腫瘤組織消化手冊

更新時間：2016-02-16

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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。

腫瘤組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
倉鼠	倉鼠, 90-100 克	Tumor	Hyaluronidase: 0.1%	Waymouth's MB	Gonzalez, A., Oberley, T., Schultz, J., Ostrom, J., and Li, J.: In VitroCharactrization of Estrogen Induced Syrian Hamster Renal Tumors: Comparison with an Immortalized Cell Line Derived from Diethylstilbestrol-Treated Adult Hamster Kidney, In Vitro Cell Dev Biol 29A, 562, 1993
倉鼠	倉鼠, 6 周大	Buccal pouch	Neutral Protease: 0.24%	CMF HBSS	Min, B., Kim, K., Cherrick, H., and Park, N.: Three Cell Lines from Hamster Buccal Pouch Tumors Induced by Topical 7,12-Dimethylbenz(a)Anthracene, Alone or in Conjunction with Herpes Simplex Virus Inoculation, In Vitro Cell Dev Biol 27A, 128, 1991
人類	人類	Non-small cell lung tumor	Collagenase Type 1: 0.1%	RPMI-1640	Liang, S., Marti, T., Dorn, P., Froment, L., Hall, S., Berezowska, S., Kocher, G., Schmid, R. and Peng, R.: Blocking the Epithelial-to-Mesenchymal Transition Pathway Abrogates Resistance to Anti-Folate Chemotherapy in Lung Cancer., Cell Death Dis 6, e1824, 2015
			Collagenase Type 2: 0.25%
人類	人類	Lung Tumor	Collagenase Type 1: 45-60 u/ml	DMEM/F12	Quatromoni, J., Singhal, S., Bhojnagarwala, P., Hancock, W., Albelda, S. and Eruslanov, E.: An Optimized Dissagregation Method for Human Lung Tumors That Perserves the Phenotype and Function of the Immune Cells, J Leukoc Biol 97, 201, 2015
			Collagenase Type 2: 15-20 u/ml
			Collagenase Type 4: 45-60 u/ml
			Elastase: 0.002%
			Deoxyribonuclease I: 0.002%
人類	人類	Glioblastoma Tumor	Trypsin: 0.025%	HBSS	Hasselbach, L., Irtenkauf, S., Lemke, N., Nelson, K., Berezovsky, A., Carlton, E., Transou, A., Mikkelsen, T. and deCarvalho, A.: Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry., J Vis Exp , e51088, 2014
			Collagenase Type 4: 500 u/ml
人類	人類	Melanoma	Collagenase Type 4: 0.1%	PBS	Welte, Y., Davies, C., Schafer, R. and Regenbrecht, C.: Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma., J Vis Exp 73, e50200, 2013
			Deoxyribonuclease I: 0.01%
人類	人類	Tumor	Collagenase Type 1: 0.1%	DMEM/F12	Chou, J., Fitzgibbon, M., Mortales, C., Towlerton, A., Upton, M., Yeung, R., McIntosh, M. and Warren, E.: Phenotypic and Transcriptional Fidelity of Patient-Derived Colon Cancer Xenografts in Immune-Deficient Mice.,PLoS ONE 8, e79874, 2013
			Hyaluronidase: 0.01%
			Deoxyribonuclease I: 0.01%
人類	人類	Colorectal cancer	Collagenase Type 4: 2%	RPMI 1640	Zhou, J, Belov, L., Solomon, M., Chan, C., Clarke, S. and Christopherson, R.: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing., J Vis Exp 55, e3322, 2011
			Deoxyribonuclease I: 0.1%
人類	人類	Tumorigenic melanoma	Collagenase Type 4: 200 u/ml	PBS	Quintana, E., Shackleton, M., Foster, H., Fullen, D., Sabel, M., Johnson, T. and Morrison, S.: Phenotypic Heterogeneity Among Tumorigenic Melanoma Cells from Patients that is Reversible and Not Hierarchically Organized., Cancer Cell 18, 510, 2010
			Trypsin: 0.05%
			Deoxyribonuclease I: 50-100 u/ml
人類	人類	Pancreatic tumor	Collagenase Type 4: 200 u/ml	RPMI-1640	Kim, M., Evans, D., Wang, H., Abbruzzese, J., Fleming, J. and Gallick, G.: Generation of Orthotopic and Heterotopic Human Pancreatic Cancer Xenografts in Immunodeficient Mice., Nat Protoc 4, 1670, 2009
