原代细胞分离中，针对骨骼组织涉及到软骨细胞、成骨细胞、骨和骨髓基质细胞、股骨近端等等的解离通常会使用酶，如何选择合适的酶来完成对应骨骼组织的解离，以及原代细胞采集的常规操作， 艾美捷针对骨骼解离分种属、不同的细胞、酶组合推荐，推荐浓度、培养基以及参考文献，详细汇总指南：
本指南总结了Worthington本技术指南描述了标准实验室程序； 为建立细胞分离方案提供了一种合乎逻辑的实验方法； 并列出了许多组织特异性参考资料，以帮助开发有效的方法。更多其他组织的解离指南，点击查看：各组织分离指南。
| 骨骼 | ||||
| 种属详情 | 细胞名称 | 酶组合推荐，含推荐浓度 | 培养基 | 参考文献 |
| Bovine | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.4% | DMEM | Buschmann, M., Gluzband, Y., Grodzinsky, A., and Hunziker, E.: Mechanical Compression Modulates Matrix Biosynthesis in Chondrocyte/Agarose Culture, J Cell Sci 108, 1497, 1995 |
| Chick, day old | 成骨细胞 / Osteoblasts | Trypsin: 0.03% | DMEM | Gay, C., Lloyd, Q., and Gilman, V.: Characteristics and Culture of Osteoblasts Derived From Avian Long Bone, In Vitro Cell Dev Biol 30A, 379, 1994 |
| Chick, Peterson/Arbor Acre, male, 4 weeks old (Gallus domesticus) | 软骨细胞 / Chondrocytes | Trypsin: 0.25% | Ham's F12 | Rosselot, G., Reginato, A.M., and Leach, R.M.: Development of a Serum-Free System to Study the Effect of Growth Hormone and Insulinlike Growth Factor-1 on Cultured Postembryonic Growth Plate Chondrocytes, In Vitro Cell Dev Biol 28A, 235, 1992 |
| Chick, embryo | 椎骨成软骨细胞 / Vertebrae chondroblasts | Trypsin: 0.25% | Simm's, CMF | Schiltz, J.R., Mayne R., and Holtzer, H.: The Synthesis of Collagen and Glycosaminoglycans by Dedifferentiated Chondroblasts in Culture, Differentiation 1, 97, 1973 |
| Human | 骨-软骨-基质细胞 / Bone-cartilage-stromal | Collagenase Type 2: 0.75% | HBSS | Agarwal, S., Loder, S., Sorkin, M., Li, S., Shrestha, S., Zhao, B., Mishina, Y., James, A. and Levi, B.: Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification., Stem Cells 34, 1692-701, 2016 |
| Human | 成骨细胞 / Osteoblasts | Collagenase Type 2: 0.2%； Trypsin: 0.25%-1.0% |
DMEM | Taylor, S., Shah, M. and Orriss, I.: Generation of Rodent and Human Osteoblasts., Bonekey Rep 3, 585, 2014 |
| Human | 成骨细胞 / Osteoblasts | Collagenase Type 3: 0.2% | MEM | Kode, A.,Manavalan1, J.,Mosialou1, I., Bhagat G., Rathinam, C, Luo, N et al.: Leukemogenesis Induced by an Activating β-catenin Mutation in Osteoblasts, Nature 506, 240, 2014 |
| Human, 22-73 year | 成骨细胞 / Osteoblasts | Trypsin: 0.5% | Basal Medium | Kneser U, Voogd A, Ohnolz J, Buettner O, Stangenberg L, Zhang YH, Stark GB, Schaefer DJ: Fibrin gel-immobilized primary osteoblasts in calcium phosphate bone cement: in vivo evaluation with regard to application as injectable biological bone substitute, Cells Tissues Organs 179, 158-69, 2005 |
| Human, 60+ year | 骨细胞，成骨细胞 / Bone Cells, Osteoblasts | Collagenase Type 2: 200-250 u/ml | DMEM | Chen X., Qian H., Neff L., Satomura K., and Horowitz M.: Thy-1 antigen Expression by Cells in the Osteoblast Lineage, J Bone Miner Res 14, 362, 1999 |
| Human | 成骨细胞 / Osteoblasts | Trypsin: 0.1% | DMEM | Meikle, M., Boyd, S., Hembry, R., Compston, J., Croucher, P., and Reynolds, J.: Human Osteoblasts in Culture Synthesize Collagenase and Other Matrix Metalloproteinases in Response to Osteotropic Hormones and Cytokines, J Cell Sci 103, 1093, 1992 |
| Human | 成骨细胞 / Osteoblasts | Collagenase Type 4: 250 u/ml | DMEM | Fedarko, N.S., Termine, J.D., Young, M.F. and Robey, P.G.: Temporal Regulation of Hyaluronan and Proteoglycan Metabolism by Human Bone Cells in Vitro, J Biol Chem 265, 12200, 1990 |
