生殖組織消化手冊

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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。

生殖組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
牛	牛	Corpus leuteal cells	Collagenase Type 4: 420 u/ml	M-199	Levy N, Gordin M, Mamluk R, Yanagisawa M, Smith M F, Hampton J H, Meidan R:Distinct cellular localization and regulation of endothelin-1 and endothelin-converting enzyme-1 expression in the bovine corpus luteum: implications for luteolysis, Endocrinology 142, 5254-60, 2001
牛	牛, 雌性	Leuteal	Collagenase Type 1: 0.2%	Ham's F-12	Tsang PC, Poff JP, Boulton EP, Condon WA: Four-day-old bovine corpus luteum: progesterone production and identification of matrix metalloproteinase activity in vitro, Biol Reprod 53, 1160-8, 1995
牛	牛犢,中晚期妊娠	Fibroblasts	Collagenase: 0.1%	Medium 199	Coplen, D., Howard, P., Duckett, J., Snyder, H., and Macarak, E.:Characterization of a Fibroblast Cell From the Urinary Bladder Wall, In Vitro Cell Dev Biol 30A, 604, 1994
牛	牛	Epithelial	Collagenase Type 2: 0.1%	DMEM/EBSS	Munson, L., Chandler, S., and Schlafer, D.: Long-Term Culture of Bovine Trophoblastic Cells, J Tiss Cul Meth 11 (3), 123, 1988
		Endometrial
牛	牛	Interna & corpus luteum	Pronase: 0.1%	Moscona's BSS	Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984
	(包括豬，人類)	Endometrium
		Ovarian
		Uterine
犬	犬	Amniotic membrane MSC	Trypsin: 0.25%	LG-DMEM	Park, S., Seo, M., Kim, H. and Kang, K.: Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells., PLoS ONE 7, e44693, 2012
			Collagenase Type 1: 0.2%
雞	雞, 家雞,20-30 周	Primary follicles	Trypsin: 0.15%	Dulbecco's phosphate buffered saline	Du Meihong, Han Haitang, Jiang Bin, Zhao Chen, Qian Changsong, Shen Haiyan, Xu Yan, Li Zandong: An efficient isolation method for domestic hen (Gallus domesticus) ovarian primary follicles, J Reprod Dev 52, 569-76, 2006
			Collagenase Type 1: 0.125%
青蛙	非洲蟾蜍光滑的	Oocytes	Collagenase: 0.2%	Barth's solution	Chatzidaki, A., D'Oyley, J., Gill-Thind, J., Sheppard, T. and Millar, N.: The Influence of Allosteric Modulators and Transmembrane Mutations on Desensitisation and Activation of alpha7 Nicotinic Acetylcholine Receptors., Neuropharmacol 97, 75-85, 2015
青蛙	非洲蟾蜍光滑的, 雌性	Oocytes	Collagenase: 0.2%	see reference	Mruk, K. and Kobertz, W.: Discovery of a Novel Activator of KCNQ1-KCNE1 K Channel Complexes., PLoS ONE 4, e4236, 2009
青蛙	非洲蟾蜍光滑的, 雌性	Oocytes	Collagenase: 0.5%	Barth's solution, CF	Cohen, S., Au, S. and Pante, N.: Microinjection of Xenopus Laevis Oocytes., J Vis Exp 24, 1106, 2009
青蛙	非洲蟾蜍光滑的, 雌性	Oocytes	Collagenase Type 1: 0.2%	see reference	Pannaccione Anna, Castaldo Pasqualina, Ficker Eckhard, Annunziato Lucio, Taglialaa Maurizio: Histidines 578 and 587 in the S5-S6 linker of the human Ether-a-gogo Related Gene-1 K+ channels confer sensitivity to reactive oxygen species, J Biol Chem 277, 8912-9, 2002
青蛙	非洲蟾蜍光滑的, 雌性	Oocytes	Collagenase Type 1: 0.2%	CF Medium	Alagem, N., Dvir, M., and Reuveny, E.: Mechanism of Ba²⁺ Block of a Mouse Inwardly Rectifying K⁺ Channel: Differential Contribution by Two Discrete Residues, J Physiol 534 (2), 381, 2001
青蛙	非洲蟾蜍	Oocytes	Collagenase Type 1: 1%	(see reference)	Tian, J., Kim, S., Heilig, E., and Ruderman, J.: Identification of XPR-1, A Progesterone Receptor Required for Xenopus Oocyte Activation, Proc Natl Acad Sci U S A 97, 14358, 2000
青蛙	非洲蟾蜍光滑的, 雌性	Oocytes	Collagenase: 0.1%	Barth's solution, CF	Karkanias, N, and Papke, R.: Subtype-Specific Effects of Lithium on Glutamate Receptor Function, J Neurophysiol 81, 1506, 1999
青蛙	非洲蟾蜍光滑的, 成熟的雌性	Oocytes	Collagenase: 0.2%	CF Medium	Moriarty, T., Gillo, B., Carty, D., Premont, R., Landau, E., Iyengar, R.: Beta gamma Subunits of GTP-Binding Proteins Inhibit Muscarinic Receptor Stimulation of Phospholipase C, Proc Natl Acad Sci U S A 85, 8865, 1988
