml of 1% BSA into a tube and shake vigorously. Coating the tube with BSA prevents adsorption of the crypts to the sides of the tube, therefore increasing yield.
    11. Filter the solution again, this time through a 70 μm filter into a BSA (1%) coated tube and then spin the filtrate at 300 x g in a tabletop centrifuge for 5 min.
    12. Aspirate the supernatant. Depending on multiple factors (animal diet, time of day of harvest, how vigorous each washing step was performed) there can be a layer of mucous that accumulates in the supernatant, sometimes near the pellet of cells. Aspirating this will increase the purity of your yield, however it can also pull the cell pellet with it during vacuum suction. Be careful to avoid losing your pellet at this step, it’s possible to try using a 10 ml pipette with a pipette aid to aspirate the supernatant here, or by using a p1000 pipette.
    13. Resuspend the cell pellet with 5 ml Basal Media containing 5% FBS and centrifuged at 100 x g for 5 min.
    14. Resuspend the cell pellet in 200 μl of Basal Media and count crypts from a 10 μl aliquot (for an example, see Figure 5). You may have to first dilute the aliquot that you count in order to get an accurate number. You want to keep the volume of media that you resuspend the crypts in as low as possible because you will aim to mix 100-1,000 crypts at a 1:10 mixture with Matrigel. In a typical isolation, we will resuspend 50 colon crypts/μl of basal media and mix this 1:10 with Matrigel.
    15. 40 μl of the resuspended mixture is plated as a bubble on the bottom of a well in a 48 well plate, being careful to avoid letting the Matrigel touch the edges of the well (see Figure 3). The plate is placed in a 37 °C incubator to allow the Matrigel to polymerize for 10-15 min.
    16. Add 250 μl of Large Intestinal Organoid Growth Media to each well.


      Figure 5. A bright field image of a 10 μl aliquot from the 200 μl resuspension of colon crypts in step A14 of Part II. Scale bar is 50 μm.

  2. Maintenance, passage, and cryogenic preservation of colon organoids
    This part is performed exactly as described for the colon organoids as it is for the small intestinal organoids.

Notes

  1. We typically test 2 or 3 lots of Matrigel and reserve a large quantity of the best performing lot. To test a lot, we plate freshly isolated stem cells into Matrigel and observe their growth after two weeks, including one passage.
  2. R-spondin1 and Wnt3a producing cell lines can be obtained from investigators who have made them previously (Sato et al., 2011) in order to condition media with these respective growth factors, greatly reducing costs.
  3. Noggin is typically added to intestinal stem cell culture growth media at a final concentration of 100 ng/ml, but we have found that 50 ng/ml is enough to support normal stem cell growth, differentiation and self-renewal.
  4. All of the growth factors that are bought commercially can be dissolved in 0.1% BSA-PBS and stored as concentrated stock solutions at -20 °C.
  5. When preparing the Collagenase Type IV (for colon organoid isolation), make a fresh 10x stock before starting. The powder is static, so weigh it on a piece of aluminum foil and place in a 15 ml conical tube, add basal media to bring to the correct concentration, and make sure everything goes into solution by vortexing or incubating at 37 °C. Then filter sterilize through a 0.45 μm filter.
  6. 10 μM Rho Kinase Inhibitor Y-27632 can be added to the growth media to increase the yield of organoid growth, although we routinely isolate and grow organoids without this inhibitor.
  7. Colon organoids should begin to grow as large hollow spheres 4-6 days after isolation, note that this is longer than typically observed for small intestinal stem cell growth.

Recipes

  1. Intestinal basal medium
    Supplement a 500 ml bottle of advanced DMEM F/12 with
    2 mM L-Glutamine
    100 units/ml Penicillin, 1,000 μg/ml Streptomycin
    1 mM N-acetylcysteine
    10 mM HEPES
  2. Small intestinal organoid growth media
    Supplement the basal medium with
    Recombinant Murine EGF 50 ng/ml
    Recombinant Murine Noggin 50 ng/ml
    Recombinant Human R-Spondin-1 500 ng/ml
  3. Large intestinal organoid growth media
    Supplement the basal medium with
    Recombinant murine EGF 50 ng/ml
    Recombinant murine noggin 50 ng/ml
    Recombinant human R-Spondin-1 500 ng/ml
    Recombinant murine Wnt-3A 100 ng/ml (Millipore GF-160)
    10 mM nicotinamide

Acknowledgments

This protocol was adapted from (Sato et al., 2009) with help from Caroline Lindemans. This work was supported by a program project grant from the NIH/NCI (CA-013106). L. E. D. was supported by a National Health and Medical Research Council (NHMRC) Overseas Biomedical Fellowship and a K22 Career Development Award from the NCI/NIH (CA 181280-01). K. P. O. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. S. W. L. is the Geoffrey Beene Chair of Cancer Biology and an investigator of the Howard Hughes Medical Institute.

