內(nèi)皮細胞組織消化手冊

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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(yǎng)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(jīng)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(jié)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(jīng)驗，而不是在*理解消化原理的基礎(chǔ)上選擇的消化方案，以及如何根據(jù)具體實驗目的修正消化方案以獲得更好的實驗結(jié)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(jié)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(yǎng)液

6、所用的酶

7、消化酶中的雜質(zhì)

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(jīng)驗決定以獲取zui適實驗結(jié)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(shù)據(jù)搞得一頭霧水。這些巨大差異來源于組織胞外基質(zhì)的復雜而動態(tài)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(zhì)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(fā)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(jié)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(guān)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(jié)腸組織	內(nèi)皮組織	上皮組織
眼	心臟	腸	腎
肝	肺	淋巴結(jié)	乳腺
其它組織	其他組織	肌組織	神經(jīng)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。

內(nèi)皮細胞組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養(yǎng)基	參考文獻
牛	牛	Bovine umbilical cord (BUVEC)	Collagenase: 0.1%	Dulbecco's/Ham F-12	Ricken Albert M, Traenkner Anja, Merkwitz Claudia, Hummitzsch Katja, Grosche Jens, Spanel-Borowski Katharina: The short prolactin receptor predominates in endothelial cells of micro- and macrovascular origin, J Vasc Res 44, 19-30, 2007
牛	牛	Pulmonary artery endothelial and smooth muscle cells	Collagenase: 0.04-0.05%	RPMI-1640	Yu, M., McAndrew, R., Al-Saghir, R., Maier, K., Medhora, M., Roman, R. and Jacobs, E.: Nitric Oxide Contributes to 20-HETE-Induced Relaxation of Pulmonary Arteries., J Appl Physiol 93, 1391, 2002
			Soybean Trypsin Inhibitor: 0.04%
牛	牛, (歐洲牛), 牛犢	Endothelial	Collagenase: 1000 u/ml	PBS, CMF	Del Vecchio, P.J., Siflinger-Birnboim, A., Belloni, P.N., Holleron, L.A., Lum, H., and Malik, A.B.: Culture and Characterization of Pulmonary Microvascular Endothelial Cell, In Vitro Cell Dev Biol 28A, 711, 1992
		Pulmonary
牛	牛犢	Endothelial	Trypsin: 0.25%	HEPES	Vender, R.: Role of Endothelial Cells in the Proliferative Response of Cultured Pulmonary Vascular Smooth Muscle Cells to Reduced Oxygen Tension, In Vitro Cell Dev Biol 28A, 403, 1992
牛	牛, 成年的	Cerebral artery	Collagenase: 0.2%	HBSS	Machi, T., Kassell, N.F., and Scheld, W.M.: Isolation and Characterization of Endothelial Cells From Bovine Cerebral Arteries, In Vitro Cell Dev Biol 26, 291, 1990
		Endothelial
牛	牛	Endothelial	Trypsin: 0.05%	Krebs Ringer solution	DeNucci, G., Gryglewski, R.J., Warner, T.D., and Vane, J.R.: Receptor-Mediated Release of Endothelium-Derived Relaxing Factor and Prostacyclin From Bovine Aortic Endothelial Cells Is Coupled, Proc Natl Acad Sci U S A 85, 2334, 1988
		Aortic
牛	牛	Endothelial, pulmonary artery	Collagenase: 0.1%	CMF-Dulbecco's PBS	Martin, T.: Formation of Diacylglycerol by a Phospholipase D-phosphatidate Phosphatase Pathway Specific for Phosphatidylcholine in Endothelial Cells, Biochim Biophys Acta 962, 282, 1988
