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Nov 2020

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Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening
基于荧光的全染色单细胞分析和清除的微组织和类器官高通量筛选   

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Abstract

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues (“organoids”), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells from their native niche and can result in the loss of 3D context during analyses. Automated workflows require parallel processing and analysis of hundreds to thousands of samples, and sectioning is mechanically complex, time-intensive, and thus less suited for automated workflows. Here, we present a simple protocol for combined whole-mount immunostaining, tissue-clearing, and optical analysis of large-scale (approx. 1 mm) 3D tissues with single-cell level resolution. While the protocol can be performed manually, it was specifically designed to be compatible with high-throughput applications and automated liquid handling systems. This approach is freely scalable and allows parallel automated processing of large sample numbers in standard labware. We have successfully applied the protocol to human mid- and forebrain organoids, but, in principle, the workflow is suitable for a variety of 3D tissue samples to facilitate the phenotypic discovery of cellular behaviors in 3D cell culture-based high-throughput screens.


Graphic abstract:



Automatable organoid clearing and high-content analysis workflow and timeline


Keywords: Organoids (类器官), 3D cell culture (三维细胞培养), Whole-mount immunostaining (整体组织免疫荧光染色), Clearing (清除), (High-content) image analysis ((高含量)图像分析), Automation (自动化)

Background

Over the past years, three-dimensional (3D) cell culture systems, in particular stem cell-based organoids, have enabled novel insights into human biology and disease (reviewed by: Rossi et al., 2018; Schutgens and Clevers, 2020; Kim et al., 2020). However, working with 3D models requires more than new cell culture techniques. Quantifying cells in complex 3D tissues in fast, unbiased, and efficient workflows requires adapting the analyses as well. So far, many studies have relied on tissue sectioning followed by immunostaining to analyze the structures and composition of their 3D aggregates in detail (Lancaster et al., 2013; Pasca et al., 2015). While this method has been invaluable, it requires ample manual intervention, is time-intensive and cumbersome, and thus not ideally suited for large-scale screening applications, including drug development campaigns. Moreover, sectioning provides only a view at a subset of sample tissue, often results in a loss of spatial information unless meticulous serial sections are prepared, and is challenging to use as a basis for 3D reconstruction.


More recently, the combination of whole-mount staining and tissue clearing allowed the analysis of entire 3D aggregates without the need for sectioning, as demonstrated by our group and others (Masselink et al., 2019; Dekkers et al., 2019; Renner et al., 2020). This approach allows rapid 3D acquisition of complex samples and preserves tissue context while providing single-cell-specific phenotypic information. Our workflow utilizes a customized immunostaining procedure, which we adapted and optimized for organoids based on a previous protocol (Lee et al., 2016). We combined this with benzyl alcohol benzyl benzoate (BABB)-based clearing (Dent et al., 1989), which clears human neural tissues quickly (minutes) and effectively (Renner et al., 2020). We specifically designed this procedure to be compatible with both manual pipetting and automated liquid handling systems, facilitating low- and high-throughput applications. Depending on the individual requirements, the samples can then be analyzed using a standard confocal microscope or a high-content imaging system. In either case, the resulting optical tissue cross sections enable the quantitative analysis of entire 3D samples down to the single-cell level. This eliminates the need to freeze/section the organoids and enables parallel staining and analysis of many samples in 96- or 384-well formats. Though we developed the workflow for the analysis of neural organoids, it is not restricted to a particular organoid system or sample type but can be applied to any 3D sample of interest.

Materials and Reagents

  1. 96-well plates, NuncTM MicroWellTM conical 96-well plates (V-bottom) (Thermo Fisher, catalog number: 277143)

  2. Screenstar microplate, 96-well, COC, F-bottom, black (Greiner, catalog number: 655866)

  3. Screw cap tubes (Sarstedt, catalog numbers: 62.547.254 [50 ml] and 62.554.502 [15 ml])

  4. Serological pipets (Falcon, catalog numbers: 356543 [5 ml], 356551 [10 ml], and 356525 [25 ml])

  5. Pipet tips (StarLab, catalog numbers: S1120-1810 [20 μl], S1120-8810 [200 μl], and S1122-1830 [1,000 μl])

  6. Conical/V-bottom 96-well plates (Thermo Fisher, catalog number: 277143)

  7. Optional: Biomek liquid handler pipette tips AP96 P250 (Beckman Coulter, catalog number: 717252)

  8. Organoids/3D samples to be stained

  9. Dulbecco's Phosphate Buffered Saline (PBS; Sigma-Aldrich, catalog number: D8662)

  10. Paraformaldehyde (PFA; VWR, catalog number: 15714-S)

  11. Triton X-100 (Roth, catalog number: 3051.2)

  12. Sodium azide (Sigma-Aldrich, catalog number: 71289)

  13. Bovine Serum Albumin (BSA, Thermo Fisher, catalog number: 15260037)

  14. Methanol (Roth, catalog number: 4627.6)

  15. Benzyl alcohol (Sigma-Aldrich, catalog number: 305197-100ML)

  16. Benzyl benzoate (Sigma-Aldrich, catalog number: B6630-250ML)

  17. Optional: Nuclear counterstain, e.g., DAPI (Sigma-Aldrich, catalog number: D9542-10MG)

  18. Primary antibodies, depending on application [here: Rabbit anti-Tyrosine Hydroxylase (Abcam, product no: ab112) and Goat anti-Sox2 (R&D Systems, catalog number: AF2018)]

  19. Secondary antibodies, depending on application [here: Donkey anti-Rabbit IgG, Alexa Fluor 647 (Thermo Fisher, catalog number: A-31573) and Donkey anti-Goat IgG, Alexa Fluor 568 (Thermo Fisher, catalog number: A-11057)]

  20. Permeabilization buffer (see Recipes, Table 1)

  21. Blocking solution (see Recipes, Table 2)

  22. BABB/methanol (see Recipes, Table 3)

  23. BABB (see Recipes, Table 4)

Equipment

  1. Humidified CO2 incubator

  2. Fridges and freezers (4°C, -20°C, and -80°C)

  3. Optional: Stereomicroscope with camera (e.g., Leica MZ10 F (microscope) and Leica DFC425 C (camera), Leica Microsystems)

  4. Mechanical pipettes

  5. Optional: Multichannel mechanical pipettes

  6. Optional: Automated liquid handling system (e.g., Biomek FXP Laboratory Automation Workstation, Beckman Coulter)

  7. Confocal microscope (e.g., LSM 700, Zeiss)

  8. Optional: High-content imaging system (e.g., Operetta high-content imager, Perkin Elmer)

Software

  1. Fiji/ImageJ (Schindelin et al., 2012)

  2. Optional: Leica application suite v 4.8 (LAS, Leica Microsystems)

  3. Optional: Biomek Software v3.3 (controlling the automated liquid handler, Beckman Coulter)

  4. Optional: High-content imaging and analysis software (e.g., Harmony v4.1, Perkin Elmer)

  5. Optional: Excel (Microsoft Corporation)

Procedure

Caution: This protocol uses hazardous substances, including PFA, Triton X-100, methanol, benzyl alcohol, and benzyl benzoate. Be aware of toxicity from multiple routes of exposure, including inhalation. Refer to MSDS information before starting the procedure and work under a fume hood.


