参见作者原研究论文

本实验方案简略版
Sep 2018

本文章节


 

Fluidigm Based Single-cell Gene Expression Library Preparation from Patient-derived Small Intestinal Organoids
基于flifgm的源于患者小肠类器官单细胞基因表达文库的制备   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

In this protocol, we describe our methods to isolate crypts from patients' biopsy samples and to culture human intestinal stem cells as it’s called “organoid.” Beyond that, we describe how to dissociate organoids cells into single cells for single-cell analysis as a further application. This protocol should provide investigators sufficient tools to generate human organoids from biopsy samples and to accomplish a stable in-vitro assay system.

Keywords: Organoid (类器官), Intestinal stem cell (肠道干细胞), Single-cell analysis (单细胞分析), Endoscopic biopsy (内窥镜活检), Multiplex PCR (多重PCR)

Background

The intestinal epithelium is a multifunctional tissue that orchestrates homeostasis and forms a physical barrier. Each intestinal epithelial cells (IECs) arising from intestinal stem cells (ISCs) renew this epithelium every 4-5 days (Crosnier et al., 2006). ISCs are located at the bottom of the crypts and express specific markers as previously reported by various papers (Muñozet al., 2012; Clevers, 2013). Studies suggested that malfunctions of proper renewals of stem cells are related to intestinal disorders, and understandings of ISCs dynamic may elucidate the pathogenesis of various disorders including Inflammatory Bowel Disease (IBD) (Okamoto et al., 2016).

However, the studies of intestinal stem cell properties had been challenging due to the lack of efficient models that recapitulates physiological intestinal epithelial layers. The epic introduction of “organoid" has overcome various obstacles (Sato et al., 2009 and 2011). Organoids can be established from a single ISC in vitro, and faithfully retain the physiological and pathological features of their tissue of origin (Middendorp et al., 2014). Organoids have been used to dissect underlying pathologic changes in various gastrointestinal disease (Fatehullah et al., 2016; Noben et al., 2017) and has shown potentials to reflect complexed mechanisms of organs.

Also, recent advances in molecular biology techniques allow us to study single-cell modalities (Stuart and Satija, 2019). These techniques developed to have insights into each single cell diversities yet had known to be homogenous populations. Studies have shown that a heterogeneous group of cells share these ISC properties, and constitute a hierarchy within the ISC population (Smith et al., 2016). Furthermore, organoids can be one of the ideal tools that are consist of mostly stem cells and transit-amplifying cells under the undifferentiated culture. In the previous report, a single-cell analysis displayed this heterogeneity among the mouse small intestinal stem cells (Li et al., 2014). Combining organoid culture technique and single-cell analysis has the potential to open a new horizon toward the understandings of the dynamics of human intestinal stem cells. In this protocol, we describe in detail the work-flow of human intestinal organoids establishment and dissociation into single cells for further various applications. Compared to those protocols using scRNA-seq (Biton et al., 2014), the present protocol using multiplex PCR enables acquiring single cell profiles in a low-cost, short-time basis, while the number of cells and genes will be limited by the capacity of the microfluid chip format.

Materials and Reagents

  1. Organoid culture
    1. 24-well Tissue Culture Plate (Corning, Falcon, catalog Number: 353226 )
    2. 50 ml and 15 ml Conical centrifuge tubes (Corning, Falcon, catalog numbers: 352096 , 352070 )
    3. 15 ml STEMFULLTM low cell adhesion tube (Sumitomo Bakelite Co., catalog number: MS-90150Z )
    4. 1.5 ml Eppendorf tube (Eppendorf Safe-Lock Tubes, catalog number: 00 30121023 )
    5. 70 μm Cell strainer (Corning, Falcon, catalog number: 352350 )
    6. Patient intestinal biopsy samples obtained by endoscopies
    7. Phosphate Buffered Saline (PBS) (Sigma, catalog number: D8537-500ML ) (stored at 4 °C)
    8. Matrigel (Corning, catalog number: 356231 ) (stored at 4 °C)
    9. Cell recovery solution (Corning, Thermo Fisher Scientific, catalog number: 354253 ) (stored at 4 °C)
    10. Advanced-DMEM (Thermo Fisher Scientific, catalog number: 12491015 ) (stored at 4 °C)
    11. Penicillin/Streptomycin (Nacalai Tesque, catalog number: 26253-84 ) (stored at 4 °C) 
    12. 1 mol/l-HEPES Buffer Solution (Nacalai Tesque, catalog number: 17557-94 ) (stored at 4 °C)
    13. GlutaMAXTM-I (100x) (Gibco, Thermo Fisher Scientific, catalog number: 35050-061 ) (stored at 4 °C)
    14. 0.5 M EDTA ((Thermo Fisher Scientific, catalog number: AM9260G )
    15. Trypan blue solution (Thermo Fisher Scientific, catalog number: 15250061 )
    16. N-acetylcysteine (Sigma, catalog number: A9165-5G ) (stored at -20 °C, 1 M,10 ml aliquot)
    17. Gastrin I (Sigma, catalog number: 3006 ) (stored at -20 °C, 100 µl, 1,000 μl aliquot)
    18. N2 supplement (R&D Systems, catalog number: AR009 ) (stored at -20 °C,100x, 1 vial)
    19. B27 supplement (R&D Systems, catalog number: AR008 ) (stored at -20 °C, 50x, 1 vial)
    20. Recombinant mouse EGF (R&D Systems, catalog number: 2028-EG-200 )
      (stored at -20 °C, 20 μg/ml, aliquot 1,000 μl)
    21. Recombinant mouse Noggin (R&D Systems, catalog number: 1967-NG-025/CF ) (stored at -20 °C, 20 μg/ml, aliquot 250 μl)
    22. Recombinant human R-spondin-1 (R&D Systems, catalog number: 4645-RS ) (stored at -20 °C, 100 μg/ml, aliquot 500 μl)
    23. Recombinant mouse Wnt-3a (R&D Systems, catalog number: 1324-WN-010/CF ) (stored at -20 °C, 10 μg/ml, aliquot 1,000 μl)
    24. Nicotinamide (R&D Systems, catalog number: 4106 ) (stored at -20 °C, 1 ml, aliquot 1,000 μl)
      Note: Stored in the RT shelf as powder. Nic 6.1 g + ddH2O 50 ml, aliquot 10 ml each into 15 ml Falcon tubes. When you thaw one tube, aliquot 1.5 ml eppentubes and store.
    25. A83-01 (Sigma-Aldrich, catalog number: 2939 ) (stored at -20 °C, 10 mM, 100 μl aliquot)
      Note: Protect from light. Aliquot 10 mM dilution into Eppendolf tubes and label them with x20, further dilute 10 mM Eppendolf tube with DMSO and aliquot 100 μl each.
    26. SB202190 (Sigma-Aldrich, catalog number: 1264 ) (stored at 4 °C, 150 μl aliquot)
      Note: SB202190 10 mg + DMSO 3,018 μl. Aliquot 150 μl each and freeze at -20 until use.
    27. Y-27632 (R&D Systems, catalog number: 1254 ) (protect from light, stored at -20 °C, 10 mM, 150 μl aliquot)
    28. Human intestinal basal culture medium (see Recipes)
    29. Human small intestinal organoid growth media (see Recipes)