牛	牛	Endothelial	Collagenase Type 2: 0.1%	PBS	Carson, M.P. and Haudenschild, C.C.: Microvascular Endothelium and Pericytes: High Yield, Low Passage Cultures, In Vitro Cell Dev Biol 22, 344, 1986
		Aorta
牛	牛	Endothelial	Collagenase Type 2: 0.1%	DMEM	Kinsella, Michael G. and Wight, Thomas N.: Modulation of Sulfated Proteoglycan Synthesis by Bovine Aortic Endothelial Cells During Migration, J Cell Biol 102, 679, 1986
		Aortic
牛	牛	Endothelial	Collagenase: 0.5%	DMEM/Ham's F-12	Gospodarowicz, D., Massoglia, S., Cheng, J., and Fujii, D.: Effect of Fibroblast Growth Factor and Lipoproteins on the Proliferation of Endothelial Cells Derived From Bovine Adrenal Cortex, Brain Cortex, and Corpus Luteum Capillaries, J Cell Physiol 127, 121, 1986
牛	牛	Endothelial	Collagenase Type 2: 0.2%	Dulbecco's PBS	Goetz, I.., Warren, J., Estrada, C., Roberts, E., and Krause, D.: Long-Term C*tion of Arterial and Capillary Endothelium From Adult Bovine Brain, In Vitro Cell Dev Biol 21, 172, 1985
		Brain arteries
牛	牛犢	Endothelial	Collagenase: 0.75%	DMEM	Voyta, J., Via, D., Butterfield, C., and Zetter, B.: Identification and Isolation of Endothelial Cells Based on Their Increased Uptake of Acetylated-Low Density Lipoprotein, J Cell Biol 99, 2034, 1984
		Smooth muscle
牛	牛	Endothelial	Trypsin: 0.2%	PBS: DMEM	Scott, D., Murray, J., and Barnes, M.: Investigation of the Attachment of Bovine Corneal Endothelial Cells, Exp Cell Res 144, 472, 1983
		Corneal
牛	牛, 2-3 周	Endothelial	Collagenase Type 1: 0.2%	RPMI 1640 w/1% Fetal Bovine Serum	Lee, S., Douglas, W., Deneke, S., and Fanburg, B.: Ultrastructural Changes in Bovine Pulmonary Artery Endothelial Cells Exposed to 80% O2 In Vitro, In Vitro 19, 714, 1983
		Pulmonary artery
牛	牛	Endothelial, Corneal	Trypsin: 0.05%	0.01M Phosphate buffer with 0.02% EDTA	Robinson, J. and Gospodarowicz, D.: Glycosaminoglycans Synthesized by Cultured Bovine Corneal Endothelial Cells, J Cell Physiol 117, 368, 1983
				0.9% NaCl (See Reference)
牛	牛	Endothelial	Collagenase Type 1: 0.10%	PBS	Olander, J., Marasa, J., Kimes, R., Johnston, G., and Feder, J.: An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells, In Vitro 18, 99, 1982
		Subclavian vein
牛	牛	Aortic	Collagenase Type 2: 0.10%	PBS	Makarski, J. S.: Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells, In Vitro 17, 450, 1981
		Pulmonary artery
牛	牛犢, 胎兒, 4-9 個月	Endothelial	Collagenase Type 1: 0.25%	PBS	Rosen, E., Mueller, S., Noveral, J., and Levine, E.: Proliferative Characteristics of Clonal Endothelial Cell Strains, J Cell Physiol 107, 123, 1981
牛	牛, 成年的	Aorta	Collagenase Type 1: 125 u/ml	Dulbecco's PBS with calcium and magnesium	Cotta-Pereira, G., Sage, H., Bornstein, P., Ross, R., and Schwartz, S.: Studies of Morphologically Atypical ("Sprouting") Cultures of Bovine Aortic Endothelial Cells. Growth Characteristics and Connective Tissue Protein Synthesis, J Cell Physiol 102, 183, 1980
牛	牛, 年幼的	Pulmonary artery	Collagenase: 0.1%	Medium 199	Ryan, U., Mortara, M., and Whitaker, C.: Methods for Microcarrier Culture of Bovine Pulmonary Artery Endothelial Cells Avoiding the Use of Enzymes, Tissue Cell12, 619, 1980