Note: All volumes mentioned here and in the following steps are optimized for samples in 96-well plates; other culture formats may require different volumes. For our setup and tissues with ca. 1 mm diameter or smaller, we use 150 μl of reagent volume per well. Larger volumes are possible and may be required for larger samples; however, we do not recommend going below 150 μl per well for fixation and washing to avoid having an insufficient amount of reagent for each tissue. For washing steps, higher volumes can increase the washing efficiency, but we found 150 μl per well to give good results.


  1. Fixation of large-scale (approx. 1 mm) 3D tissues

    Note: All steps described here can be performed either manually or using an automated liquid handling system (ALHS)/pipetting robot.

    1. To fix the samples, carefully aspirate the media and add 150 μl of freshly prepared 4% PFA in PBS per well. Incubate for 10-15 min at room temperature.

    2. Aspirate the fixative carefully and wash 3 × 5 min with 150 μl PBS per well.

    3. Either add 150 μl PBS per well and seal the plate with parafilm to store the samples at 4°C or directly proceed with the staining procedure below.

      Note: In our hands, several months of storage did not negatively impact the quality of the samples for whole-mount staining. However, we generally recommend quick turnaround times over longer storage.


  2. Whole-mount immunostaining

    Day 0
    1. Optional: Aspirate PBS carefully, add 150 µl of permeabilization buffer (see Recipes, Table 1) and incubate 1 h at 37°C.

      Note: A dedicated permeabilization step is not generally required but may improve the outcome for specific markers/sample types. This must be optimized on a case-to-case basis.

    2. Aspirate the permeabilization buffer/PBS and add 150 µl of primary antibody diluted in blocking solution (see Recipes, Table 2). Use antibody at the appropriate concentration (each antibody must be titrated and validated individually). Incubate 6 days at 37°C and 100% humidity, renewing the solution every 2nd day.

      Notes on incubation times and conditions: 1. Incubation times depend on tissue size and density and the antibodies used. We recommend a prior optimization of the incubation times with representative test samples; this will save valuable time for follow-up experiments and assure optimum outcomes. We found 6 days of incubation per antibody to work well in our samples. 2. For the antibody incubation steps, do not seal the plates with parafilm as it may “melt” and become difficult to remove from the plate after longer incubation periods at 37°C. Incubators should be humidified to prevent evaporative loss of fluids in the plates (almost all tissue culture incubators work well for this step). 3. In our hands, samples did not show any signs of deterioration over this period. If necessary, 0.1% (w/v) sodium azide in blocking and staining solutions can prevent microbial growth during this time (see Recipes).


      Note on control samples: Carefully plan control samples to be incubated without primary antibody to gauge non-specific binding of secondary antibody. Additionally, prepare control samples without antibodies to identify potential artifacts caused by autofluorescence. Quantitative assessment needs measurements of both types of background fluorescence to determine proper object-based detection thresholds for each channel.


    Day 2

    1. Renew primary antibody solution.


    Day 4
    1. Renew primary antibody solution.


    Day 6
    1. Wash samples with 150 μl 0.1% (v/v) Triton X-100 in PBS for 5 h at room temperature. Renew washing solution every hour.

      Note: In case of issues with non-specific/high background signal, washing might be extended overnight and performed at 37°C.

    2. Aspirate the washing solution carefully and add 150 µl of new blocking solution with secondary antibody 1:1,000 and DAPI (0.5 μg/ml). Incubate for 6 days at 37°C and 100% humidity. Renew solution every 2 days.

      Note: This and all following incubation steps should be performed in the dark to preserve the fluorescence signals. We have extensively optimized the conditions for various AlexaFluor-conjugated secondary antibodies and always arrived at a 1:1,000 dilution. We have not optimized the conditions for other antibodies (e.g., cyanine dye-coupled antibodies). These may require additional optimization, including testing other antibody concentrations.


    Day 8
    1. Renew secondary antibody/DAPI solution.


    Day 10
    1. Renew secondary antibody/DAPI solution


    Day 12
    1. Wash samples with 150 μl 0.1% Triton X-100 in PBS for 5 h at room temperature in the dark. Renew washing solution every hour.

      Note: In case of issues with non-specific/high background signal, washing might be extended overnight and performed at 37°C.

    2. Either replace the washing solution with 150 μl PBS per well and seal the plate with parafilm to store the samples in the dark at 4°C or directly proceed with downstream processing (e.g., BABB-based tissue clearing below).

      Note: We have not tested long-term storage of the samples before tissue clearing. We suggest performing the clearing step within approximately 1 week of finishing the whole-mount staining and then storing the cleared samples, which are stable for several months in the dark at 4 °C.


  3. BABB-based tissue clearing

    1. Carefully aspirate the PBS supernatant from samples and add 150 µl 25% methanol in PBS per well and incubate for 15 min at room temperature (RT).

      Note: For larger (> 1 mm) samples, if aggregates are not entirely cleared, incubation times might be extended to 30 min (60 min for BABB/methanol). This incubation should be optimized individually.

    2. Aspirate the 25% methanol and add 150 µl 50% methanol; incubate for 15 min at RT.

    3. Aspirate the 50% methanol and add 150 µl 75% methanol; incubate for 15 min at RT.

    4. Aspirate the 75% methanol and add 150 µl 90% methanol; incubate for 15 min at RT.

    5. Aspirate the 90% methanol and add 150 µl 100% methanol; incubate for 15 min at RT.

    6. Important: Transfer the samples in 100% methanol from standard tissue culture plates to BABB-resistant plates (e.g., "Screenstar" COC; BABB/methanol and BABB dissolve most standard tissue culture plastic within 30 min). For transfer, cut off the tip of a 1,000 μl pipette tip with ethanol-cleaned scissors. Aim to cut the conical tip about 5 mm from the opening at the narrow conical end (you can cut off more if your samples are large). This is designed to widen the pipet opening to avoid damaging large 3D samples; you can use wide-bore tips for a liquid handler.

    7. Aspirate the 100% methanol and add 150 µl BABB/methanol (1:1, (v/v), see Recipes, Table 3) and incubate for 30 min at RT. BABB consists of benzyl alcohol/benzyl benzoate at a 1:1 (v/v) mixture (see Recipes, Table 4).

