上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。

上皮細胞組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
牛	牛, fetal	Epithelial	Neutral Protease: 2%	Dissociation medium, CMF	Schumann, B.L., Cody, T.E., Miller, M.L., Leikauf, G.D.: Isolation, Characterization, and Long-Term Culture of Fetal Bovine Tracheal Epithelial Cells, In Vitro Cell Dev Biol 24, 211, 1988
		Tracheal
犬	狗	Tracheal	Pronase: 0.1%	DMEM	Virmani, A., Naziruddin, B., Desai, V., Lowry, J., Graves, D., and Sachdev, G.:Evidence for Secretion of High Molecular Weight Mucins by Canine Tracheal Epithelial Cells in Primary Culture: Effects of Select Secretagogues on Mucin Secretion, In Vitro Cell Dev Biol 28A, 120, 1992
雞	小雞, 5 天大	Intestinal mesenchymal and epithelial	Collagenase: 0.03%	DMEM	Simon-Assmann, P and Kedinger, M: Embryonic Gut-Dissagregated Culture,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12A:3.1, 1995
魚	鯊魚(海綠角鯊)	Rectal gland	Collagenase: 0.2%	Ringer's solution	Karnaky, Jr., K.J., Valentich, J.D., Currie, M.G., Oehlenschlager, W.F., and Kennedy, M.P.: Atriopeptin Stimulates Chloride Secretion in Cultured Shark Rectal Gland Cells, Am J Physiol 260, 1125, 1991
魚	鯊魚(海綠角鯊)	Rectal gland	Collagenase: 0.2%	Ringer's solution	Valentich, J: J Tiss Cul Meth 13, 149, 1991
魚	美洲擬鰈, 200-500 克	Renal tubule	Trypsin: 0.2%	CMF solution	Dickman, K.G., and Renfro, J.: Primary Culture of Flounder Renal Tubule Cells: Transepithelial Transport, Am J Physiol 251, 424, 1986
	(黃蓋美洲)
青蛙	青蛙, 有爪蟾蜍, 成年的, 雌性	Colonic epithelial	Collagenase Type 4: 0.1%	Kreb's	Heinke, B, and Clauss, W.: Potassium Conductances in Isolated Single Cells fromXenopus Laevis Colonic Epithelium, J Comp Physiol [B] 169, 148, 1999
豚鼠	Hartley豚鼠, 雌性, 200 克	Epithelial	Collagenase Type 1: 0.1%	DMEM	Rutten, M.: Use of Commerically Available Cell Culture Inserts for Primary Culture and Electrophysiologic Studies of Guinea Pig Gastric Mucous Epithelial Cells, J Tiss Cul Meth 14, 235, 1992
豚鼠	豚鼠, 哈特利白化,	Endometrial	Collagenase: 0.25%	HBSS	Chaminadas, G., Alkhalaf, M., Remy-Martin, J.P., Propper, A.Y., and Adessi, G.L.: Specific Effect of Oestrone Sulphate on Protein Synthesis and Secretion by Cultured Epithelial Cells from Guinea-pig Endometrium, J Endocrinol 123, 233, 1989
	500-600 克
倉鼠	倉鼠, 敘利亞黃金, 雄性, 100-120 克	Tracheal	Pronase: 0.1%	MEM with Hepes, CMF	Niles,R., Kim, K.C., Hyman, B., Christensen, T., Wasano, K., Brody, J.:Characterization Of Extended Primary And Secondary Cultures Of Hamster Tracheal Epithelial Cells, In Vitro Cell Dev Biol 24, 457, 1988
倉鼠	倉鼠, 敘利亞黃金 (鉻：RGH應變)	Tracheal	Trypsin: 0.1%	Ham's F-12	McDowell, E., et al.: Differentiation of Tracheal Mucociliary Epithelium in Primary Cell Culture Recapitulates Normal Fetal Development and Regeneration Following Injury in Hamsters, Am J Pathol 129, 511, 1987
倉鼠	倉鼠 (血統1516 EHS 和萊克維尤), 8-12 周	Tracheal	Trypsin: 0.05%	Medium 199	Lee, T., Wu, R., Brody, A., Barrett, J., and Nettesheim, P.: Growth and Differentiation of Hamster Tracheal Epithelial Cells in Culture, Exp Lung Res 6, 27, 1984
