干細胞組織消化手冊

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。

干細胞組織消化手冊

物種	詳細來源	細胞名稱	消化用酶	培養基	參考文獻
鳥	人	Adipose derived stem cells	Collagenase Type 1: 0.1%	DMEM	Tandon, N., Goh, B., Marsano, A., Chao, P., Montouri-Sorrentino, C., Gimble, J. and Vunjak-Novakovic, G.: Alignment and Elongation of Human Adipose-Derived Stem Cells in Response to Direct-Current Electrical Stimulation., Conf Proc IEEE Eng Med Biol Soc 2009, 6517-21, 2009
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%
犬	犬, 20-25 公斤	Adipose stem cells	Collagenase: see reference	Media-199	Fischer, L., McIlhenny, S., Tulenko, T., Golesorkhi, N., Zhang, P., Larson, R., Lombardi, J., Shapiro, I. and DiMuzio, P.: Endothelial Differentiation of Adipose-Derived Stem Cells: Effects of Endothelial Cell Growth Supplement and Shear Force., J Surg Res 152, 157, 2009
馬	馬	Adipose derived stem cells	Collagenase Type 1: 0.1%	PBS	Vidal, M., Robinson, S., Lopez, M., Paulsen, D., Borkhsenious, O., Johnson, J., Moore, R. and Gimble, J.: Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow., Vet Surg 37, 713, 2008
馬	馬, 1-5 歲	Adipose derived stem cells	Collagenase Type 1: 0.1%	PBS	Vidal, M., Kilroy, G., Lopez, M., Johnson, J., Moore, R. and Gimble, J.: Characterization of Equine Adipose Tissue-Derived Stromal Cells: Adipogenic and Osteogenic Capacity and Comparison with Bone Marrow-Derived Mesenchymal Stromal Cells., Vet Surg 36, 613, 2007
人類	人類	Mesenchymal Stromal Cells	Collagenase Type 4: 0.2%	PBS	Choudhery,M, Badowski, M, Muise, A and Harris, D: Effect of Mild Heat Stress on the Proliferative and Differentiative Ability of Human Mesenchymal Stromal Cells., Cytotherapy17, 359-68, 2015
人類	人類	Adipose derived stem cells	Collagenase Type 2: 0.1%	HBSS	Satish, L., Krill-Burger, J., Gallo, P., Etages, S., Liu, F., Philips, B., Ravuri, S., Marra, K., LaFramboise, W., Kathju, S. and Rubin, J.: Expression Analysis of Human Adipose-Derived Stem Cells During In Vitro Differentiation to an Adipocyte Lineage., BMC Med Genomics 8, 41, 2015
人類	人類	Dental mesenchymal stem	Collagenase Type 1: 0.3%	DMEM	Wu, Z., Wang, J., Dong, R., Wang, L., Fan, Z., Liu, D. and Wang, S.: Depletion of MEIS2 Inhibits Osteogenic Differentiation Potential of Human Dental Stem Cells., Int J Clin Exp Med8, 7220-30, 2015
			Neutral Protease: 0.4%
人類	人類	Decidua Mesenchymal Stem	Trypsin: 0.25%	PBS	Kusuma, G., Menicanin, D., Gronthos, S., Manuelpillai, U., Abumaree, M., Pertile, M., Brennecke, S. and Kalionis, B.: Ectopic Bone Formation by Mesenchymal Stem Cells Derived from Human Term Placenta and the Decidua., PLoS ONE 10, e0141246, 2015
			Collagenase Type 1: 1.0%
			Deoxyribonuclease I: 0.005%
人類	人類, 成年人	Adipose derived mesenchymal stem	Collagenase Type 2: 0.1%	see reference	Al-Saqi, S., Saliem, M., Asikainen, S., Quezada, H., Ekblad, A., Hovatta, O., Le Blanc, K., Jonasson, A. and Gotherstrom, C.: Defined Serum-Free Media for In Vitro Expansion of Adipose-Derived Mesenchymal Stem Cells., Cytotherapy 16, 915, 2014
人類	人類, 成年人	Adipose derived stem cells	Collagenase Type 1: 0.15%	DMEM	Koellensperger, E., Bollinger, N., Dexheimer, V., Gramley, F., Germann, G. and Leimer, U.: Choosing the Right Type of Serum for Different Applications of Human Adipose Tissue-Derived Stem Cells: Influence on Proliferation and Differentiation Abilities., Cytotherapy 16, 789, 2014