馬	馬，中國的	Ovary	Trypsin: 5%	Dialyzed fetal calf serum, 10% and 0.5M Methotrexate	Wallis, R., and Drickamer, K.: Molecular Determinants of Oligomer Formation and Complement Fixation in Mannose-Binding Proteins, J Biol Chem 274 (6), 3580, 1999
人類	人類	Endothelial colony forming	Collagenase Type 1: 0.1%	HBSS	Pa, J., Seppanen, E., Chong, M., Yeo, J., Teo, E., Chan, J., Fisk, N. and Khosrotehrani, K.: Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells from Human Term Placenta., Stem Cells Transl Med 2, 839-47, 2013
			Deoxyribonuclease I: 0.1%
			Neutral Protease: 0.075%
人類	人類	Uterine epithelial	Pancreatin: 0.34%	HBSS	Pa, M., Ghosh, M., Fahey, J. and Wira, C.: Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol., PLoS ONE 7, e35654, 2012
			Hyaluronidase: 0.01%
			Collagenase: 0.16%
人類	人類	Mesenchymal stem	Collagenase Type 1: 0.4%	DMEM/F12	Shalini, V., Pratheep, S., Muhammad, A., Sharmili, V., Elizabeth, G. and Rajesh, R.: Generation and Characterisation of Human Mesenchymal Stem Cells Derived From Umbilical Cord and Placenta, Regenerative Research 1, 48, 2012
			Deoxyribonuclease I: 0.01%
人類	人類	Mesenchymal stem	Collagenase Type 2: 10%	CMF-DPBS	Steigman, S. and Fauza, D.: Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta., Curr Protoc Stem Cell Biol Chapter 1, Unit 1E.2, 2007
			Neutral Protease: see reference
人類	人類, 女性	Decidual	Collagenase: 0.25%	DMEM/F12	Lockwood, C., Arcuri, F., Toti, P., Felice, C., Krikun, G., Guller, S., Buchwalder, L. and Schatz, F.: Tumor Necrosis Factor-Alpha and Interleukin-1 Beta Regulate Interleukin-8 Expression in Third Trimester Decidual Cells: Implications for the Genesis of Chorioamnionitis., Am J Pathol 169, 1294-302, 2006
			Deoxyribonuclease I: 6.25 u/ml
人類	人類	Extravillanous Trophoblasts and Decidual Stromal	Trypsin: 0.1-0.25%	HBSS RPMI	Spessotto, P., Bulla, R., Danussi, C., Radillo, O., Cervi, M., Monami, G., Bossi, F., Tedesco, F., Doliana, R. and Colombatti, A.: EMILIN1 Represents a Major Stromal Element Determining Human Trophoblast Invasion of the Uterine Wall., J Cell Sci119, 4574, 2006
			Collagenase Type 1: 0.1%
			Deoxyribonuclease I: 0.02-0.05%
人類	人類	Sertoli cells	Trypsin: 2.5%	DMEM/F-12	Teng Yan, Xue Wu-jun, Ding Xiao-ming, Feng Xin-shun, Xiang He-li, Jiang Ya-zhuo, Tian Pu-xun: Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro, Chin Med J (Engl) 118, 1857-62, 2005
			Collagenase Type 1: 2%
			Hyaluronidase: 1%
人類	人類, 女性	Uterine epithelial	Pancreatin: 0.34%	HBSS	Meter, R., Wira, C. and Fahey, J.: Secretion of Monocyte Chemotactic Protein-1 by Human Uterine Epithelium Directs Monocyte Migration in Culture., Fertil Steril 84, 191, 2005
			Hyaluronidase: 0.01%
			Collagenase: 0.16%
人類	人類, 女性, 34-51 歲	Endometrial epithelial and stromal cells	Collagenase Type 3: 0.03%	DMEM/F-12	Chan Rachel W S, Schwab Kjiana E, Gargett Caroline E: Clonogenicity of human endometrial epithelial and stromal cells, Biol Reprod 70, 1738-50, 2004
			Deoxyribonuclease I: 0.004%
人類	人類	Endothelial placental	Collagenase Type 1: 0.2%	DMEM	Wang Xin, Athayde Neil, Trudinger Brian: Microvascular endothelial cell activation is present in the umbilical placental microcirculation in fetal placental vascular disease, Am J Obstet Gynecol 190, 596-601, 2004
			Trypsin: 0.2%
			Deoxyribonuclease I: 0.1%
人類	人類	Amnion epithelial and fibroblast	Trypsin: 0.125%	PBS	Sun Kang, Myatt Leslie: Enhancement of glucocorticoid-induced 11beta-hydroxysteroid dehydrogenase type 1 expression by proinflammatory cytokines in cultured human amnion fibroblasts, Endocrinology 144, 5568-77, 2003