倉鼠	倉鼠,敘利亞黃金, 雄性, 6 周- 4 個月	Tracheal	Trypsin: 0.25%	PBS with EDTA	Goldman, W.E., Baseman, J.B.: Selective Isolation and Culture of a Proliferating Epithelial Cell Population from the Hamster Trachea, In Vitro 16, 313, 1980
人類	人類	Human tracheal epithelium	Protease Type XIV: 0.04%	DMEM/F12	Widdicombe JH, Sachs LA, Morrow JL, and Finkbeiner WE.: Expansion of cultures of human tracheal epithelium with maintenance of differentiated structure and function, Biotechniques 39(2), 249, 2005
人類	人類	Colonic epithelial	Collagenase:	RPMI 1640	Fukushima, K. and Fiocchi, C.: Paradoxical Decrease of Mitochondrial DNA Deletions in Epithelial Cells of Active Ulcerative Colitis Patients., Am J Physiol Gastrointest Liver Physiol 286, G804-13, 2004
			Neutral Protease: 0.3%
			Deoxyribonuclease I: 0.05%
人類	人類	Corneal limbal epithelial sheet	Neutral Protease: 5%	see reference	Espana EM, Romano AC, Kawakita T, Di Pascuale M, Smiddy R, and Tseng SC:Novel enzymatic isolation of an entire viable human limbal epithelial sheet, Inv Ophthal Visual Sci 44(10), 4275, 2003
人類	人類	Intestinal epithelial	Collagenase Type 4: 72.5 u/ml	HBSS	Fahlgren, A., Hammarstrom, S., Danielsson, A. and Hammarstrom, M.:Increased Expression of Antimicrobial Peptides and Lysozyme in Colonic Epithelial Cells of Patients with Ulcerative Colitis., Clin Exp Immunol 131, 90, 2003
人類	人類	Gastric epithelial cells	Collagenase Type 2: 200 u/ml	L-15	Smoot, D., Sewchand, J., Young, K., Desbrodes, B., Allen, C. and Naab, T.: A Method for Establishing Primary Cultures of Human Gastric Epithelial Cells,Meth Cell Sci 22, 133, 2000
			Neutral Protease: 1.2 u/ml
			Soybean Trypsin Inhibitor: 0.125%
人類	人類	Gastric	Collagenase Type 4: 0.01%	F-12 medium	Sarosiek, J, Marshall, B, Peura, D, Guerrant, L, McCallum, R and Little, C: The Isolation and Maintenance of Human Gastric Epithelial Cells in Primary Culture,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12B:10.1, 1995
人類	人類	Nasal polyp epithelial	Neutral Protease: .004%	see reference	Halbert CL, Alexander IE, Wolgamot GM, Miller AD: Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells, J Virol 69, 1473-9, 1995
			Trypsin: 0.1%
人類	人類	Epithelial	Trypsin: 0.05%	DMEM	Sabatini, L., Allen-Hoffmann, B, Warner, T., and Azen, E.: Serial C*tion of Epithelial Cells from Human and Macaque Salivary Glands, In Vitro Cell Dev Biol27A, 939, 1991
人類	人類	Epithelial	Trypsin: 0.2%	MEM, PBS	Robinson, C., and Wu, R.: Culture of Conducting Airway Epithelial Cells in Serum-Free Medium, J Tiss Cul Meth 13, 95, 1991
人類	人類	Epithelial	Collagenase: 2.0%	DMEM/Ham's F-12	Emerman, J. and Wilkinson, D.: Routine Culturing of Normal, Dysplastic and Malignant Human Mammary Epithelial Cells from Small Tissue Samples, In Vitro Cell Dev Biol 26, 1186, 1990
人類	人類	Epithelial	Pronase: 0.1%	PBS	Gruenert, D.C., Basbaum, C.B., and Widdicombe, J.H.: Long-Term Culture of Normal and Cystic Fibrosis Epithelial Cells Grown Under Serum-Free Conditions, In Vitro Cell Dev Biol 26, 411, 1990