References

  1. Association, A. P. o. E. A. V. M. (2001). 2000 report of the AVMA panel on euthanasia. J Am Vet Med Assoc 218(5): 669-696.
  2. Barker, N., van Es, J. H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P. J. and Clevers, H. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165): 1003-1007.
  3. Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., Van Es, J. H., Van den Brink, S., Van Houdt, W. J., Pronk, A., Van Gorp, J., Siersema, P. D. and Clevers, H. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141(5): 1762-1772.
  4. 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

在这个协议,我们描述我们修改分离,培养和维持鼠肠干细胞作为隐窝 - 绒毛形成类器官的方法。 从小肠或大肠分离的这些细胞随时间保持自我更新和多向分化潜能。 这为研究人员提供了培养野生型或转化的肠上皮的工具,以及用于体外干细胞组织稳态的稳健试验。

关键字:组织化, Lgr5, 肠, 结肠, 干细胞

材料和试剂

  1. 保护玻璃(Corning,目录号:2998075X25)
  2. 48孔组织培养板(Corning,Falcon ,目录号:351178)
  3. 50ml和15ml锥形离心管(Corning,Falcon ,目录号:352098和352097)。
  4. 10ml注射器(BD,目录号:309604)
  5. 21G针(BD,目录号:305165)
  6. 移液器提示
  7. 70μM细胞过滤器(Corning,Falcon ,目录号:352350)
  8. 100μM细胞过滤器(Corning,Falcon ,目录号:352360)
  9. 为本协议收获的小鼠必须获得机构动物护理和使用委员会(IACUC)批准用于赞助实验室研究的机构。
  10. 磷酸盐缓冲盐水(PBS)(Invitrogen,目录号:10010023) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:10010023" />
  11. 生长因子减少基质胶(BD,目录号:356230)
    注意:目前,"Corning,Matrigel ? ,目录号:356230"
  12. 胎牛血清(Thermo Fisher Scientific,Gibco TM ,目录号:16000-044)
  13. 胶原酶IV型(Worthington,目录号:LS004188)
  14. 牛血清白蛋白(Sigma-Aldrich,目录号:A2058)
  15. 1%BSA-PBS(无菌)
  16. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  17. 5mM EDTA-PBS
  18. 重组DNA酶I,无RNA酶(以10U /μl提供)(Sigma-Aldrich,目录号:4716728001)
  19. 任选:10μMRho激酶抑制剂Y-27632,以5mM溶液(Merck Millipore Corporation,目录号:68801)提供;
  20. Advanced DMEM F/12(Thermo Fisher Scientific,Gibco TM,目录号:12634-010)
  21. 链霉素(Gibco,目录号:15140-22)
  22. N-乙酰半胱氨酸(Sigma-Aldrich,目录号:A9165)
  23. HEPES(Sigma-Aldrich,目录号:H3375)
  24. EGF 50ng/ml(Invitrogen,目录号:PMG8043) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:PMG8043" />
  25. 重组小鼠EGF 50ng/ml(Invitrogen,目录号:PMG8043)
  26. 重组小鼠头蛋白50ng/ml(Peprotech,目录号:250-38)
  27. 重组人R-Spondin 1,500ng/ml(R& D Systems,目录号:3474-RS-050)
  28. 重组小鼠Wnt-3A 100ng/ml(Merck Millipore Corporation,目录号:GF-160)
  29. 10mM烟酰胺(Sigma-Aldrich,目录号:N3376)
  30. 肠道基础培养基(见配方)
  31. 小肠组织生长培养基(见配方)
  32. 大肠组织生长培养基(见配方)

设备

  1. 移液器,移液器和微量移液器
  2. 解剖镊子和剪刀(Fine Science Tools,目录号:11150-10和14058-09)
  3. 离心机5810R(Eppendorf,型号:5810R)
  4. 明场倒置显微镜
  5. 台式辊在4℃室(Bibby Scientific Limited,Stuart Equipment,型号:SRT9D)中
  6. 37℃,5%CO 2细胞培养箱中培养
  7. 生物安全柜(组织培养罩)
  8. 对于通过二氧化碳窒息的小鼠处死,必须根据实验室保荐机构批准的动物使用协议使用二氧化碳源,调节分配器和安乐死室。

