TITLE: 3D fluorescence staining and imaging of low amount of organoids
AUTHORS: Ami G. Toulehohoun, Carolin Bouzin, Aurelie Daumerie, Luca Maccioni, Peter Stärkel

[DESCRIPTION]
The emerging field of 3D organ modeling encounters several imaging issues in particular related to antigen retrieval and sample lost during staining processes. Due to their compact shape, several antibodies fail to penetrate intact organoids or spheroids. Histology of organoids can be approached by paraffin inclusion and sectioning at 5 µm as performed for biopsies. However, to fully understand organoid behavior, including cellular organization, extracellular matrix structure, and their response to treatments, 3D imaging is essential. Here we propose an easy workflow allowing (1) immunostaining with a HIER step, (2) preservation of the intact shape of the organoids, (3) sample immobilization in a focal plane reachable for high resolution/short working distance lenses, and (4) minimizing the risk of loss of precious material. 

[GUIDELINES]
Introduction:

Organoids are 3D in vitro models, mimicking the native patient organ (healthy or pathologic) in terms of structural and functional aspects. They offer unparalleled potential for disease modelling and large-scale therapeutic screening. Compared to animal models, organoids derived from patients’ cells translate into greater similarity in physiological and disease processes and offer a more ethically responsible approach. 

Following proper characterization and in vitro expansion, these "mini-tissues" can be established as biobanks, serving as valuable research resources for biomarker identification and novel therapeutic target development. Furthermore, organoid models hold particular promise for studying rare pathologies, where limited access to biological material delays research progress. Their versatility extends to diverse areas, including tumor biology, inflammatory diseases, regenerative medicine, and more. Importantly, unlike formalin-fixed paraffin-embedded tissues, these "living biobanks" provide a source of fresh material suitable for a wide range of analyses, including histological, protein expression, genomic, and metabolomic studies.

Histology of organoids can be approached by paraffin inclusion and sectioning at 5 µm as performed for biopsies (Mahe et al., 2013). However, to fully understand organoid behavior, including cellular organization, extracellular matrix structure, and their response to treatments, 3D imaging is essential. Different imaging technologies allow for 3D imaging of these small (50-300 µm diameter) structures, like light sheet microscopy and confocal microscopy. Light sheet microscopy offers high imaging speed and good spatial resolution (Wan et al., 2019, de Medeiros et al., 2022, Dekkers et al., 2019) while confocal microscopy is the most widely accessible device. Although sample positioning is challenging on both devices, organoid inclusion in agarose sticks for light sheet microscopy leads to a massive sample loss. For this reason, we focused on confocal microscopy.

The emerging field of 3D organ modeling requires adequate sample preparation in order to generate faithful representations of the real 3D structure. 

First, fixation with formaldehyde is known to create crosslinking by forming covalent bonds between proteins. This network can also be an obstacle to antibodies penetration. On 2D sections, a heat-induced epitope retrieval step (HIER, at acidic or basic pH) is commonly performed to brake this network and make antigens accessible to antibodies (Dunkenberger et al, 2022). Of note, fixation of organoids with methanol does not require antigen retrieval, yet our prior staining showed that this method is inappropriate for detecting some epitopes and the image quality is lower when compared to fixation performed with formaldehyde. Second, due to their compact shape and thickness, several antibodies fail to penetrate intact organoids. This can be worked around using detergents in the staining protocol. Third, light scattering and/or absorption by biological tissues is an obstacle to in depth imaging. However, optical clearing compounds are now available to render tissues transparent and straighten the light path, improving the thickness of samples that can be imaged in their entirety. Finally, when dispersed in a Matrigel dome, organoids are sometimes out of reach when using lenses with a magnification higher than x10. On the opposite, mounting organoids on a slide with a coverslip at the end of the staining leads to the flattening of the organoids and prevents from imaging them in their native shape.

Here we propose an easy workflow allowing (1) immunostaining with a HIER step, (2) preservation of the intact shape of the organoids, (3) sample immobilization in a focal plane reachable for high resolution/short working distance lenses, and (4) minimizing the risk of loss of precious material (figure 1).

 

Results:
 


 

 

 


 

Results and Discussion:

In order to assess the cell components of intestinal organoids, we performed immunostaining to detect MUC2, LYZ, OLFM4, and ZO-1 proteins, respectively expressed by goblet, paneth, stem and stem-like cells and tight junctions. Entire organoids were removed from Matrigel domes and were plated on a diluted Matrigel layer to immobilize them on a single plate for further imaging. Then, they were submitted to fixation and immunostaining without any prior antigen retrieval. They were finally imaged in a clearing solution with a confocal microscope. 

