原代细胞分离中，软骨组织分离涉及到软骨细胞、间充质、基质细胞囊泡、关节软骨细胞、骨骺和关节软骨等等的解离通常会使用酶，如何选择合适的酶来完成对应软骨组织的解离，以及原代细胞采集的常规操作， 艾美捷针对软骨组织相关的组织分类解离根据各种属、不同的细胞、酶组合推荐，推荐浓度、培养基以及参考文献汇总详细指南：
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
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| 种属 | 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 |
| 牛 | Bovine, 18-36 month | 软骨细胞 / Chondrocytes | Collagenase Type 1: 0.1% | DMEM | White, R. and Gibson, J.: The Effect of Oxygen Tension on Calcium Homeostasis in Bovine Articular Chondrocytes., J Orthop Surg Res 5, 27, 2010 |
| 牛 | Bovine, 6-8 week | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.2% | DMEM | Hwang, Y., Sangaj, N. and Varghese, S.: Interconnected Macroporous Poly(ethylene glycol) Cryogels as a Cell Scaffold for Cartilage Tissue Engineering., Tissue Eng Part A 16, 3033-41, 2010 |
| 牛 | Bovine, 1-3 month | 软骨细胞 / Chondrocytes | Collagenase Type 4: 390 u/ml | DMEM | Lima, E., Tan, A., Tai, T., Marra, K., DeFail, A., Ateshian, G. and Hung, C.: Genipin Enhances the Mechanical Properties of Tissue-Engineered Cartilage and Protects Against Inflammatory Degradation When Used as a Medium Supplement., J Biomed Mater Res 91, 692, 2009 |
| 牛 | Steers, 1-2 years | 软骨细胞 / Chondrocytes | Trypsin: 0.20% | HBSS | Mackintosh, D., and Mason, R.: Pharmacological Actions of 17 Beta-oestradiol on Articular Cartilage Chondrocytes and Chondrosarcoma Chondrocytes in the Absence of Oestrogen Receptors, Biochim Biophys Acta 964, 295, 1988 |
| 牛 | Calf, 1-14 days | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.20% | PBS | Klagsbrun, M.: Large Scale Preparation of Chondrocytes,Methods of Enzymology Vol. 58,, , 560, 1979 |
| 犬/狗 | Canine | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.3%； Trypsin: 0.25% |
Ham's F12 | Lee, J., Kim, H., Kim, J., Bae, S., Joo, D., Huh, K., Fang, Y., Jeong, J., Kim, M. and Kim, Y.: Microencapsulation of Pancreatic Islets with Canine Ear Cartilage for Immunoisolation., Transplant Proc 44, 1091-4, 2012 |
| 鸡 | Chick embryos, HH stage | 间充质 / Mesenchymal | Trypsin: 0.1% | DMEM；(see reference) | Wong, M., and Tuan, R.: Nuserum, A Synthetic Serum Replacement, Supports Chondrogenesis of Embryonic Chick Limb Bud Mesenchymal Cells in Micromass Culture, In Vitro Cell Dev Biol 29A, 917, 1993 |
| 鸡 | Chicken, broiler strain, 8-10 weeks | 基质细胞囊泡，骨骺生长板 / Matrix vesicles，Epiphyseal growth plate | Trypsin: 0.1% | Tris-buffered saline | Genge, B., Wu, L. and Wuthier, R.: Differential Fractionation of Matrix Vesicle Proteins: Further Characterization of the Acidic Phospholipid-dependent Ca2+-Binding Proteins, J Biol Chem 265, 4703, 1990 |
| 鸡 | Chicken, broiler strain, 8-10 week | 基质细胞囊泡 / Matrix vesicles | Trypsin: 0.1% | (see reference) | Genge, B.R., Wu, L.N.Y., and Wuthier, R.E.: Identification of Phospholipid-dependent Calcium-binding Proteins as Constituents of Matrix Vesicles, J Biol Chem 264, 10917, 1989 |
| 鸡 | Chick embryos, White Leghorn, 19 day old | 成纤维细胞，上皮样 / Fibroblasts，Epithelial-like | Trypsin: 0.25% | E 199 medium | Gionti, E., Capasso, O., and Cancedda, R.: The Culture of Chick Embryo Chondrocytes and the Control of Their Differentiated Functions in Vitro, J Biol Chem 258 (11), 7190, 1983 |
| 鸡 | Chick embryos, 19 day old | 软骨细胞 / Chondrocytes | Trypsin: 0.75% | Coon's modified F-12 | Capasso, O., Gionti, E., Pontarelli, G., Ambesi-Impiobato, F., Nitsch, L., Tajana, G., and Cancedda, R.: The Culture of Chick Embryo Chondrocytes and the Control of Their Differentiated Functions In Vitro, Exp Cell Res 142, 197, 1982 |