程序


图1.从小鼠结肠或小肠中分离含有干细胞的肠道的关键步骤的概述

第I部分。小肠类器官的分离,培养,维持,传代和保存


  1. 分离和培养小肠类器官的步骤(预计完成时间:2-3小时)
    1. 通过二氧化碳窒息将小鼠安乐死 ?2000年AVMA安乐死专家小组的报告(协会,2001)。
    2. 解剖并去除15厘米的近端小肠。小心 ?避免接触任何鼠标皮肤或头发到您的工具,以防止 微生物污染。解剖后,冲洗的内腔 肠用冰冷PBS,使用10ml注射器和21G针。
    3. 使用剪刀纵向打开肠,放在一个50 ml锥形管,含有25ml冰冷PBS。反转10-15次, 取出PBS,并用25ml冰冷的PBS替换。重复此操作 直到上清液不再含有任何可见的碎片
    4. 将肠切成5毫米的块,放入10毫升 冰冷的5mM EDTA-PBS。通过吸移大量研磨碎片 上下放入10ml移液管15次。让片段沉淀 重力30秒。
    5. 吸出上清液,小心 避免肠道碎片,并用10ml,5mM EDTA-PBS和 ?在4℃下在台式辊上放置10分钟
    6. 重复步骤A5 ?吸出上清液,用10ml的5mM EDTA-PBS代替,但是 现在在4℃在台式辊上放置30分钟
    7. 吸气 上清液,轻轻加入10 ml冷PBS洗涤隐窝和 吸出。然后,加入10ml冷PBS,然后剧烈研磨 ?一个10ml移液管10次
    8. 收集此10ml上清液 部分在单独的管中。加入10毫升冷PBS的隐窝, 剧烈研磨10次,然后收集该级分 单独管。重复步骤A7一次,总共收集三次 分数
    9. 在显微镜下检查每个级分10μl, 寻找完整的肠隐窝的存在和缺乏更大, ?碎片绒毛。从a。分离的完整的肠隐窝的一个例子 ?小鼠在Lgr5基因座处携带GFP敲入等位基因(Barker等人, al。,2007)如图2所示。


      图2. A 明场/epifluoresecent图像覆盖的新鲜孤立小 在Lgr5-GFP敲入等位基因的肠隐窝(Barker等, ,2007)。完整隐窝用白色箭头标记,包含Lgr5 + 干细胞由GFP(绿色)的存在标记。碎片(不可行 细胞)由白色箭头标记。比例尺为50μm。

    10. 使用含有最多隐窝的10ml部分,与10混合 ml基础培养基+ 15U/ml DNA酶I(加入30μl至10ml基础培养基)和 通过100μm过滤器过滤到BSA(1%)包被的50ml锥形中 管;涂覆管,加入1ml 1%BSA,剧烈摇动并弃去 ?过量。用BSA涂覆管防止隐窝的吸附 管的侧面,因此增加产量
    11. 过滤 溶液再次,此时通过70μm过滤器进入BSA(1%)包被 然后在台式离心机中以300×g离心滤液5分钟 ?min。
    12. 吸出上清液。取决于多个因素 (动物饮食,收获日的时间,每个洗涤步骤有多强 ?执行),可以有一层粘液积聚在 上清液,有时靠近细胞沉淀。吸引这个意志 增加您的产量的纯度,但它也可以拉细胞 在真空吸入期间与其一起沉淀。小心避免失去你的 在此步骤沉淀,可以使用10ml移液管轻轻地 在此步骤吸出上清液。
    13. 用含有5%FBS的5ml基础培养基重悬细胞沉淀,并以100×g离心5分钟。
    14. 重悬细胞沉淀在200微升的基础培养基和计数隐窝 ?从10μl等分试样。你可能需要先稀释等分试样 ?计数以获得准确的数字。保持媒体的音量 ?你重新悬挂的地穴在尽可能低,因为你会瞄准 将100-1,000个隐窝以1:10的混合物与Matrigel混合。在一个典型的 分离,我们恢复250隐窝/μl(在基础培养基中)并混合1:10 与Matrigel。
    15. 板40微升的Matrigel:crypt混合物作为 泡在48孔板的一口井,小心避免让 基质胶接触孔的边缘(对于示意图参见图3)。 ?虽然类器官仍将在Matrigel生长,已经"崩溃" 孔的侧面,如果matrigel更容易改变媒体 保持作为在井的中心的单个泡。孵育平板 ?在37℃培养箱中,使基质胶聚合10-15分钟。
    16. 每个添加250微升小肠组织生长培养基 好。在7-10 d的过程中,活的肠干细胞应该 形成具有隐窝 - 绒毛结构的类器官(例如,参见图 4)。