| Mouse | 骨和骨髓基质细胞 / Bone and bone marrow stromal | Stemxyme 1: 0.1%； Neutral Protease: 0.1% |
PBS | Mohrin, D., Koukos, G., Alabdulaaly, L., Paw, J., Hake, K., Kolumam, G., Bouxsein, M., Baron, R., Kutskova, Y., Freund, A., Liu, J., Zavala-Solorio, J., Bhargava, S., Maxwell, Trumble, J., Brito, A., Hu, D. and Brooks, D.: Inhibition of Longevity Regulator PAPP-A Modulates Tissue Homeostasis via Restraint of Mesenchymal Stromal Cells.,Aging Cell Vol. 20,, , e13313, 2021 |
| Mouse | 骨和骨髓基质细胞 / Bone and bone marrow stroma | Stemxyme 1: 0.1%； Neutral Protease: 0.1% |
Media 199 | Baryawno, N., Przybylski, D., Kowalczyk, M., Kfoury, Y., Severe, N., Gustafsson, K., Kokkaliaris, K., Mercier, F., Tabaka, M., Hofree, M., Dionne, D., Papazian, A., Lee, D., Ashenberg, O. and Subramanian, A.: A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia., Cell 177, 1915-1932.e16, 2019 |
| Mouse | 成骨细胞 / Osteoblast | Collagenase Type 1: 0.3% | HBSS | Wang, W., Majihail, G., Lui, C. and Zhou, L.: Osteoblast Sorting and Intracellular Staining of CXCL12., Bio Protoc 8, , 2018 |
| Mouse | 骨-软骨-基质细胞 / Bone-cartilage-stromal | Collagenase Type 2: 0.75% | HBSS | Agarwal, S., Loder, S., Sorkin, M., Li, S., Shrestha, S., Zhao, B., Mishina, Y., James, A. and Levi, B.: Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification., Stem Cells 34, 1692-701, 2016 |
| Mouse | 骨基质细胞 / Bone stromal | Collagenase Type 1: 0.3% | DMEM | Igarashi, H., Akahoshi, N., Ohto-Nakanishi, T., Yasuda, D. and Ishii, S.: The Lysophosphatidic Acid Receptor LPA4 Regulates Hematopoiesis-Supporting Activity of Bone Marrow Stromal Cells., Sci Rep 5, 11410, 2015 |
| Mouse | 成骨细胞 / Osteoblasts | Collagenase Type 1: 1.0%； Neutral Protease: 1.6% |
MEM | Lee, D., Choung, H., Kim, H., Gronostajski, R., Yang, Y., Ryoo, H., Lee, Z., Kim, H., Cho, E. and Park, J.: NFI-C Regulates Osteoblast Differentiation Via Control of Osterix Expression., Stem Cells 32, 2467-79, 2014 |
| Mouse, 2-5 day | 成骨细胞 / Osteoblasts | Collagenase Type 2: 0.2%； Trypsin: 0.25%-1.0% |
DMEM | Taylor, S., Shah, M. and Orriss, I.: Generation of Rodent and Human Osteoblasts., Bonekey Rep 3, 585, 2014 |
| Mouse | 成骨细胞 / Osteoblasts | Collagenase Type 3: 0.2% | MEM | Kode, A.,Manavalan1, J.,Mosialou1, I., Bhagat G., Rathinam, C, Luo, N et al.: Leukemogenesis Induced by an Activating β-catenin Mutation in Osteoblasts, Nature 506, 240, 2014 |
| Mouse, 4-22 month | 骨细胞 / Osteocytes | Collagenase Type 1: 300 u/ml | MEM | Stern, A., Stern, M., Van Dyke, M., Jahn, K., Prideaux, M. and Bonewald, L.: Isolation and Culture of Primary Osteocytes from the Long Bones of Skeletally Mature and Aged Mice., Biotechniques 52, 361, 2012 |
| Mouse | 骨内膜细胞 / Endosteal cells | Collagenase Type 1: 0.3% | DMEM | Nakamura, Y., Arai, F., Iwasaki, H., Hosokawa, K., Kobayashi, I., Gomei, Y., Matsumoto, Y., Yoshihara, H. and Suda, T.: Isolation and Characterization of Endosteal Niche Cell Populations that Regulate Hematopoietic Stem Cells., Blood 116, 1422, 2010 |