Using this protocol, organoids were attached to the plate without forcing or destroying their morphology. Furthermore, after their attachment and fixation, they were tightly stuck to the plate and did not detach during the HIER nor during the washing steps. Consequently, the number of organoids before and after the staining was approximately the same, thus avoiding excessive loss of material. 

The cellular localization of a protein of interest is an important element to assess the specificity of an immunostaining. The results represented in figure 2 showed satisfactory tight junction protein ZO-1 detection. However, OLFM4, MUC2 and LYZ staining showed unspecific binding (mainly in the nucleus) of their respective antibodies. 

We hypothesized that the lack of specific staining was due to inaccessibility of the antigens. As an antigen retrieval process, we first tried the FLASH technique proposed in a recent publication (Messal et al., 2021). Although this approach might be successful for labeling of some epitopes, it did not allow to detect MUC2, LYZ and OLFM4 proteins in intestinal organoids (data not shown). However, as illustrated in figure 3, we obtained specific staining of Muc2, OLFM4, LYZ protein in the organoids using our protocol of convenient sample preparation while avoiding sample loss and imaging at the same focal length range. 

These results support the added value of our protocol and indicates the need of appropriate antigen retrieval for detecting some epitopes by whole mount immunostaining (al., 2018; Patiño-García et al., 2019; Shi et al., 2011). 

Conclusion:

The technique suggested in this protocol allows organoids to attach to the plate while keeping their shape and structure. After fixation, the attached organoids are not removed throughout the whole staining process including Antigen retrieval and washing steps. Moreover, imaging entire organoids by acquiring the different Z-stack positions prevents losing information about each single organoid.

The advantage/ease of this technique is the use of a single plate with different experimental conditions. Moreover, no centrifugation process is required and the washing steps are straight forward. 

Limitations:

Compared to previous techniques of sample processing, fixation of the organoids on a plate does not allow Light-sheet microscopy since the organoids are strongly attached to the plate and cannot be removed after the staining. Moreover, Imaging resolution is limited to 10X microscope lenses and 96 well-plates are not suitable for this technique.   

[STEPS]
SECTION: Crypt isolation ○ Timing 3h
1. 
 

Collect duodenum biopsies in sterile PBS (without Ca2+ or Mg2+).

SECTION: Crypt isolation ○ Timing 3h
2. Wash 3x2 min under mild rotation in PBS + antibiotics and antimycotic 100X (Invitrogen, 15240062, 1/200). (1/3)

SECTION: Crypt isolation ○ Timing 3h
3. Incubate for 60 min in 2 millimolar (mM) EDTA in a 15 ml conic tube under slow rotation.

 

SECTION: Crypt isolation ○ Timing 3h
4. Allow biopsies to settle down (10 s) and discard the supernatant.

SECTION: Crypt isolation ○ Timing 3h
9. Centrifuge at 400 x g, 8 min, 4 °C. 

SECTION: Crypt isolation ○ Timing 3h
8. Add 5 mL of DMEM medium supplemented with 1% Penicillin/Streptomycin and 20% FBS. 

SECTION: Crypt isolation ○ Timing 3h
7. Repeat the previous step twice or three times to ensure total recovery of crypts.

SECTION: Crypt isolation ○ Timing 3h
6. Shake vigorously by hand. Allow biopsies to settle down (… min) and collect the supernatant containing crypts in a 15 ml conic tube. The remaining biopsies will sediment in the cryotube.

SECTION: Crypt isolation ○ Timing 3h
5. Add 1 mL chelation buffer (43.4 mM sucrose + 54.9 mM D-sorbitol in DPBS) and transfer into 2 ml cryotube. 

 

SECTION: Crypt isolation ○ Timing 3h
10. Discard the supernatant.

SECTION: Crypt isolation ○ Timing 3h
11. Wash once in 2 mL DMEM medium.

SECTION: Crypt isolation ○ Timing 3h
12. Filter through 70 µm cell strainer.

SECTION: Crypt isolation ○ Timing 3h
13. Centrifuge at 400 x g, 7 min, 4 °C.

SECTION: Crypt isolation ○ Timing 3h
14. Discard the supernatant.

SECTION: Crypt isolation ○ Timing 3h
15. Resuspend the pellet in 20 µL-30 µL cold Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free (VWR, 734-1101) and plate 25 µL-30 µL per well in flat bottom 96 well plate (96 well plates, Greiner (G) Bio-one, CELLSTAR® cat N° 655180)

SECTION: Crypt isolation ○ Timing 3h
16. Incubate at 37 °C, 5% CO2 for 10 min to allow the Matrigel to polymerize.