| 鸡 | Chick embryos, White Leghorn | 翼芽 / Wing buds | Trypsin: 0.1% | Saline G | Ahrens, P., Solursh, M., and Reiter, R.: Stage-Related Capacity for Limb Chondrogenesis in Cell Culture, Dev Biol 60, 69, 1977 |
| 马 | Equine | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.15% | DMEM | Visser, J., Levett, P., te Moller, N., Besems, J., Boere, K., van Rijen, M., de Grauw, J., Dhert, W., van Weeren, P. and Malda, J.: Crosslinkable Hydrogels Derived from Cartilage, Meniscus, and Tendon Tissue., Tissue Eng Part A 21, 1195-206, 2015 |
| 马 | Equine | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.08% | DMEM/F12 | Ley, C., Svala, E., Nilton, A., Lindahl, A., Eloranta, M., Ekman, S. and Skioldebrand, E.: Effects of High Mobility Group Box Protein-1, Interleukin-1B, and Interleukin-6 on Cartilage Matrix Metabolism in Three-Dimensional Equine Chondrocyte Cultures., Conn Tissue Res 52, 290-300, 2011 |
| 山羊 | Goat | 软骨细胞 / Chondrocytes | Collagenase Type 2: 2% | DMEM | Bekkers,J, Creemers, L, Tsuchida, A, van Rijen, M, Custers, R, Dhert, W and Saris, D.: One-Stage Focal Cartilage Defect Treatment with Bone Marrow Mononuclear Cells and Chondrocytes Leads to Better Macroscopic Cartilage Regeneration Compared to Microfracture in Goats., Osteoarthritis Cartilage 21, 950-6, 2013 |
| 人 | Human | 软骨细胞 / Chondrocytes | Trypsin: 0.25%； Hyaluronidase: 0.1%； Collagenase Type 2: 1000 u/ml； Collagenase Type 1: 400 u/ml |
Ham's F12 | Muraglia, A., Nguyen, V., Nardini, M., Mogni, M., Coviello, D., Dozin, B., Strada, P., Baldelli, I., Formica, M., Cancedda, R. and Mastrogiacomo, M.: Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support., Front Bioeng Biotechnol 5, 66, 2017 |
| 人 | Human | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.15% | DMEM | Andrews, S., Kunze, M., Mulet-Sierra, A., Williams, L., Ansari, K., Osswald, M. and Adesida, A.: Strategies to Mitigate Variability in Engineering Human Nasal Cartilage., Sci Rep 7, 6490, 2017 |
| 人 | Human | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.15% | DMEM | Capsoni, F., Ongari, A., Lonati, C., Accetta, R., Gatti, S. and Catania, A.: Alpha-Melanocyte-Stimulating-Hormone (a-MSH) Modulates Human Chondrocyte Activation Induced by Proinflammatory Cytokines., BMC Musculoskelet Disord 16, 154, 2015 |
| 人 | Human | 软骨细胞 / Chondrocytes | Collagenase: 0.3% | DMEM | Tamai, M., Nagasao, T., Yanaga, H., Hamamoto, Y., Kogure, T. and Tanaka, Y.: Correction of Secondary Deformity after Nuss Procedure for Pectus Excavatum by Means of Cultured autologous Cartilage Cell Injection., Int J Surg Case Rep 15, 70-3, 2015 |
| 人 | Human, 52-82 years | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.2%； Pronase: 0.15% |
DMEM/F12 | Pallu, S., Francin, P., Guillaume, C., Gegout-Pottie, P., Netter, P., Mainard, D., Terlain, B. and Presle, N.: Obesity Affects the Chondrocyte Responsiveness to Leptin in Patients with Osteoarthritis., Arthritis Res Ther 12, R112, 2010 |
| 人 | Human | 滑膜细胞 / Synoviocytes | Collagenase Type 1: 0.4% | DMEM | Kim Wan-Uk, Kwok Seung-Ki, Hong Kyung-Hee, Yoo Seung-Ah, Kong Jin-Sun, Choe Jongseon, Cho Chul-Soo: Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis, Arthritis Res Ther 9, R42, 2007 |
| 人 | Human, 26-68 year | 半月板和软骨 / Meniscus and cartilage | Collagenase Type 2: 0.15% | DMEM | Marsano A, Millward-Sadler SJ, Salter DM, Adesida A, Hardingham T, Tognana E, Kon E, Chiari-Grisar C, Nehrer S, Jakob M, Martin I: Differential cartilaginous tissue formation by human synovial membrane, fat pad, meniscus cells and articular chondrocytes, Osteoarthritis Cartilage 15, 48-58, 2007 |
