乳腺組織消化手冊

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Worthington組織消化指南

引言

組織消化酶主要用于組織培養(yǎng)和細胞生物學研究的原代細胞的分離和收集。盡管這些消化酶已經普遍應用多年，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(jù)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(yǎng)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(shù)據(jù)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(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。

乳腺組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養(yǎng)基	參考文獻
牛	牛, 泌乳期	Mammary epithelial	Collagenase Type 3: 400 u/ml	HBSS	Miranda, S., Wang, Y., Purdie, N., Osborne, V., Coomber, B. and Cant, J.:Selenomethionine Stimulates Expression of Glutathione Peroxidase 1 and 3 and Growth of Bovine Mammary Epithelial Cells in Primary Culture., J Dairy Sci 92, 2670, 2009
			Hyaluronidase: 100 u/ml
			Deoxyribonuclease I: 2 u/ml
牛	牛, 小母牛, 200公斤	Mammary epithelial	Collagenase Type 2: 1%	M-199	Weber M., Purup S., Vestergaard M., Ellis S., Scndergard-Andersen J., Akers R., and Sejrsen K.: Contribution of Insulin-like Growth Factor (IGF)-I and IGF-binding Protein-3 to Mitogenic Activity in Bovine Mammary Extracts and Serum, J Endocrinol 161, 365, 1999
			Hyaluronidase: 1%
			Deoxyribonuclease I: 0.03%
牛	牛	Epithelial	Hyaluronidase: 0.005%	HBSS	Gibson, C., Vega, J., Baumrucker, C., Oakley, C., and Welsch, C.: Establishment And Characterization Of Bovine Mammary Epithelial Cell Lines, In Vitro Cell Dev Biol27, 585, 1991
牛	牛	Epithelial	Deoxyribonuclease I: 0.04%	HBSS/Medium 199	Baumrucker, C., Deemer, K., Walsh, R., Riss, T., and Akers, R.: Primary Culture of Bovine Mammary Acini on a Collagen Matrix, Tissue Cell 20 (4), 541, 1988
牛	牛, 幼年的, 哺乳期, 雌性	Mammary	Collagenase: 0.30 %	HBSS or EBSS	Anderson, C., and Larson, B.: Comparative Maintenance of Function in Dispersed Cell and Organ Cultures, Exp Cell Res 61, 24, 1970
牛	牛, 牛奶場, 純血統(tǒng)的,	Secretory	Collagenase: 0.02 - 0.03%	HBSS or EBSS	Schingoethe, D., Hageman, E., and Larson, B.: Essential Amino Acids for Milk Protein Synthesis in the In Vitro Secretory Cell and Stimulation by Elevated Levels,Biochim Biophys Acta 148, 469, 1967
牛	(包括大鼠, 豪斯曼, 白化變種, 白色)	Mammary gland	Collagenase: 0.02 - 0.03%	HBSS or EBSS
山羊	山羊, 哺乳期, 1 個月	Mammary gland	Collagenase: 0.02 - 0.03%	HBSS or EBSS	Blanco, A., Rife, U., and Larson, B.: Lactate Dehydrogenase Isozymes during Dedifferentiation in Cultures of Mammary Secretory Cells, Nature 214, 1331, 1967
豚鼠	豚鼠, 懷孕的, 4-10 天	Mammary gland	Trypsin NF 1:250: 0.25%	Dulbecco phosphate	Turba, F., and Hilpert, N.: Secretion and Resorption of Proteins by Isolated Mammary Gland Cells. German, Biochem Z 334, 501, 1961
人類	人類	Mammary Epithelial	Collagenase: 200 u/ml	DMEM/F-12	Labarge, M., Garbe, J. and Stampfer, M.: Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture., J Vis Exp 71, , 2013
人類	人類	Mammary Epithelial	Hyaluronidase: 100 u/ml	DMEM/F-12
人類	人類, 正常活檢	Fibroblasts	Collagenase Type 3: 900 u/ml	DMEM/F-12	Ronnov-Jessen L., Villadsen R., Edwards J., and Petersen O.: Differential Expression of a Chloride Intracellular Channel Gene, CLIC4, in Transforming Growth Factor-beta1-mediated Conversion of Fibroblasts to Myofibroblasts, Am J Pathol 161, 471, 2002
