マウスにおけるヒト化骨髄微小環境のバイオエンジニアリングとライブイメージングによるそれらの可視化

The overall goal of this method is to implant human cell carrier scaffolds into mice to create humanized bone marrow niches and then live image the behavior of the human cells within the implants. This method will help answer key questions in the human haematopoietic field because it allows to reproduce and live image at the single cell resolution different components of the bone marrow. The versatility of this system is one of its major advantages, as it allows implantation of different niche components one by one or in combination.

This methodology could potentially be applied to other research fields, as tumor metastasis or drug screening studies. Using the appropriate sterile technique, begin by cutting a sterilized gelatin sponge into 24 pieces of similar size. Reconstitute the gelatin scaffolds by immersion in ethanol and then in PBS.

Then gently dab each scaffold on a sterile tissue to remove the excess liquid, and transfer the scaffolds into individual wells of an ultra low attachment 24-well plate. Using an insulin syringe, carefully inject one times 10 to the fifth to one times 10 to the sixth stromal cells in 50 microliters of culture medium into each scaffold, and place the plate in a cell culture incubator. When injecting the cells into the scaffold, be sure there is no leakness out from the scaffold.

After one hour, fill each well with three milliliters of fresh cell culture medium, and return the scaffolds to the incubator. After 24 hours, inject one times 10 to the fifth haematopoietic cells into the scaffolds, followed by incubation and further media addition, and 24 hour incubation as just demonstrated. For the generation of recombinant human bone morphogenetic protein two carrier scaffolds, place the reconstituted scaffolds into individual wells of a U-bottom 96-well plate, and add five microliters of recombinant human bone morphogenetic protein two to each scaffold.

Then cover the scaffolds with 20 microliters of thrombin and 20 microliters of fibrinogen. For surgical implantation of the bioengineered scaffolds, first shave the surgical area on the back of the experimental mouse, and use a cotton tip dipped in diluted chlorhexidine to clean the exposed skin surface three times in each direction. Next, use sterile forceps and a scalpel to make a 0.5 to 0.7 centimeter anterior to posterior incision in the skin, and insert the forceps under the subcutaneous tissue to make a pocket.

Insert the scaffold deep into the pocket, and close the incision with surgical glue. Allow the implanted scaffolds to develop for eight to 24 weeks before the explant. After intravenous administration of fluorescent substances and euthanization of the animals, sterilize the skin as just demonstrated and make a longitudinal skin incision on the back of the animal, near the original implantation site.

Carefully separate the skin from the subcutaneous pocket where the scaffold was implanted, then use tweezers to gently grasp the scaffold and carefully cut the residual membrane and tissues surrounding the scaffold to recover the structure. Use fast-acting adhesive glue to secure the scaffold to a 35 by 10 milliliter Petri dish. For bone morphogenetic protein two scaffolds, before filling the plate with PBS, use a surgical microdrill under a microsurgical microscope to thin the bone surface, allowing visualization of the fluorophores and high resolution capture of the images.

Be particularly careful during the drilling process because the thickness of the bone typically varies among scaffolds, and it's important not to break the surface. Fill the dish with room temperature PBS. For 2-photon microscopy imaging of the bioengineered scaffolds, place the scaffold onto the confocal microscope stage, and select the 20 times 1.0 NA water immersion lens.

Then, in the acquisition mode of the imaging software, select the show manual tools box. In the laser menu, switch on the 2-photon laser, and allow the laser to warm up and stabilize. When the laser is ready, in the imaging setup menu, activate the channel mode and switch track every frame.

In the light path menu select non descanned and main beam splitter MBS 760 plus. Activate the four non descanned detectors and set the configuration as illustrated in the schematic. In the channels menu, set the laser wavelength to 890 nanometers and the power to 50%Set the gain master to 500 to 600, the digital offset to zero, and the digital gain to 15 for each channel.

In the acquisition mode, set the appropriate parameters for obtaining high resolution images without damaging the tissue and bleaching the fluorophores. Then set the zoom to one for the initial scan of the image. When all of the parameters have been set, lower the lens until it touches the saline solution, and manually set the lens focus using a lamp as the light source.

Now activate the Z-stack menu, and select the first last function. Set the required interval between two subsequential slices and select live to image a live scan of the sample, adjusting the digital gain and digital offset as necessary for an optimal exposure. To visualize multiple channels at the same time, select the split function.

In the stage menu, while in live mode, scan the image and mark the regions of interest, such as the location of bone cavities. When the sample scan is complete, move to the first region of interest, and use the set first and set last functions in the live mode to set the top and bottom of the 3D Z-stack surrounding the area of interest. Then set the center, C, and click the start experiment button to start the Z-stack image acquisition of the region of interest.

When the acquisition is complete, save the images in the designated folder, and scan the next region of interest. In these representative images, the gross morphology of different scaffolds recovered from immunodeficient NSG mice eight weeks after implantation can be observed. Co-seeding with human endothelial cells increases the vascularization of the scaffold, while the addition of recombinant human bone morphogenetic protein two induces bone formation.

These scaffolds imaged by live 2-photon microscopy reveal the presence of blood vessels in the scaffolds and the long-term engraftment of the human haematopoietic cells in the scaffold parenchyma. Scaffolds seeded with human endothelial and mesenchymal stromal cells illustrate the participation of the human endothelial cells in the formation of vessels within the scaffold, resulting in a murine-human chymeric vasculature. The formation of bone tissue can be visualized by 2-photon microscopy, as well as the formation of cavities in humanized vasculature in the endosteal tissue, which highly resembles bone marrow endosteal tissue.

Further, in recombinant human bone morphogenetic protein two carrier scaffolds, the morphology of the tissue within the scaffold resembles mature bone marrow with adipose tissue, suggesting that the human mesenchymal stromal cells also contribute to adipose tissue formation. After watching this video, you should have a good understanding of all bioengineer and the major murine scaffolds. Always remember to maintain an ascetic environment, and take extra care while handling scaffolds.

Bear in mind the limitations associated to this method, such as human-mouse chimeras of the tissues. Remember that after imaging, samples can be used for further studies, as the scaffolds can be digest, and therefore, live cells can be retrieved from them.