Intrabone injections in mice and rats biomethodologies

Posted by: admin | January 15, 2009 | 0 Comments

Recently, a question was raised on one of the listservs regarding injection of small, minced tumors into the tibias of mice. We do not have experience in injecting minced tumors, but we do have experience with cell injections into tibias and have collected some interesting articles while putting together our protocol of this methodology for our Advanced Mouse Biomethodology workshop and course.

One of most commonly cited techniques that we found was described by Kushida¹:

“The BMCs were collected from the femurs and tibias of B6 mice. The IBM injection was carried out as follows. The region from the inguen to the knee joint was shaved of hair with a razor and a 5-mm incision was made on the thigh. The knee was flexed to 90° and the proximal side of the tibia was drawn to the anterior. A 26-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the bone marrow cavity. Using a microsyringe (50 mL; Hamilton, Reno, NV) containing the donor BMCs (3 3 107/30 mL), the donor BMCs were injected from the bone hole into the bone marrow cavity.” ¹

Intrabone marrow injection is described by Castello²:

“Injection of the BMCs or CB cells into the tibial shaft was performed through a 27-gauge needle percutaneously inserted into the tibia with the flexion of the host mouse’s right knee by a gentle but firm pressure. The correct position for the injection into the marrow cavity was reached by advancing the needle into the bone, until a sudden drop in the injection pressure was experienced with free flow of the 30 L of the cell suspension out of the needle into the bone marrow cavity.”²

Castello² also compares the seeding efficiency of intravascular (IV) injection to that of intra-bone marrow (IBM) injections of bone marrow and concludes that the IBM method is 15 times more efficient than the IV method.

Ikehara³ references intra-bone marrow Kushida. However, he also states that the injection of 1x 10⁷ BMCs/10 μL using a Hamilton syringe into the tibia is better [Note 1] than the injection of 3x 10⁷ BMCs/50 μL¹, since the injection of a high concentration of bone marrow cells is necessary to ensure that donor bone marrow cells become trapped and grow inside the bone marrow cavity.

One of the challenges with intra-bone marrow (IBM) injections is the leakage of cells into the blood circulation. Shi⁵ has described an IBM injection methodology utilizing collagen gel which leads to a significant increase in cell retention within the bone marrow. Here is the methodology:

“…the area from the inguinal region to the knee joint was shaved. The tibia was gently drilled with a 26-gauge needle through the patellar tendon into the BM cavity. BMCs
suspended in PBS or CG were aspirated into a microsyringe (50 μl; Ito, Fuji,Shizuoka, Japan, http://www.ito-ex.co.jp) and then kept at room temperature. The BMCs (10⁵, 10⁶, or 10⁷ in 10 μl) were then injected into the BM cavity using the microsyringe.” ⁵

We also have a protocol for survival bone marrow (BM) collection from mice. This protocol is useful when you need to utilize the same mouse on multiple occasions.  If anyone is interested in the details of this protocol, please send an email to szczepanb@vetbiotech.com.

Obviously, intra-bone marrow injection methodology has been utilized in rat models as well. We refer to Guo’s⁵ description of this procedure:

“BMCs (3×10⁷ or 10×10⁷ cells/60 µL) obtained from the femurs and tibias of donor F344 rats were injected intravenously (IV-BMT) or directly into the bone marrow cavity (IBM-BMT) of the left tibia of the recipient BN rats on day 0, and the cardiac allografts from F344 rats were implanted simultaneously. In terms of the IBM-BMT technique, the knee was flexed to 90 degrees and the proximal side of the tibia was drawn to the anterior. A 26-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the bone cavity.” ⁵

 Note 1 - Hamilton method⁴

“Intra-BM injection (IBMI) was carried out as reported previously with modifications.11 Briefly, after sterilization of the skin around the left knee joint, the knee was flexed to 90 degrees, and the proximal side of the tibia was drawn to the anterior. A 27-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the BM cavity. Using a Hamiltonmicrosyringe, the specified number of donor cells per 10 μL of α medium were carefully injected from the bone hole into the BM cavity.” ⁴

1. Kushida T, Inaba M, Hisha H, Ichioka N,Esumi T, Ogawa R, Iida H, Ikehara S. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood. 2001 May 15;97(10):3292-9.

1. Castello S, Podestà M, Menditto VG, Ibatici A, Pitto A, Figari O, Scarpati D, Magrassi L, Bacigalupo A, Piaggio G, Frassoni F. Intra-bone marrow injection of bone marrow and cord blood cells: an alternative way of transplantation associated with a higher seeding efficiency. Experimental  Hematology. 2004 Aug;32(8):782-7.

1. Ikehara S. A novel strategy for allogeneic stem cell transplantation: perfusion method plus intra-bone marrow injection of stem cells. Exp Hematology. 2003 Dec;31(12):1142-6

1. Wang J, Kimura T, Asada R, Harada S, Yokota S, Kawamoto Y, Fujimura Y, Tsuji T, Ikehara S, Sonoda Y. SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection. Blood. 2003 Apr 15;101(8):2924-31. Epub 2002 Dec 12.

1. Guo K, Inaba M, Li M, An J, Cui W, Song C, Wang J, Cui Y, Sakaguchi Y, Tsuda M, Omae M, Ando Y, Li Q, Wang X, Feng W, Ikehara S. Long-Term
   Donor-Specific Tolerance in Rat Cardiac Allografts by Intrabone Marrow Injection of Donor Bone Marrow Cells. Transplantation. 2008 Jan 15;85(1):93-101.

1. Shi M, Adachi Y, Shigematsu A, Koike-Kiriyama N, Feng W, Yanai S, Yunze C, Lian ZX, Li J, Ikehara S.  Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice. Stem Cells. Sep;26(9):2211-6. Epub 2008 Jul 3.