牛	牛	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
牛	牛	Saphenous Vein	Collagenase: 0.01%	PBS	Eskin, S., Sybers, H., Trevino, L., Lie, J., and Chimoskey, J.: Comparison of Tissue-Cultured Bovine Endothelial Cells from Aorta and Saphenous Vein, In Vitro 14, 903, 1978
		Aorta
牛	牛	Endothelial	Collagenase Type 2: 0.1%	PBS	Schwartz, S.M.: Selection and Characterization of Bovine Aortic Endothelial Cells,In Vitro 14, 966, 1978
		Thoracic aorta
		Saphenous veins
牛	牛	Pulmonary artery	Collagenase Type 2: 0.25%	Puck's solution	Ryan, U.S., Clements, E., Habliston, D., and Ryan, J.W.: Isolation And Culture of Pulmonary Artery Endothelial Cells, Tissue Cell 10, 535, 1978
牛	牛犢	Endothelial	Collagenase Type 2: 0.1%	DMEM	Howard, B., Macarak, E., Gunson, D., and Kefalides, N.: Characterization of the Collagen Synthesized by Endothelial Cells in Culture, Proc Natl Acad Sci U S A 73 (7), 2361, 1976
犬	狗	Microvessels	Collagenase Type 4: 0.1%	DMEM	Gerhart, D. Z., Broderius, M. A., and Drewes, L. R.: Cutlured Human and Canine Endothelial Cells from Brain Microvessels, Brain Res Bull 21, 785, 1988
	(包括人類)
犬	狗, 雜種狗, 成年的	Endothelium	Trypsin: 0.1%	Earle's PBS, CMF	Ford, J., Burkel, W., and Kahn, R.: Isolation of Adult Canine Venous Endothelium for Tissue Culture, In Vitro 17, 44, 1981
		Jugular vein
豚鼠	豚鼠, 雌性, 300-350克	Coronary endothelial	Collagenase Type 2: 0.1%	see reference	Buxton I L, Kaiser R A, Oxhorn B C, Cheek D J: Evidence supporting the Nucleotide Axis Hypothesis: ATP release and metabolism by coronary endothelium,Am J Physiol Heart Circ Physiol 281, H1657-66, 2001
人類	人類	Vascular endothelial	Collagenase Type 1: 0.1%	HBSS	Ganguly, A., Zhang, H., Sharma, R., Parsons, S. and Pa, K.: Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions., J Vis Exp 66, e4032, 2012
人類	人類, 18-68 yr	Corneal endothelial	Collagenase: 0.2%	DMEM/F12	Li Wei, Sabater Alfonso L, Chen Ying-Ting, Hayashida Yasutaka, Chen Szu-Yu, He Hua, Tseng Scheffer C G: A novel method of isolation, preservation, and expansion of human corneal endothelial cells, Inv Ophthal Visual Sci 48, 614-20, 2007
			Neutral Protease: 1.0%
人類	人類	Endothelial and vascular smooth muscle	Collagenase Type 1: 0.2%	HBSS	Moss, S., Bates, M., Parrino, P. and Woods, TC.: Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue., Ochsner J 7, 133, 2007
人類	人類	HUVEC	Collagenase Type 1: 0.1%	HBSS	Davis, J., Crampton, S. and Hughes. C.: Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)., J Vis Exp 3, 183, 2007
人類	人類	HUVEC	Collagenase Type 4: 0.1%	RPMI 1640	Silva AP, Kaufmann JE, Vivancos C, Fakan S, Cavadas C, Shaw P, Brunner HR, Vischer U, and Grouzmann E.: Neuropeptide Y expression, localization and cellular transducing effects in HUVEC, Biol Cell 97(6), 457, 2005
人類	人類	Endothelial	Collagenase Type 2: 0.1%	DMEM	Pa, V., Logan, A., Watkinson, J., Uz-Zaman, S., Sheppard, M., Ramsden, J. and Eggo, M.: Isolation and Characterization of Human Thyroid Endothelial Cells.,Am J Physiol Endocrinol Metab 284, E168, 2003