			Collagenase: 0.1%
			Deoxyribonuclease I: 0.02%
人類	人類	Uterine epithelial cells	Pancreatin: 0.34%	HBSS	Fahey John V, Wira Charles R: Effect of menstrual status on antibacterial activity and secretory leukocyte protease inhibitor production by human uterine epithelial cells in culture, J Infect Dis 185, 1606-13, 2002
			Collagenase: 0.16%
			Hyaluronidase: 0.16%
人類	人類,胚胎	Endothelial	Collagenase Type 1/2/4: 0.1%	DMEM	Oberlin, E., Tavian, M., Blazsek, I., and Peault, B.: Blood-forming potential of vascular endothelium in the human embryo, Development 129, 4147, 2002
		Hematopoietic
		Stromal
人類	人類, 女性	Mesothelial	Collagenase Type 1: 0.1%	Eagle's MEM	Witz Craig A, Allsup Karen T, Montoya-Rodriguez Iris A, Vaughn Shelby L, Centonze Victoria E, Schenken Robert S: Culture of menstrual endometrium with peritoneal explants and mesothelial monolayers confirms attachment to intact mesothelial cells, Hum Reprod 17, 2832-8, 2002
			Deoxyribonuclease I: 0.05%
人類	人類	Stem, embryonic	Neutral Protease: 0.01% - 0.02%	DMEM	Zhang, S., Wernig, M., Duncan, I., Brustle, O., and Thomson, J.: In VitroDifferentiation of Transplantable Neural Precursors from Human Embryonic Stem Cells, Nat Biotechnol 19, 1129, 2001
人類	人類, 20-40 歲	Endometrium epithelial and stromal cells	Collagenase Type 1: 0.2%	HBSS	Arnold, J., Kaufman, D., Seppala, M., and Lessey, B.: Endometrial stromal cells regulate epithelial cell grownth in vitro: a new co-culture model, Hum Reprod 16, 836, 2001
人類	人類	Chorionic villi	Trypsin: see reference	HBSS	Yusuf RZ, Naeem R: Cytogenetic studies of spontaneous miscarriages: a seven year study to compare significance of primary vs. secondary culture methods for assessment of fetal karyotype yield and maternal cell contamination, Early Pregnancy 5, 121-31, 2001
			Collagenase Type 3: 100 u/ml
人類	人類	Endometrial endothelial cells	Collagenase Type 1: 0.2%	McCoys medium	Nikitenko LL, MacKenzie IZ,Rees MC, Bicknell R: Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium, Mol Hum Reprod 6, 811, 2000
人類	人類, 女性, 20-40 歲	Microvascular endothelial cells	Collagenase Type 2: 0.2%	PBS	Gargett CE, Bucak K, Rogers PA: Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells, Hum Reprod 15, 293-301, 2000
			Deoxyribonuclease I: 0.0015%
			Trypsin: 0.05%
人類	人類,女性	Endometrial epithelial cells	Collagenase Type 3: 45 u/ml	DMEM/F-12	Zhang J, Lathbury LJ, Salamonsen LA: Expression of the chemokine eotaxin and its receptor, CCR3, in human endometrium, Biol Reprod 62, 404-11, 2000
			Deoxyribonuclease I: .00035%
人類	人類	Theca cells	Collagenase Type 1: 0.3%	PBS	Runesson E, Ivarsson K, Janson P, Brannstrom M: Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle, J Clin Endocrinol Metab 85, 4387-95, 2000
			Deoxyribonuclease I: 0.0005%
			Hyaluronidase: 0.1%
人類	人類	Corpus luteum cells	Collagenase Type 2: 0.25%	PBS	Friden BE, Runesson E, Hahlin M, Brannstrom M: Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum, Mol Hum Reprod 6, 397-403, 2000
			Deoxyribonuclease I: .005%
人類	人類, 女性, 25-45 歲	Luteal cells	Collagenase Type 2: 0.25%	PBS	Friden BE, Hagstrom H, Lindblom B, Sjoblom P, Wallin A, Brannstrom M, Hahlin M: Cell characteristics and function of two enriched fraction of human luteal cells prolonged culture, Mol Hum Reprod 5, 714-9, 1999
			Deoxyribonuclease I: 0.005%
人類	人類, 女性, 25-41 歲	Follicles	Collagenase Type 2: 0.025-0.1%	EBSS	Hovatta O, Wright C, Krausz T, Hardy K, Winston RM: Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation, Hum Reprod 14, 2519-24, 1999
人類	人類	Placental	Deoxyribonuclease I: 0.04%	PSS	Bradbury., R., Sunn, K., Crossley, M., Bai, M., Brown, E., Delbridge, D., and Conigrave, A.: Expression of the Parathyroid Ca 2+ - Sensing Receptor in Cytotrophoblasts From Human Term Placenta, J Endocrinol 156, 425-430, 1998