    8. Aspirate BABB/methanol and add 150 µl BABB.

    9. Within 5 min samples are completely cleared and ready for downstream analysis/microscopy or can be stored in the dark at 4°C for several months.

      Note: We successfully re-imaged samples that had been stored for approximately 18 months without encountering any deterioration in sample quality.


  4. Fluorescence-based single-cell analysis

    High-throughput applications, including compound screening, must process and treat a large number of samples with fast readouts. The combination of our whole-mount immunostaining and clearing workflows outlined above with high-content confocal imaging enables the automated acquisition of entire 96-well plates in a screening-compatible manner. We have described the analysis workflows necessary to analyze this kind of high-content data in detail (Renner et al., 2020). However, not all labs have access to the often highly specific hard- and software required for these kinds of analyses. Here, we detail a workflow using only standard confocal microscopes and freeware. As every 3D sample and staining has specific requirements for image analysis, we do not focus on specific parameters but rather on the general procedure, allowing anyone to apply the workflow to their own work.


    D0. Acquire images, either single optical confocal slices or entire stacks, using a confocal microscope. Depending on your analysis needs, you may prefer to image at 8, 16, or 32-bit depth with lens magnifications that suit your samples and desired lateral resolution. For our needs, we imaged whole organoids with a 10× lens magnification and 16-bit dynamic range (see Figure 1 for representative images). Adjust the distance between successive Z-planes to your analysis needs. A full 3D reconstruction will require more numerous and more closely spaced Z-planes. For quantitative comparison between samples, it is best to space the individual Z-planes further apart along the Z-axis, thus undersampling your 3D volume. This avoids data artifacts from double-counting the same physical feature in adjacent Z-planes and reduces the amount of data acquired and the acquisition times. For example, to measure a representative number of nuclei, Z-planes should be placed apart further than twice the average diameter of a nucleus. In general, empirically choose spatial increments between focal planes so that you can prevent imaging the same sample features in adjacent Z-planes, preventing double-counting.


    D1. Analysis of nuclear markers (example Sox2, see Figure 1A)

    Note: We describe all analysis procedures using Fiji/ImageJ (Schindelin et al., 2012) as it is freely available and commonly used for processing and analyzing biological imaging data (the specific commands used for the analysis here and the path to find them are described at each step below). However, the same general steps can also be performed with similar software using similar steps and parameters. Here, our raw images consisted of 16-bit grayscale data and measured 1024 pixels by 1024 pixels. Parameters below are adjusted for these conditions. Other starting points likely require the adjustment of downstream image analysis parameters.

    1. Open the 16-bit grayscale image.

    2. Duplicate the image and apply gaussian blur.

      1. Image > Duplicate.

      2. Process > Filters > Gaussian Blur (Parameter used here: 10 px).

    3. Subtract the blurred image from the original.

      Process > Image Calculator (then choose operation “subtract”).

    4. Optional: perform smoothing. This can reduce edge artifacts for thresholding operations.

      Process > Smooth.

    5. Apply threshold (the “auto threshold” function can be used, especially for batch analysis of entire stacks).

      Image > Adjust > Threshold/Auto Threshold.

    6. Optional: Use watershed to separate objects that have merged but should be separate, such as nuclei in close proximity.

      Plugins > Binary > Adjustable Watershed (Parameter used here: 0.5).

    7. Use the “analyze particles” function to measure the number of objects/nuclei.

      Note: Before measuring, use the “set measurements” function to select the parameters you want to analyze. Adjust the settings according to your requirements (e.g., increase minimum object size to exclude single-pixel noise) and enable “add to manager” (this creates regions of interest, “ROIs,” based on the identified objects and adds them to the “ROI manager”) if you also want to measure the intensity of the objects later. Larger artifacts like dust or hairs can also be excluded here by size.

      1. Analyze > Set Measurements.

      2. Analyze > Analyze Particles.

      3. The results of the analysis appear in the “Results” window and can be exported from there for further processing (e.g., to Microsoft Excel or other analysis software of choice).

    8. To measure the intensities, apply the ROIs defined before to the original image (as the raw intensity values change during thresholding/processing) via the “ROI manager” and use the “measure” function (be sure to select the appropriate parameters under “set measurements” before).

      1. Analyze > ROI Manager.

      2. Open original image (16-bit grayscale).

      3. Enable “select all” in ROI manager to show ROIs overlaid on the original image (if too many ROIs are selected, deselecting the checkbox marked “Labels” makes the image less crowded).

      4. Click “measure” in the ROI manager window to either measure single selected ROIs or all ROIs if none is selected.


    D2. Analysis of cytoplasmic/filamentous markers (example TH, see Figure 1B)
    For cytoplasmic/filamentous markers, clean segmentation of single cells can be challenging, especially in a dense 3D environment where they can span across several z-levels as thin cellular projections. Thus, it is often preferable to measure either the integrated or average intensity of positively identified structures of interest on every confocal plane. The signal for the whole organoid can then be summed for every plane within the organoid and quantitatively reflects the presence of each marker of interest contained in the 3D tissue.

    1. Steps 1-5 can be repeated as described in the Sox2 nuclear analysis above.

    2. Instead of separating the objects by watershedding, continue to define them as ROIs via the “analyze particles” function.

    3. Use the “ROI manager” to apply the previously defined ROIs to the original image and measure the intensity with the “measure” function (after adjusting “set measurements” to the sample’s specific requirements).


    D3. Measurement of the sample area for normalization (see Figure 1C for an example)

    Measuring 3D aggregates with varying diameters in different z-levels often requires normalization of the data to the total sample area to obtain comparable results between different samples.

    1. Sum all available channels to obtain an image that contains the morphological features of all visible elements of the sample across all channels.

      1. Open 16-bit grayscale images of all available channels.

      2. Process > Image Calculator (then choose the operation “add” and select all channels opened in step a). This will create a composite image containing all visible features of the sample across all fluorescent wavelengths for normalization to the total sample area.

    2. Use thresholding to create a single object from the sample, separating background and sample.

    3. Use the “measure” function to calculate the area of the object (after enabling “area” under “set measurements”).

    These procedures can be easily applied to entire aggregates by recording the workflow (with the parameters for the specific requirements of the samples) as a macro (e.g., via the “macro recorder”). These can iteratively process entire Z-stacks or even groups of Z-stacks contained in a common folder.



    Figure 1. Overview of the different steps of image analysis of whole-mount-stained and cleared 3D structures. (A-C) Representative images of the different steps from the image analysis process outlined in the Procedure Section D. (D-E) Representative results for the analysis of the nuclear marker Sox2. (D) Raw data (i.e., mean gray value for every of the identified objects) and (E) frequency distribution based on the data shown in D). Scale bars in the top image of each row (A-C) are valid for the entire column of images below. Scale bars: entire organoids = 100 μm; enlargement (A, right column) = 20 μm.