人類	人類	Epithelial	Collagenase Type 2: 0.2%	(see reference)	Wood, L. and Neufeld, E.: A Cystic Fibrosis Phenotype in Cells Cultured from Sweat Gland Secretory Coil. Altered Kinetics of 36Cl Efflux, J Biol Chem 265, 12796, 1990
		Sweat gland
人類	人類	Endometrial epithelial	Trypsin:	DMEM/Ham's F-12	Munson, L., Chandler, S., and Schlafer, D.: C*tion of Bovine Fetal and Adult Endometrial Epithelial Cells, J Tiss Cul Meth 11 (3), 129, 1988
	(包括牛)
人類	人類	Epithelial	Deoxyribonuclease I: 0.01%	HEPES with 5.9mM Glucose,5mM DTT	Widdicombe, J.H., Coleman, D.L., Finkbeiner, W.E., and Tuet, I.K.: Electrical Properties of Monolayers Cultured From Cells of Human Tracheal Mucosa, J Appl Physiol 58, 1729, 1985
人類	人類	Epithelial	Protease Type XIV: 0.1%	Eagle's MEM	Yankaskas, J., Cotton, C., Knowles, M., Gatzy, J., and Boucher, R.: Culture of Human Nasal Epithelial Cells on Collagen Matrix Supports, Am Rev Respir Dis 132, 1281, 1985
人類	人類,女人, 27-49 歲	Epithelial	Trypsin: 0.125%	HBSS, CMF	Auersperg, N., Siemens, C.H., and Myrdal, S.E.: Human Ovarian Surface Epithelium In Primary Culture, In Vitro 20, 743, 1984
		Ovary
人類	人類,嬰兒和新生兒	Epithelial	Trypsin: 0.1%	HBSS	Lechner, J., Babcock, M., Marnell, M., Narayan, K., and Kaighn, M.: Normal Human Prostate Epithelial Cell Cultures, Methods Cell Biol 21, 195, 1980
		Prostate
小鼠	小鼠, 成年的	Cilary epithelial	PDS kit: per instructions	EBSS	Gualdoni, S., Baron, M., Lakowski, J., Decembrini, S., Pearson, R., Ali, R. and Sowden, J.: Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells., Curr Protoc Stem Cell Biol Chapter 1, Unit 1H.4, 2011
小鼠	小鼠, 雄性, 8-16 周	Renal tubular epithelial	Collagenase: 200 u/ml	HBSS	Breggia, A. and Himmelfarb, J.: Primary Mouse Renal Tubular Epithelial Cells have Variable Injury Tolerance to Ischemic and Chemical Mediators of Oxidative Stress.,Oxid Med Cell Longev 1, 33, 2008
			Soybean Trypsin Inhibitor: see reference
小鼠	小鼠, 6-8 周	Lamina propria mononuclear cells	Collagenase Type 2: 0.015%	RPMI	Wirtz S., Becker C., Blumberg R., Galle P., and Neurath M.: Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA, J Immunol 168(1), 411, 2002
			Deoxyribonuclease I: 0.01%
小鼠	小鼠, 11 周	Epithelial	Collagenase Type 3: 25 u/ml	DMEM/F12	Mueller, S., Clark, J., Myers, P. and Korach, K.: Mammary Gland Development in Adult Mice Requires Epithelial and Stromal Estrogen Receptor Alpha.,Endocrinology 143, 2357, 2002
			Hyaluronidase: 0.1%
			Protease XIV: 0.05%
			Deoxyribonuclease I: 0.04%
小鼠	小鼠, 雌性	Salivary gland epithelial	Collagenase Type 1: 750 u/ml	DMEM/F12	Ishimaru N, Saegusa K, Yanagi K, Haneji N, Saito I, Hayashi Y: Estrogen deficiency accelerates autoimmune exocrinopathy in murine Sjogren's syndrome through fas-mediated apoptosis, Am J Pathol 155, 173-81, 1999
			Hyaluronidase: 500 u/ml
小鼠	小鼠, 胎兒12-13 天	Intestinal mesenchymal and epithelial	Collagenase: 0.03%	DMEM	Simon-Assmann, P and Kedinger, M: Embryonic Gut-Dissagregated Culture,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12A:3.1, 1995