			Collagenase Type 2: 0.1%
人類	人類, 女性	Endometrial stromal cells	Collagenase: 4000 u/ml	DMEM/F-12	Huang JC, Liu DY, Dawood MY: The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17beta-oestradiol, Mol Hum Reprod 4, 603-7, 1998
人類	人類	Stromal endometrial	Collagenase Type 3: 45 u/ml	DMEM/Ham's F12	Zhang J, Nie G, Jian W, Woolley DE, Salamonsen LA: Mast cell regulation of human endometrial matrix metalloproteinases: A mechanism underlying menstruation, Biol Reprod 59, 693-703, 1998
			Deoxyribonuclease I: .00035%
人類	人類	Epithelial, fallopian tube	Collagenase Type 1: 1%	Medium 199	Takeuchi, K., Maruyama, I., Yamamoto, S., Oki, T., Nagata, Y.: Isolation and Monolayer Culture of Human Fallopian Tube Epithelial Cells, In Vitro Cell Dev Biol 27, 720, 1991
人類	人類	Trophoblasts, placental	Trypsin: 0.25%	EBSS, CMF	Branchaud, C.L., Goodyer, C.G., Guyda, H.J. and Lefebvre, Y.: A Serum-Free System for Culturing Human Placental Trophoblasts, In Vitro Cell Dev Biol 26, 865, 1990
人類	人類	Trophoblasts, placental	Trypsin: 0.25%	PBS	Jie, Z., Fey, S., Hager, H., Hollsberg, P., Ebbesen, P., and Larsen, P.: Markers For Human Placental Trophoblasts in Two-Dimensional Gel Electrophoresis, In Vitro Cell Dev Biol 26, 937, 1990
人類	人類	Chorionic, placental	Deoxyribonuclease I: 0.003%	HBSS	Egan, D., Grzegorczyk, V., Tricarico, K., Rueter, A.H., Olleman, W., and Marcotte, P.: Human Placental Chorionic Renin: Production, Purification and Characterization, Biochim Biophys Acta 965, 68, 1988
人類	人類, 女性	Endometrial	Collagenase: 2%	RPMI 1640	Rinehart, C., Lyn-Cook, B., and Kaufman, D.: Gland formation from human endometrial epithelial cells in vitro, In Vitro Cell Dev Biol 24 (10), 1037, 1988
人類	人類	Placental	Trypsin: 0.25%	DMEM	Morrish, D., and Siy, O.: Critical Factors in Establishing Monolayer Cultures of Normal Human Placental Cells in Serum-Free Medium, Endocr Res 12 (3), 229, 1986
人類	人類	Interna & corpus luteum	Pronase: 0.1%	Moscona's BSS	Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984
	(包括豬，牛)	Endometrium
		Ovarian
		Uterine
人類	人類,女性, 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	Epithelial	Collagenase: 0.25%	See reference	Siegfried, J., Nelson, K., Martin, J., and Kaufman, D.: Histochemical Identification of Cultured Cells From Human Endometrium, In Vitro 20 (1), 25, 1984
		Stromal
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
人類	人類, 女性, 27 歲	Smooth muscle, uterine	Trypsin: 0.05%	EBSS	Rifas, L., Fant, J., Makman, M., and Seifter, S.: The Characterization of Human Uterine Smooth Muscle Cells in Culture, Cell Tissue Res 196, 385, 1979
人類	人類	Epithelial	Collagenase Type 1: 180 u/ml	DMEM	Kirk, D., King, R., Heyes, J., Peachey, L., Hirsch, P., and Taylor, W.: Normal Human Edometrium in Cell Culture, In Vitro 14 (8), 651, 1978
		Stromal
昆蟲	果蠅	Ovarian	Collagenase Type 2: 0.1%	Grace's	Ma, X., Wang, S., Do, T., Song, X., Inaba, M., Nishimoto, Y., Liu, L., Gao, Y., Mao, Y., Li, H., McDowell, W., Park, J., Malanowski, K., Peak, A., Perera, A., Li, H. et al.: Piwi is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary., PLoS ONE 9, e90267, 2014
昆蟲	果蠅, 胚胎	Cardiac differentiating	Collagenase Type 1: 20 u/ml	Schneider	Salmand, P. Iche-Torres, M. and Perrin, L.: Tissue-Specific Cell Sorting from Drosophila Embryos: Application to Gene Expression Analysis.,Fly (Austin) Vol. 5,, , 261,
			Trypsin: 0.25%
			Deoxyribonuclease I: 4 u/ml
小鼠	小鼠, 成年的	Spermatogonial stem	Trypsin: 0.05%	DMEM	Martin, L. and Seandel, M.: Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-Term Adult Mouse SSC Lines., J Vis Exp 72, e50017, 2013