人類	人類	Tumor	Collagenase Type 4: 0.1%	see reference	Sauvageot, C., Weatherbee, J., Kesari, S., Winters, S., Barnes, J., Dellagatta, J., Ramakrishna, N., Stiles, C., Kung, A., Kieran, M. and Wen, P.: Efficacy of the HSP90 Inhibitor 17-AAG in Human Glioma Cell Lines and Tumorigenic Glioma Stem Cells.,Neuro Oncol Vol. 11,, , 109, 2009
			Hyaluronidase: 0.07%
			Deoxyribonuclease I: 0.04%
人類	人類	Colon cancer	Collagenase Type 1: 300 u/ml	DMEM/F12	Varnat, F., Duquet, A., Malerba, M., Zbinden, M., Mas, C., Gervaz, P. and Ruiz i Altaba, A.: Human Colon Cancer Epithelial Cells Harbour Active HEDGEHOG-GLI Signalling that is Essential for Tumour Growth, Recurrence, Metastasis and Stem Cell Survival and Expansion., EMBO Mol Med 1, 338-51, 2009
			Hyaluronidase: 100 u/ml
人類	人類	Breast epithelial	Collagenase Type 3: 200 u/ml	HBSS	Liu, R., Wang, X., Chen, G., Dalerba, P., Gurney, A., Hoey, T., Sherlock, G., Lewicki, J., Shedden, K. and Clarke, M.: The Prognostic Role of a Gene Signature from Tumorigenic Breast-Cancer Cells., N Engl J Med 356, 217, 2007
人類	人類	Prostate stromal cells	Collagenase Type 1: 0.1%	RPMI 1640	Nakashiro Koh-Ichi, Hara Shingo, Shinohara Yuji, Oyasu Miho, Kawamata Hitoshi, Shintani Satoru, Hamakawa Hiroyuki, Oyasu Ryoichi: Phenotypic switch from paracrine to autocrine role of hepatocyte growth factor in an androgen-independent human prostatic carcinoma cell line, CWR22R, Am J Pathol 165, 533-40, 2004
人類	人類, 成年人	Human synovial sarcoma	Collagenase Type 2: 200 u/ml	DMEM/F-12	Nishio Jun, Iwasaki Hiroshi, Ishiguro Masko, Ohjimi Yuko, Fujita Chikako, Isayama Teruto, Naito Masatoshi, Oda Yoshinao, Kaneko Yasuhiko, Kikuchi Masahiro: Establishment of a new human synovial sarcoma cell line, FU-SY-1, that expresses c-Met receptor and its ligand hepatocyte growth factor, Int J Oncol 21, 17-23, 2002
人類	人類	Colonocytes	Collagenase:	DMEM/F12	Emenaker N, Calaf G, Cox D, Basson M and Qureshi N: Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes, J Nutr 46, 96-105, 2001
人類	人類	Epithelial, fibroblasts	Trypsin: 0.25%	Ham's F-12	MacLeod, R: Rapid Monolayer Primary Cell Culture from Tissue Biopsy,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons Ltd, 3E:2.1, 1995
人類	人類	Colon adenocarcinoma	Hyaluronidase: 100 u/ml	DMEM	Hague, A and Paraskeva, C: Colon Adenocarcinoma Cells,Cell & Tissue Culture: Laboratory Procedures Vol. 1,Doyle, A., Griffiths, J., and Newell, D., John Wiley and Sons, Ltd., 12C:1.1, 1995
人類	人類	Tumor, breast	Hyaluronidase: 100 u/ml	DMEM	Beaupain, R., Mainquene, C., Brouty-Boye, D., Planchon, P., and Magniew, V.: "Normal" Breast Cells Adjacent to a Tumor Grown in Long-term Three Dimensional Culture, In Vitro Cell Dev Biol 29, 100, 1993
人類	人類	Glioma	Hyaluronidase: 0.01%	HBSS	Kruse, C., Mitchell, D., Kleinschmidt-DeMasteis, B., Franklin, W., Morse, H., Spector, E., and Lillehei, K.: Characterization of a Continuous Human Glioma Cell Line DBTRG-OSMG: Growth Kinetics, Karyotype, Receptor Expression and Tumor Suppressor Gene Analyses, In Vitro Cell Dev Biol 28, 609, 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	Neutral Protease: 0.24%	DMEM/Ham's F-12	Boyd, J., Rinehart Jr., C., Walton, L., Siegal, G. and Kaufman, D.:Ultrastructural Characterization of Two New Human Endometrial Carcinoma Cell Lines and Normal Human Endometrial Epithelial Cells Cultured on Extracellular Matrix, In Vitro Cell Dev Biol 26, 701, 1990
