Murino Hind Limb Ossos Longos Dissecção e Medula Óssea O isolamento
Summary
Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.
Abstract
Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.
Introduction
The study of long bones and the cells of the bone marrow is central to a myriad of research disciplines, including, but not limited to, bone biology, cancer biology, immunology, hematology, and biomechanics. The bone is a highly dynamic organ that together with the cartilage forms the skeleton to provide mechanical support against loading and protection of the internal organs. In addition, the mineral components of bone are a storage sink for the critical signaling molecules calcium and phosphorus, as well as other factors1. Finally, bones house the bone marrow and, together with metabolically active bone forming osteoblasts and bone resorbing osteoclasts, provide the stem cell niche necessary for the maintenance of hematopoietic and lymphoid cell populations.
Bone and bone marrow are affected in many disorders, often leading to bone marrow dysfunction, severe bone pain, and pathologic fracture. Bone is a common site of metastasis in many solid tumors, most notably breast cancer and prostate cancer, where tumor cells directly engage the bone marrow niche to initiate the vicious cycle of bone metastasis and displace hematopoietic stem cells2,3. Hematopoietic malignancies including myeloma and leukemia are characterized by bone marrow dysfunction as well as deregulation of healthy bone remodeling1. Other non-malignant skeletal disorders are also active areas of research, such as osteoarthritis, osteoporosis, scoliosis, and rickets. Even in an otherwise healthy individual, biomechanical failure in a bone leads to a painful fracture. All of these disorders represent active areas of research with the goal of identifying new preventative measures and treatment regimens to reduce morbidity and mortality.
To research the plethora of roles of the bone and the bone marrow, both under physiologic and pathologic conditions, it is critical for researchers to have a simple and efficient standardized method for dissection of the mouse long bones for rapid processing of large in vivo experiments. The dissection protocol outlined here is suitable for all long bone analyses including ex vivo imaging, histology, histomorphometry, and strength testing, among others. Similarly, a standardized bone marrow isolation method with high bone marrow cell recovery and low inter-user variability is important for experimental analysis such as fluorescence-activated cell sorting (FACS) or quantitative PCR (qPCR) as well as downstream applications such as primary cell culture of bone marrow cells.
Protocol
Representative Results
Discussion
We present a simple and efficient method for removal of mouse hind long bones and subsequent bone marrow isolation. This method maintains the high structural and cellular integrity of the bones and bone marrow and has low handling time, minimizing the likelihood of user-induced fracture or bone scoring that may influence downstream analyses. In addition, the centrifugation method for isolating bone marrow does not require cutting the bone to expose the bone marrow space or fluid to flush the bone marrow, reducing potential points of contamination. Moreover, the centrifuge technique is relatively high-throughput with lower hands-on time than other methods, thus reducing processing time.
High variation is inherent to in vivo mouse studies due to high mouse-to-mouse phenotypic variation. In order to maximize the research impact of expensive and labor-intensive mouse studies, it is critical to minimize technical experimental error9,10. Time from animal sacrifice to downstream analysis or tissue fixation introduces experimental variation that may overcome subtle changes and reduce large differences between groups. Therefore, rapid processing of samples is essential for accurate data analysis. The long bone dissection and bone marrow isolation techniques described here are optimized for rapid processing of animals and samples to reduce technical variation.
This protocol can be widely applied to many research fields, including investigation of the bone tissue itself or interrogation of the cells of the bone marrow. In addition, this straightforward approach to long bone dissection will enable researchers in related fields to directly interrogate bone contributions in order to expand our knowledge of bone marrow dysfunction in otherwise understudied pathologies.
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by NCI grant nos. U54CA143803, CA163124, CA093900, and CA143055 to K.J.P. The authors thank the current and past members of the Weilbaecher lab, especially Katherine Weilbaecher, Michelle Hurchla, and Hongju Deng, and members of the Brady Urological Institute, especially members of the Pienta laboratory for critical reading of the manuscript.
Materials
| pinboard | |||
| pins | |||
| 70% ethanol | |||
| dissection sissors | |||
| dissection forceps | |||
| Kimwipes | Kimberly-Clark | 34120 | |
| 16 guage needle | |||
| 1.5 ml microcentrifuge tube | |||
| 0.5 ml microcentrifuge tube | |||
| microcentrifuge |
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