人類	人類	Esophageal microvascular endothelial	Collagenase Type 2: 0.2%	MCDB-131	Rafiee, P., Ogawa, H., Heidemann, J., Li, M., Aslam, M., Lamirand, T., Fisher, P., Graewin, S., Dwinell, M., Johnson, C., Shaker, R. and Binion, D.: Isolation and Characterization of Human Esophageal Microvascular Endothelial Cells: Mechanisms of Inflammatory Activation., Am J Physiol Gastrointest Liver Physiol285, G1277, 2003
人類	人類	Colonic endothelial cells	Collagenase Type 2: 0.25%	HBSS/5%FBS	Wang D., Lehman R., Donner D., Matli M., Warren R., and Welton M.: Expression and Endocytosis of VEGF and Its Receptors in Human Colonic Vascular Endothelial Cells, Am J Physiol/Gastro 282, G1088, 2002
人類	人類	Pulmonary vascular endothelial cells	Neutral Protease: 1.18 u/ml	M199	Muller AM, Hermanns MI, Skrzynski C, Nesslinger M, Muller KM, and Kirkpatrick CJ.: Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro, Exp Mol Pathol 72, 221, 2002
			Elastase: 10 u/ml
人類	人類	HUVEC	Collagenase Type 2: 0.1%	PBS	Takano Manabu, Meneshian Avedis, Sheikh Emran, Yamakawa Yasuhiko, Wilkins Kirsten Bass, Hopkins Elise A, Bulkley Gregory B: Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation, Am J Physiol Heart Circ Physiol 283, H2054-61, 2002
人類	人類	Endothelial	Trypsin: 2%	PBS	Goolcharran, C., Cleland, J., Keck, R., Jones, A., and Borchardt, R.: Comparsion of the Rates of Deamidation, Diketopiperazine Formation and Oxidation in Recombinant Human Vascular Endothelial Growth Factor and Model Peptides,AAPS PharmSci 2 (1), 5, 2000
人類	人類	HUVEC, porcine pulmonary arterial endothelial cells	Collagenase Type 2: 0.2%	DMEM	Kwak HJ, Lee SJ, Lee YH, Ryu CH, Koh KN, Choi HY, and Koh GY.: Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells, Circulation 101(19), 2317, 2000
人類	人類	Vascular endothelial cells	Collagenase: 0.1%	DMEM	Schonbeck U, Sukhova GK, Graber P, Coulter S, Libby P: Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions, Am J Pathol 155, 1281-91, 1999
人類	人類	Hepatic endothelial cells	Collagenase: 0.2%	DMEM	Sanyal AJ, and Mirshahi F.: A simplified method for the isolation and culture of endothelial cells from pseudointima of transjugular intrahepatic portasystemic shunts, Lab Invest 78(11), 1469, 1998
人類	人類	Foreskin microvascular endothelial	Trypsin: 0.3%	HBSS	Wojta J, Gallicchio M, Zoellner H, Filonzi E, Hamilton J, McGrath K: Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells, Blood 81, 3285-92, 1993
人類	人類	Endothelial	Trypsin: 0.3.%	HBSS	Lee, K, Lawley, T, Xu, Y, and Swerlick, R.: VCAM-1-, ELAM-1-, and ICAM-1-Independent Adhesion of Melanoma Cells to Cultured Human Dermal Microvascular Endothelial Cells, J Invest Dermatol 98, 79, 1992
				(see reference)
人類	人類	Vascular endothelial	Neutral Protease: 0.15%	M-199	Farber HW, Antonov AS, Romanov YA, Smirnov VN, Scarfo LM, Beer DJ: Cytokine secretion by human aortic endothelial cells is related to degree of atherosclerosis,Am J Physiol 262, H1088-95, 1992
			Trypsin: 0.25%