			Collagenase Type 1: 0.03%
			Deoxyribonuclease I: 80 u/ml
小鼠	小鼠	Ovarian	Collagenase Type 4: 800 u/ml	HBSS	White, Y., Woods, D., Takai, Y., Ishihara, O., Seki, H. and Tilly, J.: Oocyte Formation by Mitotically Active Germ Cells Purified from Ovaries of Reproductive-Age Aomen., Nat Med 18, 413, 2012
小鼠	小鼠, 雌性	Lymphocytes	Collagenase: 450 u/ml	RPMI	Jiang, J. and Kelly, K.: Isolation of Lymphocytes from Mouse Genital Tract Mucosa.,J Vis Exp 67, e4391, 2012
小鼠	小鼠	Vaginal smooth muscle	Collagenase Type 2: 175 u/ml	DMEM/F12	Venkataraman, L., Lenis, A., Couri, B., Damaser, M. and Ramarurthi, A: Induced Regenerative Elastic Matrix Repair in LOXL 1 Knockout Mouse Cell Cultures: Towards Potential Therapy for Pelvic Organ Prolapse, J Tissue Sci Eng 3, 3, 2012
			Elastase: 0.025%
小鼠	小鼠, 雄性	Testis, meiotic	Collagenase Type 1: 120 u/ml	Gey's BSS	Getun, I., Torres, B. and Bois, P.: Flow Cytometry Purification of Mouse Meiotic Cells., J Vis Exp 50, 2602, 2011
			Deoxyribonuclease I: 0.001%
			Trypsin: 0.1%
小鼠	小鼠	Uterine stomal	Neutral Protease: 0.6%	DMEM/F12	Chen, L., Belton, R. and Nowak, R.: Basigin-Mediated Gene Expression Changes in Mouse Uterine Stromal Cells During Implantation., Endocrinology 150, 966, 2009
			Pancreatin: 0.25%
			Collagenase Type 3: 0.05%
小鼠	小鼠	Germ cells	Collagenase Type 1: 100 u/ml	HBSS	Breault, D., Min, I., Carlone, D., Farilla, L., Ambruzs, D., Henderson, D., Algra, S., Montgomery, R., Wagers, A. and Hole, N.: Generation of mTert-GFP Mice as a Model to Identify and Study Tissue Progenitor Cells., Proc Natl Acad Sci U S A 105, 10420, 2008
小鼠	小鼠, 胚胎	Hematopoietic stem cells	Collagenase: 0.1%	PBS	Gekas, C., Rhodes, K. and Mikkola, H.: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta., J Vis Exp 16, e742, 2008
小鼠	小鼠, 6 天	Sertoli	Collagenase Type 2: 500 u/ml	DMEM	Nalbandian Angele, Dettin Luis, Dym Martin, Ravindranath Neelakanta:Expression of vascular endothelial growth factor receptors during male germ cell differentiation in the mouse, Biol Reprod 69, 985-94, 2003
			Hyaluronidase: 0.1%
			Deoxyribonuclease I: .0005%
小鼠	小鼠, 成年的, 新生兒與胎兒	Testicular cells	Collagenase Type 1: 0.1%	DMEM/F-12	O'Shaughnessy PJ, Fleming LM, Jackson G, Hochgeschwender U, Reed P, Baker PJ: Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis, Endocrinology 144, 3279-84, 2003
小鼠	小鼠, 雄性, 3.5 個月大	Leydig	Collagenase: 0.06%	Medium E 199	Faldikova, L., Diblikova, I., Canderle, J., Zraly, Z., Veznik, Z., and Sulcova, A.: Effects of Nutrition, Social Factors and Chronic Stress on the Mouse Leydig Cell Testosterone Production, Vet Med 46 (6), 160, 2001
小鼠	小鼠, 雄性, 12-14-周大	Seminiferous tubules	Trypsin: 0.05%	DMEM	Lin, Q., Sirotkin, A., and Skoultchi, A.: Normal Spermatogenesis in Mice Lacking the Testis-Specific Linker Histone H1t, Mol Cell Biol 20 (6), 2122, 2000
小鼠	小鼠, 6-12 天	Oocyte-granulosa	Collagenase Type 1: 0.1%	Waymouth	Eppig JJ, O'Brien MJ: Development in vitro of mouse oocytes from primordial follicles, Biol Reprod 54, 197-207, 1996
			Deoxyribonuclease I: 0.02%
小鼠	小鼠, 雌性,6-8 周	Uterine	Trypsin: 0.2%	HBSS	Ghosh, D., Danielson, K., Alston, J., Heyner, S.: Functional Differentiation of Mouse Uterine Epithelial Cells Grown On Collagen Gels Or Reconstituted Basement Membranes, In Vitro Cell Dev Biol 27, 713, 1991
小鼠	小鼠, 雌性, 21 天, 10-12 克	vaginal epithelial	Collagenase Type 3: 38 u/ml	DMEM	Tsai, P., Uchima, F., Hamamoto, S., and Bern, H.: Proliferation and Differentiation of Prepubertal Mouse Vaginal Epithelial Cells In Vitro and the Specificity of Estrogen-Induced Growth Retardation, In Vitro Cell Dev Biol 27A, 461, 1991
