肌組織消化手冊

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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(xì)胞生物學(xué)研究的原代細(xì)胞的分離和收集。盡管這些消化酶已經(jīng)普遍應(yīng)用多年，但是對其組織消化和細(xì)胞收集的作用機(jī)制卻沒有全面的理解。由此導(dǎo)致的結(jié)果便是選擇一種消化方法大多是隨機(jī)選擇或基于過去的經(jīng)驗(yàn)，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(shí)驗(yàn)?zāi)康男拚桨敢垣@得更好的實(shí)驗(yàn)結(jié)果。

細(xì)胞分離程序的目的是zui大程度的獲得功能活性的單個細(xì)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(shí)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(yàn)決定以獲取zui適實(shí)驗(yàn)結(jié)果。

通過查閱文獻(xiàn)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(shí)驗(yàn)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(fù)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(xì)胞收集知識和方法，描述了標(biāo)準(zhǔn)的實(shí)驗(yàn)步驟，以便于制定一個*的細(xì)胞分離方案，同時(shí)列舉了一些相關(guān)的參考文獻(xiàn)。

下表列出多種組織的適宜消化酶（含參考文獻(xiàn)，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(xì)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(xiàn)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。

肌組織消化手冊

物種	詳細(xì)來源	細(xì)胞名稱	消化用酶	培養(yǎng)基	參考文獻(xiàn)
牛	牛	Muscle salite cells	Collagenase Type 2: 300 u/ml	Krebs-Ringer Bicarbonate	Lee, E., Choi, J., Hyun, J., Cho, K., Hwang, I., Lee, H., Chang, J. and Choi, I.: Steroid Effects on Cell Proliferation, Differentiation and Steroid Receptor Gene Expression in Adult Bovine Salite Cells., AJAS 20, 501, 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	Smooth muscle	Trypsin: 0.25%	DMEM	Absher, M., Woodcock-Mitchell, J., Mitchell, J., Baldor, L., Low, R., and Warshaw, D.: Characterization of Vascular Smooth Muscle Cell Phenotype in Long-Term Culture, In Vitro Cell Dev Biol 25 (2), 183, 1989
牛	牛	Vascular smooth muscle	Elastase Type 3: 50 u/ml	PSS	Warshaw, D., Szarek, J., Hubbard, M., and Evans, J.: Pharmacology and Force Development of Single Freshly Isolated Bovine Cartoid Artery Smooth Muscle Cells,Circ Res 58, 399, 1986
牛	牛	Smooth muscle, fibroblasts	Trypsin: 0.055%	DMEM	Davies, P. and Kerr, C.: Modification of LDL Metabolism by Growth Factors in Cultured Vascular Cells and Human Skin Fibroblasts, Biochim Biophys Acta 712, 26, 1982
犬	犬	skeletal muscle cells	Collagenase Type 4: 200 u/ml	DMEM	Parker, M., Loretz, C., Tyler, A., Snider, L., Storb. R. and Tapscott, S.: Inhibition of CD26/DPP-IV Enhances Donor Muscle Cell Engraftment and Stimulates Sustained Donor Cell Proliferation., Skelet Muscle 2, 4, 2012
			Neutral Protease: 1 u/ml
犬	狗	Smooth muscle	Elastase: 50 u/ml	PSS	Subramanian, M., Madden, J., and Harder, D.: A Method for the Isolation of Cells from Arteries of Various Sizes, J Tiss Cul Meth 13, 13, 1991
犬	狗, 獵兔犬, 成年的	Smooth muscle	Elastase: 34 u/ml	Tyrode's solution w/ calcium	Wilde, D., and Lee, K.: Outward Potassium Currents in Freshly Isolated Smooth Muscle Cell of Dog Coronary Arteries, Circ Res 65, 1718, 1989
		Vascular
犬	狗	Artery	Elastase: 80 u/ml	PSS	Dobrin, P., and Canfield, T.: Elastase, Collagenase, and the Biaxial Elastic Properties of Dog Carotid Artery, Am J Physiol Heart Circ Physiol 247 (16), H124, 1984
		Carotid
雞	雞, 1-2 天	Gizzard and aorta smooth muscle	Collagenase Type 1: 0.15%	HBSS	Dirksen W., Vladic F., and Fisher S.: A Myosin Phosphatase Targeting Subunit Isoform Transition Defines a Smooth Muscle Developmental Phenotypic Switch, Am J Physiol/Cell 278(3), C589, 2000
