Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

All animal experiments were performed under PPL 70/8904, approved by the U.K. Home Office and in accordance with Cancer Research UK guidelines. The use of human umbilical cord blood (UCB) and primary human acute myeloid leukemia (AML) samples was approved by the East London Ethical Committee after receiving consent and was carried out in accordance with the Declaration of Helsinki.

1. Bioengineering Collagen-based Scaffolds with Human Hematopoietic and Stromal Cells

NOTE: The entire protocol should be performed in sterile conditions and with sterile material. Cell culture medium 1 corresponds to hMSC medium (MEM-α, P/S, and 10% hMSC-FBS); cell culture medium 2 corresponds to EC medium (M199, 20% FBS, P/S, 10mM HEPES, 50 µg/mL heparin, 2 mM glutamine and 50 μg/mL ECGS) and cell culture medium 3 corresponds to hematopoietic cell medium (H5100 and P/S).

1. Prepare umbilical cord blood mononuclear cells (CB-MNCs) or primary AML (bone marrow or peripheral blood MNCs) using a Ficoll-Paque density gradient, according to well-established protocols⁵³.
   1. For AML, deplete the T cells using OKT.3 antibody. Incubate 4 µg of OKT.3 per 1 x 10⁶ AML MNCs for 30 min at room temperature before washing the cells in PBS. If required, cryopreserve the MNCs in FBS with 10% DMSO at 2 x 10⁸ cells per mL.
2. Prepare hMSC (culture medium 1) and EC (culture medium 2) cell cultures. Plate hMSC cells (see the Table of Materials) on regular cell culture flasks in hMSC medium. Plate ECs (see the Table of Materials) on collagen 1-coated surfaces in EC medium.
3. At 85-90% cell confluence remove the medium, wash twice with PBS, and add trypsin-EDTA solution (20 µL per cm²).
   1. After 5 min, check that cells are detached. Recover the cells by diluting 1:3 with cell culture medium. Centrifuge at 300 x g for 5 min and re-suspend in the corresponding medium for each cell type and count the cells using a Neubauer chamber.
   2. Use cells at 2 x 10⁶ – 10⁷ cells per mL. If both cell types need to be used together, mix the hMSC and EC suspensions in a 1:1 ratio.
4. Transfer the cell suspension to an insulin syringe.
5. Using a scalpel, cut the sterilized gelatin sponge (e.g., gelfoam) initial scaffold (20 mm x 60 mm x 7 mm) into 24 pieces of similar size (6.6 mm x 7.5 mm x 7 mm; Figure 1A and B).
6. Reconstitute gelatin scaffolds by immersion in PBS (5 min).
7. One by one, gently put the scaffolds on a sterile tissue to remove excess PBS. Transfer the scaffolds to one well of a 24-well plate (ultra-low attachment surface) and use the syringe to inject 50 µL containing 10⁵ – 10⁶ cells (hMSC alone or in combination with hEC; Figure 1C).
8. Repeat step 1.7 with each scaffold until all required scaffolds are seeded with stromal cells.
9. Incubate for 1 h inside a cell culture incubator (37 °C and CO₂ 5%).
10. Fill each well with 3 mL of cell culture medium 1 (Figure 1D) and return the scaffolds to a cell culture incubator (37 °C and CO₂ 5%) for overnight incubation. Use cell culture medium 2 if ECs are used in the scaffolds.
11. Thaw CB-MNCs and isolate CD34⁺ cells from them following an appropriate CD34⁺ section kit protocol. Suspend the selected CD34⁺ cells in medium 3 and count them in a Neubauer chamber.
    NOTE: Here, cells were used at a concentration of 2 x 10⁶ cells per mL.
    1. If AML patient-derived primary samples are used, thaw the cells, dilute them 1:10 in FBS, centrifuge them for 5 min at 300 x g, resuspend the cells in PBS-2% FBS, add anti-human CD3 antibody (2 – 4 µg per 10⁶ cells), and incubate at room temperature for 30 min. Centrifuge for 5 min at 300 x g and resuspend in hematopoietic cell medium supplemented with cytokines (20 ng/mL granulocyte-colony stimulating factor (G-CSF), 20 ng/mL IL-3, and 20 ng/mL thrombopoietin (TPO)).
12. Repeat steps 1.7 – 1.9 but in this case seed 1 x 10⁵ human hematopoietic cells instead of stromal components, as above.
    1. Fill the well with 3 mL of cell culture medium 3 (Figure 1D) and return the scaffolds to a cell culture incubator (37 °C and CO₂ 5%) for overnight incubation. Use a 1:1 mix of cell culture media 2 and 3 If ECs are used in the scaffolds.
13. For only recombinant human bone morphogenetic protein-2 (rhBMP-2) carrier scaffolds, gently put the scaffolds one by one on a sterile tissue to remove excess PBS. Transfer the scaffolds to one well of a u-bottom, 96-well plate (Figure 1E) and add 5 µL of rhBMP-2, thoroughly covering the scaffold.
    1. Add 20 µL of thrombin followed by 20 µL of fibrinogen, thoroughly covering the scaffold each time (Figure 1F). Repeat the procedure for each scaffold until all required scaffolds are treated and then incubate for 5 – 10 min at 37 °C. Check if coagulation has successfully formed.