小鼠	小鼠, CF1, 雌性	Cumulus, one-cell embryos	Hyaluronidase: 0.1%	PBS, CMF	Spindle, A.: In vitro Development of One-Cell Embryos from Outbred Mice: Influence of Culture Medium Composition, In Vitro Cell Dev Biol 26, 151, 1990
小鼠	小鼠 (白變種實驗室老鼠CRGL), 雄性, 2-3 個月	Epithelial, prostate gland	Hyaluronidase: 0.1%	Medium 199	Turner, T., Bern, H., Young,P., and Cunha, G.: Serum-Free Culture of Enriched Mouse Anterior and Ventral Prostatic Epithelial Cells in Collagen Gel, In Vitro Cell Dev Biol 26, 722, 1990
小鼠	小鼠 (C57B/T), 胎兒, 16-17 天大	Prostate	Trypsin: 1.0%	HBSS/ DMEM	Thompson, T.C., Southgate, J., Kitchener, G., and Land, H.: Multistage Carcinogenesis Induced by ras and myc Oncogenes in a Reconstituted Organ, Cell56, 917, 1989
小鼠	小鼠, outbred, CD-1, 21-23 天s old	Epithelial	Trypsin: 0.5%	Medium 199	Tomooka, Y., DiAugustine, R., and McLachlan, J.: Proliferation of Mouse Uterine Epithelial Cells in Vitro, Endocrinology 118 (3), 1011, 1986
小鼠	小鼠, 白變種實驗室老鼠	Epithelial	Trypsin: 1%	DMEM	Bigsby, R., Cooke, P., and Cunha, G.: A Simple Efficient Method For Separating Murine Uterine Epithelial and Mesenchymal Cells, Am J Physiol 251, E630, 1986
		Mesencymal
小鼠	小鼠, 白變種實驗室老鼠Crg, 40 天大	Epithelial	Trypsin: 1%	Medium 199	Cooke, P., Uchima, F., Fujii, D., Bern, H., and Cunha, G.: Restoration of Normal Morphology and Estrogen Responsiveness in Cultured Vaginal and Uterine Epithelia Transplanted with Stroma, Proc Natl Acad Sci U S A 83, 2109, 1986
小鼠	小鼠, 雄性, 10-13 周	Leydig	Deoxyribonuclease I: 0.001%	Medium 199 w/ BSA	Stalvcey, J. and Payne, A.: Luteinizing Hormone Receptors and Testosterone Production in Whole Testes and Purified Leydig Cells from the Mouse: Differences among Inbred Strains, Endocrinology 112, 1696, 1983
		Testis
小鼠	小鼠 (白變種實驗室老鼠Crgl), 雌性, 50 - 60 天	Epithelial, vagina	Collagenase Type 3: 0.1%	HBSS	Iguchi, T., Uchima, F.A., Ostrander, P., and Bern, H.: Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels, Proc Natl Acad Sci U S A 80, 3743, 1983
綿羊	羊	Epithelial	Collagenase: 125 - 190 u/ml	DMEM	Salamonsen, L., Sum O, W., Doughton, B., and Findlay, J.: The Effects of Estrogen and Progesterone In Vivo on Protein Synthesis and Secretion by Cultured Epithelial Cells from Sheep Endometrium, Endocrinology 117 (5), 2148, 1985
豬	豬, 雄性, 8 天	Seminiferous epithelial cells	Collagenase: 0.15%	DMEM/F12	Dirami G, Ravindranath N, Pursel V, Dym M: Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM, Biol Reprod 61, 225-30, 1999
			Deoxyribonuclease I: .0001%
			Hyaluronidase: 0.15%
			Trypsin: 0.05%
豬	豬, 雌性	Corpus Leuteum	Collagenase Type 4: 600 u/ml	Medium 199	Ciereszko, R., Petroff, B., Ottobre, A., Guan, Z., Stokes, B., and Ottobre, J.:Assessment of the Mechanism by Which Prolactin Stimulates Progesterone Production by Early Corpora Lutea of Pigs, J Endocrinol 159, 201, 1998
豬	豬 (包括牛，人類)	Interna & corpus luteum	Pronase: 0.1%	Moscona's BSS	Marcus, G., Connor, L., Domingo, M., Tsang, B., Downey, B., and Ainsworth, L.:Enzymatic Dissociation of Ovarian and Uterine Tissues, Endocr Res 10, 151, 1984
		Endometrium
		Ovarian
		Uterine
豬	豬, 3-4 周	Leydig	Trypsin: 0.0003%	Lebovitz L-15 Medium	Mather, J., Saez, J., and Haour, F.: Regulaton of Gonadotropin Receptors and Steroidogenesis in Cultured Porcine Leydig Cells, Endocrinology 110, 933, 1982
		Testis
兔	兔, 新西蘭, 3 個月	Testicular germ	Collagenase Type 1: 0.1%	HBSS	Kubota, H., Wu, X., Goodyear, S., Avarbock, M. and Brinster, R.: Glial Cell Line-Derived Neurotrophic Factor and Endothelial Cells Promote Self-Renewal of Rabbit Germ Cells with Spermatogonial Stem Cell Properties., FASEB J 25, 2604, 2011
			Trypsin: 0.25%
			Deoxyribonuclease I: 0.7%