雞	小雞	Smooth muscle	Trypsin: 0.05% - 0.1%	HBSS	Chamley-Campbell, J., Campbell, G., and Ross, R.: The Smooth Muscle in Cell Culture, Physiol Res 59, 1, 1979
雞	小雞,白來杭雞, 12 天	Muscle	Trypsin: 0.25%	Puck's saline A	Bullaro, J., and Brookman, D.: Comparison of Skeletal Muscle Monolayer Cultures Initiated With Cells Dissociated by the Vortex and Trypsin Methods, In Vitro 12, 564, 1976
雞	小雞, 白來杭雞, 胚胎, 11 天	Muscle	Trypsin: 0.05%	Saline G	Tepperman, K., Morris, G., Essien, F., and Heywood, S.M.: A Mechanical Dissociation Method For Preparation of Muscle Cell Cultures, J Cell Physiol 86, 561, 1975
雞	小雞胚胎	Thyroid	Collagenase: 0.25%	Tyrode's saline, potassium free	Hilfer, S., and Brown, J.: Collagenase. Its Effectiveness as a Dispersing Agent for Embryonic Chick Thyroid and Heart, Exp Cell Res 65, 246, 1971
		Muscle
		Heart
雞	小雞, 胚胎	Muscle	Trypsin: 0.1%	CMF HBSS	Hilfer, S.: Collagenase Treatment of Chick Heart and Thyroid,Tissue Cult Methods & Applications,Kruse, P., and Patterson, M., , 246, 1971
雞	小雞胚胎	Various tissues (heart, liver, skeletal, cardiac)	Trypsin: various grades	CMF Tyrode's solution	Rinaldini, L.: An Improved Method for the Isolation and Quantitative C*tion of Embryonic Cells, Exp Cell Res 16, 477, 1959
貓科動物	貓, 成年雜種, 任一性別, 2.5-4.0 公斤	Cerebral arteries	Elastase: 50 u/ml	Puck's solution	Madden, J., Vadula, M., and Kurup, V.: Effects of Hypoxia and Other Vasoactive Agents on Pulmonary and Cerebral Artery Smooth Muscle Cells, Am J Physiol 263, L384, 1992
貓科動物	貓, 雜交的, 成年的, 2-4 公斤	Myocytes	Collagenase: 0.12%	Kreb's Henseleit, CF	Follmer, C.H., Ten Eick, R.E., and Yeh, J.Z.: Sodium Current Kinetics in Cat Atrial Myocytes, J Physiol 384, 169, 1987
魚	角鯊,海綠角鯊 , 2-6 公斤	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠，兔, 青蛙)			(see reference)
青蛙	青蛙, 有爪蟾蜍, 胚胎期 17 & 19	Muscle	Collagenase: 0.10%	Steinberg's solution	Stollberg, J. and Fraser, S.: Acetylcholine Receptors and Con A-Binding Sites onXenopus Muscle Cells, J Cell Biol 107, 1397, 1988
青蛙	青蛙	Myocytes	Trypsin: 0.1%	CF Ringer	Shepherd, N. and Kavaler, F.: Direct Control of Contraction Force of Single Frog Atrial Cells by Extracellular Ions, Am J Physiol 251, C653, 1986
青蛙	青蛙, 美洲豹蛙(50-100 克)	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠, 兔, 角鯊,海綠角鯊)			(see reference)
青蛙	青蛙, 有爪蟾蜍	Muscle	Trypsin: 0.5%	L15 medium	Anderson, M.J., Cohen, M.W., and Zorychta, E.: Effects of Innervation on the Distribution of Acetylcholine Receptors on Cultured Muscle Cells, J Physiol 268, 731, 1977
				(see reference)
豚鼠	豚鼠	Bladder smooth muscle	Collagenase Type 2: 0.1-0.2%	Krebs-Ringer bicarbonate	Shieh CC, Feng J, Buckner SA, Brioni JD, Coghlan MJ, Sullivan JP, Gopalakrishnan M: Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells, J Pharmacol Exp Ther 296, 669-75, 2001
豚鼠	豚鼠, 200-380 克	Capillaries	Collagenase Type 2: 0.15%	CF solution	Schnitzler, M., Derst, C., Daut, J., and Preisig-Muller, R.: ATP-Sensitive Potassium Channels in Capillaries Isolated From Guinea-Pig Heart, J Physiol 525 (2), 307, 2000
		Myocytes
豚鼠	豚鼠, 2-4 周大, 雄性, 雌性	Smooth muscle	Papain: 0.1%	Krebs solution	Firth, T., Mawe, G., and Nelson, M.: Pharmacology and Modulation of K_ATPChannels by Protein Kinase C and Phosphatates in Gallbladder Smooth Muscle, Am J Physiol Cell Physiol 278, C1031, 2000
		Gallbladder