  2. Single-cell analysis
    1. C1 preamp IFC (10-17 μm, Fluidigm, catalog number: 100-5480 )
    2. 48 x 48 IFC chip (Fluidigm, catalog number: BMK-M-48.48 )
    3. TrypLE Select (Thermo Fisher Scientific, catalog number: 12563011 ) (stored at 4 °C)
    4. C1 Single-Cell Reagent Kit for Preamp (including Module 1, Module 2: Fluidigm, catalog number; 100-5319 ) (stored at -20 °C)
    5. Single Cell-to-CT kit (Thermo Fisher Scientific, catalog number: 4458236 ) (stored at -20 °C)
    6. LIVE/DEAD viability/cytotoxicity kit (Thermo Fisher Scientific, catalog number: L3224 ) (stored at -20 °C)
    7. Pooled primers (see Recipes) 
    8. Lysis final mix (see Recipes)
    9. RT final mix (see Recipes)
    10. PreAmp final mix (see Recipes)
    11. LIVE/DEAD cell staining (see Recipes)

  3. Biomark HD
    1. 2x Assay Loading Reagent, 1.5 ml (Fluidigm, catalog number: 100-7611 )
    2. 20x TaqMan Gene Expression Assay (Thermo Fisher Scientific, catalog number: 4351372 )
    3. 2x Master Mix (Thermo Fisher Scientific, catalog number: 4369514 )
    4. 20x GE Sample Loading Reagent (Fluidigm, catalog number: 100-7610 )

Equipment

  1. Pipettes (5 ml, 10 ml, 25 ml, 50 ml), micro-pipettes (10 μl, 20 μl, 200 μl, 1,000 μl)
  2. Multi-channel pipette (Mettler Toledo, RAININ, model: E8-20 XLS+, catalog number: 17013798 )
  3. Centrifuge
  4. Vortex mixer
  5. Hemacytometer (Burker-Turk, Fujirika Co., A114)
  6. 37 °C, 5% CO2 cell culture incubator
  7. Fluorescence microscope (Keyence, model: BZ-X700 ,)
  8. C1 single cell auto prep system (Fluidigm, San Francisco, CA, USA)
  9. C1 single cell auto prep array IFCs (MX) (Fluidigm, San Francisco, CA, USA)
  10. Biomark HD system (Fluidigm, San Francisco, CA, USA)

Software

  1. Singular Analysis Toolset Software v3.5.2 (Fluidigm, San Francisco, CA, USA)
  2. R software
  3. The Partek Genomic Suite (Version 6.6-6.16.0812, Partek, Chesterfield, MO, USA)

Procedure

Part I: Crypt isolation and epithelial organoid culture


  1. Procedure to isolate and culture small intestinal organoids from patient-derived biopsy samples
    1. Small intestinal enteroscopic biopsy samples are obtained from patients undergoing evaluation for diseases such as small intestinal tumors, occult bleeding, or Crohn’s disease. Up to 8 biopsies from each patient are taken from a region approximately 100 cm proximal to the ileocecal valve. The Ethics Committee approval and written informed consent should be obtained from each patient.
    2. Using a 50 ml Falcon tube, wash the fragments with cold PBS until the supernatant is clear.
    3. Incubate the samples in 15 mM EDTA/PBS solution (rotating) at 4 °C for 30 min (10 ml PBS + 300 µl 0.5 M EDTA).
    4. After removing the EDTA buffer, add 10 ml of PBS and voltex for 2 min to isolate intestinal crypts (Figure 1).


      Figure 1. Freshly embedded of small intestinal crypts from patients. Scale bars = 1 mm (left) and 100 μm (right).

    5. Confirm intact crypt isolation by observation under a microscope (see Notes).
    6. After settling down the specimen in the tube, collect the supernatant and transfer to a 50 ml Falcon tube filtering through a 70 μm filter.
    7. Repeat the Step A5 of Part I about 2-3 times till an approximately 30 ml volume of crypts containing solution is collected.
    8. Centrifuge the tube at 300 x g for 3 min to pellet.
    9. Resuspend the cell pellet with10 ml of Advanced-DMEM/F12 and transfer to one STEMFULLTM tube.
    10. Centrifuge the tube at 300 x g for 3 min to pellet.
    11. After examining the pellet under a microscope, carefully aspirate the supernatant and add the desired amount of Matrigel (30-50 µl per well) to embed the crypts (20-30 crypts per well).
    12. After gently pipetting within the Matrigel, gently apply 30 µl of Matrigel on the 24-well plate. Incubate 30-60 min to allow Matrigel to polymerize.
    13. Add the 500 µl of human intestinal growth medium to each well. Add 10 µM of Y-27632 to the culture medium for the initial 3 days.

  2. Procedure to maintain and passage small intestinal organoids
    1. After isolation, change the small intestinal growth medium every two days.
    2. After 13 days of isolation, you will be able to see the growth of organoids, and they start to form crypt-like structures called "budding" (Figure 2). In order to subculture, remove the growth media and add 800 µl of cold cell recovery solution to the well. Using cell recovery solution, scrape off the Matrigel containing organoids by 1,000 µl pipette.


      Figure 2. Establishment of small intestinal organoids from patients. Scale bars = 1 mm (left) and 100 μm (right).

    3. Transfer the resuspension to a 15 ml STEMFULLTM tube. Using a P100 pipette, pipette up and down 50-100 times vigorously to mechanically disassociate the organoids into smaller fragments. After examining the fragments under the microscope, add 9 ml of cold Advanced-DMEM/F12 to the mixture.
    4. Centrifuge the cells at 300 x g for 3 min.
    5. Carefully aspirate the supernatant and resuspend the pellet in Matrigel. Usually, split at a 1:5-6 ratio. On the around day 13, established organoids start budding.

Part II: Single cell-level gene expression analysis


  1. Single-cell dissociations of cultured organoid cells
    1. Remove the entire volume of the growing medium from each well (use 2-3 wells to obtain a sufficient number of cells).
    2. Using the same method as its described in passing cultured organoids, collect the organoids in a 15 ml STEMFULLTM tube, pipette up and down 50-100 times vigorously to mechanically disassociate the organoids into smaller fragments. Add 9 ml of cold Advanced-DMEM/F12 to the mixture.
    3. Centrifuge at 300 x g for 3 min and aspirate the supernatant.
    4. Add 5 ml of TrypLE select to the pellet and vortex for 10 min at 600 rpm (add Hoechst 10 μl).
    5. After 10 min, shake the tube vigorously for 10 times to further dissociate them into single cells.
    6. Centrifuge at 500 x g for 3 min and confirm the palette.
    7. Resuspend with 1,000 μl of Advanced-DMEM/F12 and transfer them to a 1.5 ml Eppendorf tube and count the live cell number by using conventional Trypan Blue staining (optimized cell concentration: 3 x 105 cells/ml, expected cell viability > 70%).
    8. Take out the desired amount of cells containing solution and centrifuge at 500 x g for 3 min and aspirate the supernatant.
    9. Resuspend with 1,000 μl of PBS and proceed to C1 protocol.

