軟骨組織消化手冊

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Worthington組織消化指南

引言

組織消化酶主要用于組織培養和細胞生物學研究的原代細胞的分離和收集。盡管這些消化酶已經普遍應用多年，但是對其組織消化和細胞收集的作用機制卻沒有全面的理解。由此導致的結果便是選擇一種消化方法大多是隨機選擇或基于過去的經驗，而不是在*理解消化原理的基礎上選擇的消化方案，以及如何根據具體實驗目的修正消化方案以獲得更好的實驗結果。

細胞分離程序的目的是zui大程度的獲得功能活性的單個細胞，而影響結果的原因有很多，包括（不限于）以下：

1、組織類型

2、物種來源

3、動物年齡

4、遺傳改造情況（如knockouts）

5、所用的消化培養液

6、所用的酶

7、消化酶中的雜質

8、所用消化酶的工作濃度

9、溫度

10、孵育時間

通常前四個原因沒辦法選擇，而剩下的原因一般由經驗決定以獲取zui適實驗結果。

通過查閱文獻來選擇*組織消化酶和zui適消化條件的研究人員通常被互相矛盾的實驗數據搞得一頭霧水。這些巨大差異來源于組織胞外基質的復雜而動態的特性，以及過去常用的粗制、未提純的組織分離酶。另外，組織胞外基質由各種蛋白、糖蛋白、脂類和糖脂組成，這些成分的組成比例在不同物種、不同組織、甚至不同發育（年齡）之間存在明顯的差異，這些因素都極大的影響了消化方案的制定。目前通常使用的粗制酶，如鏈霉蛋白酶、NF1:250、以及含有幾種其他不同濃度蛋白酶的膠原酶、以及各種多糖酶、核酶和脂酶。

本指南總結了目前常用組織消化酶的zui適消化和細胞收集知識和方法，描述了標準的實驗步驟，以便于制定一個*的細胞分離方案，同時列舉了一些相關的參考文獻。

下表列出多種組織的適宜消化酶（含參考文獻，以組織類型和物種分類，詳見鏈接）

脂肪組織	腎上腺組織	骨組織	腦組織
軟骨組織	結腸組織	內皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結	乳腺
其它組織	其他組織	肌組織	神經組織
胰腺	腮腺	垂體	前列腺
生殖	動物鱗片	皮膚	脾臟
干細胞	胸腺	甲狀腺/甲狀旁腺	扁桃體
腫瘤