豚鼠	豚鼠, 成年的, 250-350 克	Smooth muscle	Papain: 0.1%	NaCl, sodium glutamate, MgCl, KCl, glucose, Kreb's, and HEPES	Jennings, L., Xu, Q., Firth, T., Nelson, M., and Mawe, G.: Cholesterol Inhibits Spontaneous Action Potentials and Calcium Currents in Guinea Pig Gallbladder Smooth Muscle, Am J Physiol 277, G1017, 1999
		Gallbladder
豚鼠	Dunkin-Hartley豚鼠, 雌性	Myocytes	Protease:	DMEM	Ryder, K., Bryant, S., and Hart, G.: Membrane Current Changes in Left Ventricular Myocytes Isolated From Guinea-Pigs After Abdominal Aortic Coarctation, Cardiovasc Res 27, 1278, 1993
豚鼠	豚鼠	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括大鼠, 兔)
豚鼠	豚鼠, 200-300 克	Smooth muscle	Collagenase: 0.3%	CF solution	Ohya, Y. and Sperelakis, N.: ATP Regulation of the Slow Calcium Channels in Vascular Smooth Muscle Cells, Circ Res 64, 145, 1989
		Mesenteric artery
豚鼠	豚鼠, 200-400克	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括兔, 青蛙, 角鯊)			(see reference)
豚鼠	豚鼠, 青春期前的	Smooth muscle	Trypsin: 0.05%	Dulbecco-Vogt modification of Eagle's	Ross, R.: The Smooth Muscle Cell . II. Growth of Smooth Muscle in Culture and Formation of Elastic Fibers, J Cell Biol 50, 172, 1971
		Aortic
倉鼠	倉鼠, 雄性, 60-70 天	Salite	Trypsin: 0.25%	DMEM	Nakamura, T., Iwata, Y., Sampaolesi, M., Hanada, H., Saito, N., Artman, M., Coetzee, W., and Shigekawa, M.: Stretch-Activated Cation Channels in Skeletal Muscle Myotubes From Sarcoglycan-Deficient Hamsters, Am J Physiol Cell Physiol281, C690, 2001
人類	人類	Skeletal muscle stem cells	Collagenase Type 2: 750 u/ml	Ham's F10	Charville, G., Cheung, T., Yoo, B., Santos, P., Lee, G., Shrager, J. and Rando, T.:Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells.,Stem Cell Reports Vol. 5,, , 621, 2015
			Neutral Protease: 2 u/ml
人類	人類	Smooth muscle	Collagenase Type 1: 0.1%	DMEM	Lu, S., Sun, X., Hong, T., Song, K., Yang, S. and Wang, C.: Isolation and Culture of Smooth Muscle Cells from Human Acute Type A Aortic Dissection., J Cardiothorac Surg 8, 83, 2013
人類	人類, 男性	Myogenic	Collagenase Type 4: 0.1%	HBSS	Stadler, G., Chen, J., Wagner, K., Robin, J., Shay, J., Emerson, C. and Wright, W.: Establishment of Clonal Myogenic Cell Lines from Severely Affected Dystrophic Muscles - CDK4 Maintains the Myogenic Population., Skelet Muscle 1, 12, 2011
			Neutral Protease: 2.4 u/ml
人類	人類	Muscle derived multiprogenitor cells	Collagenase Type 2: 0.05%	DMEM	Nesti, L., Jackson, W., Shanti, R., Koehler, S., Aragon, A., Bailey, J., Sracic, M., Freedman, B., Giuliani, J. and Tuan, R.: Differentiation Potential of Multipotent Progenitor Cells Derived from War-Traumatized Muscle Tissue., J Bone Joint Surg Am90, 2390, 2008
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	Urinary tract smooth muscle	Collagenase Type 4: 100 u/ml	DMEM	Kimuli, M., Eardley, I. and Southgate, J.: In Vitro Assessment of Decellularized Porcine Dermis as a Matrix for Urinary Tract Reconstruction., BJU Int 94, 859, 2004
人類	人類, 女性	Smooth muscle	Deoxyribonuclease I: 0.015% and 0.007%	HBSS	Richardson, M., Taylor, D., Casey, M., MacDonald, P., and Stull, J.: Biochemical Markers of Contraction in Human Myometrial Smooth Muscle Cells in Culture, In Vitro Cell Dev Biol 23, 21, 1987
		Myometrial
人類	人類, 女性	Smooth muscle	Deoxyribonuclease I: 0.12%	HBSS	Casey, M., MacDonald, P., Mitchell, M., and Snyder, J.: Maintenance and Characterization of Human Myometrial Smooth Muscle Cells in Monolayer Culture, In Vitro 20, 396, 1984