      图3.示意图说明了适当电镀的40μl 'matrigel bubble'在48孔板的一个孔的底部。250μl 的生长培养基在聚合反应后添加在气泡的顶部。 确保基质胶不接触孔边缘允许a 移液管放置在井的侧面以移除介质 媒体更改。


      图4.高分辨率明视场显微镜 图片的小肠组织器,伴有示意图 描述在所观察到的各个细胞类型 结构体。 比例尺为50μm。

  2. 保持,通过和低温保存小肠组织的程序
    1. 分离后,每两天更换小肠生长培养基
    2. 生长5-7天后,或当类器官显示黑暗,坏死 核心,它们是传代的。为此,去除生长培养基 将500μl冷PBS加入孔中,并将Matrigel分解 移液管,小心避免在悬浮液中形成气泡 混合物
    3. 转移重悬浮到15毫升锥形管。使用 ?一个p200移液管,上下移液器50-100次,机械 将类固醇分解为更小的片段。完成后, 再向混合物中加入另外7ml冷PBS,用移液管吸取 下降20次。
    4. 在台式离心机中以200×g离心细胞5分钟。
    5. 小心吸出上清液,小心不要除去 颗粒。颗粒可能不是肉眼可见的,这是 尤其是如果只传代1井真。重悬沉淀 Matrigel,并且每孔重复40μl到48孔板中。对于 非转化器官培养物,我们通常以1:4的比例分裂
    6. 如果准备储存在液氮中的细胞,重悬 在含有10%FBS和10%DMSO的基础培养基中沉淀。允许冻结 缓慢地在-80℃冰箱中,然后移至液氮中 不定存储。

第二部分。分离,培养,维持,传代和低温保存大肠内器官


  1. 分离和培养大肠类器官的步骤(视频1)

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    视频1. 分离和培养大肠道器官的步骤
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    1. 小鼠按照AVMA安乐死面板2000年报告(Association,2001)的建议进行安乐死。
    2. 解剖并去除近端大肠5-7厘米。是 小心避免接触任何鼠标皮肤或头发到你的工具 ?防止微生物污染。解剖后,冲洗内腔 肠用冷PBS。
    3. 使用剪刀,打开冒号 纵向。使用玻璃载片,轻轻刮擦管腔 肠道清除粪便和粘液。放置在50ml圆锥形 管用25ml冰冷PBS。倒转10-15次,取出PBS和 用25ml冰冷的PBS替换。重复此过程,直到 上清液不再含有任何可见的碎片
    4. 切割 肠5毫米,置于10ml冷5mM中 EDTA-PBS。通过向上和向下移液来强力研磨碎片 移入10ml移液管15次。让片段通过重力沉降 30秒。
    5. 吸出上清液,小心避免 肠片段,并且用10ml,5mM EDTA-PBS替换并置于 ?4℃在台式辊上15分钟
    6. 从中取出EDTA 管抽吸。用PBS洗涤2次。然后加入3ml的基础培养基 含有500U/ml胶原酶IV型。吸移上下5次 使用5ml移液管。置于37℃水浴中30分钟。
    7. 加入10毫升冰冷的PBS,然后用10毫升吸管大力研磨10次
    8. 在单独的管中收集该10ml上清液级分。加入10ml ?的冷PBS加入到隐窝中,剧烈研磨10次,然后 将该级分收集在单独的管中。重复步骤B7一次 共收集三个分数。
    9. 吸移管各10μl 部分在载玻片上并在显微镜下检查, 寻找完整的结肠隐窝的存在
    10. 到10毫升 含有最多隐窝的部分,加入10ml基础培养基 ?15U/ml DNA酶I(加入30μl至10ml基础培养基),然后过滤 通过100μm过滤器进入BSA(1%)包被的50ml Falcon管。至 包被管,移取1ml的1%BSA到管中并剧烈摇动。 用BSA涂覆管防止了隐窝吸附到侧面 ?的管,因此增加产量
    11. 过滤解决方案 再次,这次通过70μm过滤器进入BSA(1%)包被管和 然后在台式离心机中以300×g离心滤液5分钟
    12. 吸出上清液。取决于多种因素(动物 饮食,收获日的时间,每个洗涤步骤有多强 执行),可以有一层粘液积聚在 上清液,有时靠近细胞沉淀。吸引这个意志 增加您的产量的纯度,但它也可以拉细胞 在真空吸入期间与其一起沉淀。小心避免失去你的 沉淀在这一步,可以尝试使用10毫升移液器与 吸移助剂吸出上清液,或通过使用p1000 吸管。
    13. 用含有5%FBS的5ml基础培养基重悬细胞沉淀,并以100×g离心5分钟。
    14. 重悬细胞沉淀在200μl的基础培养基和计数隐窝 ?从10μl等分试样(例如,参见图5)。你可能需要 首先稀释您计数的等分,以获得准确 数。您想要保留重新悬挂的媒体的音量 隐藏在尽可能低的地方,因为你会瞄准混合100-1,000 与基质胶1:10混合物的隐窝。在典型的隔离,我们会 重悬50结肠隐窝/μl的基础培养基,并与1:10混合 基质胶。
    15. 将40μl重悬的混合物平板接种 泡在48孔板的孔底部,小心 避免让基质胶接触孔的边缘(见图3)。 将板置于37℃培养箱中以允许基质胶 聚合10-15分钟
    16. 向每个孔中加入250μl大肠组织生长培养基。