人類	人類	Umbilical vein	Collagenase: 0.1%	Cord Buffer	Grant, D.S., Lelkes, P.I., Fukuda, K., Kleinman, H.K.: Intracellular Mechanisms Involved in Basement Membrane Induced Blood Vessel Differentiation In Vitro, In Vitro Cell Dev Biol 27, 327, 1991
		HUVEC		(See Reference)
人類	人類	Crypt cells	Collagenase: 125 u/ml	RMPI 1640	Whitehead, R., and Eeden, P.: A Method For the Prolonged Culture of Colonic Epithelial Cells, J Tiss Cul Meth 13, 103, 1991
人類	人類	Endothelial	Collagenase Type 1: 0.2%	Medium 199	Fischer, E., Stingl, A., and Kirkpatrick, C.: Migration Assay for Endothelial Cells in Multiwells Application to Studies on the Effect of Opioids, J Immunol Methods 128, 235, 1990
人類	人類	Human umbilical vein endothelial cells	Collagenase Type 2: 75 u/ml	M199	Muller WA, Ratti CM, McDonnell SL, Cohn ZA: A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions, J Exp Med 170, 399-414, 1989
人類	人類	Microvessels	Collagenase Type 4: 0.1%	DMEM	Gerhart, D. Z., Broderius, M. A., and Drewes, L. R.: Cutlured Human and Canine Endothelial Cells from Brain Microvessels, Brain Res Bull 21, 785, 1988
人類	人類	Endothelial/HUVEC	Trypsin: 0.3%	HBSS/PBS, Medium 199	Kubota, Y., Kleinman, H., Martin, G., and Lawley, T.: Role of Laminin and Basement Membrane in Morphological Differentiation of Human Endothelial Cells into Capillary-like Structures, J Cell Biol 107, 1589, 1988
		Foreskin & umbilical cord		(see reference)
人類	人類	Umbilical cord	Collagenase: 0.1%	HEPES	Hoshi, H., Kan, M., Chen, J., and McKeehan, W.: Comparative Endocrinology-Paracrinology-Autocrinology of Human Adult Large Vessel Endothelial and Smooth Muscle Cells, In Vitro Cell Dev Biol 24 (4), 309, 1988
		Smooth muscle
人類	人類	Endothelial	Collagenase Type 2: 0.1%	PBS, CMF	Sharefkin, J.B., Fairchild, K.D., Albus, R.A., Cruess, D.F., and Rich, N.M.: The Cytoxic Effect of Surgical Glove Powder Particles on Adult Human Vascular Endothelial Cell Cultures: Implications for Clinical Uses of Tissue Culture Techniques, J Surg Res 41, 463, 1986
		Saphenous vein
人類	人類	Endothelial	Collagenase: 0.1%	HEPES	Hoshi, H., and McKeehan, W.: Isolation, Growth Requirements, Cloning, Prostacylclin Production and Life-Span of Human Adult Endothelial Cells in Low Serum Culture Medium, In Vitro Cell Dev Biol 22 (1), 51, 1986
人類	人類	Endothelial	Trypsin: 0.3%	PBS	Marks, R.M., Czerniecki, M., and Penny, R.: Human Dermal Microvascular Endothelial Cells: An Improved Method for Tissue Culture and Description of Some Singular Properties in Culture, In Vitro Cell Dev Biol 21, 627, 1985
		Dermal
人類	人類	Fibroblasts	Hyaluronidase: 0.10%	DMEM	Gordon, P., Sussman, I., and Hatcher, V.: Long-Term Culture of Human Endothelial Cells, In Vitro 19, 661, 1983
		Foreskin
人類	人類	Iliac arteries	Collagenase: 0.25%	PBS w/Ca⁺⁺, Mg⁺⁺, & BSA	Glassberg, M., Bern, M., Coughlin, S., Haudenschild, C., Hoyer, L., and Antoniades, H.: Cultured Endothelial Cells Derived from the Human Iliac Arteries,In Vitro 18, 859, 1982
				(see reference)
人類	人類, 成年的	Peripheral blood mononuclear	Collagenase: 0.25%	RPMI 1640	Ashida, E., Johnson, A., and Lipsky, P.: Human endothelial cell-lymphocyte interaction. Endothelial cells function as accessory cells necessary for mitogen-induced human T lymphocyte activation in vitro, J Clin Invest 67, 1490, 1981