人類	人類	Mammary epithelial cells	Collagenase Type 1: 0.1%	DMEM	Huss, F. and Kratz, G.: Mammary Epithelial Cell and Adipocyte Co-Culture in a 3-D Matrix: The First Step Towards Tissue-Engineered Human Breast Tissue., Cells Tissues Organs 169, 361-7, 2001
人類	人類	Epithelial	Collagenase Type 1: 450 IU/ml	DMEM/Ham's F-12	Ogmundsdottir, H., Petursdottir, I., Gudmundsdottir, I., Amundadottir, L., Ronnov-Jessen, L., and Petersen, O.: Effects of Lymphocytes and Fibroblasts on the Growth of Human Mammary Carcinoma Cells Studied in Short-Term Primary Cultures, In Vitro Cell Dev Biol 29A, 936, 1993
人類	人類	Fibroblasts	Collagenase Type 1: 450 IU/ml	DMEM/Ham's F-12
人類	人類	Myofibroblasts	Collagenase: 900 IU/ml	DME - F12	Ronnov-Jessen, L., VanDeurs, B., Nielsen, M., and Petersen, O.W.: Identification, Paracrine Generation, and Possible Function of Human Breast Carcinoma Myofibroblasts in Culture, In Vitro Cell Dev Biol 28, 273, 1992
人類	人類, 女性, 15-61 歲大	Epithelial	Hyaluronidase: 150 IU/ml	DMEM	Berthon, P., Pancino, G., Cremoux, P., Roseto, A., Gespach, C., and Calvo, F.:Characterization of Normal Breast Epithelial Cells in Primary Cultures: Differentiation and Growth Factor Receptors Studies, J Tiss Cul Meth 28A, 716, 1992
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	Tumor, breast	Neuraminidase: 0.8 u/ml	HBSS	Leung, C., and Shiu, R.: Morphological and Proliferative Characteristics of Human Breast Tumor Cells Cultured on Plastic and in Collagen Matrix, In Vitro 18, 476, 1981
人類	人類	Epithelial	Hyaluronidase: 100 u/ml	DMEM/Ham's F-12	Stampfer, M., Hallowes, R., and Hackett, A.: Growth of Normal Human Mammary Cells in Culture, In Vitro 16 (5), 415, 1980
小鼠	小鼠	Mammary tumor	Collagenase: 0.15%	DMEM/F12	Liu, X., Johnson, S., Liu, S., Kanojia, D., Yue, W., Singh, U., Wang, Q, Wang Qi, Nie, Q. and Chen H.: Nonlinear Growth Kinetics of Breast Cancer Stem Cells: Implications for Cancer Stem Cell Targeted Therapy., Sci Rep 3, 2473, 2013
小鼠	小鼠	Mammary tumor	Hyaluronidase: 0.020%	DMEM/F12
小鼠	小鼠, 10 周	Fibroblasts, carcinoma	Collagenase Type 4: 0.5%	DMEM	Cheng, N. and Lambert, D.: Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice., J Vis Exp 54, e2716, 2011
			Trypsin: 0.2%
			Deoxyribonuclease I: 0.004
			Hyaluronidase: 1,000 u/ml
小鼠	小鼠, 12 周	Mammary epithelial	Collagenase: 0.3%	see reference	Taddei Ilaria, Deugnier Marie-Ange, Faraldo Marisa M, Petit Valerie, Bouvard Daniel, Medina Daniel, Fsssler Reinhard, Thiery Jean Paul, Glukhova Marina:Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells, Nat Cell Biol 10, 716-22, 2008
			Hyaluronidase: 100 u/ml
			Trypsin: 0.25%
			Neutral Protease: 0.5%
			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	Mammary epithelial stem	Collagenase Type 3: 0.1%	DMEM	Boulanger CA, Smith GH: Reducing mammary cancer risk through premature stem cell senescence, Oncogene 20, 2264-72, 2001
			Hyaluronidase: 0.1%
			Pronase: 1.25%
			Deoxyribonuclease I: 0.2%
小鼠	小鼠, 白變種實驗室老鼠	Epithelium	Collagenase: 250 u/ml	HBSS	Kanazawa, T., and Hosick, H.: Transformed Growth Phenotype of Mouse Mammary Epithelium in Primary Culture Induced by Specific Fetal Mesenchymes, J Cell Physiol153, 381, 1992