兔	兔, 新西蘭白, 4-6 個月大,	Ovarian	Collagenase Type 1: 300 u/ml	HBSS	Setrakian, S., Oliveros-Saunders, B., and Nicosia, S.: Growth Stimulation of Ovarian and Extraovarian Mesothelial Cells by Corpus Luteum Extract, In Vitro Cell Dev Biol 29A, 879, 1993
		Mesothelial
兔	兔, 新西蘭白, 成年的, 雌性 (非妊娠的),	Myocytes, uterine	Deoxyribonuclease I: 200 礸/ml	HBSS-HEPES buffer	Phillippe, M., Saunders, T., and Bangalore, S.: Alpha-1,Alpha-2,and Beta Adrenergic Signal Transduction in Cultured Uterine Myocytes, In Vitro Cell Dev Biol26, 369, 1990
	3-4公斤
兔	兔, 新西蘭, 成年的, 雌性, 4-5公斤	Ovarian surface epithelial and peritoneal mesothelial	Collagenase Type 1: 300 IU/ml (280 IU/mg)	HBSS, CMF	Piquette, G., and Timms, B.: Isolation and Characterization of Rabbit Ovarian Surface Epithelium, Granulosa Cells, and Peritoneal Mesothelium in Primary Culture,In Vitro Cell Dev Biol 26, 471, 1990
兔	兔, 混合品種	Endometrial epithelial	Collagenase Type 1: 0.005%	DMEM	Mulholland, J., Winterhager, E., and Beier, H.: Changes in Proteins Synthesized by Rabbit Endometrial Epithelial Cells Following Primary Culture, Cell Tissue Res 252, 123, 1988
兔	兔, 新西蘭白, 成年的, 3-4 公斤	Myometrial	Trypsin: 0.02%, 0.03%, 0.0375%	HBSS	Boulet, A., and Fortier, M.: Preparation and Characterization of Rabbit Myometrial Cells in Primary Culture: Influence of Estradiol and Progesterone Treatment, In Vitro Cell Dev Biol 23, 93, 1987
兔	兔, 新西蘭白發情的, 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
大鼠	SD大鼠, 350-450 克	Leydig cells	Collagenase: .05-0.1%	Medium 199	Sharma RS, Pal PC, Rajalakshmi M.: Isolation and Culture of Leydig Cells from Adult Rats, Indian J Clinical Biochem 21, 27, 2006
大鼠	SD大鼠, 成年的, 雄性, 8 - 10 周	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	SD大鼠, 未成熟的	Ovary	Collagenase Type 1: 144 u/ml	McCoy's	Ando, M., Kol, S., Irahara, M., Sirois, J., and Adashi, E.: Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) Block the Late, Prostanoid-Dependent/Ceramide-Independent Component of Ovarian IL-1 Action: Implications for the Ovulatory Process, Mol Cell Endocrinol 157, 21, 1999
大鼠	Han-Wistar大鼠, 成年的, 雄性, 250 克	Testes	Trypsin: 0.1%	Medium 199 w/ Hank's salts	Leckie, C., Welberg, L., and Seckl, J.: 11B-Hydroxysteroid Dehydrogenase is a Predominant Reductase in Intact Rat Leydig Cells, J Endocrinol 159, 233, 1998
大鼠	SD大鼠, 雌性, 未成熟的, 26 天	Corpus luteum	Neutral Protease: 2.4 u/ml Deoxyribonuclease: 200u/ml	Serum-free medium	lieria, C., Ou, J., Sugino, N., Ferguson, S., and Gibori, G.: The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid, Endocrinology 139 (8), 3597, 1998
	(包括大鼠, 妊娠和假孕)			(see reference)
大鼠	SD大鼠, 25 天	Ovarian theca-interstitial	Collagenase Type 1: 0.5%	Medium 199	Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR: Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells, Biol Reprod 56, 891-7, 1997
			Deoxyribonuclease I: 0.02%
大鼠	SD大鼠, 雄性, 90 天	Sertoli	Hyaluronidase: 0.1%	DMEM	Onoda, M. and Djakiew, D.: Pachytene Spermatocyte Protein(s) Stimulate Sertoli Cells Grown in Bicameral Chambers: Dose-Dependent Secretion of Ceruloplasmin, Sulfated Glycoprotein-1, Sulfated Glycoprotein-2, and Transferrin, In Vitro Cell Dev Biol 27, 215, 1991
大鼠	SD大鼠, 雄性	Leydig	Trypsin: 0.02%	DMEM	Abayasekara, D., Kurlak, L., Band, A., Sullivan, M., and Cooke, B.: Effect of Cell Purity, Cell Concentration, and Incubation Conditions on Rat Testis Leydig Cell Steroidogenesis, In Vitro Cell Dev Biol 27, 253, 1991
大鼠	大鼠, 新生的	Uterine	Trypsin: 1%	HBSS	Branham, W., Lyn-Cook, B., Andrews, A., McDaniel, M., Sheehan, D.: Growth of Neonatal Rat Uterine Luminal Epithelium on Extracellular Matrix, In Vitro Cell Dev Biol27, 442, 1991
大鼠	SD大鼠, 雄性, 120-160 克	Leydig	Collagenase Type 2: 0.03% (adrenal)	Krebs Ringer bicarbonate buffer	Ng, T. and Liu, W.: Toxic Effect of Heavy Metals on Cells Isolated from the Rat Adrenal and Testis, In Vitro Cell Dev Biol 26, 24, 1990