人類	人類, 9-74 歲	Neurofibroma	Neutral Protease: 1.25 u/ml	L-15	Sheela S, Riccardi VM, Ratner N: Angiogenic and invasive properties of neurofibroma Schwann cells, J Cell Biol 111, 645-53, 1990
			Collagenase Type 1: 0.05%
			Hyaluronidase: 0.1%
人類	人類	Tumor	Trypsin: 0.05%	DMEM	Sacks, P., Parnes, S., Gallick, G., Mansouri, Z., Lichtner, R., Satya-Prakash, K., Pathak, S, and Parsons, D.: Establishment and Characterization of Two New Squamous Cell Carcinoma Cell Lines Derived from Tumors of the Head and Neck, Cancer Res 48, 2858, 1988
人類	人類	Tumor, colon	Trypsin: 0.25%	McCoy's	Brattain, M., Marks, M., McCombs, J., Finely, W., and Brattain, D.:Characterization of Human Colon Carcinoma Cell Lines Isolated From a Single Primary Tumour, Br J Cancer 47, 373, 1983
人類	人類	Epthelial and tumor	Collagenase: 300 u/ml	PBS medium 199 or medium F 12	Friedman, E., Higgins, P., Lipkin, M., Shinya, H., and Gelb, A.: Tissue Culture of Human Epithelial Cells from Benign Colonic Tumors, In Vitro 17, 632, 1981
		Colon
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	Melanoma	Collagenase Type 3: 0.10%	DMEM	Creasey, A., Smith, H., Hackett, A., Fukuyama, K., Epstein, W., and Madin, S.: Biological Properties of Human Melanoma Cells in Culture, In Vitro 15, 342, 1979
		Metastatic tumors
人類	人類	Mammary tumors, hard	Collagenase: 0.10%	RPMI-1640 w/ 5% Fetal Calf Serum	Lasfargues, E.: Tissue Culture Methods/Applications,Kruse, P., and Patterson, M., Academic Press, 45, 1973
小鼠	小鼠	Fibroblasts	Collagenase Type 2: 0.25%	DMEM	Sharon, Y., Alon, L., Glanz, S., Servais, C. and Erez, N.: Isolation of Normal and Cancer-Associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)., J Vis Exp 71, e4425, 2013
			Collagenase Type 4: 0.25%
			Deoxyribonuclease I: 0.05%
小鼠	小鼠	Prostate tumor	Collagenase Type 4: 1,600 u/ml	DMEM/F12	Mazzoleni, S., Jachetti, E., Morosini, S., Grioni, M., Piras, I., Pala, M., Bulfone, A., Freschi, M., Bellone, M. and Galli, R.: Gene Signatures Distinguish Stage-Specific Prostate Cancer Stem Cells Isolated From Transgenic Adenocarcinoma of the Mouse Prostate Lesions and Predict the Malignancy of Human Tumors., Stem Cells Transl Med 2, 678, 2013
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
			Hyaluronidase: 0.020%
小鼠	小鼠	Rhabdomycosarcoma tumor	Trypsin: 0.012%	DMEM	Chen, X., Stewart, E., Shelat, A., Qu, C., Bahrami, A., Hatley, M., Wu, G., Bradley, C., McEvoy, J., Pappo, A., Spunt, S., Valentine, M., Valentine, V., Krafcik, F., Lang, W., Wierdl, M. and Tsurkan, L.:Targeting Oxidative Stress in Embryonal Rhabdomyosarcoma., Cancer Cell24, 710-24, 2013
			Collagenase Type 2: 0.1%
小鼠	小鼠	Tumor endothelial	Collagenase Type 1: 0.2%	HBSS	Kazerounian, S., Gerald, D., Huang, M., Chin, R, Udayakumar, D, Zheng, N., O'Donnell, R., Perruzzi, C., Mangiante, L., Pourat, J., Phung, T., Bravo-Nuevo, A., Shechter, S., McNamara, S. and Duhadaway, K.:RhoB Differentially Controls Akt Function in Tumor Cells and Stromal Endothelial Cells During Breast Tumorigenesis., Cancer Res 73, 50, 2013