  2. Loading single cells into the chip (C1 pre-amplification process)
    1. Prepare single-cell suspension for C1 loading by mixing previously prepared cell suspension (A9) and C1 Cell suspension reagent while thawing other reagents.
      Components
      Volume (μl)
      Cells 166-250K/ml
      30 μl
      C1 Cell Suspension Reagent (Fluidigm)
      20 μl
      Total
      50 μl
    2. C1 chip priming
      1. Turn on the C1 machine, place a new C1 single-cell auto prep IFC and apply each reagent as shown in the below (Figure 3).


        Figure 3. Application of a plate priming (Fluidigm C1 to Taqman Primers protocol, PN100-6117)

      2. After loading each reagent, place the place on the stage and select Prime program and run (it takes 10min).
        Important: After the priming, apply the sample within 1 h to prevent the priming reagents from drying out.
    3. While priming the plate, prepare Lysis Final Mix, Reverse Transcription (RT) Final Mix, and PreAmp Final Mix and keep them on ice (Recipes 4-6).
    4. Cell application and staining
      1. After the priming, remove the blocking solution as shown below (Figure 4).
      2. Pipet prepared cell mix for 10 times and apply 20 μl to the blue coded well. 
      3. Prepare the C1 LIVE/DEAD (see Recipes) and apply 20 μl to the pink coded well.


        Figure 4. Cell application and staining to a C-1 plate (Fluidigm C1 to Taqman Primers protocol, PN100-6117)

      4. Set the plate on the stage, select Cell Load & Stain program and run (this process takes 60-80 min)
      5. After the program is over, take out the plate and confirm the single-cell capture (Figure 5) and cell viabilities of 96 wells under a microscope (BX-X700, Keyence, Osaka, Japan).


        Figure 5. Examples of a viable single-cell capture positively stained with calcein AM and a dead cell stained with Ethidium homodimer-1. Scale bar = 100 μm.

    5. After confirming the cells, apply each solution as shown in the below (Figure 6) (Recipes 4-6)


      Figure 6. Application of pre-amplification solutions onto a C-1 plate (Fluidigm C1 to Taqman Primers protocol, PN100-6117)

    6. Set the plate on the stage and select the PreAmp program and set the estimated end time (it is an over-night process).
    7. It is important to collect the sample within an hour of completion; otherwise, the samples will be evaporated.
    8. Prepare a new 96-well plate and aliquot 25 μl of C1 DNA Dilution reagent into each well of the labeled Diluted Harvest Plate
    9. Harvest the Pre-Amplified products showed in the below and mix with each well with DNA dilutions using an 8-channel pipette (each well has approximately 3.6 μl) (Figure 7).
    10. Store the harvested plate in -20 °C.
      Components
      Volume (μl)
      C1 DNA Dilution Reagent (Fluidigm)
      25
      C1 harvest amplicons
      3
      Total
      28


      Figure 7. Pipetting steps of pre-amplified products from a C1-plate to a 96-wells plate (Fluidigm C1 to Taqman Primers protocol, PN100-6117)

  3. Biomark HD workflow
    Further amplification and data acquirement of the multiplex quantitative PCR analysis will be performed using the Biomark 48.48. Dynamic array IFC and Biomark HD system (Fluidigm, San Francisco, CA, USA). Dynamic array IFC system operates nanofluidic IFC circuits that automatically combines harvested samples with sets of gene assays.
    1. Preparing
      1. Prepare both “10x assay mix” and “Sample mix” as they are shown below.
        TaqMan primers 10x assay mix
        Components
        Volume per Inlet (μl)
        Volume per Islet with Overage (μl)
        Volume for 48.48 (μl)
        Volume for 96.96 (μl)
        2x Assay Loading Reagent
        2.5
        6.0
        X 60 = 360
        X 120 = 600
        20x TaqMan Gene Expression Assay
        2.5
        6.0


        Total volume
        5.0
        6.0
        300.0
        600.0
        Final concentration at 10x
        Primers: 9 μM Probe: 2.5 μM
        Store at -20°C

        Sample Mix
        Components
        Volume per Inlet (μl)
        Volume per Islet with Overage (μl)
        Volume for 48.48 (μl)
        Volume for 96.96 (μl)
        2x Mater Mix
        2.5
        3.0
        X 60 = 180.0
        X 120 = 36.0
        20x GE Sample Loading Reagent
        0.25
        0.3
        X 60 = 18.0
        X 120 = 36.0
        cDNA
        2.25
        2.7


        Total
        5.0
        6.0
        198
        396.0

    2. Priming IFC and sample loading
      Make sure to use IFC Controller MX for 48.48 Dynamic Array IFC, and IFC Controller HX for 96.96 Dynamic Array IFC.
      1. Inject control line fluid into each accumulator on the IFC.
      2. Remove and discard the protective film from the bottom of the IFC.
      3. Place the IFC into the appropriate controller, then run the appropriate “Prime” script to prime the control line fluid into the IFC. Each IFC type corresponds to a distinct script number, and the plate ID number will be identified by the controller (Priming takes up to 30 min).
        Important: Please make sure to run “Load” within 1 h after completing the priming as the plate will dry out.
      4. Remove the IFC from the controller, and pipette samples and assay solutions into the inlets on the IFC (5 μl each).
      5. Return the IFC to the controller.
      6. Run the appropriate “Load Mix” script. Air pressure forces samples and gene assay solutions into the IFC where they mix.
      7. Remove the loaded IFC from the controller.
      8. Place the IFC into the Biomark HD instrument, making sure the A1 corner on the IFC aligns with the A1 on the instrument tray.
        Important: Make sure to run Biomark HD within 1 h after completing the “Load Mix” script as the plate will dry out.
    3. Running Biomark HD
      1. Turn on the Biomark HD and the computer.
      2. Click the icon named “Biomark Data Collection”.
      3. Wait until the application shows “Ready” when the temperature of the CCD camera reaches to -5 °C.
      4. Click “Start a New Run” of the default desktop. Confirm that the “Data Collection” screen appears in response.
      5. Place the pre-loaded IFC on the tray and click “Load”. The tray retracts and the system scans the barcode and identifies the IFC.
      6. Click “Next”. Browse and select a file that you desire to save.
      7. Select the Gene Expression application. Select the Passive Reference (ROX). Select Probe and select “Next”.
      8. Select Browse and Protocol file. Start the Run.

Data analysis

Data acquired from multiplex single-cell gene expression analysis were processed using the Singular Analysis Toolset Software v3.5.2 (Fluidigm, San Francisco, CA, USA) and the Partek Genomic Suite (Version 6.6-6.16.0812, Partek, Chesterfield, MO, USA) by following standard workflows.