		Myometrial
人類	人類, 胎兒	Smooth muscle, fibroblasts	Trypsin: 0.055%	DMEM	Davies, P. and Kerr, C.: Modification of LDL Metabolism by Growth Factors in Cultured Vascular Cells and Human Skin Fibroblasts, Biochim Biophys Acta 712, 26, 1982
	(包括牛)
人類	人類	Smooth muscle	Trypsin: 0.25%	DMEM	Eskin, S., Sybers, H., Lester, J., Navarro, L., Gotto, A., and DeBakey, M.: Human Smooth Muscle Cells Cultured From Atherosclerotic Plaques and Uninvolved Vessel Wall, In Vitro 17 (8), 713, 1981
人類	人類	Smooth muscle	Trypsin: 0.05% - 0.1%	HBSS	Chamley-Campbell, J., Campbell, G., and Ross, R.: The Smooth Muscle in Cell Culture, Physiol Res 59, 1, 1979
	(包括大鼠, 豚鼠, 小雞, 猴)
蜥蜴	蜥蜴(安樂蜥)	Myoblasts, tail	Collagenase: 0.2%	GM III	Cox, P., and Simpson, Jr., S.: A Microphotometric STudy of Myogenic Lizard Cells Grown in Vitro, Dev Biol 23, 433, 1970
				(see reference)
猴	猴 (豚尾猴)	Smooth muscle	Trypsin: 0.05%	Dulbecco-Vogt	Chait, A., Ross, R., Albers, J., and Bierman, E.: Plaet-Derived Growth Factor Stimulates Activity of LDL Receptors, Proc Natl Acad Sci U S A 77, 4084, 1980
猴	獼猴, 1 歲	Smooth muscle, saphenous vein	Elastase: 0.05%	BSS	Chamley, J., Campbell, G., McConnell, J., and Groschel-Stewart, U.: Comparison of Vascular Smooth Muscle Cells from Adult Human, Monkey and Rabbit in Primary Culture and in Subculture, Cell Tissue Res 177, 503, 1977
	(包括人類, 兔)
小鼠	小鼠, 6-8 周	Myofibers	Collagenase Type 1: 0.2%	DMEM	Pasut, A., Jones, A. and Rudnicki, M.: Isolation and Culture of Individual Myofibers and their Salite Cells from Adult Skeletal Muscle., J Vis Exp 73, e50074, 2013
小鼠	小鼠	Vascular Smooth Muscle	Collagenase Type 2: 0.14%	Basal Culture	Metz, R., Patterson, J. and Wilson, E.: Vascular Smooth Muscle Cells: Isolation, Culture, and Characterization., Methods Mol Biol 843, 169, 2012
小鼠	小鼠, 10 周	Disphragm cells	Collagenase Type 2: 100 u/ml	PBS	Rezk, B., Yoshida, T., Semprun-Prieto, L., Higashi, Y., Sukhanov, S. and Delafontaine, P.: Angiotensin II Infusion Induces Marked Diaphragmatic Skeletal Muscle Atrophy., PLoS ONE 7, e30276, 2012
			Pronase: 0.125%
小鼠	小鼠	Skeletal muscle fiber	Collagenase Type 1: 0.2%	Tyrode	Weisleder, N., Zhou, J. and Ma, J.: Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers., Methods Mol Biol 798, 395, 2012
小鼠	小鼠	Myoblast	Collagenase Type 2: 0.2%	DMEM	Shi, H., Boadu, E., Mercan, F., Le, A., Roth Flach, R., Zhang, L., Tyner, K., Olwin, B. and Bennett, A.: MAP Kinase Phosphatase-1 Deficiency Impairs Skeletal Muscle Regeneration and Exacerbates Muscular Dystrophy., FASEB J 24, 2985, 2010
小鼠	小鼠, 1-25 個月	Myocytes, endothelial	Neutral Protease: 1.2 u/ml	PBS	Ieronimakis Nicholas, Balasundaram Gayathri, Reyes Morayma: Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential, PLoS ONE 3, e0001753, 2008
			Collagenase Type 4: 0.2%
小鼠	小鼠, 15-20 天	Intersitial cells of Cajal	Collagenase Type 2: 0.13%	M199	Li CX, Liu BH, Tong WD, Zhang LY, and Jiang YP: Dissociation, culture and morphologic changes of interstitial cells of Cajal in vitro, World J Gastroenterol 11, 2838, 2005
小鼠	小鼠,新生的	Skeletal muscle myotubes	NCIS kit: per instructions	L-15	Johnson, B., Scheuer, T. and Catterall, W.: Convergent Regulation of Skeletal Muscle Ca2+ Channels by Dystrophin, the Actin Cytoskeleton, and cAMP-Dependent Protein Kinase., Proc Natl Acad Sci U S A 102, 4191, 2005