    Data analysis

    The manual analysis of images acquired with a confocal microscope is described under “Procedure section D: Fluorescence-based single-cell analysis.” The analysis of high-content imaging data is described in detail in the original publication (Renner et al., 2020) under “Materials and Methods,” “General workflow for high-content imaging and analysis,” and the following sections.

    Notes

    The BABB-based clearing procedure tends to quench the native fluorescence of fluorescent fusion proteins, including GFP, RFP, and all the mFruits. However, fluorescent dyes, including the Alexa fluors, are not affected. Thus, we recommend performing the whole-mount staining procedure outlined above using specific antibodies raised against the fluorescent protein of interest (e.g., anti-GFP) to analyze them in BABB-cleared structures. This has the additional benefit of signal amplification via multivalent secondary antibodies and better photostability, as commercial dyes commonly surpass the bleaching resistance of native fusion proteins.

    Several steps of the protocol can be optimized to save antibody solution, especially in large-scale experiments. Two options that have worked well in our hands are:

    1. Reducing the volume of the antibody solution used to a minimum of 50 μl (for large samples, it can also be increased to 200 μl or more to yield better results).

    2. Only replacing the antibody solution once after 3 days of incubation (instead of twice, after 2 and 4 days).

    Note: This is highly specific for every individual experiment and dependent, amongst other factors, on the size of the aggregates and antibodies used. Therefore, this should be carefully validated before performing larger experiments or using valuable samples.

    Recipes

    1. Permeabilization buffer (Table 1)


      Table 1. Permeabilization buffer

      Reagent Amount for 10 ml Supplier Product no. Dilution factor Stock conc.*
      PBS 9.5 ml Sigma-Aldrich D8662
      Triton X-100 500 μl Roth 3051.21 20 10% (v/v)

    2. Blocking solution (Table 2)


      Table 2. Blocking solution

      Reagent Amount for 10 ml Supplier Product no. Dilution factor Stock conc.*
      PBS 1.4 ml Sigma-Aldrich D8662
      BSA 8 ml Thermo Fisher 15260037 1.25 7.5%
      Triton X-100 500 μl Roth 3051.21 20 10% (v/v)
      Sodium azide 100 μl Sigma-Aldrich 71289 100 10%(w/v)

      *Whereas not required, we recommend preparing intermediary stock solutions of Triton X-100 (10% (v/v) in PBS) and sodium azide (10% (w/v) in PBS) as facilitates handling.


    3. BABB/methanol (Table 3)


      Table 3. BABB/methanol

      Reagent Amount for 10 ml Supplier Product no.
      Methanol 5 ml Roth 4627.6
      Benzyl alcohol 2.5 ml Sigma-Aldrich 305197-100ML
      Benzyl benzoate 2.5 ml Sigma-Aldrich B6630-250ML

    4. BABB (Table 4)


      Table 4. BABB

      Reagent Amount for 10 ml Supplier Product no.
      Benzyl alcohol 5 ml Sigma-Aldrich 305197-100ML
      Benzyl benzoate 5 ml Sigma-Aldrich B6630-250ML

      Note: The permeabilization buffer can be stored at room temperature for several months. The blocking solution should be stored at 4°C and can be used for approximately 1 month. BABB/methanol and BABB should be freshly prepared right before use.

    Acknowledgments

    This work was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No [669168]). HR is supported by the International Max Planck Research School - Molecular Biomedicine, Münster, Germany. This protocol is based on our previous publication (Renner et al., 2020) (DOI: 10.7554/eLife.52904).

    Competing interests

    The work presented here is the subject of the patent application EP 18 19 2698.0-1120 to the European Patent Office, where all of the authors are inventors.

    References

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    2. Dent, J. A., Polson, A. G. and Klymkowsky, M. W. (1989). A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105(1): 61-74.
    3. Kim, J., Koo, B. K. and Knoblich, J. A. (2020). Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol 21(10): 571-584.
    4. Lancaster, M. A., Renner, M., Martin, C. A., Wenzel, D., Bicknell, L. S., Hurles, M. E., Homfray, T., Penninger, J. M., Jackson, A. P. and Knoblich, J. A. (2013). Cerebral organoids model human brain development and microcephaly. Nature 501(7467): 373-379.
    5. Lee, E., Choi, J., Jo, Y., Kim, J. Y., Jang, Y. J., Lee, H. M., Kim, S. Y., Lee, H. J., Cho, K., Jung, N., Hur, E. M., Jeong, S. J., Moon, C., Choe, Y., Rhyu, I. J., Kim, H. and Sun, W. (2016). ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.Sci Rep 6: 18631.
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简介

[摘要]三维 (3D) 细胞培养,尤其是以器官样微组织(“类器官”)的形式,已成为一种新工具,可能比标准二维培养更接近地模仿人体组织生物学。通常,组织切片是免疫组织化学分析的标准方法。然而,它会从它们的原生生态位中移除细胞,并可能导致分析过程中 3D 上下文的丢失. 自动化工作流程需要对成百上千个样本进行并行处理和分析,而且切片在机械上复杂且耗时,因此不太适合自动化工作流程。在这里,我们提出了一个简单的协议,用于对具有单细胞级分辨率的大型(约 1 毫米)3D 组织进行组合全贴装免疫染色、组织清除和光学分析。虽然该协议可以手动执行,但它专门设计为与高通量应用程序和自动液体处理系统兼容。这种方法可自由扩展,并允许在标准实验室器具中并行自动处理大量样品。我们已经成功地应用了协议,以人中期和前脑组织体,但在原则上,工作流是适合于各种3D的组织样本,方便的细胞行为的三维细胞表型的发现文化为基础的高-通量筛选。



图文摘要:





自动化类器官清算和高-内容分析的工作流程和时间表


[背景]在过去的几年中,三维 (3D) 细胞培养系统,特别是基于干细胞的类器官,使人们对人类生物学和疾病有了新的见解(审阅:Rossi等人,2018 年;Schutgens 和 Clevers ,2020 年) ;Kim等人,2020 年)。然而,使用 3D 模型需要的不仅仅是新的细胞培养技术。定量在复杂的三维组织的细胞中快速,无偏,和有效的工作流程需要适应的分析以及。到目前为止,许多研究都依赖于组织切片和免疫染色来详细分析其 3D 聚集体的结构和组成(Lancaster等人,2013 年;Pasca等人,2015 年)。虽然这种方法非常宝贵,但它需要大量的人工干预,耗时且繁琐,因此不适合大规模筛选应用,包括药物开发活动。此外,切片仅提供样本组织子集的视图,通常会导致空间信息的丢失,除非准备细致的连续切片,并且难以用作 3D 重建的基础。