Notes

Crypts that can be harvested from endoscopic biopsy samples are very limited in number. After step A4, take out 1ml of the crypt suspension and spread it onto a 24-well plate to observe under a microscope and confirm existence of viable crypts.

Recipes

  1. Human intestinal basal medium
    Components
    Stock Con.
    Final con.
    /500 ml medium
    Advanced-DMEM/F12

    1x
    500 ml
    GlutaMAX-1
    200 mM
    2 mM
    5 ml
    HEPES
    1 M
    10 mM
    5 ml
    Penicillin/Streptomycin
    10,000 U/ml
    100 units/ml
    5 ml
    N-acetylcysteine
    500 mM
    1 mM
    1 ml
    Gastrin
    100 μM
    10 mM
    50 μl
    N2 supplement
    100x
    1x
    5 ml
    B27 supplement
    50x
    1x
    10 ml
  2. Human small intestinal organoid growth media
    Components
    Stock Cons
    Final Cons
    Unit ml
    1 well
    2 wells
    Basal Medium-
    - - ml 0.25
    0.5
    m EGF
    20 μg/ml
    50 ng/ml
    μl
    1.25
    2.5
    m Noggin
    20 μg/ml
    100 ng/ml
    μl
    2.5
    5
    m R-spondin-1
    100 μg/ml
    1 μg/ml
    μl
    5 10
    m Wnt-3A
    10 μg/ml
    300 ng/ml
    μl
    15 30
    μl
    1 M
    10 mM
    μl
    5 10
    A83-01
    500 μM
    500 nM
    μl
    0.5
    1
    SB202190
    10 mM
    10 μM
    μl
    0.5
    1
  3. Pooled primers
    Components
    Volume (μl)
    1 μl each primer pair (100 μM each)
    1.0 (x 93 = 93 μl)
    Optional RNA Spike primers
    1.0 (x 3 = 3 μl)
    C1 DNA Dilution Reagent
    104.0
    Total
    200.0
    Can save up to 6 months (-20 °C)
  4. Lysis final mix
    Components
    Volume (μl)
    C1 DNA Dilution Reagent (Fluidigm)
    0.90
    Single-Cell Lysis Solution (Thermo Fisher Scientific)
    12.75
    C1 Lysis Plus Reagent (Fluidigm)
    4.35
    Total
    18.0
  5. RT final mix
    Components
    Volume (μl)
    Single-Cell VOLO RT Mix (Thermo Fisher Scientific)
    5.84
    Single-Cell SuperScript RT (Thermo Fisher Scientific)
    3.62
    Stop solution (Thermo Fisher Scientific)
    1.94
    C1 Loading Reagent (Fluidigm)
    0.60
    Total
    12.00
  6. PreAmp final mix
    Components
    Volume (μl)
    Single-Cell PreAmp Mix (Thermo Fisher Scientific)
    12.0
    C1 Loading Reagent (Fluidigm)
    3.0
    DNA-Free Water (DPEC)
    30.0
    Pooled Primers (500 nM)
    15.0
    Total
    60.0
  7. LIVE/DEAD cell staining
    Components
    Volume (μl)
    C1 Cell Wash Buffer (Fluidigm)
    1250.0
    Ethidium homodimer-1 (LIVE/DEAD kit, Thermo Fisher Scientific)
    2.5
    Calcein AM (LIVE/DEAD kit, Thermo Fisher Scientific)
    0.625
    Total
    1253.125

Acknowledgments

This protocol was adapted from Sato et al. (2009) and Suzuki et al. (2018) (Fluidigm, C1 microRNA PreAmp protocol). This work was supported by MEXT/JSPS KAKENHI (18K15774, 18K15743, 19H03634, 19K17484); the Research Center Network Program for Realization of Regenerative Medicine from AMED (18bm03041h0006, 18bk0104008h0001, 19bm0304001h0007, 19bk0104008h0002, 19bm0404055h000).

Competing interests

The authors declare that they have no conflict of interest.

Ethics

The Ethics Committees of Tokyo Medical & Dental University and Yokohama Municipal Hospital approved our study (M2000-2093 and M2000-1176); and written informed consent forms were obtained from each patient.

References

  1. Biton, M., Haber, A. L., Rogel, N., Burgin, G., Beyaz, S., Schnell, A., Ashenberg, O., Su, C. W., Smillie, C., Shekhar, K., Chen, Z., Wu, C., Ordovas-Montanes, J., Alvarez, D., Herbst, R. H., Zhang, M., Tirosh, I., Dionne, D., Nguyen, L. T., Xifaras, M.E., Shalek, A. K., von Andrian, U. H., Graham, D. B., Rozenblatt-Rosen, O., Shi, H. N., Kuchroo, V., Yilmaz, O. H., Regev, A. and Xavier, R. J. (2018). T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation. Cell 175(5):1307-1320.
  2. Clevers, H. (2013). The intestinal crypt, a prototype stem cell compartment. Cell 154(2): 274-284.
  3. Crosnier, C., Stamataki, D. and Lewis, J. (2006). Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 7(5): 349-359.
  4. Fatehullah, A., Tan, S. H. and Barker, N. (2016). Organoids as an in vitro model of human development and disease. Nat Cell Biol 18(3): 246-254.
  5. Li, N., Yousefi, M., Nakauka-Ddamba, A., Jain, R., Tobias, J., Epstein, J. A., Jensen, S. T. and Lengner, C. J. (2014). Single-cell analysis of proxy reporter allele-marked epithelial cells establishes intestinal stem cell hierarchy. Stem Cell Reports 3(5): 876-891.
  6. Middendorp, S., Schneeberger, K., Wiegerinck, C. L., Mokry, M., Akkerman, R. D., van Wijngaarden, S., Clevers, H. and Nieuwenhuis, E. E. (2014). Adult stem cells in the small intestine are intrinsically programmed with their location-specific function. Stem Cells 32(5): 1083-1091.
  7. Muñoz, J., Stange, D. E., Schepers, A. G., van de Wetering, M., Koo, B. K., Itzkovitz, S., Volckmann, R., Kung, K. S., Koster, J., Radulescu, S., Myant, K., Versteeg, R., Sansom, O. J., van Es, J. H., Barker, N., van Oudenaarden, A., Mohammed, S., Heck, A. J. and Clevers, H. (2012). The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers. EMBO J 31(14): 3079-3091.
  8. Noben, M., Vanhove, W., Arnauts, K., Santo Ramalho, A., Van Assche, G., Vermeire, S., Verfaillie, C. and Ferrante, M. (2017). Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology. United European Gastroenterol J 5(8): 1073-1081. 
  9. Okamoto, R. and Watanabe, M. (2016). Role of epithelial cells in the pathogenesis and treatment of inflammatory bowel disease. J Gastroenterol 51(1): 11-21.
  10. 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.
  11. 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.
  12. Smith, N. R., Gallagher, A. C. and Wong, M. H. (2016). Defining a stem cell hierarchy in the intestine: markers, caveats and controversies. J Physiol 594(17): 4781-4790.
  13. Stuart, T. and Satija, R. (2019). Integrative single-cell analysis. Nat Rev Genet 20(5): 257-272.
  14. Suzuki, K., Murano, T., Shimizu, H., Ito, G., Nakata, T., Fujii, S., Ishibashi, F., Kawamoto, A., Anzai, S., Kuno, R., Kuwabara, K., Takahashi, J., Hama, M., Nagata, S., Hiraguri, Y., Takenaka, K., Yui, S., Tsuchiya, K., Nakamura, N., Ohtsuka, K., Watanabe, M., Okamoto, R. (2018). Single cell analysis of Crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties. J Gastroenterol 53: 1035-1047.