小鼠	小鼠, 雄性, 6-14 周	Precursor cells	Collagenase Type 2: 0.5%	DMEM/F12	Winitsky, S., Gopal, T., Hassanzadeh, S., Takahashi, H., Gryder, D., Rogawski, M., Takeda, K., Yu, Z., Xu, Y. and Epstein, N.: Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro., PLoS Biol3, e87, 2005
小鼠	小鼠, 6-8 周	Skeletal muscle progenitor	Collagenase Type 2: 0.2%	DMEM	Majka S., Jackson K., Kienstra K., Majesky M., Goodell M., and Hirschi K.: Distinct Progenitor Populations in Skeletal Muscle Are Bone Marrow Derived and Exhibit Different Cell Fates During Vascular Regeneration, J Clin Invest 111, 71, 2003
小鼠	小鼠, neonatal	Myocytes	Collagenase Type 1: 0.5%	DMEM	Fukada S., Miyagoe-Suzuki Y., Tsukihara H., Yuasa K., Higuchi S., Ono S., Tsujikawa K., Takeda S., and Yamamoto H.: Muscle Regeneration by Reconstitution with Bone Marrow or Fetal Liver Cells from Green Fluorescent Protein-gene Transgenic Mice, J Cell Sci 115, 1285, 2002
小鼠	小鼠, 6-8 week	Myocytes	Collagenase Type 2: 0.2%	HBSS	McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA.: Muscle-derived hematopoietic stem cells are hematopoietic in origin, Proc Natl Acad Sci U S A 99, 1341, 2002
			Trypsin: 0.25%
綿羊	綿羊, 成年和新生的	Tracheal smooth muscle cells	Papain: 0.2%	MOPS-PSS	Driska S., Laudadio R., Wolfson M., and Shaffer T.: A Method for Isolating Adult and Neonatal Airway Smooth Muscle Cells and Measuring Shortening Velocity, J Appl Physiol 86(1), 427, 1999
			Deoxyribonuclease I: 0.1%
豬	豬, 2個月	Skeletal muscle	Collagenase: 10%	HBSS	Lewis, F., Henning, B., Marazzi, G., Sassoon, D., Ellison, G. and Nadal-Ginard, B.: Porcine Skeletal Muscle-Derived Multipotent PW1pos/Pax7neg Interstitial Cells: Isolation, Characterization, and Long-Term Culture., Stem Cells Transl Med 3, 702-12, 2014
			Neutral Protease: 0.3%
豬	豬, 雄性, 25-40 公斤	Coronary myocytes	Collagenase Type 2: 294 u/ml	low calcium physiological saline	Korzick, D., Laughlin, M., and Bowles, D.: Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries, J Appl Physiol 96, 1425-32, 2004
			Elastase: 6.5 u/ml
			Deoxyribonuclease I: 0.04%
			Soybean Trypsin Inhibitor: 0.1%
豬	豬, 成年的, 35-45 公斤	Arterial smooth muscle	Collagenase Type 2: 294 u/ml	MEM	Wamhoff BR, Dixon JL, Sturek M: Atorvastatin treatment prevents alterations in coronary smooth muscle nuclear Ca2+ signaling in diabetic dyslipidemia, J Vasc Res 39, 208, 2002
			Elastase: 6.5 u/ml
			Deoxyribonuclease I: 0.4 mg/ml
			Soybean Trypsin Inhibitor: 1 mg/ml
豬	約克夏豬, 30公斤	Coronary smooth muscle	Collagenase Type 2: 150 u/ml	HBSS	Sirous ZN, Fleming JB, Khalil RA: Endothelin-1 enhances eicosanoids-induced coronary smooth muscle contraction by activating specific protein kinase C isoforms, Hypertension 37, 497-504, 2001
			Elastase: 0.05%
豬	豬	Bladder smooth muscle	Collagenase Type 2: 0.1-0.2%	Krebs-Ringer bicarbonate	Shieh CC, Feng J, Buckner SA, Brioni JD, Coghlan MJ, Sullivan JP, Gopalakrishnan M: Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells, J Pharmacol Exp Ther 296, 669-75, 2001
豬	豬, 少年的, 30公斤	Coronary smooth muscle cells	Collagenase: 0.3%	HBSS	Huckle WR, Drag MD, Acker WR, Powers M, McFall RC, Holder DJ, Fujita T, Stabilito II, Kim D, Ondeyka DL, Mantlo NB, Chang RS, Reilly CF, Schwartz RS, Greenlee WJ, Johnson RG: Effects of subtype-selective and balanced angiotensin II receptor antagonists in a porcine coronary artery model of vascular restenosis,Circulation 93, 1009-19, 1996