小鼠	小鼠, 白變種實驗室老鼠, 處女, 雌性, 4 個月	Adipocytes	Trypsin: 50 礸/ml	DMEM	Beck, J., Hosick, H., and Watkins, B.: Growth of Epithelium From a Preneoplastic Mammary Outgrowth on Response to Mammary Adipose Tissue, In Vitro Cell Dev Biol25 (5), 409, 1989
小鼠	小鼠 (白變種實驗室老鼠Crgl), 成年的	Epithelial	Pronase: 0.01%	Hepes buffered medium 199	Bandyopadhyay, G., Imagawa, W., Wallace, D., and Nandi, S.: Proliferative effects of insulin and epidermal growth factor on mouse mammary epithelial cells in primary culture, J Biol Chem 263, 7567, 1988
小鼠	小鼠, 白變種實驗室老鼠, 雌性, 6-8 周	Epithelial	Collagenase Type 3: 0.1%	DMEM	Ehmann, U., Peterson, W., and Misfeldt, D.: To Grow Mouse Mammary Epithelial Cells in Culture, J Cell Biol 98, 1026, 1984
小鼠	小鼠 (C3H/HeN), 雌性	Epithelial	Deoxyribonuclease I: 0.0001%	Medium 199	Taketani, Y., and Oka, T.: EGF Stimulates Cell Proliferation and Inhibits Functional Differentiation of Mouse Mammalian Dipithelial Cells in Culture, Endocrinology 113, 871, 1983
小鼠	小鼠, (白變種實驗室老鼠/Crgl Me), 雌性, 懷孕的	Epithelial	Pepsin: 0.1% and 0.05%	HBSS with 0.2% EDTA, CMF	Riser, M., Huff, B., and Medina, D.: Pepsin Can Be Used To Subculture Viable Mammary Epithelial Cells, In Vitro 19, 730, 1983
小鼠	小鼠, 雌性	Epithelial	Deoxyribonuclease I: 0.1%	DMEM	Jones, W., and Hallowes, R.: Isolation of the Epithelial Subcomponents of the Mouse Mammary Gland for Tissue-Level Culture Studies, J Tiss Cul Meth 8 (1), 17, 1983
小鼠	小鼠 (NMuMG), 雌性, 2 months	Epithelial	Collagenase Type 3: 0.1%	DMEM	Ehmann, U., and Misfeldt, D.: Mouse Mammary Cells in D-Valine Medium, In Vitro 18, 407, 1982
小鼠	小鼠, 白變種實驗室老鼠Cr1, 雌性	Epithelial	Collagenase Type 2: 0.2%	HBSS/DMEM	Asch, B., Burstein, N., Vidrich, A., and Sun, T.: Identification of Mouse Mammary Epithelial Cells by Immunofluorescence With Rabbit and Guinea-Pig Antikeratin Antisera, Proc Natl Acad Sci U S A 78, 5643, 1981
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
小鼠	小鼠 (白變種實驗室老鼠fC3H)	Epithelial	Collagenase: 1.0%	HBSS
小鼠	小鼠, 懷孕的 8-12 天	Mammary	(see reference):	HBSS	Yang J, Richards J, Guzman R, Imagawa W, and Nandi S.: Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels, Proc Natl Acad Sci U S A 77, 2088, 1980
小鼠	小鼠 (白變種實驗室老鼠Crgl), 處女, 雌性, 4-8 個月	Epithelial	Collagenase Type 3: 0.1%	HBSS	White, M., Hu, A., Hamamoto, S. and Nandi: In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: fibroblast-free growth and serial passage, In Vitro 14, 271, 1978
小鼠	小鼠 (白變種實驗室老鼠Crgl), 處女, 雌性, 4-8 個月	Mammary gland	Collagenase Type 3: 0.1%	HBSS
小鼠	小鼠 (白變種實驗室老鼠), 懷孕的, 60-80 天	Mammary	Hyaluronidase: 0.1%	CMF medium	Ceriani, R., Peterson, J., and Abraham, S.: Removal of Cell Surface Material by Enzymes Used to Dissociate Mammary Gland Cells, In Vitro 14, 887, 1978
小鼠	小鼠, 白變種實驗室老鼠fC3H/Crgl, 處女, 雌性	Nodule-transformed	Hyaluronidase: 0.1%	Medium 199	DeOme, K., Miyamoto, M., Osborn, R., Guzman, R., and Lum, K.: Detection of Inaparent Nodule-transformed Cells in the Mammary Gland Tissues of Virgn 雌性白變種實驗室老鼠fC3H Mice, Cancer Res 38, 2103, 1978