最近,组合整个安装染色和组织清除允许编整个3D聚集体的分析,而不需要用于切片,由我们的基团作为表现出与他人(Masselink等人,2019; Dekkers的。等人,2019;伦纳等人,2020 年)。此方法允许快速3D获取复采样和蜜饯组织上下文的同时提供单-细胞特异性表型信息。我们的工作流程使用定制的免疫染色程序,我们根据之前的协议(Lee等人,2016 年)对类器官进行了调整和优化。我们将其与基于苯甲醇苯甲酸苄酯 (BABB) 的清除(Dent等人,1989 年)相结合,可快速(分钟)有效地清除人类神经组织(Renner等人,2020 年)。我们专门设计日是程序能够同时与手动移液和自动液体处理系统,有利于低和高吞吐量的应用兼容。根据个体需求,样品然后可以使用标准的共焦显微镜或高分析-含量成像系统。在任一情况下,所得到的光学组织的横截面使整个三维样品的定量分析下至单个-细胞水平。这消除了对类器官进行冷冻/切片的需要,并且可以对 96 孔或 384 孔格式的许多样品进行平行染色和分析。虽然我们开发了用于分析神经类器官的工作流程,但它并不限于特定的类器官系统或样本类型,而是可以应用于任何感兴趣的 3D 样本。

关键字:类器官, 三维细胞培养, 整体组织免疫荧光染色, 清除, (高含量)图像分析, 自动化

 


材料和试剂

 

1. 96 孔板,Nunc TM MicroWell TM锥形 96 孔板(V 型底)(Thermo Fisher,目录号:277143)      

2. Screenstar 微孔板,96 孔,COC,F 底,黑色(Greiner,目录号:655866)      

3.螺丝帽试管(Sarstedt的,目录号小号:62.547.254 [ 50 ml的]和62.554.502 [ 15 ml的] )      

4.血清学吸管中(Falcon,目录号小号:356543 [ 5 ml的] ,356551 [ 10 ml的] ,和356525 [ 25 ml的] )      

5.枪头(STARLAB,目录号š :S1120-1810 [ 20 μ升〕,S1120-8810 [ 200 μ升〕,和S1122-1830 [ 1 ,000 μ升〕)      

6.锥形/V形底96孔板(Thermo Fisher,目录号:277143)      

7.可选:BIOMEK升iquid ħ andler移液管尖端AP96 P250(Beckman Coulter公司,目录号:717252)      

8.待染色的类器官/3D 样品      

9. Dulbecco's磷酸盐缓冲盐水(PBS ;Sigma-Aldrich,目录号:D8662)      

10.多聚甲醛(PFA ;VWR,目录号:15714-S)   

11. Triton X-100(Roth,目录号:3051.2)   

12.叠氮化钠(Sigma-Aldrich,目录号:71289)   

13.牛血清白蛋白(BSA,Thermo Fisher,目录号:15260037)   

14.甲醇(Roth,目录号:4627.6)   

15.苯甲醇(Sigma-Aldrich,目录号:305197-100ML)   

16.苯甲酸苄酯(Sigma-Aldrich,目录号:B6630-250ML)   

17.可选:核复染剂,例如,DAPI(Sigma-Aldrich公司,目录号:D9542-10MG)   

18. 一抗,取决于应用[此处:兔抗酪氨酸羟化酶(Abcam,产品编号:ab112)和山羊抗 Sox2(R&D Systems,目录号:AF2018)]   

19.二抗,取决于应用[此处:驴抗兔 IgG,Alexa Fluor 647(Thermo Fisher,目录号:A-31573)和驴抗山羊 IgG,Alexa Fluor 568(Thermo Fisher,目录号:A- 11057) ]   

20.透化缓冲液(见配方,表 1 )   

21.封闭液(见配方,表 2 )   

22. BABB/甲醇(见配方,表 3)   

23. BABB(见食谱,表 4)   

 

设备

 

加湿CO 2我ncubator
冰箱和冰柜(4°C、-20°C 和 -80°C)
可选:带摄像头的立体显微镜(例如,Leica MZ10 F(显微镜)和 Leica DFC425 C(摄像头)、Leica Microsystems)
机械移液器
可选:多通道机械移液器
可选:自动液体处理系统(例如,Biomek FX P实验室自动化工作站、Beckman Coulter)
共聚焦显微镜(例如,LSM 700、Zeiss)
可选:高-含量成像系统(例如,歌剧高-含量成像仪,Perkin Elmer)中
 

软件

 

斐济/ImageJ (Schindelin et al. , 2012)
可选:Leica 应用程序套件 v 4.8(LAS、Leica Microsystems)
可选:Biomek Software v3.3(控制自动液体处理机,Beckman Coulter)
可选:高-内容成像和分析软件(例如,和谐V4.1,珀金埃尔默)
可选:Excel(微软公司)
 

程序

 

注意:本协议使用有害物质,包括 PFA、Triton X-100、甲醇、苯甲醇和苯甲酸苄酯。注意多种接触途径的毒性,包括吸入。在开始该程序并在通风橱下工作之前,请参阅 MSDS 信息。

 

注意:此处和以下步骤中提到的所有体积均针对 96 孔板中的样品进行了优化;其他文化格式可能需要不同的卷。对于我们的设置和组织与 ca。1个毫米直径或更小,我们用150 μ升每孔试剂体积。更大的体积是可能的,并且可能需要更大的样品;然而,我们不建议去低于150 μ升每孔用于固定和洗涤,以避免为每个组织试剂的量不足。用于洗涤步骤,更高体积小号可以增加的洗涤效率,但我们发现150个μ升每孔,得到良好的结果。

 

大型(约 1 毫米)3D 组织的固定
注意:此处描述的所有步骤都可以手动执行,也可以使用自动液体处理系统(ALHS)/移液机器人执行。

以固定的样品,小心吸媒体,并添加150 μ升每孔新鲜制备的4%PFA的PBS中。在室温下孵育 10-15 分钟。
吸出固定液仔细洗3 × 5分钟,用150 μ升每孔PBS。
要么添加150 μ升PBS每孔和密封板用封口膜的采样存储在4℃下,或直接与下面的染色过程进行。
注:在我们的手中,储存数月没有负面样品整体质量的影响-安装染色。^ h H但是,我们摹enerally recommen d快速的周转时间较长的存储。

整体免疫染色
第 0 天

可选:吸PBS小心,加入150微升的透化缓冲液(见配方,表1 ),并孵育在37 1个小时℃。
注意:通常不需要专门的透化步骤,但可能会改善特定标记/样本类型的结果。这必须根据具体情况进行优化。

