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(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?zāi)康男拚桨敢垣@得更好的實驗結(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(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)的實驗步驟，以便于制定一個*的細(xì)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(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(xì)來源	細(xì)胞名稱	消化用酶	培養(yǎng)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(fā)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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