人類	人類	Umbilical mesenchymal stem	Collagenase Type 2: 0.1%	DMEM	Hang, H., Yu, Y., Wu, N., Huang, Q., Xia, Q. and Bian, J.: Induction of Highly Functional Hepatocytes from Human Umbilical Cord Mesenchymal Stem Cells by HNF4偽 Transduction.,PLoS ONE 9, e104133, 2014
			Trypsin: 0.25%
人類	人類	Cardiac stem	Collagenase Type 2: 0.25%	Joklik modified Eagle抯	Avolio, E., Gianfranceschi, G., Cesselli, D., Caragnano, A., Athanasakis, E., Katare, R., Meloni, M., Palma, A., Barchiesi, A., Vascotto, C., Toffoletto, B., Mazzega, E., Finato, N., Aresu, G. and Livi, U.: Ex vivo Molecular Rejuvenation Improves the Therapeutic Activity of Senescent Human Cardiac Stem Cells in a Mouse Model of Myocardial Infarction., Stem Cells 32, 2373, 2014
人類	人類	Adipose stromal stem	Collagenase animal free: 200 u/ml	DMEM/Hams F-12	Carvalho, P., Gimble, J., Dias, I., Gomes, M. and Reis, R.: Xenofree Enzymatic Products for the Isolation of Human Adipose-Derived Stromal/Stem Cells., Tiss Eng 19, 473-8, 2013
人類	人類, 成年人	Epidermal stem cells	Neutral Protease: 2 u/ml	DMEM	Nagel, S., Rohr, F., Weber, C., Kier, J., Siemers, F., Kruse, C., Danner, S., Brandenburger, M. and Matthiessen, A.: Multipotent Nestin-Positive Stem Cells Reside in the Stroma of Human Eccrine and Apocrine Sweat Glands and can be Propagated Robustly In Vitro., PLoS ONE 8, e78365, 2013
			Trypsin: 2.5%
人類	人類	Dental pulp stem	Collagenase Type 1: 0.3%	DMEM/F12	Bonnamain, V., Thinard, R., Sergent-Tanguy, S., Huet, P., Bienvenu, G., Naveilhan, P., Farges, J. and Alliot-Licht, B.: Human Dental Pulp Stem Cells Cultured in Serum-Free Supplemented Medium., Front Physiol 4, 357, 2013
			Neutral Protease: 0.4%
人類	人類	Umbilical Cord Mesenchymal Stem	Collagenase: 0.1-0.2%	DMEM	Han, Y., Tao, R., Sun, T., Chai, J., Xu, G. and Liu, J.: Optimization of Human Umbilical Cord Mesenchymal Stem Cell Isolation and Culture Methods., Cytotechnology 65, 819, 2013
			Trypsin: 0.1%
人類	人類	Dental pulp derived stem cell	Collagenase Type 1: 0.3%	DMEM	Sakai, K., Yamamoto, A., Matsubara, K., Nakamura, S., Naruse, M., Yamagata, M., Sakamoto, K., Tauchi, R., Wakao, N., Imagama, S., Hibi, H., Kadomatsu, K., Ishiguro, N. and Ueda, M: Human Dental Pulp-Derived Stem Cells Promote Locomotor Recovery After Complete Transection of the Rat Spinal Cord by Multiple Neuro-Regenerative Mechanisms., J Clin Invest 122, 80, 2012
			Neutral Protease: 0.4%
人類	人類	Adipose-derived stem cells	Collagenase Type 1: 0.1%	PBS	Cervelli, V., Scioli, M., Gentile, P., Doldo, E., Bonanno, E., Spagnoli, L. and Orlandi, A.:Plaet-Rich Plasma Greatly Potentiates Insulin-Induced Adipogenic Differentiation of Human Adipose-Derived Stem Cells Through a Serine/Threonine Kinase Akt-dependent Mechanism and Promotes Clinical Fat Graft Maintenance., Stem Cells Transl Med 1, 206-20, 2012
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	Wharton jelly mesenchymal stem	Collagenase Type 2: 0.1%	DMEM	Salehinejad, P., Alitheen, N., Ali, A., Omar, A., Mohit, M., Janzamin, E., Samani, F., Torshizi, Z. and Nematollahi-Mahani, S.: Comparison of Different Methods for the Isolation of Mesenchymal Stem Cells from Human Umbilical Cord Wharton's Jelly., In Vitro Cell Dev Biol Anim 48, 75, 2012
			Trypsin: 0.25%