简介

[摘要]在此协议中,我们描述了从患者的活检样本中分离隐窝并培养人类肠干细胞(称为“类器官”)的方法。除此之外,我们还介绍了如何将类器官细胞分解为单细胞以进行单细胞分析,作为进一步的应用。该方案应为研究人员提供足够的工具,以从活检样品中产生人类器官并完成稳定的体外测定系统。

[背景]肠上皮是一个多功能组织即编排动态平衡并形成物理屏障。由肠干细胞(ISC)产生的每个肠上皮细胞(IEC)每4-5天更新一次该上皮(Crosnier等,2006 )。ISC位于隐窝的底部,并表达各种文献先前报道的特定标记(Muñoz等,2012 ;Clevers ,2013 )。研究表明,干细胞正确更新的功能障碍与肠道疾病有关,对ISCs动态的了解可能阐明了包括炎症性肠病(IBD)在内的各种疾病的发病机制(Okamoto et al。,2016 )。

然而,由于缺乏能概括生理性肠上皮层的有效模型,因此对肠干细胞特性的研究具有挑战性。史诗般的“类器官”的引入克服了种种障碍(Sato等人,2009和2011 ),可以从单个ISC体外建立类器官,并忠实地保留其起源组织的生理和病理特征(Middendorp等人)。 。,2014 )。类器官已被用于各种胃肠道疾病解剖基础病理变化(Fatehullah 。等人,2016; Noben等人,2017 ),并已示出的电势,以反映组织的络合机制ANS。

此外,分子生物学技术的最新进展使我们能够研究单细胞模式(Stuart和Satija ,2019 )。这些技术的发展是对每个单个细胞的多样性有深刻的了解,但已知它们是同质的种群。研究表明,一组异质性细胞共享这些ISC属性,并在ISC群体内构成一个层次结构(Smith等,2016 )。此外,类器官可能是理想的工具之一,该工具主要由未分化培养物中的干细胞和转运扩增细胞组成。在以前的报告中,单细胞分析显示了小鼠小肠干细胞之间的这种异质性(Li等,2014 )。结合类器官培养技术和单细胞分析,有可能为了解人类肠道干细胞的动力学开辟新的视野。在此协议中,我们详细描述了人类肠道类器官的建立和解离为单个细胞以进一步进行各种应用的工作流程。与使用scRNA -seq的那些协议相比(Biton等人,2014 ),使用多重PCR的本协议能够以低成本,短时间的基础获取单细胞谱,而细胞和基因的数量将受到限制微流体芯片形式的容量。

关键字:类器官, 肠道干细胞, 单细胞分析, 内窥镜活检, 多重PCR


材料和试剂
类器官文化
24孔组织培养板(Corning,Falcon,目录号353226)
50 ml和15 ml锥形离心管(Corning,Falcon,目录号s :352096、352070)
15毫升STEMFULL TM低细胞粘附管(住友电木Ç Ò 。,目录号:MS-90150Z)
1.5 ml Eppendorf管(Eppendorf Safe-Lock Tubes,货号:0030121023)
70 μ米细胞过滤器(康宁公司,Falcon,目录号:352350)
通过内窥镜检查获得的患者肠内活检样本
磷酸盐缓冲盐水(PBS)(Sigma,目录号:D8537-500ML)(储存在4°C)
基质胶(康宁,目录号:356231 )(š在4℃tored)
细胞回收解决方案(Corning,Thermo Fisher Scientific ,目录号:354253)(存储在4°C下)
Advanced-DMEM (Thermo Fisher Scientific,目录号:12491015)(存储在4°C下)
青霉素/链霉素(Nacalai Tesque ,目录号:26253-84)(储存于4°C)                           
1 mol / l-HEPES缓冲溶液(Nacalai Tesque ,目录号:17557-94)(储存在4°C)             
GlutaMAX TM -I(100 x )(Gibco,Thermo Fisher Scientific ,目录号:35050-061)(存储在4°C下)
0.5 M EDTA((Thermo Fisher Scientific,目录号:AM9260G)
台盼蓝溶液(Thermo Fisher Scientific ,目录号:15250061)
N-乙酰半胱氨酸(Sigma,目录号:A9165-5G )(储存在-20°C,1 M,10 ml等分试样)                           
胃泌素I (Sigma,目录号:3006) (储存在-20℃下,100微升,1 ,000微升等分试样)                           
N2补充剂(R&D Systems ,目录号:AR009)(储存在-20°C,100x,1小瓶)             
B27补充品(R&D Systems,货号:AR008)(储存在-20°C,50x,1小瓶中)             
重组小鼠EGF(R&D Systems,目录号:2028-EG-200 )
(储存在-20℃下,20微克/ ml时,等分试样1 ,000微升)

重组小鼠成头蛋白(R&d系统,目录号:1967-NG-025 / CF )(储存在-20℃下,20微克/ ml时,等分试样250微升)
重组人R-脊椎蛋白-1(R&d系统,目录号:4645-RS )(保存在-20℃,100微克/ ml时,等分试样500微升)
重组小鼠的Wnt-3A(R&d系统,目录号:1324-WN-010 / CF )(存储在- 20℃,10微克/ ml时,等分试样1 ,000微升)
烟酰胺(R&d系统,目录号:4106 )(保存在-20℃,1米升,等分试样1 ,000微升)             
注意:以粉末形式存放在RT货架中。Nic 6.1 g + ddH 2 O 50 ml,等分试样各10 ml放入15 ml F alcon管中。解冻一支试管时,等分1.5毫升的戊烷并保存。

A83-01(Sigma-Aldrich,目录号:2939 )(存储在-20°C,10 mM,100μl等分试样中)
注意:避光。等分试样10毫稀释成Eppen dolf管中,用20倍对其进行标记,进一步稀释10毫Eppendolf管与DMSO和等分试样100微升每。

SB202190(Sigma-Aldrich公司,目录号:1264) (储存在4℃,150 μ升等分试样)             
注意:SB 202190 10毫克+ DMSO 3 ,018微升。等分试样150微升每并冷冻于-20,直到使用。

Y-27632(R&d系统,目录号:1254 )(p从光rotect,储存在-20℃,10毫米,150微升等分试样)
人肠基础培养基(请参阅食谱)
人小肠类器官生长介质(请参阅食谱)
 