			Elastase: 0.05%
豬	豬	Smooth muscle, aorta	Collagenase: 0.3%	DMEM	Xiong, Yimin, Xu, Shangzhe, and Slakey, Linda L: Modulation of Response to Adenosine in Vascular Smooth Muscle Cells Cultured in Defined Medium, In Vitro Cell Dev Biol 27, 355, 1991
豬	豬	Smooth muscle	Trypsin: 0.05%	EDTA 0.02%	Breton, M., Berrou, E., Deudon, E. and Picard, J.: Changes in Proteoglycans of Cultured Pig Aortic Smooth Muscle Cells During Subculture, In Vitro Cell Dev Biol26, 157, 1990
		Aorta
豬	豬	Smooth muscle	Collagenase: 0.30%	DMEM	Fehr, T., Dickinson, E., Goldman, S. and Slakey, L.: Cyclic AMP Efflux is Regulated by Occupancy of The Adenosine Receptor in Pig Aortic Smooth Muscle Cells, J Biol Chem 265, 10974, 1990
		Aortic medial tissue
鵪鶉	鵪鶉, 胚胎, 10 天	Myoblasts	Collagenase Type 2: 0.1%	Puck's solution	Konigsberg, I.: Skeletal Myoblasts in Culture, Methods Enzymol LVIII, 511, 1979
兔	兔,新西蘭白	Aortic smooth muscle	Collagenase Type 2: 300 u/ml	F10 Ham's	Croons Valerie, Martinet Wim, Herman Arnold G, Timmermans Jean-Pierre, De Meyer Guido R Y: Selective clearance of macrophages in atherosclerotic plaques by the protein synthesis inhibitor cycloheximide, J Pharmacol Exp Ther 320, 986-93, 2007
			Elastase: 5 u/ml
兔	兔, 新西蘭, 雄性, 2.5-3.5 公斤	Enterocytes	Trypsin: 0.1%	RPMI 1640 w/ 1% fetal bovine serum	Santos, M., Nguyen, B.., Thompson, J.: Factors Affecting in Vitro Growth of Harvested Enterocytes, Cell Transplant 1, 299, 1992
				PBS
兔	兔	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括大鼠, 豚鼠)
兔	兔,成年的, 1-2 公斤	Smooth muscle	Elastase: 0.17 - 0.25%	Saline	Benham, C., Bolton, T., Byrne, N., and Large, W.: Action of Extremely Applied Adenosine Triphosphate O Single Smooth Muscle Cells Dispersed From Rabbit Ear Artery, J Physiol 387, 473, 1987
兔	兔, 新西蘭白, 1500 克	Smooth muscle, aortic	Trypsin: 0.038%	MEM	Knodle, S., Anderson, S., and Papaioannou, S.: Large Scale Preparation of Rabbit Aortic Smooth Muscle Cells For Use in Calcium Uptake Studies, In Vitro Cell Dev Biol 22, 23, 1986
兔	兔, 成年的, 1-2 公斤	Smooth muscle, ear artery	Trypsin: 0.1%	CF solution	Benham, C.D., Bolton, T.B.: Spontaneous Transient Outward Currents in Single Visceral and Vascular Smooth Muscle Cells of the Rabbit, J Physiol 381, 385, 1986
				(see reference)
兔	兔, 0.5-1 公斤	Myocytes, heart and stomach	Protease XIV: 0.028%	Solution C	Mitra, R. and Morad, M.: A Uniform Enzymatic Method for Dissociation of Myocytes from Hearts and Stomachs of Vertebrates, Am J Physiol 249, H1056, 1985
	(包括豚鼠, 青蛙, 角鯊)			(see reference)
兔	兔, 新西蘭白, 成年的, 雄性, 2 公斤	Smooth muscle, aorta	Trypsin: 0.1%	Krebs Ringer HEPES solution	Ives, H., Schultz, G., Galardy, R., and Jamieson, J.: Preparation of Functional Smooth Muscle Cells from the Rabbit Aorta, J Exp Med 148, 1400, 1978
兔	兔, 新西蘭白白化, 5-6 個月	Smooth muscle, saphenous vein	Elastase: 0.05%	BSS	Chamley, J., Campbell, G., McConnell, J., and Groschel-Stewart, U.: Comparison of Vascular Smooth Muscle Cells from Adult Human, Monkey and Rabbit in Primary Culture and in Subculture, Cell Tissue Res 177, 503, 1977
	(包括人類, 獼猴, 兔)
兔	兔, 南美洲栗鼠, 5-8 月, 處女, 雌性, 2-3 公斤	Smooth muscle, aorta	Hyaluronidase: 800 u/ml	HBSS	Peters, T., Muller, M., and deDuve, C.: Lysosomes of the Arterial Wall. I. Isolation and Subcellular Fractionation of Cells from Normal Rabbit Aorta, J Exp Med 136, 1117, 1972