SECTION: Crypt isolation ○ Timing 3h
17. Add 37 °C heated IntestiCult OGM Human (STEMCELL technologies, #06010) containing 1% Penicillin/Streptomycin.

SECTION: Organoid passaging and culture ○ Timing 7-14d
18. Remove medium from the Matrigel dome.

SECTION: Organoid passaging and culture ○ Timing 7-14d
19. Add 200 µL/well of Corning® Cell Recovery Solution (VWR, #734-0107).

SECTION: Organoid passaging and culture ○ Timing 7-14d
26. Discard the supernatant.

SECTION: Organoid passaging and culture ○ Timing 7-14d
20. Incubate on ice for40 min.

SECTION: Organoid passaging and culture ○ Timing 7-14d
25. Centrifuge 400 x g, 7 min, 4 °C.

SECTION: Organoid passaging and culture ○ Timing 7-14d
24. Wash once with 2 mL cold DMEM medium.

SECTION: Organoid passaging and culture ○ Timing 7-14d
23. Discard the supernatant.

SECTION: Organoid passaging and culture ○ Timing 7-14d
22. Centrifuge at 400 x g, 7 min.

SECTION: Organoid passaging and culture ○ Timing 7-14d
21. Pull the enteroids into a 15 ml conic tube.

SECTION: Organoid passaging and culture ○ Timing 7-14d
30. Pipette up and down 10 times to dissociate remaining organoids clusters.

SECTION: Organoid passaging and culture ○ Timing 7-14d
29. Neutralize with 3 mL DMEM medium containing FBS (1/20).

SECTION: Organoid passaging and culture ○ Timing 7-14d
28. Incubate for 25 min at 37 °C. 

SECTION: Organoid passaging and culture ○ Timing 7-14d
27. Add 2 mL Gibco™ TrypLE™ Express Enzyme (1X), phenol red (Thermofisher, #12605010) to the pellet.

SECTION: Organoid passaging and culture ○ Timing 7-14d
31. Centrifuge as previously and discard the supernatant.

SECTION: Organoid passaging and culture ○ Timing 7-14d
35. Change medium every 2 days until day 6.

SECTION: Organoid passaging and culture ○ Timing 7-14d
34. Add heated IntestiCult OGM Human (STEMCELL technologies, #06010) containing 1% Penicillin/Streptomycin.

SECTION: Organoid passaging and culture ○ Timing 7-14d
33. Resuspend the pellet in cold Matrigel and plate approximately 15000 single cells in 20 µL-30 µL per well in a flat bottom 96 well plate (Greiner (G) Bio-one, CELLSTAR ® cat N° 655180).

 

SECTION: Organoid passaging and culture ○ Timing 7-14d
32. Wash once with DMEM.

SECTION: Organoid passaging and culture ○ Timing 7-14d
36. Change medium every day from day 7.

SECTION: Organoid preparation for imaging
37. Plate coating ○ Timing 30 min

Dilute Matrigel 50x with cold PBS.

SECTION: Organoid preparation for imaging
38. Plate 200 µL/well of diluted Matrigel in a 24 well plate (Greiner (G) Bio-one, CELLSTAR ® cat N° 62210).

 

SECTION: Organoid preparation for imaging
39. Incubate at least 20 min-30 min at 37 °C to allow the Matrigel to polymerize.

SECTION: Enteroids retrieving ○ Timing 3h
40. Remove culture medium from the organoids.

SECTION: Enteroids retrieving ○ Timing 3h
43. Incubate for 40 min on ice.

SECTION: Enteroids retrieving ○ Timing 3h
42. Place the plate on ice.

SECTION: Enteroids retrieving ○ Timing 3h
41. Add 200 µL/well of Corning® Cell Recovery Solution (CRC, 354253).

SECTION: Enteroids retrieving ○ Timing 3h
48. Wash 1x with DMEM medium.

SECTION: Enteroids retrieving ○ Timing 3h
47. Discard the supernatant. 

SECTION: Enteroids retrieving ○ Timing 3h
46. Centrifuge at 400 x g, 7 min, 4 °C.

SECTION: Enteroids retrieving ○ Timing 3h
45. Wash each well 2X with Cell Recovery Solution and add this to the 15 ml tube.

SECTION: Enteroids retrieving ○ Timing 3h
44. Transfer the detached organoids in a 15 ml conic tube. 