单细胞分析
C1前置放大器IFC(10-17微米,Fluidigm公司,目录号:100-5480)
48 x 48 IFC芯片(Fluidigm ,目录号:BMK-M-48.48)
TrypLE Select(瑟莫·费舍尔科学公司(Thermo Fisher Scientific),目录号:12563011)(存储在4°C下)
用于前置放大器的C1单细胞试剂盒(包括模块1,模块2:Fluidigm ,目录号; 100-5319)(存储在-20°C下)
单细胞至CT试剂盒(Thermo Fisher Scientific ,目录号:4458236)(存储在-20°C)
LIVE / DEAD生存力/细胞毒性试剂盒(Thermo Fisher Scientific ,目录号:L3224)(储存在-20°C)
汇总的底漆(请参阅食谱)
裂解最终混合物(请参阅食谱)
RT最终混合物(请参阅食谱)
PreAmp最终混音(请参阅食谱)
活/死细胞染色(请参见食谱)
 

碧欧马克高清
2x上样试剂1.5 ml(Fluidigm ,目录号:100-7611)
20x TaqMan基因表达测定(Thermo Fisher Scientific,目录号:4351372)
2x预混液(Thermo Fisher Scientific,目录号4369514)
20x GE样品加载试剂(Fluidigm ,目录号:100-7610)
 

设备

 

移液管(5毫升,10毫升,25毫升,50ml)中,微移液器(10微升,20微升,200微升,1000微升)
多通道移液器(Mettler Toledo ,RAININ,型号:E 8-20 XLS +,目录号:17013798)
离心机
涡旋混合器
血细胞计数器(Burker -Turk,Fujirika Co.,A114)
37 °C ,5%CO 2细胞培养培养箱
荧光显微镜(Keyence,型号:BZ-X700,)
C1单细胞自动制备系统(Fluidigm ,美国加利福尼亚州旧金山)
C1单细胞自动制备阵列IFC(MX)(Fluidigm ,美国加利福尼亚州旧金山)
Biomark HD系统(Fluidigm ,美国加利福尼亚州旧金山)
 

软件

 

奇异分析工具集软件v3.5.2(Fluidigm,美国加利福尼亚州旧金山)
R软件
该帕特克的基因组套件(6.6-6.16.0812版本,帕特克,切斯特菲尔德,密苏里州,美国)
 

程序

 

第一部分:隐窝分离和上皮类器官培养

 

从患者活检样本中分离和培养小肠类器官的步骤
小肠肠镜活检样本取自进行小肠肿瘤,隐匿性出血或克罗恩病等疾病评估的患者。从每个患者的最多8个活检样本取自回盲瓣近端约100 cm的区域。应当从每个患者那里获得伦理委员会的批准和书面知情同意。
使用50 ml Falcon管,用冷PBS洗涤片段,直到澄清上清液为止。
将样品在15 mM EDTA / PBS溶液(旋转)中于4°C孵育30分钟(10 ml PBS + 300 µl 0.5 M EDTA)。
除去EDTA缓冲液后,添加10 ml PBS和涡旋2分钟以分离肠隐窝(图1)。




图1.新鲜嵌入患者的小肠隐窝。标度b ar s = 1 mm(左)和100μm (右)。

 

通过在显微镜下观察确认完整的隐窝隔离(参见注s )。
在管沉淀下来的试样后,收集上清液并转移至50ml ˚F爱尔康管通过70过滤μ微米的过滤器。
重复第I部分的步骤A5约2-3次,直到收集到大约30 ml体积的含有溶液的隐窝。
将试管以300 xg离心3分钟以沉淀。
用10 ml Advanced-DMEM / F12重悬细胞沉淀,并转移到一个STEMFULL TM管中。
离心管中,在300 ×g下FO ,R 3分钟以沉淀。
在显微镜下检查沉淀后,小心吸出上清液,并加入所需量的Matrigel(每孔30-50 µl)以嵌入隐窝(每孔20-30隐窝)。
后轻轻移液管吨我纳克基质胶内,轻轻放在24孔板应用30μl的基质胶。孵育30-60分钟以使Matrigel聚合。
向每个孔中添加500 µl人肠生长培养基。在最初的3天中,向培养基中添加10 µM的Y-27632。
 

维持和通过小肠类器官的程序
隔离后,每两天更换一次小肠生长培养基。
隔离13天后,您将能够看到类器官的生长,它们开始形成称为“萌芽”的隐窝状结构(图2)。为了继代培养,除去生长培养基,并向孔中加入800 µl冷细胞回收溶液。使用细胞回收液,通过1,000 µl移液管刮掉含有类器官的Matrigel。
 



图2.从患者身上建立小肠类器官。标度b ar s = 1 mm(左)和100μm (右)。

 

将再悬浮液转移到15 ml STEMFULL TM试管中。使用P100移液器,剧烈吸移50-100次,以将类器官机械分解为较小的碎片。在显微镜下检查碎片后,向混合物中加入9 ml冷的Advanced-DMEM / F12。
将细胞以300 x g离心3分钟。
小心吸出上清液,然后将沉淀重新悬浮在Matrigel中。通常,以1:5-6的比例分割。在大约第13天,已建立的类器官开始萌芽。
 

第二部分:单细胞水平的基因表达分析

 

培养的类器官细胞的单细胞解离
从每个孔中移出全部体积的生长培养基(使用2-3个孔以获得足够数量的细胞)。
使用与通过培养的类器官中所述相同的方法,将类器官收集在15 ml STEMFULL TM管中,用力吸打50-100次,以机械方式将类器官分解为较小的片段。向混合物中加入9毫升冷Advanced-DMEM / F12。
以300 xg离心3分钟,然后吸出上清液。
将5 ml的TrypLE select加入沉淀并以600 rpm涡旋10分钟(加入Hoechst 10μl )。
10分钟后,剧烈摇动试管10次,以将其进一步分离成单个细胞。
以500 xg离心3分钟,然后确认调色板。
用千重悬微升的高级-DMEM / F12 ,并将它们传送到1.5ml Eppendorf管中,并通过使用常规的台盼蓝染色计数活细胞数目(优化的细胞浓度:3×10 5个细胞/ ml ,预期细胞存活率> 70% )。
取出所需量的含溶液细胞,以500 xg离心3分钟,然后吸出上清液。
与1000重悬微升PBS中,并继续进行到C1协议。
 

将单个单元格加载到芯片中(C1前置放大过程)
PR epare单细胞悬浮液C1装载通过混合预先制备的细胞悬浮液(A9)的d C1细胞悬浮试剂而解冻其他试剂。
组分体积(微升)             

细胞166-250K / ml的30微升             

C1细胞悬浮试剂(Fluidigm公司)20微升             

共有50微升             

C1芯片灌注
打开C1机器,放置一个新的C1单电池自动制备IFC并按如下所示使用每种试剂(图3 )。
 



图3 。一个板启动的应用程序(Fluidigm公司C1到的Taqman引物协议,PN100-6117)

 