      图5.来自200μl的10μl等分试样的明视场图像 在第II部分的步骤A14中重悬结肠隐窝。比例尺为50μm。

  2. 保持,通过和低温保存结肠组织
    这部分完全按照结肠类器官的描述进行,因为它是小肠类器官

笔记

  1. 我们通常测试2或3批Matrigel,并保留大量最好的性能。为了测试很多,我们将新鲜分离的干细胞涂布到基质胶中,并在两周后观察它们的生长,包括一次。
  2. R-spondin1和Wnt3a产生细胞系可以从先前已经制备它们的研究者获得(Sato等人,2011),以便调节具有这些相应生长因子的培养基,大大降低了成本。 />
  3. 头蛋白通常以100ng/ml的终浓度加入到肠干细胞培养生长培养基中,但是我们发现50ng/ml足以支持正常的干细胞生长,分化和自我更新。
  4. 所有商业购买的生长因子可以溶解在0.1%BSA-PBS中,并在-20℃下作为浓缩储备溶液储存。
  5. 当准备胶原酶类型IV(结肠组织分离),在开始之前,做一个新的10x股票。粉末是静态的,因此将其称重在一片铝箔上并放置在15ml锥形管中,加入基础培养基以达到正确的浓度,并确保通过在37℃下涡旋或孵育使一切进入溶液。然后通过0.45μm过滤器过滤灭菌。
  6. 可以将10μMRho激酶抑制剂Y-27632加入到生长培养基中以增加类器官生长的产量,尽管我们常规地分离并生长没有该抑制剂的类器官。
  7. 分离后4-6天,结肠类器官应该开始生长为大空心球体,注意这比小肠干细胞生长通常观察到的更长。

食谱

  1. 肠道基础培养基

    补充500 ml高级DMEM F/12瓶 2mM L-谷氨酰胺 100单位/ml青霉素,1,000μg/ml链霉素 1mM N-乙酰半胱氨酸 10 mM HEPES
  2. 小肠组织生长培养基

    补充基础培养基 重组小鼠EGF 50ng/ml
    重组小鼠头蛋白50 ng/ml
    重组人类R-Spondin-1 500ng/ml
  3. 大肠组织生长培养基

    补充基础培养基 重组鼠EGF 50ng/ml
    重组鼠noggin 50 ng/ml
    重组人类R-Spondin-1500ng/ml
    重组鼠Wnt-3A 100ng/ml(Millipore GF-160)
    10mM烟酰胺

致谢

该协议在Caroline Lindemans的帮助下改编自(Sato et al。,,2009)。这项工作得到了来自NIH/NCI(CA-013106)的计划项目赠款的支持。 L. E. D.得到国家卫生和医学研究委员会(NHMRC)海外生物医学奖学金和NCL/NIH(CA 181280-01)的K22职业发展奖的支持。 K.P. O.得到NIH的国家综合医学科学研究所授予的医学科学家培训计划资助,授予T32GM07739授予Weill Cornell /洛克菲勒/Sloan-Kettering三机构MD-PhD计划。 S. W. L.是Geoffrey Beene癌症生物学主席和霍华德休斯医学研究所的研究员。