		Monocytes
		T cells
		Endothelial
人類	人類	Microvascular endothelial	Neutral Protease: at 1000 u/ml	Konigsberg's modification of HBSS	Sherer, G., Fitzharris, T., Faulk, W., and LeRoy, E.: C*tion of Microvascular Endothelial Cells from Human Preputial Skin, In Vitro 16, 675, 1980
		Neonatal foreskins		(See Reference)
人類	人類 (成年人和小孩)	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
人類	人類	Umbilical vein	Trypsin: 100 礸/ml	Tris-HCl,0.2 M	Jaffe, E.A., Minick, C.R., Adelman, B., Becker, C.G., and Nachman, R.: Synthesis of Basement Membrane Collagen By Cultured Human Endothelial Cells, J Exp Med 144, 209, 1976
人類	人類	Umbilical vein	Collagenase Type 1: 125 u/ml	Dulbecco's PBS	Gimbrone Jr., M.A.: Culture of Vascular Endothelium, Prog Hemost Thromb 3, 1, 1976
人類	人類	Umbilical cord	Collagenase: 0.1%	Dulbecco's PBS	Gimbrone, M.A., Cotran, R.S., and Folkman, J.: Human Vascular Endothelial Cells in Culture: Growth and DNA Synthesis, J Cell Biol 60, 673, 1974
人類	人類	Umbilical cord	Collagenase: 0.2%	Cord buffer	Jaffe, E., Nachman, R., Becker, C., and Minick, C.: Culture of Human Endothelial Cells Derived from Umbilical Veins. Identification by Morphologic and Immunologic Criteria, J Clin Invest 52, 2745, 1973
				(See Reference)
人類	人類	Umbilical cord	Trypsin NF 1:250: 0.125%	Saline, normal	Lewis, L.J., Haok, J.C., Maca, R.D., and Fry, G.L.: Replication of Human Endothelial Cells in Culture, Science 181, 452, 1973
人類	人類	Umbilical cord	Trypsin NF 1:250: 0.25%	CMF solution	Fryer, D.G., Birnbaum, G., and Luttrell, C.N.: Human Endothelium in Cell Culture, J Atheroscler Res 6, 151, 1966
小鼠	小鼠	Endothelial lung	Collagenase Type 1: 0.2%	DMEM	Wang, J., Sun, C., Gerdes, N., Liu, C., Liao, M., Liu, J., Shi, M., He, A., Zhou, Y., Sukhova, G., Chen, H., Cheng, X., Kuzuya, M., Murohara, T., Zhang, J., Cheng, X., Jiang, M., Shull, G. and Rogers, S.: Interleukin 18 Function in Atherosclerosis is Mediated by the Interleukin 18 Receptor and the Na-Cl Co-Transporter., Nat Med 21, 820-6, 2015
小鼠	小鼠	Tumor endothelial	Collagenase Type 1: 0.2%	HBSS	Kazerounian, S., Gerald, D., Huang, M., Chin, R, Udayakumar, D, Zheng, N., O'Donnell, R., Perruzzi, C., Mangiante, L., Pourat, J., Phung, T., Bravo-Nuevo, A., Shechter, S., McNamara, S. and Duhadaway, K.: RhoB Differentially Controls Akt Function in Tumor Cells and Stromal Endothelial Cells During Breast Tumorigenesis., Cancer Res 73, 50, 2013
小鼠	小鼠, 8-14 week	Endothelial	Collagenase Type 4: 0.2%	HBSS	Imoukhuede, P. and Popel, A.: Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle., PLoS ONE 7, e44791, 2012
小鼠	小鼠, 7-10 周	Liver endothelial	Collagenase: 0.03%	DMEM	Follenzi, A., Benten, D., Novikoff, P., Faulkner, L., Raut, S. and Gupta, S.:Transplanted Endothelial Cells Repopulate the Liver Endothelium and Correct the Phenotype of Hemophilia A Mice., J Clin Invest 118, 935, 2008
