Femoral Bone Marrow Aspiration in Live Mice

The overall goal of this procedure is to safely aspirate the bone marrow hematopoietic cell contents from the femoral marrow space of a living mouse. This is accomplished by first wedding, a tuberculin syringe with PBS. In the second step, the mouse's leg is carefully arranged at the optimal angle for bone marrow retrieval.

The hematopoietic cells are then aspirated from the femur marrow space and the red blood cells are depleted. Ultimately, the successful aspiration of the bone marrow contents can be visualized by flow cytometric and cytological analyses. Demonstrating the procedure will be young Rock Chung, a very talented technician from our laboratory.

The main advantage of this technique over existing method, like conventional bone marrow harvest, is that this technique allows the retrieval of a portion of bone marrow's hematopoietic cells while leaving the animal alive for continued observation and serial analysis. F, after confirming sedation by toe pinch, apply ophthalmic ointment to the eyes of the anesthetized mouse to prevent corneal drying and shave the leg to expose the skin. Sterile surgical preparation of skin is then performed by scrubbing the area with chlorhexidine disinfectant, followed by alcohol swab.

This process is completed three times and a fresh, unopened sterile syringe and needle are used for each animal. Confirm that a suitable anesthetic plane has been attained. Next wet, a 0.5 milliliter tuberculin syringe with sterile PBS and then aspirate and immediately expunge 200 to 500 milliliters of the saline solution two to three times.

Then use one of the last two fingers from one hand to gently bend the tibia away from the femur to facilitate exposure of the condylar space of the femur and the insertion of the needle. Now insert the needle through the patellar tendon so that it is lodged securely between the two condyles of the femur. Use the thumb and index finger of the other hand to hold the diaphysis close to the epiphysis of the femur to allow the needle to be easily inserted into the shaft of the bone.

If no blood is seen in the syringe, it is likely that a small bone or tissue fragment is stuck in the needle. This can be removed from the needle by moving the plunger up and down in PBS. If the tissue cannot be dislodged from the syringe, use a new needle and syringe.

Again, wet the syringe with 200 to 500 microliters of PBS. Then swivel the needle clockwise and counterclockwise while pushing it against the condylar space so that it is parallel with the shaft of the femur. To facilitate the retrieval of the bone marrow contents from the femur shaft.

Turn the needle clockwise and counterclockwise while pushing it slowly into the femoral marrow cavity. Confirm the correct positioning of the needle by gently moving the syringe laterally. Then gently pull the needle plunger back, creating negative pressure while moving the needle back and forth within the bone marrow cavity.

Successful aspiration will be confirmed by the appearance of blood in the top of the needle at the base of the syringe. Once the bone marrow has been successfully aspirated from the femur, remove the needle from the bone and transfer the aspirated bone marrow to a micro fuge prefilled with 500 microliters of PBS on ice. After administering analgesic, allow the mouse to recover under a heat lamp before returning mice to the housing area.

Ensure they are able to ambulate and reach food and water. Then return the mouse to its cage and observe the animal for signs of distress or infection post procedure for the next 24 hours. In the meantime, spin down the bone marrow cells, aspirate out PBS carefully and resuspend the pellet.

In 500 microliters of red blood cell lysis buffer after 10 minutes, wash the cells in one milliliter of PBS. The pellet contains bone marrow of mononuclear cells that can be used for experimental analysis. These first figures illustrate how similar types and proportions of bulk cells and hematopoietic stem and progenitor cells can be observed by morphologic and flow cytometric analyses using either femoral bone marrow aspiration, or conventional bone marrow harvest.

Here the percentage of lineage negative SKA one positive C kit positive and myeloid progenitor subpopulations as a frequency of total live cells in three independent aspirations and conventional marrow harvest is shown. These data reveal a decrease in lineage negative SKA one, positive C kit positive and myeloid progenitors subpopulations from bone marrow collected by femoral aspiration compared with conventional marrow harvest. This difference was not statistically significant for population.

However, sorting of lineage negative ska one positive cki positive cells, followed by ex vivo culture in Methylcellulose results in similar colony numbers and cell types obtained by femoral bone marrow aspiration. As with conventional bone marrow harvest, the colony types observed and enumerated in both cultures include granulocyte, erythrocyte, monocyte, and megakaryocyte colony forming units, granulocyte and monocyte colony forming units and erythroid burst forming units One master. This technique can be done in 10 minutes per mouse if it is performed properly Following this procedure.

Other methods like intra femoral bone marrow injection can be performed to answer additional questions such as what is the effect of xenograft in human hematopoietic cells into immunocompromised mice?