In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

We have long been engaged in biomaterials'research, introducing the pioneering concept of material biologists under developing virus, active-bone repair materials, and the technologists for growth factor, preparation and activation. We developed an In Vivo Osteo-Organoid approach using bioactive materials to achieve on-demand production of auto nodules, tissues and the cells for disease treatment. The traditional technology of individual expansion of stem cells faces limitations in maintaining the physiological functions of the stem cells.

Our protocol constructs an individual cultures'microenvironment to ensure an abundant quantity of stem cells while preserving their stimulus. To begin, place the recombinant human stock solution and unopened gelatin sponge on a sterile clean bench. Use sterilized scissors to cut the gelatin sponge into 48 uniformly-sized pieces and transfer them to a 48-well plate.

Pipette the recombinant human stock solution several times to mix it thoroughly. Aspirate 30 microliters of the solution and add it dropwise to the gelatin sponge. Then, seal the 48-well plate with a layer of parafilm.

Remove the plate from the clean bench and place it at a temperature of minus 20 degrees Celsius. To begin, transfer the anesthetized mouse to the surgical area, and position it in the right lateral recumbent position for easy implantation of the bioactive scaffold into the left leg. Using a shaving machine, shave the hair of the left leg and disinfect the shaved area using alternating rounds of chlorhexidine and alcohol, three times each.

After making the incision, insert ophthalmic scissors into the muscle of the pouch in the direction of the tibia to provide space for subsequent material implantation. Now, take the prepared bioactive scaffold loaded with recombinant human and using ophthalmic forceps, pinch it into a cylindrical shape. Implant the scaffold into the muscle pocket along the slit created earlier.

After 12 weeks of implantation, anesthetize the mouse and carefully separate the hind limb from the trunk. Using miniature scissors, meticulously remove the muscle tissue from the osteo-organoids. Eliminate any attached soft tissues using gauze to ensure a clean osteo-organoid sample.

Fix a portion of the osteo-organoids in 4%Paraformaldehyde solution for 24 hours at room temperature for histological analysis. To begin, harvest osteo-organoids from mice implanted with a scaffold containing recombinant human Place osteo-organoids into a mortar, and add one milliliter of HBSS, without calcium and magnesium ions. Use surgical scissors to chop the samples and then gently crush them in a mortar and pestle.

Pass the result in cell suspension through a 40-micrometer cell strainer. Then, centrifuge it at 300 G for five minutes at four degrees Celsius. Afterward, discard the supernatant.

Re-suspend the cell pellets in 300 microliters of HBSS and mix well to achieve a uniform cell suspension. Place the anesthetized sub lethally irradiated mouse on a heating pad set at 37 degrees Celsius. After ensuring full anesthesia, position the mouse in left lateral recumbency with its head oriented to the right.

Using a one-milliliter syringe with a 25-gauge needle, aspirate 200 microliters of the prepared cell suspension. To expose the eye for the procedure, place a finger on the top of the head and along the jawline, and gently pull the skin back and down. Carefully insert the needle with the bevel facing downwards into the medial canthus of the eye at an approximate 30-degree angle.

Gently, follow the contour of the eyeball with the needle until it reaches the base of the eye. Inject the cell suspension smoothly and slowly into the designated area. After completing the injection, return the mouse to its cage to allow for recovery.