加载每种试剂后,将其放置在平台上并选择Prime程序并运行(需要10分钟)。
重要提示:灌注后,应在1小时内涂抹样品,以防止灌注剂变干。

在对板进行底涂时,准备Lysis Final Mix,逆转录(RT)Final Mix和PreAmp Final Mix,并将m放在冰上(食谱4-6)。
细胞应用和染色
灌注后,如下所示除去封闭溶液(图4 )。
移取准备的细胞混合物10次,然后将20μl涂在蓝色编码的孔中。
准备C1 LIVE / DEAD(参见ř ecipe小号)和申请20微升到粉红色编码井。
 



图4 。细胞的应用和染色给C1板(Fluidigm公司C1到的Taqman引物协议,PN100-6117)

 

将板放在舞台上,选择“ Cell Load&Stain”程序并运行(此过程需要60-80分钟)
程序结束后,取出板并确认单细胞captur È (图5 )和96个孔的细胞生存力显微镜(BX-X700,基恩士,大阪,日本)下。
 



图5 。用钙黄绿素AM阳性染色的活细胞捕获实例和用Ethidium homodimer-1染色的死细胞实例。规模b AR = 100 μ米。

 

确认细胞后,应用每种溶液中所示的贝洛W(图6 )(RECIP上课4-6)
 



图6 。的预放大的解决方案到应用C1板(Fluidigm公司C1到的Taqman引物协议,PN100-6117)

 

将盘子放在舞台上,然后选择PreAmp程序并设置估计的结束时间(这是一整夜的过程)。
重要的是在完成后的一个小时内收集样品;否则,样品将蒸发。
准备一个新的96孔板中和等分试样25微升C1 DNA稀释试剂的进入每个标记的稀释收获板以及
哈尔韦斯吨预扩增产物表明在下面并与每个拌匀与使用8通道移液器DNA稀释液(每孔具有约3.6微升)(图7 )。
将收获的板保存在-20°C下。
组分体积(微升)             

C1 DNA稀释试剂(Fluidigm )25             

C1收获扩增子3             

总计28             

 



图7 。吸移的预扩增产物的步骤从一个C1-板至96孔板(Fluidigm公司C1到的Taqman引物协议,PN100-6117)

 

Biomark HD工作流程
多重定量PCR分析的进一步扩增和数据获取将使用Biomark 48.48进行。动态阵列IFC和Biomark HD系统(Fluidigm ,美国加利福尼亚州旧金山)。动态阵列IFC系统运行纳米流体IFC电路,该电路可自动将收获的样品与一组基因测定结合起来。

准备中
如下所示准备“ 10 x分析混合液”和“样品混合液”。


TaqMan底漆10 x分析混合物

组件

每体积入口(μ升)

每胰岛体积超龄(μ升)

为48.48体积(μ升)

为96.96体积(μ升)

2 x上样试剂

2.5

6.0

X 60 = 360

X 120 = 600

20 x TaqMan基因表达分析

2.5

6.0

 

 

总容积

5.0

6.0

300.0

600.0

最终浓度为10倍

引物:9μM探针:2.5μM

储存在-20 °C

 

样品混合

组件

每体积入口(μ升)

每胰岛体积超龄(μ升)

为48.48体积(μ升)

为96.96体积(μ升)

2 x材料混合

2.5

3.0

X 60 = 180.0

X 120 = 36.0

20 x GE样品加载试剂

0.25

0.3

X 60 = 18.0

X 120 = 36.0

基因

2.25

2.7

 

 



5.0

6.0

198

396.0

 

启动IFC和样品加载
确保对48.48动态阵列IFC使用IFC控制器MX,对于96.96动态阵列IFC使用IFC控制器HX。

一种。将控制管路流体注入IFC上的每个蓄能器。       

b。取下并丢弃IFC底部的保护膜。      

C。将IFC放入适当的控制器中,然后运行适当的“灌注”脚本以将控制管路流体灌注至IFC中。每种IFC类型都对应一个不同的脚本编号,并且印版ID号将由控制器标识(灌注最多需要30分钟)。       

重要提示:请确保在完成底漆后1小时内运行“加载”,因为板将变干。

d。从控制器上取下IFC,然后将样品和测定溶液吸移至IFC的入口中(每个5μl )。      

e。将IFC返还给控制器。       

F。运行适当的“ Load Mix”脚本。气压迫使样品和基因测定溶液进入IFC,在此混合。        

G。从控制器中卸下已装载的IFC。      

H。将IFC放入Biomark HD仪器中,确保IFC上的A1角与仪器托盘上的A1对齐。      

重要提示:确保完成“ Load Mix”脚本后1小时内运行Biomark HD,因为板将变干。

运行Biomark HD
打开Biomark HD和计算机。
单击名为“ Biomark Data Collection”的图标。
CCD摄像机的温度达到-5 °C时,请等待应用程序显示“就绪” 。
单击默认桌面的“开始新运行” 。确认Ť他“数据收集”屏幕出现响应。
将预装的IFC放在托盘上,然后单击“加载”。托盘缩回,系统扫描条形码并识别IFC。
点击下一步”。浏览并选择您想要保存的文件。
选择基因表达应用程序。选择被动参考(ROX)。选择探针,然后选择“下一步” 。
选择浏览和协议文件。开始运行。
 

数据分析

 

使用Singular Analysis Toolset Software v3.5.2(Fluidigm ,旧金山,加利福尼亚州,美国)和Partek Genomic Suite(版本6.6-6.16.0812,Partek ,Partek ,Chesterfield,MO )处理从多重单细胞基因表达分析获得的数据。遵循标准工作流程。

 

笔记

 

可以从内窥镜活检样本中收获的地穴数量非常有限。在步骤A4之后,取出1ml隐窝悬浮液并将其铺在24孔板上,在显微镜下观察并确认存在隐窝。

 

菜谱

 

1.人肠道基础培养基      

组件

股票骗局。

最后的骗局。

/ 500毫升培养基

先进的DMEM / F12

 

1倍

500毫升

GlutaMAX-1

200毫米

2毫米

5毫升

HEPES

1 M

10毫米

5毫升

青霉素/链霉素

10 ,000 U / ml的

100单位/毫升

5毫升

N-乙酰半胱氨酸

500毫米

1毫米

1毫升

胃泌素

100微米

10毫米

50微升

N2补充

100倍

1倍

5毫升

B27补充

50倍

1倍

10毫升

 



2.人类小肠类器官生长介质       

组件

股票缺点

最终缺点

单元

1口

2口

毫升

0.5

1个

基础培养基

--

--

毫升

0.25

0.5

生长因子

20微克/毫升

50 ng /毫升

微升

1.25

2.5

米·诺金

20微克/毫升

100 ng /毫升

微升

2.5

5

m R-spondin-1

100微克/毫升

1微克/毫升

微升

5

10

m Wnt-3A

10微克/毫升

300 ng / ml

微升

15

30

烟酰胺

1 M

10毫米

微升

5

10

A83-01

500微米

500 nM

微升

0.5

1个

SB202190

10毫米

10微米

微升

0.5

1个

 

3.合并引物       

组件

体积(微升)

1微升的每个引物对(100 μM每个)

1.0(x 93 = 93微升)