		Adrenal
大鼠	Wistar大鼠, 成年的	Uterine	Trypsin: 0.5%	PBS	Pampfer, S., Vanderheyden, I., Michiels, B., and DeHertogh, R.: Co-Culture of Two-Cell Rat Embryos on Cell Monolayers, In Vitro Cell Dev Biol 26, 944, 1990
大鼠	SD大鼠, 10 天	Sertoli cells	Collagenase Type 2: 500 u/ml	PBS	Hadley MA, Weeks BS, Kleinman HK, Dym M: Laminin promotes formation of cord-like structures by Sertoli cells in vitro, Dev Biol 140, 318-27, 1990
			Deoxyribonuclease I: 0.0005%
			Hyaluronidase: 0.1%
大鼠	大鼠, 未成熟的	Luteal, ovaries	Collagenase: 0.3%	McCoy's	Rajan, V. and Menon, K.: Differential Uptake and Metabolism of Free and Esterified Cholesterol from High-density Lipoproteins in the Ovary, Biochim Biophys Acta 959, 206, 1988
大鼠	大鼠, 雄性, 20 天	Sertoli	Trypsin: 0.15%	(see reference)	Skinner, M., Fetterolf, P., and Anthony, C.: Purification of a Paracrine Factor, P-Mod-S,Produced by Testicular Peritubular Cells That Modulates Sertoli Cell Function,J Biol Chem 263, 2884, 1988
大鼠	SD大鼠, 雌性	Luminal epithelial	Trypsin: 0.5%	HBSS	Glasser, S., Julian, J., Decker, G., Tang, J., and Carson, D.: Development of Morphological and Functional Polarity in Primary Cultures of Immature Rat Uterine Epithelial Cells, J Cell Biol 107 (6), 2409, 1988
大鼠	Wistar大鼠, 處女, 雌性, 250 克	Vaginal epithelial	Trypsin: 0.5%	PBS	Conti, C., and Tasat, D.: Regulation of Cultured Rat Vaginal Epithelial Cells By 17B-Estradiol and Progesterone, J Steroid Biochem 24 (3), 747, 1986
大鼠	SD大鼠, 雌性, 21 天	Luteal, ovaries	Deoxyribonuclease I: 0.0004%	McCoy's	Rajan, V. and Menon, K.: Involvement of Microtubules in Lipoprotein Degradation and Utilization for Steroidogenesis in Cultured Rat Luteal Cells, Endocrinology 117, 2408, 1985
大鼠	SD大鼠, 雄性, 10 天	Sertoli	Trypsin: 0.025%	DMEM	Hadley, M., Byers, S., Suarez-Quian, C., Kleinman, H., and Dym, M.: Extracellular Matrix Regulates Seratoli Cell Differentiation, Testicular Cord Formation and germ cell development in vitro, J Cell Biol 101, 1511, 1985
大鼠	Holtzman大鼠, 雌性, 未成熟的	Uterine	Deoxyribonuclease I: 0.025%	DMEM buffered with HEPES	Kassis, J., Walent, J., and Gorski, J.: Estrogen Receptors in Rat Uterine Cell Cultures: Effects of Medium on Receptor Concentration, Endocrinology 115, 762, 1984
大鼠	SD大鼠, 成年的,雄性, 50-70 天	Testicular	Deoxyribonuclease I: 10 礸/ml	HEPES	Hsueh, A., Bambino, T., Zhuang, L., Welsh, T., and Ling, N.: Mechanism of the Direct Action of Gonadotropin Releasing Hormone and Its Antagonist on Androgen Biosynthesis by Cultured Rat Testicular Cells, Endocrinology 112, 1653, 1983
大鼠	SD大鼠, 雌性, 假妊娠的, 21 天	Luteal, ovaries	Hyaluronidase: 0.1%	EBSS	Rajendran, K., Hwang, J., and Menon, K.: Binding, Degradation an Utilization of Plasma High Density and Low Density Lipoproteins for Progesterone Production In Cultured Rat Luteal Cells, Endocrinology 112, 1746, 1983
大鼠	SD大鼠, 4-6 天, 雄性	Sertolli, seminiforous tubules	Collagenase: 0.03%	Serum-free medium	Rich, K., Bardin, C., Gunsalus, G., and Mather, J.: Age-Dependent Pattern of Androgen-Binding Protein Secretion from Rat Sertoli Cells in Primary Culture,Endocrinology 113, 2284, 1983
大鼠	SD大鼠, 雌性, 26 天	Luteal	Deoxyribonuclease I: 0.0004%	Medium 199	Azhar, S. and Reaven, E.: Effect of Antimicrotubule Agents on Microtubules and Steroidogenesis in Luteal Cells, Am J Physiol 243, E380, 1982
大鼠	SD大鼠, 雄性	Leydig	Collagenase: 0.1%	Krebs Ringer bicarbonate buffer	Ramachandran, J., and Sairam, M.R.: The Effects Of Interstitial Cell-Stimulating Hormone, Its Subunits,and Recombinants on Isolated Rat Leydig Cells, Arch Biochem Biophys 167, 294, 1975

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