小鼠	小鼠	Submandibular salivary	Collagenase Type 2 or 3: 0.16%	DMEM	Durban, E: Submandibular Salivary Epithelial Cells,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12B:2.1, 1995
小鼠	小鼠	Esophageal	Trypsin: 0.25%	PBS, CMF	Katayama, M., Kan, M.: Heparin-Binding (Fibroblast) Growth Factors re Potential Autocrine Regulators of Esophageal Epithelial Cell Proliferation, In Vitro Cell Dev Biol 27, 533, 1991
小鼠	小鼠, 成年的, 雌性, 6-8周大	Uterine	Trypsin: 0.2%	HBSS	Ghosh, D., Danielson, K., Alston, J., and Heyner, S.: Functional Differentiation of Mouse Uterine Epithelial Cells Grown on Collagen Gels or Reconstituted Basement Membranes, In Vitro Cell Dev Biol 27A, 713, 1991
小鼠	小鼠, 雌性, 18 - 20天	Uterine	Trypsin: 0.25%	HBSS	Fukamachi, H., and McLachlan, J.: Proliferation and Differentiation of Mouse Uterine Epithelial Cells in Primary Serum-Free Culture: Estradiol-17B Suppresses Uterine Epithelial Proliferation Cultured on a Basement Membrane-Like Substratum,In Vitro Cell Dev Biol 27A, 907, 1991
	(包括 20 - 22 天)
小鼠	小鼠, (白變種實驗室老鼠), 雄性, 3-5 月	Epithelial	Collagenase Type 3: 0.16% , 1:1 v/v	DMEM with 15 mM HEPES	Durban, E.M.: Mouse Submandibular Salivary Epithelial Cell Growth and Differentiation in Long-Term Culture:Influence of the Extracellular Matrix, In Vitro Cell Dev Biol 26, 33, 1990
		Submandibular salivary gland
小鼠	小鼠, 雌性	Epithelial	Pepsin: 0.1%	HBSS	Reiser, M., Huff, B., and Medina, D.: Pepsin Can be Used to Subculture Viable Mammary Epithelial Cells, In Vitro 19 (9), 730, 1983
小鼠	小鼠, (白變種實驗室老鼠fC3H or白變種實驗室老鼠) 8-12 天中孕	Epithelial	Collagenase Type 3: 0.1%	HBSS	Yang, J., Flynn, D., Larson, L., and Hamamoto, S.: Growth in Primary Culture of Mouse Submandibular Epithelial Cells, In Vitro 18, 435, 1982
		Submandibular gland
小鼠	小鼠 (白變種實驗室老鼠fC3H)	Mammary tumors	Collagenase: 1.0%	HBSS	Yang, J., Guzman, R., Richards, J., and Nandi, S.: Primary Cultures of Mouse Mammary Tumor Epithelial Cells Embedded in Collagen Gels, In Vitro 16, 502, 1980
		Epithelial
小鼠	小鼠, C3H, 6-8 周	Epithelial	Collagenase: 0.10%	DMEM	Lillehaug, J., Mondal, S., and Heidelberger, C.: Establishment of Epithelial Cell Lines from Mouse Regenerating Liver, In Vitro 15, 910, 1979
豬	豬, 5-60 公斤	Retinal pigment epithelial cells	Collagenase: 2%	DMEM	Wiencke, A., Kiilgaard, J., Nicolini, J., Bundgaard, M., Ropke, C., and La Cour, M.: Growth of Cultured Porcine Retinal Pigment Epithelial Cells, Acta Opthalmol Scand 81(2), 170, 2003
豬	豬	Trachea	Neutral Protease: 0.2%	HBSS	De Buysscher, E., Kennedy, J., and Mendicino, J.: Synthesis of Mucin Glycoproteins by Epithelial Cells Isolated from Swine Trachea by Specific Proteolysis, In Vitro 20, 433, 1984
兔	兔, 新西蘭白, 成年的	Colon	Neutral Protease: 0.3%	PBS	Vidrich, A., Racindranath, R., Farsi, K., and Targan, S.: A Method for the Rapid Establishment of Normal Adult Mammalian Colonic Epithelial Cell Cultures, In Vitro Cell Dev Biol 24 (3), 188, 1988
兔	兔, 新西蘭白, 雄性, 4-5磅	Gastric	Collagenase Type 2: 0.08%	Sodium phosphate buffer	Chew, C.S., Brown,M.R.: Release of Intracellular Ca2+ and Elevation of Inositol Triphosphate by Secretagogues in Parietal and Chief Cells Isolated from Rabbit Gastric Mucosa, Biochim Biophys Acta 888, 116, 1986