兔	兔, 新英格蘭, 白化變種	Thoracic aorta	Elastase: 0.008%	Kreb's Ringer	天, A., Phil, D., and Newman, H.: Synthesis of Phospholipid by Foam Cells Isolated from Rabbit Atherosclerotic Lesions, Circ Res XIX, 122, 1966
大鼠	SD大鼠,	Pulmonary arterial smooth muscle	Collagenase Type 2: 0.1%	Buffer	Zeng, Y., Liu, H., Kang, K., Wang, Z., Hui, G., Zhang, X., Zhong, J., Peng, W., Ramchandran, R., Raj, J. and Gou, D.: Hypoxia Inducible Factor-1 Mediates Expression of miR-322: Potential Role in Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells., Sci Rep 5, 12098, 2015
			Elastase: 0.05%
大鼠	大鼠	Skeletal muscle fiber	Collagenase Type 1: 0.2%	Tyrode	Weisleder, N., Zhou, J. and Ma, J.: Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers., Methods Mol Biol 798, 395, 2012
大鼠	大鼠, 胎兒 17-18 天	Myotubes	Trypsin: 0.05%	see reference	Das, M., Rumsey, J., Bhargava, N., Stancescu, M. and Hickman, J.: Skeletal Muscle Tissue Engineering: A Maturation Model Promoting Long-Term Survival of Myotubes, Structural Development of the Excitation-Contraction Coupling Apparatus and Neonatal Myosin Heavy Chain Expression., Biomaterials 30, 5392, 2009
大鼠	大鼠	Myogenic	Collagenase Type 2: 1.0%	Ham's F-10	Pacak, C. and Cowan, D.: Fabrication of Myogenic Engineered Tissue Constructs., J Vis Exp 27, e1137, 2009
			Neutral Protease: 2.4 u/ml
大鼠	Lewis大鼠, 初生的	Myoblasts	Collagenase Type 2: 1.0%	Ham's F-10	Kim, J., Hadlock, T., Cheney, M., Varvares, M. and Marler, J.: Muscle Tissue Engineering for Partial Glossectomy Defects., Arch Facial Plast Surg 5, 403, 2003
			Neutral Protease: 2.4 u/ml
大鼠	SD大鼠, 成年和出生一天的	Myooids	Neutral Protease: 4 u/ml	Ham's F-12	Dennis, R., Kosnik II, P., Gilbert, M., and Faulkner, J.: Excitability and Contractility of Skeletal Muscle Engineered from Primary Cultures and Cell Lines, Am J Physiol Cell Physiol 280, C288, 2001
大鼠	SD大鼠, 3 個月	Smooth muscle cells	Collagenase Type 2: 0.2%	M-199	Su E., Stevenson S., Rollence M., Marshall-Neff J., and Liau G.: A Genetically Modified Adenoviral Vector Exhibits Enhanced Gene Transfer of Human Smooth Muscle Cells, J Vasc Res 38(5), 471, 2001
			Elastase: 0.04%
			Soybean Trypsin Inhibitor: 0.1%
大鼠	SD大鼠, 250 克	Arterial smooth muscle	Papain: 0.03%	see reference	Jaggar JH: Intravascular pressure regulates local and global Ca(2+) signaling in cerebral artery smooth muscle cells, Am J Physiol Cell Physiol 281, C439-48, 2001
			Collagenase: 0.1%
大鼠	Wistar大鼠, 成年的, 雄性	Smooth & skeletal muscle	Protease: 0.01%	PSS	Wellman, G., Barrett-Jolley, R., Koppel, H. Everitt, D., and Quayle, J.: Inhibition of Vascular K_ATP Channels by U-37883A: A Comparison with Cardiac and Skeletal Muscle, Br J Pharmacol 128, 909, 1999
		Cardiac myocytes
大鼠	SD大鼠, 雄性, 250-350 克	Vascular smooth muscle	Collagenase Type 2: 0.1%	DMEM	Hrometz, S., Edelmann, S., McCune, D., Olges, J., Hadley, R., Perez ,D., and Piascik, M.: Expression of Multiple Alpha1-Adrenoceptors on Vascular Smooth Muscle: Correlation with the Regulation of Contraction, J Pharmacol Exp Ther 290(1), 452, 1999
			Elastase: 0.0125%
大鼠	SD大鼠, 雄性, 150-175 克	Smooth muscle, endothelial	Trypsin: 0.04%	MEM	Redmond, E., Cahill, P., and Sitzmann, J.: Perfused Transcapillary Smooth Muscle and Endothelial Cell Co-Culture-A Novel In Vitro Model, In Vitro Cell Dev Biol Anim 31, 601, 1995