小鼠	小鼠, 白變種實驗室老鼠Crgl, 雌性	Swiss 3T3	Collagenase Type 3: 0.2%	PBS CMF	Asch, B., and Medina, D.: Concanavalin A-Induced Agglutinability of Normal, Preneoplastic, and Neoplastic Mouse Mammary Cells, J Natl Cancer Inst 61 (6), 1423, 1978
小鼠	(包括豚鼠)	Swiss 3T3	Collagenase Type 3: 0.2%	PBS CMF
小鼠	小鼠, (C3H/Crgl or 白變種實驗室老鼠Crgl), 8-10 天懷孕的	Epithelial	Collagenase Type 3: 0.12%	HBSS	Emerman, J., Enami, J., Pika, D., and Nandi, S.: Hormonal Effects on Intracellular and Secreted Casein in Cultures of Mouse Mammary Epithelial Cells on Floating Collagen Membranes, Proc Natl Acad Sci U S A 74 (10), 4466, 1977
小鼠	小鼠, 哺乳（期）	Parenchymal	Collagenase Type 1: 0.3%	Kreb's Ringer bicarbonate buffer	Kerkof, P., and Abraham, S.: Preparation of Adipose Cell-Free Suspensions of Mammary Gland Parenchymal Cells from Lactating Mice, Methods Enzymol 69, 693, 1976
小鼠	小鼠, 雌性, 早期妊娠小于10 天	Epithelial	Collagenase: 0.1%	Eagle's MEM	Moore, D., and Lasfargues, E.: Method for the Continuous C*tion of Mammary Epithelium, In Vitro 7, 21, 1971
小鼠	小鼠, CBA, 處女, 9/10 周	Mammary	Hyaluronidase: 0.1%	BSS, CMF	Prop, F., and Wiepjes, G.: Improved method for preparation of single-cell suspensions from mammary glands of adult virgin mouse, Exp Cell Res 61, 451, 1970
小鼠	小鼠, 哺乳期, 14-18 天	Parenchymal	Collagenase: 0.33%	Kreb's buffer	Pika, D., Kerkof, P., Gagne, H., Smith, S., and Abraham, S.: Characteristics of Cells Dissociated from Mouse Mammary Glands. I. Method of Separation and Morphology of Parenchymal Cells from Lactating Glands, Exp Cell Res 57, 43, 1969
小鼠	小鼠, 哺乳期, 14 天	Mammary	Trypsin NF 1:250: 0.25%	HBSS	Kopelovich, L., Abraham, S., McGrath, H., DeOme, K., Chaikoff, I.: Metabolic Characteristics of a Naturally Ocurring Preneoplastic Tissue. I. Glycolytic Enzyme Activators of Hyperplastic Alveolar Nodule Outgrowths and Adenocarcinomas of Mouse Mammary Gland, Cancer Res 26, 1534, 1966
小鼠	小鼠, 懷孕的, 14-17 天	Mammary	Collagenase: 0.05% - 0.1%	HBSS	Daniel, C., and DeOme, K.: Growth of Mouse Mammary Glands In Vivo After Monolayer Culture, Science 149, 634, 1965
小鼠	小鼠, adult, 1-10 天s 懷孕的	Epithelial	Collagenase: 0.02%	Simm's	Lasfargues, E.: C*tion and Behavior In Vitro of the Normal Mammary Epithelium of the Adult Mouse. II. Observations on the Secretory Activity, Exp Cell Res 13, 553, 1957
小鼠	小鼠, adult, 1-10 天s 懷孕的	Mammary	Collagenase: 0.02%	Simm's
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, 雌性, 50 天	Mammary epithelial	Collagenase Type 3: 0.15%	DMEM/F12	Maffini M., Soto A., Calabro J., Ucci A., and Sonnenschein C.: The Stroma as a Crucial Target in Rat Mammary Gland Carcinogenesis, J Cell Sci 117, 1495, 2004
大鼠	大鼠, 雌性	Mammary gland epithelial	Collagenase Type 3: 0.35%	see reference	Djuric Z., Lewis S., Lu M., Mayhugh M., Naegeli L., Tang N., and Hart R.: Effect of Varying Caloric Restriction Levels on 雌性 Rat Growth and 5-hydroxymethyl-2'-deoxyuridine in DNA, Toxicol Sci 66, 125, 2002
大鼠	SD大鼠,	Mammary fibroblasts	Collagenase Type 3: 0.2%	DMEM/F-12	Brake P., Zhang L., and Jefcoate C.: Aryl Hydrocarbon Receptor Regulation of Cytochrome P4501B1 in Rat Mammary Fibroblasts: Evidence for Transcriptional Repression by Glucocorticoids, Mol Pharmacol 54, 825, 1998
			Neutral Protease: 0.2%