2. Surgical Implantation of Bioengineered Scaffolds

NOTE: Here, either male or female, 6- to 12-week-old NSG mice were used. As the animals are immunodeficient, all procedures should be done in sterile conditions. Steps 2.10 – 2.13 are related to survival strategies and post-surgical care.

1. 60 – 120 min before surgery, subcutaneously administer pain medication (carprofen, 5 mg/kg bodyweight/mouse).
2. Induce anesthesia in a chamber with 0.5% isoflurane and 2 L/min O₂.  Mice should be monitored continuously during the procedure. Transfer the animal to the surgical area in prone position in order to have easy access to the back. Keep the animal under anesthesia using a nose cone supplying 1.5% isoflurane and 2 L/min O₂. Keep the mouse under anesthesia during the surgical procedure and frequently check the animal state.
3. While it is under anesthesia, use ophthalmic gel on the eyes of the mouse to prevent dryness, and keep the mouse at 37 °C.
4. Shave the surgical area on the back of the mouse using an electric trimmer. To sterilize the skin, dip a cotton tip in diluted clorexidine (diluted 1:10 in PBS) and use this tip to clean the skin surface. Repeat this procedure twice.
5. Using sterile forceps and a scalpel (or scissors), make a 0.5- to 0.7-cm anterior-to-posterior full incision of the skin. Use the forceps inserted under the subcutaneous tissue to make a pocket.
6. Insert the scaffold subcutaneously, making sure that it is placed deep within the pocket (Figure 1G and H). Close the incision with surgical glue (Figure 1I and J).
7. To treat post-surgical pain, subcutaneously administer buprenorphine (0.1 mg/kg bodyweight).
8. During recovery, place the animal on its side in a pre-warmed cage and monitor recovery until normal behavior is observed.
9. Dilute pain medication in water (carprofen, 0.1 mg/mL of water) and provide it to animals as drinking water for 4 days after surgery.
10. Check the animal and the wound frequently during the 48-h post-surgery period for possible adverse effects.

3. Mouse Treatments, Euthanasia, and Sample Retrieval for Imaging

NOTE: Analysis of the scaffolds is performed between 8 and 24 weeks post-implantation.

1. 60 min before imaging, keep the implanted mouse warm in a heating box at 37 °C and intravenously administer 100 µL of human immunoglobulin to block unspecific sites.
2. 30 min before imaging, intravenously administer 10 µg (per mouse) of specific antibodies to label the cells of interest.
3. 5 min before imaging, intravenously administer 15 µL (diluted in 100 µL of PBS) of 655-nm fluorophore-labeled, vessel-pooling agent (655-VPA) to visualize vascular structures.
4. Euthanize the mouse via cervical dislocation.
5. Using sharp scissors, make a longitudinal skin incision on the back of the mouse, near the original implantation site.
6. With the help of tweezers and scissors, carefully separate the skin from the subcutaneous pocket where the scaffold has been implanted.
7. Hold the scaffold with tweezers and gently explant it from the skin by cutting the residual membrane and tissue surrounding the scaffold using scissors. See examples of scaffolds to be recovered in Figure 2.
8. Secure the scaffold with fast-acting adhesive glue to an imaging plate (a 35 mm x 10 mm Petri dish) and fill with saline solution (PBS) at room temperature.
9. For BMP scaffolds, before filling the plate with PBS, use a surgical microdrill to thin the bone surface under a microsurgical microscope; this allows fluorophore visualization and high-resolution image capture. Use either the 1.2- or 1.6-mm burrs, depending upon the size of the scaffold.
   NOTE: The user will realize how much to drill depending upon the thickness of the bone. In general, as the BMP scaffolds are vascularized, the bone will slightly change color and become more red when approaching the correct thickness for imaging.
10. Insert the plate onto the stage of the confocal microscope.

4. Live-imaging Using Two-photon Microscopy

NOTE: When using non-descanned detectors (NDD), always use the NDD slider for imaging to direct the fluorescence to the NDD. The microscope configuration is provided in Figure 3.