SECTION: Enteroids retrieving ○ Timing 3h
50. Add 200 µL/well culture medium on each pellet (containing treatment if applicable).

 

SECTION: Enteroids retrieving ○ Timing 3h
49. Centrifuge as previously and discard the medium.

SECTION: Enteroids retrieving ○ Timing 3h
51. Remove the 24 well coated plate from the incubator and remove the excess PBS with 200 µl tips.

SECTION: Enteroids retrieving ○ Timing 3h
52. Plate 200 µL/well of cell suspension.

SECTION: Enteroids retrieving ○ Timing 3h
53. Incubate 90 min-120 min at 37 °C to allow the organoids to attach to the plate.

SECTION: Fixation ○ Timing 1h
54. Remove the excess of medium.

SECTION: Fixation ○ Timing 1h
55. Wash 1x with heated PBS (37 °C).

SECTION: Fixation ○ Timing 1h
56. Fix with cold 4% formaldehyde for 45 min at 4 °C. 

SECTION: Fixation ○ Timing 1h
57. Wash 1x with cold PBS (4 °C).

 

SECTION: Fixation ○ Timing 1h
58. Proceed directly to permeabilization if the antibody does not require an antigen retrieval step.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
59. Remove PBS.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
60. Prepare 250 ml of citrate buffer:

Add 25 mL of citrate buffer stock solution 10x concentrated (3.78 gcitric acid and 24.12 g sodium citrate dihydrate in 100 mL distilled water, pH adjusted at 5.6, volume adjusted at 1 L, storage at 4 °C) and 675 µL of 20% Triton-X100 solution (diluted in distilled water) in 225 mL distilled water.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
61. Fill each well with citrate buffer at Room temperature. 

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
62. Boil the remaining buffer in microwave at 900 Watt and keep it aside.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
63. Put the plate in the microwave.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
64. Heat at 900W until boiling (approximately 30 s).

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
65. Replace citrate buffer by hot citrate buffer.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
66. Repeat three times by filling the well before each boiling.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
67. Heat at 90 Watt for 15 min. 

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
68. Boil 3 times at 900 Watt.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
69. Let the plate cool down for 10 min-15 min.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
70. Wash 1X with distilled water.

SECTION: Antigen retrieval, permeabilization, blocking ○ Timing 1 day
71. Permeabilize and Block with Organoid Washing Buffer (OWB) 120 min (Dekkers et al., 2019).

 

SECTION: Antibody staining ○ Timing 2 days
72. Dilute primary antibody in OWB solution.

SECTION: Antibody staining ○ Timing 2 days
73. Incubate organoids with 300 µL of primary antibody solution 15 min at 4 °C.

SECTION: Antibody staining ○ Timing 2 days
78. Wash twice with OWB.

SECTION: Antibody staining ○ Timing 2 days
77. Incubate with 300 µL of DAPI (ref, stock 1µg/ml diluted 1:1000) for at least 60 min. 
SECTION: Antibody staining ○ Timing 2 days
76. Wash twice with OWB as described previously.

SECTION: Antibody staining ○ Timing 2 days
75. Incubate with 300 µL of secondary antibody in OWB solution 15 min at 4 °C.

SECTION: Antibody staining ○ Timing 2 days
74. Wash 2x 5 min with 500 µl OWB.

SECTION: Antibody staining ○ Timing 2 days
80. Remove completely the water.

SECTION: Antibody staining ○ Timing 2 days
79. Wash with distilled water.

SECTION: Clearing and imaging
81. Add 3 drops/well of RapiClear 1.49 (Sunjin Lab #RC147001).
 
 

SECTION: Clearing and imaging
82. Image after 120 min or the next day.

SECTION: Crypt isolation ○ Timing 3h
2.1. Wash 2 min under mild rotation in PBS + antibiotics and antimycotic 100X (Invitrogen, 15240062, 1/200). (1/3)

SECTION: Crypt isolation ○ Timing 3h
2.2. Wash 2 min under mild rotation in PBS + antibiotics and antimycotic 100X (Invitrogen, 15240062, 1/200). (2/3)

SECTION: Crypt isolation ○ Timing 3h
2.3. Wash 2 min under mild rotation in PBS + antibiotics and antimycotic 100X (Invitrogen, 15240062, 1/200). (3/3)

SECTION: Antibody staining ○ Timing 2 days
74.1. Wash 5 min with 500 µL OWB. (1/2)

SECTION: Antibody staining ○ Timing 2 days
74.2. Wash 5 min with 500 µL OWB. (2/2)