可选的RNA Spike引物

1.0(x 3 = 3微升)

C1 DNA稀释试剂

104.0



200.0

最多可保存6个月(-20°C)

 

4.裂解最终混合物       

组件

体积(微升)

C1 DNA稀释试剂(Fluidigm )

0.90

单细胞裂解液(Thermo Fisher Scientific )

12.75

C1 Lysis Plus试剂(Fluidigm )

4.35



18.0

 

5. RT最终混合物       

组件

体积(微升)

单细胞VOLO RT混合液(Thermo Fisher Scientific )

5.84

单细胞SuperScript RT(Thermo Fisher Scientific )

3.62

停止解决方案(赛默飞世尔科技)

1.94

C1加载试剂(Fluidigm ) 

0.60



12.00

 



6. PreAmp最终混音       

组件

体积(微升)

单节PreAmp混音(Thermo Fisher Scientific )

12.0

C1加载试剂(Fluidigm )

3.0

无DNA水(DPEC)

30.0

混合引物(500 nM )

15.0



60.0

 

7.活/死细胞染色       

组件

体积(微升)

C1细胞洗涤缓冲液(Fluidigm )

1250.0

Ethidium homodimer-1(LIVE / DEAD试剂盒,赛默飞世尔科技)

2.5

钙黄绿素AM(LIVE / DEAD试剂盒,赛默飞世尔科技)

0.625



1253.125

 

致谢

 

该协议改编自Sato等人。(2009年)一第二铃木等人。(2018)(Fluidigm ,C1 microRNA PreAmp协议)。这项工作得到了MEXT / JSPS KAKENHI(18K15774、18K15743、19H03634、19K17484)的支持;来自AMED的研究中心网络计划以实现再生医学(18bm03041h0006、18bk0104008h0001、19bm0304001h0007、19bk0104008h0002、19bm0404055h000)。

 

利益争夺

 

作者宣称他们没有利益冲突。

 

伦理

 

东京医科牙科大学和横滨市立医院伦理委员会批准了我们的研究(M2000-2093和M2000-1176);并从每位患者获得书面知情同意书。

 

参考文献

 

Biton ,M.,Haber,A. L.,Rogel ,N.,Burgin,G.,Beyaz ,S.,Schnell,A.,Ashenberg ,O.,Su,C.W.,Smillie ,C.,Shekhar ,K.,Chen,Z.,Wu,C.,Ordovas-Montanes ,J.,Alvarez,D.,Herbst,R.H.,Zhang,M.,Tirosh,I.,Dionne,D.,Nguyen, L. T.,Xifaras ,ME,Shalek ,A. K.,冯安德里安,U. H.,格雷厄姆,D. B.,Rozenblatt -Rosen,O.,施,H. N.,Kuchroo ,V., Yilmaz,O.H. ,Regev,A.和Xavier,R.J .(2018)。T辅助细胞细胞因子调节肠道干细胞的更新和分化。单元格175(5):1307-1320。
Clevers ,H.(2013年)。肠道隐窝,干细胞隔室的原型。细胞154(2):274-284。
Crosnier ,C.,Stamataki ,D.和Lewis,J.(2006)。在肠道内组织细胞更新:干细胞,信号和组合控制。Nat Rev Genet 7(5):349-359。
Fatehullah ,A.,Tan,SH和Barker,N.(2016年)。类器官作为人类发育和疾病的体外模型。Nat Cell Biol 18(3):246-254。
Li,N.,Yousefi ,M.,Nakauka-Ddamba ,A.,Jain,R.,Tobias,J.,Epstein,JA,Jensen,ST和Lengner ,CJ(2014)。代理记者等位基因标记的上皮细胞的单细胞分析建立了肠道干细胞的层次结构。干细胞报告3(5):876-891。
Middendorp ,S.,Schneeberger ,K.,Wiegerinck ,CL,Mokry ,M.,Akkerman ,RD,van Wijngaarden ,S.,Clevers ,H.和Nieuwenhuis ,EE(2014)。小肠中的成年干细胞通过其位置特定功能进行内在编程。干细胞32(5):1083-1091。
亩ñ盎司,J.,施坦格,DE,SCHEPERS ,AG,范·德Wetering ,M.,辜,BK,Itzkovitz ,S.,Volckmann ,R.,功夫,KS,科斯特,J.,Radulescu ,S.,Myant ,K.,Versteeg,R.,Sansom,OJ,van Es,JH,Barker,N.,van Oudenaarden ,A.,Mohammed,S.,Heck,AJ和Clevers ,H.(2012)。Lgr5肠干细胞签名:提议的静态“ +4”细胞标记物的稳定表达。EMBO J 31(14):3079-3091。
Noben ,M.,Vanhove ,W.,Arnauts,K.,Santo Ramalho ,A.,Van Assche ,G.,Vermeire ,S.,Verfaillie ,C.和Ferrante,M.(2017)。菜中人肠上皮:胃肠道病理生理研究的当前模型。联合欧洲胃肠J 5(8):1073-1081。              
冈本R.和渡边M.(2016)。上皮细胞在炎症性肠病的发病机理和治疗中的作用。胃肠病学杂志51(1):11-21。
佐藤,T.,施坦格,DE,费兰特,M.,弗里斯,RG,范ES,JH,凌科,S.,范Houdt ,WJ,普龙,A.,范GORP ,J.,Siersema ,PD和Clevers ,H.(2011年)。人结肠,腺瘤,腺癌和巴雷特上皮的上皮类器官的长期扩张。胃肠病学141(5):1762-1772。
Sato,T.,Vries,RG,Snippert ,HJ,van de Wetering ,M.,Barker,N.,Stange ,DE,van Es,JH,Abo,A.,Kujala ,P.,Peters,PJ和Clevers , H.(2009年)。单个Lgr5干细胞在体外建立隐窝-绒毛结构,而没有间充质位。自然459(7244):262-265。
史密斯(NR),加拉格尔(Gallagher)和黄(MH)(2016)。定义肠道中的干细胞等级:标记,注意事项和争议。Ĵ生理学594(17):4781-4790。
Stuart,T.和Satija ,R.(2019年)。集成单细胞分析。Nat Rev Genet 20(5):257-272。
铃木,肯塔基州,穆拉诺,T 。,清水,H。,伊藤,G。,中田,T。,藤井,S。,石桥,F。,川本,A。,安西,S.,Kuno ,R.,Kuwabara ,K。,高桥,J。 ,哈马,M.,永田,S.,Hiraguri ,Y.,竹中,K.,衣,S.,土屋,K.,中村,N.,大冢,K.,渡边,M.,冈本,R. (2018)。对克罗恩氏病患者来源的小肠类器官的单细胞分析显示,疾病活性依赖于干细胞特性的修饰。ĴGastroenterol 53 :1035 - 1047 。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Suzuki, K. and Okamoto, R. (2020). Fluidigm Based Single-cell Gene Expression Library Preparation from Patient-derived Small Intestinal Organoids. Bio-protocol 10(19): e3775. DOI: 10.21769/BioProtoc.3775.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。