小鼠	小鼠, 雄性	Dendritic, macrophages	Collagenase (1 or 4): 100-200 u/ml	RPMI	Watkins, S., Zhu, Z., Watkins, K. and Hurwitz, A.: Isolation of Immune Cells from Primary Tumors., J Vis Exp 64, e3952, 2012
			Deoxyribonuclease I: 0.01%
小鼠	小鼠	Lung tumor	Neutral Protease: 50 u/ml	DMEM	Vaughan, A., Halbert, C., Wootton, S. and Miller, A.: Lung Cancer in Mice Induced by the Jaagsiekte Sheep Retrovirus Envelope Protein is not Maintained by Rare Cancer Stem Cells, but Tumorigenicity does Correlate with Wnt Pathway Activation., Mol Cancer Res 10, 86, 2012
			Collagenase: 400 u/ml
			Deoxyribonuclease I: 50 u/ml
小鼠	小鼠	Macrophages	Collagenase Type 4: 0.2%	PBS	Hamzah, J., Kotamraju, V., Seo, J., Agemy, L., Fogal, V., Mahakian, L., Peters, D., Roth, L., Gagnon, M., Ferrara, K. and Ruoslahti, E.: Specific Penetration and Accumulation of a Homing Peptide Within Atherosclerotic Plaques of Apolipoprotein E-deficient Mice., Proc Natl Acad Sci U S A 108, 7154, 2011
小鼠	小鼠	Pancreatic Cancer Stem	Collagenase Type 4: 200 u/ml	DMEM	Rasheed, Z., Wang, Q. and Matsui, W.: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts., J Vis Exp 43, 2169, 2010
			Neutral Protease: 0.6 u/ml
小鼠	小鼠	Tumor infiltration lymphocytes	Collagenase Type 1: 0.25%	RPMI 1640	Kwong, B., Roberts, S., Silberzahn, T., Filler, R., Neustadter, J., Galan, A., Reddy, S., Lin, W., Ellis, P., Langford, C., Hayday, A. and Girardi, M.:Molecular Analysis of Tumor-Promoting CD8+ T cells in Two-Stage Cutaneous Chemical Carcinogenesis., J Invest Dermatol 130, 1726, 2010
			Collagenase Type 2: 0.15%
			Collagenase Type 4: 0.1%
			Hyaluronidase: 0.025%
小鼠	小鼠, 4-6 周	Pancreatic tumor	Collagenase Type 4: 200 u/ml	RPMI-1640	Kim, M., Evans, D., Wang, H., Abbruzzese, J., Fleming, J. and Gallick, G.: Generation of Orthotopic and Heterotopic Human Pancreatic Cancer Xenografts in Immunodeficient Mice., Nat Protoc 4, 1670, 2009
小鼠	小鼠	Tumor	Collagenase Type 3: 200 u/ml	RPMI-1640	Prince, M., Sivanandan, R., Kaczorowski, A., Wolf, G., Kaplan, M., Dalerba, P., Weissman, I., Clarke, M. and Ailles, L.: Identification of a Subpopulation of Cells with Cancer Stem Cell Properties in Head and Neck Squamous Cell Carcinoma., Proc Natl Acad Sci U S A 104, 973, 2007
小鼠	小鼠, 30 周	Tumor endothelial	Collagenase Type 4: 500 u/ml	PBS	Berger, M., Bergers, G., Arnold, B., Hammerling, G. and Ganss, R.:Regulator of G-protein Signaling-5 Induction in Pericytes Coincides With Active Vessel Remodeling During Neovascularization, Blood 105, 1094, 2005
			Collagenase Type 2: 550 u/ml
			Deoxyribonuclease I: 3 u/ml
小鼠	小鼠	Granule cell precursors, pre-neoplastic and tumor cells	Papain: 10 u/ml	Neurobasal/B27	Oliver, T., Read, T., Kessler, J., Mehmeti, A., Wells, J., Huynh, T., Lin, S. and Wechsler-Reya, R.: Loss of Patched and Disruption of Granule Cell Development in a Pre-Neoplastic Stage of Medulloblastoma.,Development 132, 2425, 2005
			Deoxyribonuclease I: 250 u/ml
小鼠	小鼠, 8-10 周	Tumor associated endothelial cells	Collagenase Type 2:	see reference	Hida Kyoko, Hida Yasuhiro, Amin Dhara N, Flint Alan F, Panigrahy Dipak, Morton Cynthia C, Klagsbrun Michael: Tumor-associated endothelial cells with cytogenetic abnormalities, Cancer Res 64, 8249-55, 2004