大鼠	Wistar京都大鼠, 10-15 周	Smooth muscle, mesenteric artery	Trypsin: 0.05%	MEM	McGuire, P., Walker-Caprioglio, H., Little, S., and McGuffee, L.: Isolation and Culture of Rat Superior Mesenteric Artery Smooth Muscle Cells, In Vitro Cell Dev Biol 29, 135, 1993
大鼠	大鼠	Smooth muscle	Trypsin: 0.1%	Potassium buffer solution	Hu, S., and Kim, H.: Activation of K+ Channel in Vascular Smooth Muscle by Cytochrome P-450 Metabolites of Arachidonic Acid, FASEB J 6, A383, 1992
	(包括兔, 豚鼠)
大鼠	SD大鼠, 19 天	Smooth muscle, myometrial	Trypsin: 150 礸/ml	HBSS or PSS, CMF	Loch-Caruso, R., Pahl, M., and Juberg, D.: Rat Myometrial Smooth Muscle Cells Show High Levels of Gap Junctional Communication Under a Variety of Culture Conditions, In Vitro Cell Dev Biol 28, 97, 1992
大鼠	SHRs 和 WKY大鼠, 雄性, 10 - 14 周	Smooth muscle, tail arteries	Papain: 0.1%	HEPES buffer	Bolzon, B. and Cheung, D.: Isolation and Characterization of Single Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats, Hypertension 14, 137, 1989
				(see reference)
大鼠	大鼠, 1-3 天	Smooth muscle, aortic	Elastase: 0.0125%	DMEM	Barone, L., Wolfe, L., Faris, B., and Franzblau, C.: Elastin mRNA Levels and Insoluble Elastin Accumulation in Neonatal Rat, Biochemistry 27, 3175, 1988
大鼠	大鼠	Muscle, mesenteric arteries	Trypsin: 0.05%	HEPES KG solution	Bean, B., Sturek, M., Puga, A., and Hermsmeyer, K.: Calcium Channels in Muscle Cells Isolated From Rat Mesenteric Arteries: Modulation by Dihydropyridine Drugs,Circ Res 59, 229, 1986
				(see reference)
大鼠	SHR和WKY大鼠, 任一性別, 12-19 天, 3 個月, 和退休飼養(yǎng)者	Endothelial, aortic	Elastase: 0.05%	Waymouth's culture medium	Gordon, D., Mohai, L., and Schwartz, S.: Induction of Polyploidy in Cultures of Neonatal Rat Aortic Smooth Muscle Cells, Circ Res 59, 633, 1986
大鼠	大鼠, 雄性, 150-250 克	Endothelial, aortic	Trypsin:	RPMI 1640	Cole, O., Fan, T., and Lewis, G.: Isolation, Characterization, Growth and Culture of Endothelial Cells From the Rat Aorta, Cell Biol Int Rep 10 (6), 399, 1986
大鼠	Wistar大鼠, 雌性, 10 周	Myocytes	Collagenase: 0.1%	HBSS	Boulanger-Saunier, C., Kattenburg, D., and Stoclet, J.: Cyclic AMP-dependent Phosphorylation of a 16kDa Protein in a Plasma Membrane-enriched Fraction of Rat Aortic Myocytes, F.E.B.S. Lett. 193, 283, 1985
大鼠	SD大鼠, 雄性, 200 - 250 克	Smooth muscle, thoracic aorta	Trypsin: 0.0375%	Eagle's MEM with calcium	Brock, T., Alexander, R., Ekstein, L., Atkinson, W., and Gimbrone, M.: Angiotensin Increases Cytosolic Free Calcium in Cultured Vascular Smooth Muscle Cells,Hypertension 7, 105, 1985
大鼠	SD大鼠, 225-250克	Mesenteric artery smooth muscle cells	Elastase: .0125%	HBSS	Gunther S, Alexander RW, Atkinson WJ, and Gimbrone MA Jr.: Functional angiotensin II receptors in cultured vascular smooth muscle cells, J Cell Biol 92, 289, 1982
			Soybean Trypsin Inhibitor: 0.025%
			Collagenase Type 1: 0.1%
大鼠	大鼠, 3-4 天	Myocardial	Trypsin NF 1:250: 0.125%	HBSS CMF	Kasten, F.: Rat Myocardial Cells In Vitro: Mitosis and Differentiated Properties, In Vitro 8, 128, 1972
大鼠	大鼠, 200 克	Muscle	Trypsin: 0.05%	Kreb's Henseleit bicarbonate buffer	Kono, T.: Roles of Collagenases and Other Proteolytic Enzymes in the Dispersal of Animal Tissues, Biochim Biophys Acta 178, 397, 1969
大鼠	Wistar大鼠, 3-10 天	Heart	Trypsin NF 1:250: 250: 0.1%	Saline A	Harary, I., and Farley, B.: In Vitro Studies on Single Beating Rat Heart Cells, Exp Cell Res 29, 451, 1963
				(See reference)

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