| 人 | Human, 64-83 yr | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.08% | DMEM/F12 | Tallheden T, Bengtsson C, Brantsing C, Sjogren-Jansson E, Carlsson L, Peterson L, Brittberg M, and Lindahl A.: Proliferation and Differentiation Potential of Chondrocytes from Osteoarthritic Patients, Arthritis Res Ther 7(3), R560, 2005 |
| 人 | Human, adult | 滑膜 / Synovial | Collagenase: 0.15%； Hyaluronidase: 0.1%； Deoxyribonuclease I: 0.015% |
DMEM | Liagre B, Vergne-Salle P, Corbiere C, Charissoux JL, and Beneytout JL.: Diosgenin, a Plant Steroid, Induces Apoptosis in Human Rheumatoid Arthritis Synoviocytes with Cyclooxygenase-2 Overexpression, Arthritis Res Ther 6(4), R373, 2004 |
| 人 | Human, adult | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.15% | DMEM | Jakob M, Demarteau O, Schafer D, Stumm M, Heberer M, and Martin I.: Enzymatic Digestion of Adult Human Articular Cartilage Yields a Small Fraction of the Total Available Cells, Conn Tissue Res 44, 173, 2003 |
| 人 | Human, adults | 滑膜细胞 / Synoviocytes | Collagenase Type 1: 0.1% | RPMI | McEvoy A., Murphy E., Ponnio T., Conneely O., Bresnihan B., FitzGerald O., and Murphy E.: Activation of Nuclear Orphan Receptor NURR1 Transcription by NF-kappa B and Cyclic Adenosine 5'-Monophosphate Response Element-binding Protein in Rheumatoid Arthritis Synovial Tissue, J Immunol 168(6), 2979, 2002 |
| 人 | Human, 15-60 years | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.2% | DMEM | Rotter N, Bonassar LJ, Tobias G, Lebl M, Roy AK, Vacanti CA: Age dependence of cellular properties of human septal cartilage: implications for tissue engineering, Arch Otolaryngol Head Neck Surg 127, 1248-52, 2001 |
| 人 | Human | 滑膜成纤维细胞 / Synovial fibroblasts | Collagenase: 0.4% | DMEM | Sarkissian M, Lafyatis R: Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts, J Immunol 162, 1772-9, 1999 |
| 人 | Human | 隔膜软骨细胞 / Septal chondrocytes | Collagenase Type 2: 0.2%； Hyaluronidase: 0.01%； Deoxyribonuclease I: 0.015% |
DMEM/F12 | Dunham B P, Koch R J: Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment, Arch Otolaryngol Head Neck Surg 124, 1325-30, 1998 |
| 人 | Human w/ rheumatoid arthritis | 滑膜组织 / Synovial tissue | Trypsin: 0.05% | DMEM (see reference) | Dayer, J., Krane, S., Russell, R., and Robinson, D.: Production of Collagenase and Prostaglandins by Isolated Adherent Rheumatoid Synovial Cells, Proc Natl Acad Sci U S A 73 (3), 945, 1976 |
| 人 | Human, 13-62 years | 关节软骨细胞 / Articular chondrocytes | Trypsin: 0.2% | BSS | Srivastava, V.M.L., Malemud, C.J., Hough, A.J., Bland, J.H., and Sokoloff, L.: Preliminary Experience with Cell Culture of Human Articular Chondrocytes, Arthritis Rheum 17, 165, 1974 |
| 人 | Human, 26-84 years | 软骨细胞 / Chondrocytes | Collagenase: | GBSS | Manning, W.K., and Bonner, W.M.: Isolation and Culture of Chondrocytes From Human Adult Articular Cartilage, Arthritis Rheum 10, 235, 1967 |
| 小鼠 | Mouse, 2-5 day | 软骨细胞 / Chondrocytes | Trypsin: 0.25%； Collagenase Type 1: 86.5 u/ml |
DMEM | Otsuru, S., Hofmann, T., Raman, P., Olson, T., Guess, A., Dominici, M. and Horwitz, E.: Genomic and Functional Comparison of Mesenchymal Stromal Cells Prepared Using Two Isolation Methods., Cytotherapy 17, 262-70, 2015 |
| 小鼠 | Mouse, 1 day | 软骨细胞 / Chondrocytes | Collagenase: 0.2% | DMEM | Terpstra, L, Prud'homme, J, Arabian, A, Takeda, S, Karsenty, G, Dedhar, S, and St-Arnaud, R.: Reduced Chondrocyte Proliferation and Chondrodysplasia in Mice Lacking the Integrin-linked Kinase in Chondrocytes, J Cell Biol 162, 139, 2003 |