小鼠	小鼠	Pancreatic tumor	Collagenase Type 2: 0.5%	PBS	Bergers Gabriele, Song Steven, Meyer-Morse Nicole, Bergsland Emily, Hanahan Douglas: Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors, J Clin Invest 111, 1287-95, 2003
			Collagenase Type 4: 0.5%
			Deoxyribonuclease I: 0.2%
小鼠	小鼠	Tumor-infiltrating lymphocyte	Collagenase: 200 u/ml	RPMI 1640	Uekusa Yasuhiro, Yu Wen-Gong, Mukai Takao, Gao Ping, Yamaguchi Nobuya, Murai Masako, Matsushima Kouji, Obika Satoshi, Imanishi Takeshi, Higashibata Yuji, Nomura Shintaro, Kitamura Yukihiko, Fujiwara Hiromi, Hamaoka Toshiyuki: A pivotal role for CC chemokine receptor 5 in T-cell migration to tumor sites induced by interleukin 12 treatment in tumor-bearing mice, Cancer Res 62, 3751-8, 2002
小鼠	小鼠, 雄性	Melanoma tumor cells	Collagenase Type 2: 0.5%	DMEM	Arbiser JL, Raab G, Rohan RM, Paul S, Hirschi K, Flynn E, Price ER, Fisher DE, Cohen C, Klagsbrun M: Isolation of mouse stromal cells associated with a human tumor using differential diphtheria toxin sensitivity, Am J Pathol 155, 723-9, 1999
小鼠	小鼠 (BALB/cfC3H)	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, BALB/cfC3H/Crgl	Neoplastic	Trypsin:	DMEM	Hosick, H.: A Note on Growth Patterns of Epithelial Tumor Cells in Primary Culture, Cancer Res 34, 259, 1974
		Epithelial tumor
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
大鼠	大鼠, 成年的	Tumor	Collagenase Type 1: 0.16%	DMEM/F12	Duarte, S., Momier, D., Baque, P., Casanova, V., Loubat, A., Samson, M., Guigonis, J., Staccini, P., Saint-Paul, M., De Lima, M., Carle, G. and Pierrefite-Carle, V.: Preventive Cancer Stem Cell-Based Vaccination Reduces Liver Metastasis Development in a Rat Colon Carcinoma Syngeneic Model., Stem Cells 31, 423-32, 2013
			Hyaluronidase: 0.002%
			Deoxyribonuclease I: 0.006%
大鼠	大鼠, 雄性 12 周	Sponge infiltrating cells	Collagenase Type 4: 0.15%	RPMI 1640	Sharma N, Luo J, Kirschmann DA, O'Malley Y, Robbins ME, Akporiaye ET, Lubaroff DM, Heidger PM, Hendrix MJ: A novel immunological model for the study of prostate cancer, Cancer Res 59, 2271-6, 1999
			Deoxyribonuclease I: 0.02%
大鼠	大鼠 (ACI/NMs X BUF/Mna) F1, 雄性, 28 個月, 大鼠, (ACI/MNs) 雄性, 8 周	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	大鼠	Yolk sac tumor	Trypsin: 0.01%	DMEM	Brennan, M., Oldberg, A., Hayman, E., and Ruoslahti, E.: Effect of a Proteoglycan Produced by Rat Tumor Cells on Their Adhesion to Fibronectin-Collagen Substrata, Cancer Res 43, 4302, 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	大鼠	Tumor, islet	Trypsin: 0.05%	Medium 199	Gazdar, A., Chick, W., Oie, H., Sims, H., King, D., Weir, G., and Lauris, V.: Continuous, Clonal, Insulin-and Somatostatin-Secreting Cell Lines Established from a Transplantable Rat Islet Cell Tumor, Proc Natl Acad Sci U S A 77 (6), 3519, 1980
大鼠	大鼠	Ascites hepatoma	Trypsin: 0.1%	Phosphate buffer (see reference)	Essner, E.: Experiments on an Ascites Hepatoma. I. Enzymatic Digestion and Alkaline Degradation of the Cementing Substance and Separation of Cells, in Tumor Islands, Exp Cell Res 7, 430, 1954

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