吸出透化缓冲液/PBS 并加入 150 µl 在封闭溶液中稀释的一抗(参见配方,表 2 )。使用适当浓度的抗体(每个抗体必须单独滴定和验证)。孵育6天在37℃和100%湿度,更新荷兰国际集团的溶液,每2次一天。
注意š上的温育时间和条件:1.孵育时间取决于组织的大小和密度以及该抗体使用。我们建议一个事先优化的具有代表性的测试样品的培养时间; 这将节省宝贵的时间用于后续-起来实验,并保证最佳的效果。我们发现每种抗体孵育 6 天在我们的样品中效果很好。2.对于抗体孵育步骤,不密封的封口膜板,因为它可能“熔体”,成为难以从板后较长的孵育时间,在37℃,以除去℃。培养箱应加湿以防止板中液体的蒸发损失(几乎所有组织培养箱都适用于这一步)。3. 在我们手中,样品在此期间没有出现任何变质迹象。如有必要,封闭和染色溶液中的 0.1% (w/v) 叠氮化钠可以在此期间防止微生物生长(参见食谱)。

 

关于对照样品的注意事项:仔细计划在没有一抗的情况下孵育的对照样品,以衡量二抗的非特异性结合。另外,制备对照样品,而不antibod IES到鉴定引起自发荧光潜在伪像。定量评估需要测量两种类型的背景荧光,以确定每个通道的基于对象的检测阈值。

 

第 2 天

更新一抗溶液。
 

第 4 天

更新一抗溶液。
 

第 6 天

洗液样品用150 μ升0.1%(V / V)的Triton X-100的PBS于室温下5小时。每小时更新一次洗涤液。
注意:如果出现非特异性/高背景信号的问题,洗涤可能会延长过夜并在 37°C 下进行。

吸的清洗液小心,并添加150微升的新的阻塞溶液与二次抗体1:1 ,000和DAPI(0.5 μ微克/毫升)。孵育对湿度6天在37℃和100%。更新的解决方案每2天小号。
注:这和所有后续孵化步骤应在黑暗中进行保存的荧光信号。我们进行了广泛的优化了在各种条件下的AlexaFluor缀合的二抗,并始终以1抵:1 ,000稀释。我们还没有优化的用于条件的其他抗体(例如,花青染料偶联的抗体)。这些可能需要额外的优化,包括测试其他抗体浓度。

 

第 8 天

更新二抗/DAPI 解决方案。
 

第 10 天

更新二抗/DAPI 解决方案
 

第 12 天

用150洗样品μ升0.1%的Triton X-100的PBS用于在黑暗中在室温下5小时。每小时更新一次洗涤液。
注意:如果出现非特异性/高背景信号的问题,洗涤可能会延长过夜并在 37°C 下进行。

任一取代的具有150洗涤液μ升PBS每孔和密封板用封口膜在黑暗中的采样存储在4℃下或直接与下游处理继续进行(例如,在下面基于BABB组织清除)。
注意:我们没有在组织清除之前测试样品的长期储存。我们建议大约一周内进行清除步骤完成整个的-安装染色,然后存储清除样本,这是稳定的几个在暗月在4 ℃下。

 

基于 BABB 的组织清除
小心地从样品中吸出PBS 上清液,每孔加入 150 µl 25% 甲醇的 PBS,并在室温 (RT) 下孵育 15 分钟。
注意:对于较大的(> 1mm)的试样,我˚F聚集体没有被完全清除,温育时间可以被延长至30分钟(60分钟为BABB /甲醇)。这种孵化应该单独优化。

抽吸的25%甲醇,并加入150微升50%的米乙醇; 在 RT 中孵育 15 分钟。
抽吸的50%甲醇,并添加150μl的75%的米乙醇; 在 RT 中孵育 15 分钟。
抽吸的75%甲醇,并加入150微升90%米乙醇; 在 RT 中孵育 15 分钟。
抽吸的90%甲醇,并添加150μl的100%的米乙醇; 在 RT 中孵育 15 分钟。
重要的是:传输的在100%的样品米从标准组织培养板乙醇小号到BABB耐板(例如,“ Screenstar ” COC; BABB /甲醇和BABB溶解在30分钟内最标准的组织培养塑料制品)。对于传输,截止1000的尖端μ升用移液管尖端乙醇清洗剪刀。将锥形尖端从窄锥形端的开口处切割约 5 毫米(如果您的样品很大,您可以切割更多)。这旨在加宽移液器开口以避免损坏大型 3D 样品;您可以使用大口径吸头作为液体处理机。
吸出100% 甲醇并加入 150 µl BABB/甲醇(1:1 ,(v/v ),参见配方,表 3 )并在室温下孵育 30 分钟。BABB 由 1:1 (v/v) 混合物的苯甲醇/苯甲酸苄酯组成(参见配方,表 4)。
吸出 BABB/甲醇并加入 150 µl BABB。
样品在 5 分钟内完全清除并准备好进行下游分析/显微镜检查,或者可以在 4°C 的暗处储存数月。
注意:我们成功地对储存了大约18 个月的样品进行了重新成像,而没有遇到样品质量的任何下降。

 

荧光基于单-细胞分析
高通量应用,包括化合物筛选,必须处理和处理了一大批具有快速读出样品。我们的整个的组合-安装免疫染色和结算工作流小号上面概述的高-含量共焦成像,能够使自动获取在筛选兼容的方式整个96孔板的。我们所描述的工作流程分析,必要分析这种高-详细内容数据(雷纳等人,2020年)。然而,并非所有实验室都可以使用此类分析所需的通常高度特定的硬件和软件。在这里,我们详细介绍了仅使用标准共聚焦显微镜和免费软件的工作流程。由于每个 3D 样本和染色对图像分析都有特定要求,我们不关注特定参数,而是关注一般程序,允许任何人将工作流程应用于自己的工作。

 

D0 。使用共焦显微镜获取图像,无论是单个光学共焦切片还是整个堆栈。根据您的分析需求,您可能更喜欢使用适合您的样品和所需横向分辨率的镜头放大率以 8、16 或 32 位深度成像。对于我们的需要,我们成像为具有10个整类器官×透镜的放大倍率和16位的动态范围(参见图1为代表性图像)。根据您的分析需要调整连续 Z 平面之间的距离。完整的 3D 重建将需要更多且间隔更近的 Z 平面。对于样本之间的定量比较,最好沿 Z 轴将各个 Z 平面间隔得更远,从而对3D 体积进行欠采样。这从重复计算在相邻的Z-平面相同的物理特征避免数据伪影并降低数据量获取和所述采集时间。例如,要测量具有代表性数量的原子核,Z 平面的间距应大于原子核平均直径的两倍。一般情况下,根据经验选择的焦平面之间的空间的增量,以便可以防止在相邻的Z-平面成像在同一样品的特征,防止荷兰国际集团双-counting。

 

D1 。核标记分析(例如 Sox2,见图 1A)