小鼠	小鼠, 4 周	Endothelial kidney	Collagenase Type 1: 0.1%	DMEM	Kondo, S., Scheef, E., Sheibani, N. and Sorenson, C.: PECAM-1 Isoform-Specific Regulation of Kidney Endothelial Cell Migration and Capillary Morphogenesis., Am J Physiol Cell Physiol 292, C2070, 2007
小鼠	小鼠, 胚胎或卵黃囊	Endothelial	Collagenase Type 3: 200 u/ml	PBS	Braren Rickmer, Hu Huiqing, Kim YungHae, Beggs HilaryE, Reichardt LouisF, Wang Rong: Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation, J Cell Biol 172, 151-62, 2006
			Deoxyribonuclease I: 0.001%
小鼠	小鼠, 初生的	Microvascular endothelial	Neutral Protease: 0.005%	DMEM	Cha, S., Talavera, D., Demir, E., Nath, A. and Sierra-Honigmann, M.: A Method of Isolation and Culture of Microvascular Endothelial Cells from Mouse Skin.,Microvasc Res 70, 198, 2005
			Collagenase Type 1: 4%
小鼠	小鼠, 4 周	Retinal endothelial cells	Collagenase Type 1: 0.1%	DMEM	Su X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003
小鼠	小鼠, 雄性	Endothelial cells from lymph node	Collagenase Type 1: 0.1%	PBS	Izawa D, Tanaka T, Saito K, Ogihara H, Usui T, Kawamoto S, Matsubara K, Okubo K, and Miyasaka M: Expression profile of active genes in mouse lymph node high endothelial cells, Int Immunol 11(12), 1989, 1999
小鼠	小鼠, 2-4 周	Cerebrovascular Endothelial	Collagenase/Dispase: 0.1%	PBS	Sapatino, B., Welsh, C., Smith, C., Bebo, B., and Linticum, D.: Cloned Mouse Cerebrovascular Endothelial Cells That Maintain Their Differentiation Markers for Factor VIII, Low Density Lipoprotein, and Angiotensin-Converting Enzyme, In Vitro Cell Dev Biol 29A, 923, 1993
豬	豬, 6 個月	Valvular endothelial	Neutral Protease: 2 u/ml	PBS	Balaoing, L., Post, A., Lin, A., Tseng, H., Moake, J. and Grande-Allen, K.: Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation., PLoS ONE 10, e0130749, 2015
			Collagenase Type 2: 60 u/ml
豬	豬	Valvular endothelial	Collagenase Type 2: 300-600 u/ml	DMEM	Gould, R. and Butcher, J.: Isolation of Valvular Endothelial Cells., J Vis Exp 46, e2158, 2010
豬	豬	Endothelial	Collagenase Type 1:	DMEM w/ 10% calf serum	Nugent, H., and Edelman, E.: Endothelial Implants Provide Long-Term Control of Vascular Repair in a Porcine Model of Arterial Injury, J Surg Res 99, 228, 2001
	(包括牛)
豬	豬	HUVEC, porcine pulmonary arterial endothelial cells	Collagenase Type 2: 0.2%	DMEM	Kwak HJ, Lee SJ, Lee YH, Ryu CH, Koh KN, Choi HY, and Koh GY.: Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells, Circulation 101(19), 2317, 2000
豬	豬, 6-7 個月	Porcine pulmonary endothelial	Collagenase Type 1: 0.3%	RPMI 1640	Hill-Kapturczak N, Kapturczak MH, Block ER, Pa JM, Malinski T, Madsen KM, and Tisher CC.: Angiotensin II-stimulated nitric oxide release from porcine pulmonary endothelium is mediated by angiotensin IV, J Am Soc Nephrol 10(3), 481, 1999
豬	豬	Endothelial	Trypsin: 0.25%	Medium 199	Shasby, S.: Endothelial Cells Grown On Permeable Membrane Supports, J Tiss Cul Meth 14, 247, 1992
豬	豬	Endothelial	Trypsin: 0.05%	DMEM	Vischer, P., and Buddecke, E.: Alteration of Glycosytransferase Activities during Proliferation of C*ted Arterial Endothelial Cells and Smooth Muscle Cells, Exp Cell Res 158, 15, 1985