			Deoxyribonuclease I: 0.01%
大鼠	SD大鼠, 50 天	Mammary epithelial	Collagenase Type 3: 0.2%	DMEM/F12	Varela L., Darcy K., and Ip M.: The Epidermal Growth Factor Receptor is not Required for Tumor Necrosis Factor-alpha Action in Normal Mammary Epithelial Cells,Endocrinology 138, 3891, 1997
大鼠	SD大鼠, 50 天	Mammary epithelial	Neutral Protease: 0.2%	DMEM/F12
大鼠	大鼠	Epithelial	Collagenase Type 1: 0.4%	DMEM	Soriano, J., Pepper, M., Nakamura, T., Orci, L., and Montesano, R.: Hepatocyte Growth Factor Stimulates Extensive Development of Branching Duct-like Structures by Cloned Mammary Gland Epithelial Cells, J Cell Sci 108, 413-430, 1995
大鼠	(包括人類)	Epithelial	Collagenase Type 1: 0.4%	DMEM
大鼠	SD大鼠, 雌性, 60-90 天	Epithelial	Collagenase Type 3: 0.5%	EBSS	Laduca, J., and Sinha, D.: In Vitro Carcinogenesis of Mammary Epithelial Cells by N-Nitroso-N-Methylurea Using a Collagen Gel Matrix Culture, In Vitro Cell Dev Biol 29A, 789, 1993
大鼠	Lewis大鼠, 雌性, 90 天	Epithelial	Collagenase: 0.05%	Medium 199	Lin, T., Hom, Y.K., Richards, J. and Nandi, S.: Effects of Antioxidants and Reduced Oxygen Tension on Rat Mammary Epithelial Cells in Culture, In Vitro Cell Dev Biol 27A, 191, 1991
大鼠	Lewis大鼠, 雌性, 90 天	Mammary	Collagenase: 0.05%	Medium 199
大鼠	SD大鼠, 雌性, 55 天	Epithelial	Neutral Protease: 3 u/ml	Medium 199	Ehmann, U., Osborn, R., Guzman, R., and Fajardo, L.: Cultured Proliferating Rat Mammary Epithelial Cells, In Vitro Cell Dev Biol 27, 749, 1991
大鼠	大鼠, 50-60 天	Epithelial	Neutral Protease: 0.2%	EBSS	Hahm, H.A., Ip, M.M.: Primary Culture of Normal Rat Mammary Epithelial Cells Within a Basement Matrix. 1. Regulation of Proliferation by Hormones and Growth Factors, In Vitro Cell Dev Biol 26, 791, 1990
大鼠	大鼠, 食魚動物 344, 處女, 80-100 天	Epithelial	Hyaluronidase: 0.1%	Medium 199	McGrath, M., Palmer, S., and Nandi, S.: Differential Response of Normal Rat Mammary Epithelial Cells to Mammogenic Hormones and EGF, J Cell Physiol 125, 182, 1985
大鼠	大鼠, LEW, 處女, 雌性, 45-50 天	Epithelial	Collagenase: 0.1%	Medium 199	Ethler, S.: Primary Culture and Serial Passage of Normal and Carcinogen-Treated Rat Mammary Epithelial Cells In Vitro, J Natl Cancer Inst 74 (6), 1307, 1985
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	大鼠, 近親繁殖的LEW, 雌性, 50-60 天大	Epithelial	Collagenase Type 3: 0.1%	Medium 199	Richards, J., and Nandi, S.: Primary Culture of Rat Mammary Epithelial Cells. I. Effect of Plating Density, Horomones, and Serum on DNA Synthesis, J Natl Cancer Inst 61 (3), 765, 1978
大鼠	Wistar大鼠, 產后的13-18 天	Acini	Collagenase: 0.05%	HBSS	Katz, J., Wals, P. and Van de Velde, R: Lipogenesis by Acini from Mammary Gland of Lactating Rats, J Biol Chem 249, 7348, 1974
大鼠	SD大鼠, 產后的15-20 天	Mammary	Collagenase: 0.2%	Kreb's Ringer bicarbonate buffer	Martin, R., and Baldwin, R.: Effects of Insulin on Isolated Rat Mammary Cell Metabolism: Glucose Utilization and Metabolite Patterns, Endocrinology 89, 1263, 1971
大鼠	大鼠, 處女	Mammary	Collagenase: 0.35%	Medium 199	Moon, R., Janns, D., and Young, S.: Preparation of Fat Cell-"Free" Rat Mammary Gland, J Histochem Cytochem 17 (3), 182, 1969

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