参考文献

  1. Association,A.P.O。 (2001)。 AVMA专家小组关于安乐死的2000年报告 J Am Vet Med Assoc 218(5):669-696。
  2. Barker,N.,van Es,JH,Kuipers,J.,Kujala,P.,van den Born,M.,Cozijnsen,M.,Haegebarth,A.,Korving,J.,Begthel,H.,Peters,PJ和Clevers,H。(2007)。 通过标记基因Lgr5鉴定小肠和结肠中的干细胞。自然 449(7165):1003-1007。
  3. Sato,T.,Stange,DE,Ferrante,M.,Vries,RG,Van Es,JH,Van den Brink,S.,Van Houdt,WJ,Pronk,A.,Van Gorp,J.,Siersema, Clevers,H.(2011)。 人结肠,腺瘤,腺癌和Barrett上皮的上皮组织的长期扩张。 a> Gastroenterology 141(5):1762-1772。
  4. 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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:O’Rourke, K. P., Ackerman, S., Dow, L. E. and Lowe, S. W. (2016). Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells. Bio-protocol 6(4): e1733. DOI: 10.21769/BioProtoc.1733.
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Kaviyarasi Kandasamy
Madras Veterinary College
Sir, can you please tell the size of crypt cells during different culture days
6/14/2017 5:35:15 AM Reply
Kaviyarasi Kandasamy
Madras Veterinary College
Hello, Am a M.V.Sc student from Madras Veterinay College currently working in Chicken intestinal crypt cells. After isolation while pelleting the cells its forming like a mucous and i could not able to see the crypt cells in the pellet. Please kindly help me to troubleshoot the problem.
2/15/2017 6:13:54 AM Reply
Kevin O’Rourke
Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program

Hi Kaviyarasia,

I do not have experience working in chicken crypts. I would recommend the following steps to prevent mucous accumulation:
1- during the initial wash (Part I, A.3) perform the PBS wash thoroughly. There should be no visible debris in your tube after washing. This is usually 5 washes per mouse colon.
2- Be sure to add DNAse to your suspension after EDTA/Collagenase treatment. This will help prevent DNA from clogging the filter and increase your yield.

Let me know if this helps! Best Regards, Kevin.

3/28/2017 8:41:14 AM


Kaviyarasi Kandasamy
Madras Veterinary College

Sir, Thank you so much. Actually after filtering only it is getting clumped before mixing it with matrigel. am adding the cells to the matrigel without pelleting it, so the number of cells is very less and it varies with each well. what may be the reason for the cells to get clumped?

5/7/2017 7:34:15 AM


Kevin O’Rourke
Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program

I'm not sure I completely understand the step at which you are seeing clumps. Is there a reason you are not pelleting? There will always be some debris and clumping in the prep, but after you pellet, you can resuspend the clumped cells into media (see Part 1. A. Step 14) into as much or as little media as you need to make a dilution that gets you an ideal number of cells per well after mixing with matrigel.

5/8/2017 7:10:13 AM


Kaviyarasi Kandasamy
Madras Veterinary College

I'll follow your instructions. Thank you sir.

5/8/2017 8:11:21 AM


Minglin Lin
SIU
In part II. "10.To the 10 ml fraction that contains the most crypts, add 10 ml Basal Media containing 15 U/ml DNase I (add 30 μl to 10 ml Basal Media) and then filter through a 100 μm filter into a BSA (1%) coated 50 ml Falcon tube". What is the exact role of DNase I here? Is it essential? If we use the Basal Media without DNase I, What will happen?
Also, I notice that other references use the Collagenase type IX to digest mucosa, and your protocol here is Collagenase Type IV. What is the different? Thank you!
10/5/2016 7:37:41 PM Reply
Kevin O’Rourke
Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program

The DNase is used to digest the free floating DNA that arises as a result of cell lysis during the incubation and trituration steps. If you don't add the DNAse, you will notice that your fraction will be "soupy" and will not easily pass through the filter, and so after you clog one filter you will have to replace it with a new one and repeat. So, while it is not essential, it will increase your yield and decrease the number of filters required to finish filtering your fraction. I am not sure why other protocols use Type IX Collagenase, if they report success with this reagent and you have it readily in your lab then I don't see why you can't give it a try. Historically, we've had success with Type IV collagenase and so that's what we choose to use. Good luck with your protocol and let me know if you have any other questions! -Kevin O'Rourke

10/6/2016 9:57:08 AM


Minglin Lin
SIU

Ok, get it. Thank you very much!

10/6/2016 1:03:48 PM