		Parietal and chief
兔	兔, 新西蘭白動情期的,雌性, 4-5 個月	Mesothelial and surface epithelial	Trypsin: 0.125%-0.5%	Medium 199	Nicosia, S., Johnson, J., and Streibel, E.: Isolation and Ultrastructure of Rabbit Ovarian Mesothelium(Surface Epithelium), Int J Gynecol Pathol 3, 348, 1984
		Ovaries
兔	兔, 胎兒的	Epithelial	Collagenase Type 3: 0.10%	HBSS	Logsdon, C.D., Bisbee, C.A., Rutten, M.J. and Machen, T.E.: Fetal Rabbit Gastric Epithelial Cells Cultured on Floating Collagen Gel, In Vitro 18, 233, 1981
		Gastric
大鼠	大鼠	Mammary epithelial	Collagenase Type 3: 0.35%	HBSS	Mei, N., McDaniel, L., Dobrovolsky, V., Guo, X., Shaddock, J., Mitstaedt, R., Azuma, M., Shelton, S., McGarrity, L., Doerge, D. and Heflich, R.: The Genotoxicity of Acrylamide and Glycidamide in Big Blue Rats., Toxicol Sci 115, 412, 2010
大鼠	大鼠, SD, 成年的,男性, 8-10 周大	Seminiferous tubules	Trypsin: 0.05%	Krebs-Ringer bicarbonate buffer	Abou-Haila, A., and Tulsiani, D.: Acid Glycohydrolases in Rat Spermatocytes, Spermatids and Spermatozoa: Enzyme Activities, Biosynthesis and Immunolocalization, Biol Proced Online 3 (1), 35, 2001
				(see reference)
大鼠	大鼠, 男性	Alveolar epithelial	Elastase: 40 u/ml	DMEM	Planus, E., Galiacy, S., Matthay, M., Laurent, V., Gavrilovic, J., Murphy, G., Clerici, C., Isabey, D., Lafuma, C., and d'Ortho, M.: Role of Collagenase in Mediating in Vitro Alveolar Epithelial Wound Repair, J Cell Sci 112, 243, 1999
大鼠	大鼠, 胚胎	Tracheal epithelial	Collagenase Type 4: 0.05%	DMEM/F12	Shannon JM, Gebb SA, and Nielsen LD: Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture,Development 126, 1675, 1999
			Neutral Protease:
			Deoxyribonuclease I:
大鼠	大鼠, fetal, 14-15 day	Intestinal mesenchymal and epithelial	Collagenase: 0.03%	DMEM	Simon-Assmann, P and Kedinger, M: Embryonic Gut-Dissagregated Culture,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12A:3.1, 1995
大鼠	大鼠, 4-14 周	Retinal pigment epithelial cells	Collagenase Type 1: 65 u/ml	CF Hanks with EDTA	Wang N, Koutz CA, and Anderson RE.: A method for the isolation of retinal pigment epithelial cells from adult rats, Inv Ophthal Visual Sci 34(1), 101, 1993
			Hyaluronidase: 220 u/ml
大鼠	大鼠, SD, 成年的, 90-120 日大, 350-450克	Epididymal epithelial	Collagenase Type 2: 0.1%	HBSS	Klinefelter, G.: A Novel System for the Co-Culture of Epididymal Epithelial Cells and Sperm From Adult Rats, J Tiss Cul Meth 14, 195, 1992
大鼠	大鼠, 6 天大	Rat intestinal epithelial	Neutral Protease: 0.01%	DMEM	Evans GS, Flint N, Somers AS, Eyden B, and Potten CS.: The development of a method for the preparation of rat intestinal epithelial cell primary cultures, J Cell Sci 101, 219, 1992
			Collagenase: 300 u/ml
大鼠	大鼠, SD, 雄性, 150 - 250克	Epithelial	Pronase: 0.15%	Medium 199	Dial, E., Kao, Y., and Lichtenberger, L.: Effects of 16,16-Dimethyl Prostaglandin E2 On Glycoprotein And Lipid Synthesis of Gastric Epithelial Cells Grown in a Primary Culture, In Vitro Cell Dev Biol 27, 39, 1991