| Mouse | 骨髓 / Bone marrow | Collagenase: 0.2% | DMEM | Morikawa, S., Mabuchi, Y., Kubota, Y., Nagai, Y., Niibe, K., Hiratsu, E., Suzuki, S., Miyauchi-Hara, C., Nagoshi, N., Sunabori, T., Shimmura, S., Miyawaki, A., Nakagawa, T., Suda, T., Okano, H. and Matsuzaki, Y.: Prospective Identification, Isolation, and Systemic Transplantation of Multipotent Mesenchymal Stem Cells in Murine Bone Marrow., J Exp Med 206, 2483-96, 2009 |
| Mouse, male, 6-8 week old | 破骨细胞 / Osteoclasts | Collagenase Type 3: 0.1% | DMEM | Sakai, E., Miyamoto, H., Okamoto, K., Kato, Y., Yamamoto, K., and Sakai, H.: Characterization of Phagosomal Subpopulations Along Endocytic Routes in Osteoclasts and Macrophages, J. Biochem. 130, 823, 2001 |
| Mouse, 3-5 day, 6-8 week | 骨细胞，成骨细胞 / Bone Cells, Osteoblasts | Collagenase Type 2: 200-250 u/ml | DMEM | Chen X., Qian H., Neff L., Satomura K., and Horowitz M.: Thy-1 antigen Expression by Cells in the Osteoblast Lineage, J Bone Miner Res 14, 362, 1999 |
| Mouse, BALB/c | 成骨细胞样细胞，基质细胞系，造血母细胞 / Osteoblast-like Cells，Stromal Cell Lines，Hematopoietic Blast Cells | Trypsin: 0.1% | Eagle's MEM | Takanashi, H., Matsuishi, T., and Yoshizato, K.: Establishment and Characterization of Stromal Cell Lines That Support Differentiation of Murine Hematopoietic Blast Cells into Osteoblast-like Cells, In Vitro Cell Dev Biol 30A, 384, 1994 |
| Mouse, Swiss-Webster | 新生儿骨 / Neonatal bone | Collagenase Type 2: 0.20% | Tris-buffered saline | Chen,T. and Feldman,D.: Regulation of 1,25-Dihydroxyvitamin D3 Receptors in Cultured Mouse Bone Cells, J Biol Chem 256, 5561, 1981 |
| Rat, 2 day | 成骨细胞 / Osteoblasts | Collagenase Type 2: 0.1%； Trypsin: 0.25% |
DMEM | Jeon, J., Lee, M. and Yang, H.: Differentiated Osteoblasts Derived Decellularized Extracellular Matrix to Promote Osteogenic Differentiation., Biomater Res 22, 4, 2018 |
| Rat, Wistar, 250g | 股骨近端 / Proximal femur stem | Collagenase Type 1: 0.075% | HBSS | Jacobs, F., Gijsen, H., Van de Vyver, M. and Ferris, W.: Vanadate Impedes Adipogenesis in Mesenchymal Stem Cells Derived from Different Depots within Bone., Front Endocrinol 7, 108, 2016 |
| Rat, 2-3 day | 成骨细胞 / Osteoblasts | Collagenase Type 2: 0.2%； Trypsin: 0.25%-1.0% |
DMEM | Taylor, S., Shah, M. and Orriss, I.: Generation of Rodent and Human Osteoblasts., Bonekey Rep 3, 585, 2014 |
| Rat, fetal, 21 days of gestation | 卡尔瓦利亚 / Calvaria | Collagenase: 0.2% | MEM | Owen, T., Aronow, M., Shalhoub, V., Barone, L., Wilming, L., Tassinari, M., Kennedy, M., Pockwinse, S., Lian, J., and Stein, G.: Progressive Development of the Rat Oseoblast Phenotype In Vitro: Reciprocal Relationships in Expression of Genes Associated with Osteoblast Proliferation and Differentiation During Formation of the Bone Extracellular Matrix, J Cell Physiol 143, 420, 1990 |
| Rat, newborn | 成骨细胞样细胞 / Osteoblastlike cells | Collagenase Type 2: 0.3% | MEM | Ernst, M., and Froesch, E.: Osteoblastlike Cells in a Serum-Free Methylcellulose Medium Form Colonies: Effects of Insulin and Insulinlike Growth Factor I, Calcif Tissue Int 40, 27, 1987 |
| Rat, fetus, 17-21 day | 卡尔瓦利亚 / Calvaria | Collagenase: 0.01%-0.6% | Tris-buffered saline | Peck, W., Birge, S., and Fedak, S.: Bone Cells: Biochemical and Biological Studies After Enzymatic Isolation, Science 146, 1476, 1964 |