注：使用Worthington公司消化酶的文獻不限于上表鏈接中所列，一般而言，Worthington公司的組織消化酶可用于大多組織類型的消化。

軟骨組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
牛	牛, 18-36個月	Chondrocytes	Collagenase Type 1: 0.1%	DMEM	White, R. and Gibson, J.: The Effect of Oxygen Tension on Calcium Homeostasis in 牛 Articular Chondrocytes., J Orthop Surg Res 5, 27, 2010
牛	牛, 6-8 周	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	牛, 1-3 個月	Chondrocytes	Collagenase Type 4: 390 u/ml	hgDMEM	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	控制, 1-2 歲	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	牛犢, 1-14 天	Chondrocytes	Collagenase Type 2: 0.20%	PBS	Klagsbrun, M.: Large Scale Preparation of Chondrocytes,Methods of Enzymology Vol. 58,, , 560, 1979
犬	犬	Chondrocytes	Collagenase Type 2: 0.3%	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
犬	犬	Chondrocytes	Trypsin: 0.25%	Ham's F12
雞	小雞embryos, HH stage	Mesenchymal	Trypsin: 0.1%	DMEM	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
雞	小雞embryos, HH stage	Mesenchymal	Trypsin: 0.1%	(see reference)
雞	雞肉,肉雞應變, 8-10 周	Matrix vesicles	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
雞	雞肉,肉雞應變, 8-10 周	Epiphyseal growth plate	Trypsin: 0.1%	Tris-buffered saline
雞	雞肉,肉雞應變, 8-10 周	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
雞	小雞胚胎, 白來杭雞, 19 天大的	Fibroblasts	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
雞	小雞胚胎, 白來杭雞, 19 天大的	Epithelial-like	Trypsin: 0.25%	E 199 medium
雞	小雞胚胎, 19 天大的	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
雞	小雞胚胎, 白來杭雞	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
馬	馬	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	山羊	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
人類	人類	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
人類	人類	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 Vol. 15,, , 70-3, 2015
人類	人類, 52-82 歲	Chondrocytes	Collagenase Type 2: 0.2%	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
人類	人類, 52-82 歲	Chondrocytes	Pronase: 0.15%	DMEM/F12
人類	人類	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
人類	人類, 26-68 歲	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
人類	人類, 64-83 歲	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
人類	人類, 成年的	Synovial	Collagenase: 0.15%	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
			Hyaluronidase: 0.1%
			Deoxyribonuclease I: 0.015%
人類	人類, 成年的	Chondrocytes	Collagenase Type 2: 0.15%	DMEM	Jakob M, Demarteau O, Schafer D, Stumm M, Heberer M, and Martin I.: Enzymatic Digestion of 成年的 Human Articular Cartilage Yields a Small Fraction of the Total Available Cells, Conn Tissue Res 44, 173, 2003
人類	人類, 成年的	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
人類	人類, 15-60 歲	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
人類	人類	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
人類	人類	Septal chondrocytes	Collagenase Type 2: 0.2%	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
			Hyaluronidase: 0.01%
			Deoxyribonuclease I: 0.015%
人類	人類 ,風濕性關節炎	Synovial tissue	Trypsin: 0.05%	DMEM	天er, 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
人類	人類 ,風濕性關節炎	Synovial tissue	Trypsin: 0.05%	(see reference)
人類	人類, 13-62 歲	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
人類	人類, 26-84 歲	Chondrocytes	Collagenase:	GBSS	Manning, W.K., and Bonner, W.M.: Isolation and Culture of Chondrocytes From Human 成年的 Articular Cartilage, Arthritis Rheum 10, 235, 1967
小鼠	小鼠, 2-5 天	Chondrocytes	Trypsin: 0.25%	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	小鼠, 2-5 天	Chondrocytes	Collagenase Type 1: 86.5 u/ml	DMEM
小鼠	小鼠, 1 天	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
綿羊	綿羊, 2 個月	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
豬	豬, 1歲	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
豬	豬, 2-4 個月	Chondrons	Neutral Protease: 0.3%	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
豬	豬, 2-4 個月	Chondrons	Collagenase: 0.2%	PBS
兔	兔, 新西蘭, 1.2-1.4公斤	Chondrocytes	Collagenase Type 2: 0.025%	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	兔, 新西蘭, 1.2-1.4公斤	Chondrocytes	Pronase: 0.2%	DMEM
兔	兔, 新西蘭, 1.8-2.3公斤	Chondrocytes	Hyaluronidase: .05%	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
			Collagenase Type 2: 0.2%
			Trypsin: 0.2%
兔	兔, 新西蘭, 白色, 4-6 周 & 22-25 周	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	兔, 白色, 雄性, 8 周	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	兔, 新西蘭白或荷蘭的, 1 周	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	(還有人類, 初生)	Chondrocytes	Trypsin: 0.1%	Saline G, CMF
兔	兔, 新西蘭白, 幼小的, 2.25 - 3.3 公斤	Articular chondrocytes	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	兔, 新西蘭白, 幼小的, 2.25 - 3.3 公斤	Hyaline	Trypsin: 0.2%	Gey's BSS
兔	兔, 新西蘭白, 雄性, 250-350 克	Epiphyseal	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
兔	兔, 新西蘭白, 雄性, 250-350 克	Articular cartilage	Trypsin: 0.25%	Eagle's basal medium
大鼠	大鼠, SD, 年幼的, 100-120 克	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

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軟骨組織消化手冊