1. Switch on the microscope and the computer, start the software by clicking "Start System," and go to the "Acquisition" mode.
2. Tick the "Show manual tools" box. In the "Laser" menu switch "on" the two-photon laser and allow it to warm up and stabilize.
3. In the "Imaging Setup" menu, simultaneously activate "Channel Mode" and "Switch track every Frame." In the "Light Path" menu, select "Non Descanned'" and "Main Beam Splitter MBS 760+." Tick to activate the four NDDs and set the configuration as illustrated in Figure 3.
   NOTE: With this configuration, the collagen signal from bone structures (second harmonic generation, SHG) is collected at 380 – 485 nm, FITC-hCD31+ human endothelial cells and AF488- hCD45+ human hematopoietic cells at 500-550 nm, and 655-VPA at 640 – 690 nm.
4. In the "Channels" menu, set the "laser wavelength" to 890 nm and the power to 50%. Set the "Gain (Master)" to 500-600, the "Digital Offset" to 0, and the "Digital Gain" to 15 for each channel. Adjust these values once the acquisition has started.
5. In "Acquisition Mode," set up the required parameters to obtain high-resolution images without damaging the tissue and bleaching the fluorophores. Set "Scan Mode" to "Frame," "Frame Size" to "x512 y512," Line Step" to 1, "Speed" to 9, "Averaging number" to 8, "Bit Depth" to "8 Bit," "Mode" to "line," "Direction" to "bidirectional," and "Method" to "mean."
   1. Set the "Zoom" to 1 for the initial scan of the image, and increase it if required to focus on particular areas.
6. Place the plate containing the scaffold on the microscope stage under the 20X, 1.0 NA water-immersion lens and lower the lens until it touches the saline solution. Set the focus of the lens on the scaffold using the microscope eyepieces, using a lamp as the light source.
7. Activate the "Z-stack" menu, select the "First/Last" function, and set the required interval between two sub-sequential slices (e.g., 2-µm Z-stack). Keep the intervals constant within the Z-stack.
8. Select "Live" to image a live scan of the sample and adjust the "Digital Gain" and "Digital Offset" for optimal exposure. To visualize multiple channels at the same time, select the "Split" function.
9. In the "Stage" menu, while in "Live" mode, scan the image and "Mark" regions of interest (ROI), such as the location of human hematopoietic cells and vascular structures. When the scan of the sample is complete, move to the first ROI to start imaging.
10. In "Live" mode, set the top and bottom of the 3D Z-stack surrounding the area of interest using the "Set first" and "Set last" functions. Once finished, set the center, "C." Start the acquisition of the ROI with the "start experiment" button. See examples of images in Figure 4, Figure 5, and Figure 6.
11. Once acquisition is complete, save the image in the designated folder. Move to the next ROI and repeat step 4.10. Once the experiment is complete, remove the imaging plate from the microscope, carefully detach the sample from the plate, and clean any residual glue.
12. Prepare the sample for the next analysis technique.

5. Sample Processing for Histology and Immunostaining

NOTE: Samples are processed according to the protocol described in the JoVE general laboratory techniques⁵⁴ describing sample fixation, embedding, and sectioning processes. Bone-forming samples should be treated for 7 days in an EDTA-based decalcifying agent between the fixation and embedding processes. The blocking/permeabilization solution is 10 mM PBS pH 7.4 buffer with 1% Triton X-100, 1% bovine serum albumin (BSA), and 10% normal goat serum (NGS).

1. Put the slices in xylene for 10 min), xylene for 5 min, 100% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and H₂O for 5 min.
2. Transfer the slices to a citrate-based antigen unmasking working solution.
3. Boil the slices for 15 min and allow them to cool to room temperature.
4. Wash the slices in 10 mM PBS pH 7.4 solution with 1% Triton X-100 (5 min, 3 times).
5. Transfer the samples to the blocking/permeabilization solution and incubate for 30 min.
6. Add primary antibody diluted in blocking/permeabilization solution and incubate overnight at 4 °C.
7. Wash the slices in 10 mM PBS pH 7.4 solution with 1% Triton X-100 (5 min, 3 times).
8. Add secondary antibody diluted in blocking/permeabilization solution for 1 h in the dark at room temperature.
9. Wash with H₂O (5 min, 3 times).
10. To reduce background fluorescence, immerse the slices in Sudan Black working solution for 10 min, in the dark and at room temperature.
11. Wash the slices in H₂O (5 min, 3 times).
12. Mount the slices using fluorescent mounting medium with DAPI (0.5 µg/mL).
13. Store the slices at 4 °C and check that the mounting medium is dry before conducting fluorescent imaging. See example images in Figure 7.