豬	豬	Endothelial	Collagenase Type 2: 0.1%	Dulbecco-Vogt MEM w/o serum	Dickinson, E. and Slakey, L.: Plasma-derived Serum as a Selective Agent to Obtain Endothelial Cultures from Swine Aorta, In Vitro 18, 63, 1982
		Aortic
豬	豬, 30-40 公斤	Endothelia	Collagenase Type 4: 0.025%	Medium 199	Merrilees, M.J., and Scott, L.: Interaction of Aortic Endothelial and Smooth Muscle Cells in Culture, Atherosclerosis 39, 147, 1981
		Aortic
豬	豬, 20-30 周	Endothelial	Collagenase: 0.1%	Medium 199 w/BSS and HEPES or NaHCO₃	Slater, D.N., and Sloan, J.M.: The Porcine Endothelial Cell in Tissue Culture,Atherosclerosis 21, 259, 1975
		Aortas
		Veins
豬	豬, 60-100 天	Aorta	Trypsin: 0.1%	Phosphate buffer	Coulson, W.F.: The Effect Of Proteolytic Enzymes on the Tensile Strength of Whole Aorta and Isolated Aortic Elastin, Biochim Biophys Acta 237, 378, 1971
				(see reference)
兔	兔	Corneal endothelial cells (CEC)	Hyaluronidase: 0.05%	DMEM	Choi, J., Ko, M., and Kay, E.: Subcellular Localization of the Expressed 18 kDa FGF-2 Isoform in Corneal Endothelial Cells, Mol Vis 6, 222, 2000
兔	兔, 2-3公斤	Endothelial, aortic	Elastase: 0.2%	Hanks solution	Haley, N., Shio, H., Fowler, S.: Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. I. Resolution of aortic cell populations by metrizamide density gradient centrifugation, Lab Invest 37, 287, 1977
大鼠	大鼠, SD, 雄性, 250-300 克	Smooth muscle, aorta	Soybean Trypsin Inhibitor: 0.25%	HBSS with 0.2 mM Ca++	Schwertschlag, U.S., and Whorton, A.R.: Plaet-Activating Factor-Induced Homologous and Heterologous Desensitization In Cultured Vascular Smooth Muscle Cells, J Biol Chem 263, 13791, 1988
大鼠	大鼠, SD, 雄性, 350 - 450 g	Lipocytes	Collagenase: 0.015%	DMEM/Ham's F-12	Friedman, S. and Roll, F.: Isolation and Culture of Hepatic Lipocytes,Kupffer Cells, and Sinusoidal Endothelial Cells by Density Gradient Centrifugation with Stractan,Anal Biochem 161, 207, 1987
		Kupffer
		Sinusoidal endothelial
大鼠	wistar大鼠, 雄性, 3 個月	Endothelial	Pronase: 0.25%	HBSS	Nagelkenke, J., Barto, K., and VanBerkel, T.: In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells, J Biol Chem 258, 12221, 1983
		Kupffer
		Parenchymal
大鼠	京都大鼠, 雄性, 100 - 200 克	Endothelial	Collagenase Type 2: 0.05%	HBSS	Diglio, C.A., Grammas, P., Filiberto Giacomelli, M.S., and Wiener, J.: Primary Culture of Rat Cerebral Microvascular Endothelial Cells, Lab Invest 46, 554, 1982
		Cerebral
大鼠	大鼠, 300 克, 和豬, 30-40 公斤	Endothelial	Trypsin: 0.05%	Medium 199 and 0.01M EDTA	Merrilees, M.J., and Scott, L.: Interaction of aortic endothelial and smooth muscle cells in culture. Effect on glycosaminoglycan levels, Atherosclerosis 39, 147, 1981
		Thoracic aorta
大鼠	大鼠	Foreskin	Collagenase: 0.5%	Dulbecco's MEM w/10% calf serum	Folkman, J., Haudenschild, C. C., and Zetter, B. R.: Long-term Culture of Capillary Endothlial Cells, Proc Natl Acad Sci U S A 76, 5217, 1979
大鼠	wistar大鼠, 成年的, 170 克	Endothelial	Trypsin: 0.5%	BSS	Phillips, P., Kumar, P., Kumar, S., and Waghe, M.: Isolation And Characterization of Endothelial Cells From Rat And Cow Brain White Matter, J Anat 129, 261, 1979

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