注意:我们将介绍使用斐济/ ImageJ的所有分析程序(Schindelin等,2012) ,因为它是免费提供的,并常用于处理和ANALY懋生物成像数据(用于这里的分析和路径的特定命令找到他们在下面的每个步骤中描述)。然而,同样的一般步骤也可以使用类似的软件使用类似的步骤和参数来执行。这里,我们的原始图像小号由16位灰度数据和由1024个像素测量1024个像素。以下参数针对这些条件进行了调整。其他的出发点可能需要在下游的图像分析参数的调整。

打开的16位灰度图像。
复制的图像并应用高斯模糊。
图像 > 复制。
处理 > 滤镜 > 高斯模糊(此处使用的参数:10 像素)。
减去了从原来的模糊图像。
处理 > 图像计算器(然后选择操作“减法”)。             

可选:执行平滑。这可以减少阈值操作的边缘伪影。
过程 > 平滑。

应用阈值(可以使用“自动阈值”功能,特别是对整个堆栈的批量分析)。
图像 > 调整> 阈值/自动阈值。             

可选:使用分水岭分离已合并但应分离的对象,例如靠近的原子核。
              插件 > 二进制> 可调分水岭(此处使用的参数:0.5)。             

使用的“分析粒子”功能来测量物体/核的数目。
注意:测量前,请使用“设置测量”功能选择要分析的参数。根据您的要求(例如,增加最小物体的大小,排除单一调整设置-像素噪声),并启用“添加到管理员”(这会产生利益的区域,“投资回报率,根据所识别的对象”,并将其添加到“ ROI manager”),如果您稍后还想测量对象的强度。在这里也可以按尺寸排除较大的工件,如灰尘或毛发。

              分析> 设置测量。             
分析> 分析粒子。                           
分析结果出现在“结果”窗口中,可以从那里导出以供进一步处理(例如,到 Microsoft Excel 或其他选择的分析软件)。
为了测量的强度,通过“ROI管理”应用之前定义为原始图像的ROI(如阈值处理过程中的原始强度值改变/处理),并使用“测量”功能(是一定要选择“设定下的相应参数测量”之前)。
分析 > ROI 管理器。
打开原始图像(16 位灰度)。
在 ROI 管理器中启用“全选”以显示覆盖在原始图像上的ROI (如果选择的 ROI 过多,取消选中标记为“标签”的复选框会使图像不那么拥挤)。
单击 ROI 管理器窗口中的“测量”以测量单个选定的 ROI 或所有 ROI(如果未选择)。
 

D2 。细胞质/丝状标记的分析(例如 TH,见图 1B)

对于细胞质/丝状标记,单细胞的干净分割可能具有挑战性,特别是在密集的 3D 环境中,它们可以跨越几个 z 水平作为薄细胞投影。因此,通常最好在每个共焦平面上测量正确定的感兴趣结构的综合强度或平均强度。然后可以将整个类器官的信号对类器官内的每个平面求和,并定量反映 3D 组织中包含的每个感兴趣标记的存在。

可以按照上述 Sox2 核分析中的说明重复步骤 1-5。
不是通过分水岭分离对象,而是继续通过“分析粒子”功能将它们定义为 ROI。
使用“ROI经理”到先前定义的ROI应用到原始图像和测量的强度与“测量”功能(调整“组测量”到样品之后“的具体要求)。
 

D3 。用于归一化的样品面积的测量(参见图1C中的示例)

在不同 z 水平测量具有不同直径的 3D 聚集体通常需要将数据归一化到总样本面积,以获得不同样本之间的可比较结果。

对所有可用通道求和以获得包含所有通道中样本的所有可见元素的形态特征的图像。
打开所有可用通道的 16 位灰度图像。
过程>图像计算器(然后选择该“add”操作,并选择在步骤a中打开的所有信道)。这将创建包含样品的所有荧光波长的所有可见特征进行归一化,以合成图像的总样本区域。             
使用阈值从样本中创建单个对象,将背景和样本分开。
使用“测量”功能计算物体的面积(在“设置测量”下启用“面积”后)。
通过将工作流程(带有样品特定要求的参数)记录为宏(例如,通过“宏记录器”),可以轻松地将这些程序应用于整个集合。这些可以迭代地处理整个Z堆栈或什至包含在公共文件夹中的 Z 堆栈组。

 

 

图1的不同步骤概述的全图像分析-贴装染色和清除3D结构。(AC)程序部分D中概述的图像分析过程中不同步骤的代表性图像。(DE)核标记 Sox2 分析的代表性结果。(d)- [R AW的数据(即,对于每一个所标识的对象的平均灰度值)和(E)˚F基于在d中示出的数据requency分布)。 每行 (AC) 顶部图像中的比例尺对下面的整列图像都有效。比例尺:整个有机体s = 100 μm ;放大(A,右列)= 20 μm。

数据分析

 

用共聚焦显微镜获得的图像的手动分析“方法部分中所述基于荧光的单:d -细胞分析。”高的分析-内容的成像数据被详细描述在原出版物中描述(雷纳等人,2020)材料和方法下‘ ’‘用于高含量成像和分析的一般工作流,’和下面的部分。

 

笔记

 

基于 BABB 的清除程序倾向于淬灭荧光融合蛋白的天然荧光,包括 GFP、RFP和所有 mFruits。然而,荧光染料,包括荧光染料Alexa的,不会受到影响。因此,我们建议进行全-安装上述染色使用针对感兴趣的荧光蛋白提出了具体的概述抗体的程序(例如,抗GFP),以分析它们在BABB清除的结构。这具有信号放大的多价经由二级抗体和更好的光稳定性的额外益处,因为商业染料通常超过漂白性的天然融合蛋白。

  小号协议的everal步骤可以优化吨ö节省抗体溶液,尤其是在大规模的实验。在我们手中运行良好的两个选项是:

降低抗体溶液的体积使用为最小50的μ升(用于大的样品,也可以增加至200 μ升或更多,以产生更好的结果)。
孵育 3 天后仅更换一次抗体溶液(而不是在 2 天和 4 天后更换两次)。
注意:这对于每个单独的实验都是高度特异性的,并且取决于所使用的聚集体和抗体的大小等因素。因此,在进行大型实验或使用有价值的样本之前,应仔细验证这一点。

 


食谱

 

透化缓冲液(表 1)
 

表 1.透化缓冲液

试剂

10毫升的量

供应商

产品编号

稀释因子

股票浓度*

PBS

9.5 毫升

西格玛奥德里奇

D8662

 

 

海卫 X-100

500微升

罗斯

3051.2 1

20

10% (v/v)

 

阻塞解决方案(表 2)
 

表 2.阻塞解决方案

试剂

10毫升的量

供应商

产品编号

稀释因子

股票浓度*

PBS

1. 4毫升

西格玛奥德里奇

D8662

 

 

BSA

8毫升

赛默飞世尔

1 5260037

1.25

7.5%

海卫 X-100

500微升

罗斯

3051.2 1

20

10% (v/v)