| 绵羊 | Sheep, 2 month | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.3% | Ham's F-12 | Kojima Koji, Bonassar Lawrence J, Roy Amit K, Mizuno Hirokazu, Cortiella Joaquin, Vacanti Charles A: A Composite Tissue-Engineered Trachea Using Sheep Nasal Chondrocyte and Epithelial Cells, FASEB J 17, 823-8, 2003 |
| 猪 | Porcine, 1 year | 软骨细胞 / Chondrocytes | Collagenase Type 1: 0.2% | DEMEM | Chowdhury, T., Schulz, R., Rai, S., Thuemmler, C., Wuestneck, N., Bader, A and Homandberg, G: Biomechanical Modulation of Collagen Fragment-Induced Anabolic and Catabolic Activities in Chondrocyte/Agarose Constructs., Arthritis Res Ther 12, R82, 2010 |
| 猪 | Porcine, 2-4 month | 软骨 / Chondrons | Neutral Protease: 0.3%； Collagenase: 0.2% |
PBS | Graff RD, Lazarowski ER, Banes AJ, Lee GM: ATP release by mechanically loaded porcine chondrons in pellet culture, Arthritis Rheum 43, 1571-9, 2000 |
| 兔 | Rabbit, New Zealand, 1.2-1.4 kg | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.025%； Pronase: 0.2% |
DMEM | Ju, X., Deng, M., Ao, Y., Yu, C., Wang, J., Yu, J., Cui, G. and Hu, Y.: Protective Effect of Sinomenine on Cartilage Degradation and Chondrocytes Apoptosis., Yakugaku Zasshi 130, 1053-60, 2010 |
| 兔 | Rabbit, New Zealand, 1.8-2.3kg | 软骨细胞 / Chondrocytes | Hyaluronidase: .05%； Collagenase Type 2: 0.2%； Trypsin: 0.2% |
Gey's solution | Mehraban F, Tindal MH, Proffitt MM, Moskowitz RW.: Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion, Ann Rheum Dis 56, 108, 1997 |
| 兔 | Rabbit, New Zealand, white, 4-6 wk & 22-25 wk | 软骨细胞 / Chondrocytes | Protease XIV: 5 mg/g of tissue | Ham's F-12 | Plaas, A., Sandy, J., and Kimura, J.: Biosynthesis of Cartilage Proteoglycan & Link Protein Articular Chondrocytes, J Biol Chem 263, 7560, 1988 |
| 兔 | Rabbit, white, male, 8 weeks | 软骨细胞 / Chondrocytes | Trypsin: 0.2% | Gey's BSS | Benya, P.D., Padilla, S.R., and Nimni, M.E.: The Progeny of Rabbit Articular Chondrocytes Synthesize Collagen Types I and III and Type I Trimer, but Not Type II, Biochemistry 16, 865, 1977 |
| 兔 | Rabbit, New Zealand white or Dutch, 1 week (also human, newborn) | 软骨细胞 / Chondrocytes | Trypsin: 0.1% | Saline G, CMF | Schindler, F.H., Ose, M.A., and Solursh, M.: The Synthesis of Cartilage Collagen by Rabbit and Human Chondrocytes in Primary Cell Culture, In Vitro 12, 44, 1976 |
| 兔 | Rabbit, New Zealand white, immature, 2.25 - 3.3 Kg | 关节软骨细胞，透明 / Articular chondrocytes，Hyaline | Trypsin: 0.2% | Gey's BSS | Green, J.R., and William, T.: Articular Cartilage Repair. Behavior of Rabbit Chondrocytes During Tissue Culture and Subsequent Allografting, Clin Orthop Relat Res , 237, 1976 |
| 兔 | Rabbit, New Zealand white, male, 250-350 g | 骨骺，关节软骨 / Epiphyseal，Articular cartilage | Trypsin: 0.25% | Eagle's basal medium | Bentley, G., and Greer , R.: Homotransplantation of Isolated Epiphyseal and Articular Cartilage Chondrocytes into Joint Surfaces of Rabbits, Nature 230, 385, 1971 |
| 大鼠 | Rat | 软骨细胞 / Chondrocytes | Collagenase Type 2: 0.15%； Trypsin: 0.25% |
DMEM/F12 | Mei, L., Shen, B., Ling, P., Liu, S., Xue, J., Liu, F., Shao, H., Chen, J., Ma, A. and Liu, X.: Culture-expanded Allogenic Adipose Tissue-derived Stem Cells Attenuate Cartilage Degeneration in an Experimental Rat Osteoarthritis Model., PLoS ONE 12, e0176107, 2017 |
| 大鼠 | Rat, SD, young, 100-120 g | 软骨细胞 / Chondrocytes | Trypsin: 0.2% | Ham's F-12 medium | Shimomura, Y., Yoneda, T., and Suzuki, F.: Osteogenesis by Chondrocytes from Growth Cartilage of Rat Rib, Calcif Tissue Res 19, 179, 1975 |