		Stomach
大鼠	大鼠, 成年的	Interlobular duct fragments	Papain:	DMEM/Ham's F-12	Heimann, T., and Githens, S.: Rat Pancreatic Duct Epithelium Cultured on a Porous Support Coated with Extracellular Matrix, Pancreas 6 (5), 514, 1991
	(包括倉鼠)
大鼠	大鼠, SD, 200 克	Colon	Deoxyribonuclease I: 10 礸/ml	(see reference)	Yassin, R., Clearfield, H., Katz, S., and Murthy, S.: Gastrin Induction of mRNA Expression in Rat Colonic Epithelium In Vitro, Peptides 12, 63, 1991
大鼠	Wistar大鼠, 新生的	Epithelial	Trypsin: 0.1%	HBSS	Jassal, D., Han, R., Caniggia, I., Post, M., and Tanswell, A.: Growth of Distal Fetal Rat Lung Epithelial Cells in a Defined Serum-Free Medium, In Vitro Cell Dev Biol27A, 625, 1991
大鼠	嚙齒目動物, 各種各樣的 (參考)	Epithelial	Trypsin: 0.2%	MEM, PBS	Robinson, C., and Wu, R.: Culture of Conducting Airway Epithelial Cells in Serum-Free Medium, J Tiss Cul Meth 13, 95, 1991
大鼠	大鼠, 6-8 天	Retinal pigment epithelial	Neutral Protease: 2%	DMEM	Chang CW, Roque RS, Defoe DM, and Caldwell RB.: An improved method for isolation and culture of pigment epithelial cells from rat retina, Curr Eye Res10(11), 1081, 1991
大鼠	大鼠 (ACI/NMs X BUF/Mna) F1, 雄性, 28個月	Epthelial	Collagenase Type 3: 0.1%	Eagle's MEM Serum-free	Masuda, A., Ohtsuka, K., and Matsuyama, M.: Establishment of Functional Epithelial Cell Lines from a Rat Thyoma and a Rat Thymus, In Vitro Cell Dev Biol 26, 713, 1990
	大鼠 (ACI/MNs) 雄性, 8周
大鼠	大鼠, 費希爾344, 雄性, 8 周大,	Tracheal epithelial	Pronase: 0.5%	DMEM	Chang, L., Wu, R., and Nettesheim, P.: Morphological Changes in Rat Tracheal Cells During The Adaptive and Early Growth Phase in Primary Cell Culture, J Cell Sci74, 283, 1985
	250-300 克
大鼠	大鼠,費希爾, 雄性, 4-6 周	Epithelial	Hyaluronidase: 0.1%	HEPES BSS	Babcock, M., Marino, M., Gunning, W., and Stoner, G.: Clonal Growth and Serial Propagation of Rat Esophageal Epithelial Cells, In Vitro 19, 403, 1983
		Esophagus
大鼠	大鼠, 費希爾, Lewis and SD, 雄性, 10-18 個月	Epithelial	Trypsin: 0.05%	HBSS CMF	Herring, A., Raychaudhuri, R., Kelley, S., and Iybe, P.: Repeated Establishment of Diploid Epithelial Cell Cultures from Normal and Partially Hepatectomized Rats,In Vitro 19, 576, 1983
大鼠	大鼠, SD, 雌性	Epithelial, cancer and tumor	Collagenase: 0.1%	Eagles's MEM	Cohen, L.: Isolation and Characterization of a Serially C*ted, Neoplastic, Epithelial Cell Line from the N-nitrosomethylurea Induced Rat Mammary Adenocarcinoma, In Vitro 18, 565, 1982
大鼠	Wistar大鼠, 12 天	Epithelial	Trypsin: 0.05%	HBSS, CMF	Malan-Shibley, L., and Iype, P.: Influence of Cultures on Cell Morphology/Tyrosine Aminotransferase Levels, Exp Cell Res 131, 363, 1981
大鼠	大鼠,費希爾, 成年的, 200-250 克	Epithelial	Hyaluronidase: 0.0075%	KCl-NaCl HEPES Buffer	Williams, G., and Gunn, J.: Long-Term Culture of Adult Rat Liver Epithelial Cells,Exp Cell Res 89, 139, 1974
大鼠	Fischer大鼠, 10 天	Epithelial-like	Trypsin: 0.25%	PBS	Williams, G., Weisburger, E., and Weisburger, J.: Isolation and Long-Term Cell Culture of Epithelial-Like Cells from Rat Liver, Exp Cell Res 69, 106, 1971

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