叠氮化钠

100微升

西格玛奥德里奇

71289

100

10%(w/v)

 

*虽然不是必需的,我们建议准备的的Triton X-100的中间储备液(10%(V / V)的PBS)和小号裂果叠氮化物(10%(W / V)在PBS)的功能有助于处理。

 

BABB/甲醇(表3 )
 

表 3. BABB/甲醇

试剂

10毫升的量

供应商

产品编号

甲醇

5毫升

罗斯

4627.6

苯甲醇

2.5 毫升

西格玛奥德里奇

305197-100ML

苯甲酸苄酯

2.5 毫升

西格玛奥德里奇

B6630-250ML

 

BABB(表 4)
 

表 4. BABB

试剂

10毫升的量

供应商

产品编号

苯甲醇

5毫升

西格玛奥德里奇

305197-100ML

苯甲酸苄酯

5毫升

西格玛奥德里奇

B6630-250ML

 

注:在P ermeabilization缓冲区可以在室温下保存数月。在B锁定溶液应储存在4℃和可用于约1个月。 BABB/甲醇和 BABB 应在使用前新鲜制备。

 



致谢

 

这项工作是由欧洲研究委员会(ERC)欧盟资助的联盟展望2020研究和创新编程的,是米(赠款协议否[669168])。HR 得到德国明斯特国际马克斯普朗克研究学院 - 分子生物医学的支持。该协议基于我们之前的出版物(Renner et al. , 2020) ( DOI : 10.7554/eLife.52904) 。

 

利益争夺

 

此处介绍的工作是向欧洲专利局提交的专利申请 EP 18 19 2698.0-1120 的主题,其中所有作者都是发明人。

 

参考

 

Dekkers, JF, Alieva, M., Wellens, LM, Ariese, HCR, Jamieson, PR, Vonk, AM, Amatngalim, GD, Hu, H., Oost, KC, Snippert, HJG, Beekman, JM, Wehrens, EJ, Visvader, JE、Clevers, H. 和 Rios, AC(2019 年)。固定和清除类器官的高分辨率 3D 成像。国家议定书 14(6):1756-1771。
Dent, JA, Polson, AG 和Klymkowsky , MW (1989)。非洲爪蟾中间丝蛋白波形蛋白表达的整体免疫细胞化学分析。发展105(1):61-74。              
Kim, J., Koo, BK 和 Knoblich, JA (2020)。人体类器官:人类生物学和医学的模型系统。Nat Rev Mol Cell Biol 21(10): 571-584。
Lancaster, MA, Renner, M., Martin, CA, Wenzel, D., Bicknell, LS, Hurles, ME, Homfray, T., Penninger, JM, Jackson, AP 和 Knoblich, JA (2013)。大脑类器官模拟人脑发育和小头畸形。自然501(7467):373-379。
Lee, E., Choi, J., Jo, Y., Kim, JY, Jang, YJ, Lee, HM, Kim, SY, Lee, HJ, Cho, K., Jung, N., Hur, EM, Jeong , SJ, Moon, C., Choe, Y., Rhyu, IJ, Kim, H. 和 Sun, W. (2016)。ACT-PRESTO:用于 3 维 (3D) 成像的快速且一致的组织清除和标记方法。科学代表6:18631。              
Masselink, W., Reumann, D., Murawala, P., Pasierbek, P., Taniguchi, Y., Bonnay, F., Meixner, K., Knoblich, JA 和 Tanaka, EM (2019)。简化的基于肉桂酸乙酯的清除程序的广泛适用性。发展146(3)。
Pasca, AM, Sloan, SA, Clarke, LE, Tian, Y., Makinson, CD, Huber, N., Kim, CH, Park, JY, O'Rourke , NA, Nguyen, KD, Smith, SJ, Huguenard, JR、Geschwind、DH、Barres、BA 和 Pasca,SP(2015 年)。3D 培养中来自人类多能干细胞的功能性皮质神经元和星形胶质细胞。Nat 方法12(7): 671-678。
Renner, H., Grabos, M., Becker, KJ, Kagermeier, TE, Wu, J., Otto, M., Peischard, S., Zeuschner, D., TsyTsyura, Y., Disse, P., Klingauf, J., Leidel, SA, Seebohm, G., Scholer, HR和 Bruder, JM (2020)。用于人类中脑类器官中基于 3D 的化学筛选的全自动高通量工作流程。Elife 9 :e52904。
Rossi, G.、Manfrin, A. 和 Lutolf, MP (2018)。类器官研究的进展和潜力。Nat Rev Genet 19(11): 671-687。
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B ., Tinevez, JY, White, DJ, Hartenstein, V., Eliceiri, K., Tomancak, P. 和 Cardona, A. (2012)。斐济:一个用于生物图像分析的开源平台。Nat 方法9(7): 676-682。              
Schutgens, F. 和 Clevers, H.(2020 年)。人体类器官:了解生物学和治疗疾病的工具。Annu Rev Pathol 15:211-234。
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Copyright Renner et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Renner, H., Otto, M., Grabos, M., Schöler, H. R. and Bruder, J. M. (2021). Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening. Bio-protocol 11(12): e4050. DOI: 10.21769/BioProtoc.4050.
  2. Renner, H., Grabos, M., Becker, K. J., Kagermeier, T. E., Wu, J., Otto, M., Peischard, S., Zeuschner, D., TsyTsyura, Y., Disse, P., Klingauf, J., Leidel, S. A., Seebohm, G., Scholer, H. R. and Bruder, J. M. (2020). A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids. Elife 9: e52904.
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Anu Sebin
Anu Sebin
Hi,
I came across this protocol and had a couple of questions - Does each well has only one organoid? If yes, how can one make sure, the organoid is not lost during so many wash steps?

Thanks!
2021/7/12 14:23:15 回复
Henrik Renner
Max Planck Institute for Molecular Biomedicine Münster

Dear Anu Sebin,

Thank you very much for your question and interest in our protocol. Using our standard workflow for the generation of organoids each well does, indeed, contain exactly one organoid. Please also refer to our previous publications (https://elifesciences.org/articles/52904 and https://bio-protocol.org/e4049) for more information on organoid generation and culture. If your culture system generates several organoids per well, this should also be compatible with the procedure we outlined here. However, we have not optimized this approach.

In order to prevent the loss of samples during the various washing steps, careful liquid handling is crucial. To facilitate the aspiration of liquids, we recommend slightly tilting the plate and performing all liquid handling by pipetting (not by vacuum pumps or similar devices). Leave a small amount of liquid (≤ 10 μl) in the well so as to not damage the organoids by extensive direct contact with a pipet tip.

Best regards,
Henrik Renner & Jan Bruder

2021/7/14 2:15:20 回复