人類	人類	Placental Mesenchymal Stem	Collagenase Type 2: 0.4%	DMEM	Vellasamy, S., Sandrasaigaran, P., Vidyadaran, S., George, E. and Ramasamy, R.:Isolation and Characterisation of Mesenchymal Stem cells Derived from Human Placenta Tissue., World J Stem Cells 4, 53-61, 2012
			Deoxyribonuclease I: 0.01%
人類	人類, 男性 40-60 years	Adipose derived stem cells	Collagenase: 0.25%	PBS	Blasi, A., Martino, C., Balducci, L., Saldarelli, M., Soleti, A., Navone, S., Canzi, L., Cristini, S., Invernici, G., Parati, E. and Alessandri, G.: Dermal Fibroblasts Display Similar Phenotypic and Differentiation Capacity to Fat-Derived Mesenchymal Stem Cells, but Differ in Anti-Inflammatory and Angiogenic Potential, Vasc Cell 3, 5, 2011
			Deoxyribonuclease I: 0.002%
人類	人類	Umbilical cord stromal stem	Collagenase Type 4: 0.08%	DMEM	Farias, V., Linares-Fernandez, J., Penalver, J., Paya Colmenero, J., Ferron, G., Duran, E., Fernandez, R., Olivares, E., O'Valle, F., Puertas, A., Oliver, F. and Ruiz de Almodovar, J.:Human Umbilical Cord Stromal Stem Cell Express CD10 and Exert Contractile Properties.,Placenta 32, 86, 2011
			Neutral Protease: 0.138%
			Hyaluronidase: 0.02%
人類	人類	Vascular wall-resident multipotent stem cells	Collagenase Type 2: 0.2%	see reference	Klein, D., Weisshardt, P., Kleff, V., Jastrow, H., Jakob, H., and Ergun, S.: Vascular Wall-Resident CD44+ Multipotent Stem Cells Vive Rise to Pericytes and Smooth Muscle Cells and Contribute to New Vessel Maturation., PLoS ONE 6, e20540, 2011
			Elastase: 5 u/ml
人類	人類	Adipose derived stem	Collagenase Type 1: 0.1%	DMEM/Ham's F-12	Yu, G. Floyd, ZE., Wu, X., Halvorsen, Y. and Gimble, J.: Isolation of Human Adipose-Derived Stem Cells from Lipoaspirates., Methods Mol Biol 702, 17-27, 2011
人類	人類	Glioma stem cells	PDS kit: per instructions	neurobasal medium	Hjelmeland, A., Wu, Q., Wickman, S., Eyler, C., Heddleston, J., Shi, Q., Lathia, J., Macswords, J., Lee, J., McLendon, R. and Rich, J.: Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth., PLoS Biol 8, e1000319, 2010
人類	人類	Dental pulp and apical papilla stem cells	Collagenase Type 1: 0.3%	MEM	Huang, G., Yamaza, T., Shea, L., Djouad, F., Kuhn, N., Tuan, R. and Shi, S.:Stem/Progenitor Cell-Mediated De Novo Regeneration of Dental Pulp with Newly Deposited Continuous Layer of Dentin in an In Vivo Model., Tissue Eng Part A 16, 605, 2010
			Neutral Protease: 0.4%
人類	人類	Corneal stromal stem	Neutral Protease: 1.2 u/ml	DMEM	Du, Y., Roh, D., Funderburgh, M., Mann, M., Marra, K., Rubin, J., Li, X. and Funderburgh, J.: Adipose-Derived Stem Cells Differentiate to Keratocytes In Vitro., Mol Vis 16, 2680, 2010
			Collagenase: 0.1%
人類	人類	Adipose tissue-derived stem	Collagenase Type 2: 1.0%	DMEM/F12	Tan, H., DeFail, A., Rubin, J., Chu, C. and Marra, K.: Novel Multiarm PEG-Based Hydrogels for Tissue Engineering., J Biomed Mater Res 92, 979, 2010
人類	人類	Umbilical cord mesenchymal	Hyaluronidase: 0.05%	DMEM	Zeddou, M., Briquet, A., Relic, B., Josse, C., Malaise, M., Gothot, A., Lechanteur, C. and Beguin, Y.: The Umbilical Cord Matrix is a Better Source of Mesenchymal Stem Cells (MSC) Than the Umbilical Cord Blood., Cell Biol Int 34, 693-701, 2010
			Collagenase: 0.08%
人類	人類	Adipose stem cells	Collagenase Type 1: 0.1%	DMEM	Hareendran, S., Sathishkumar, S., Abbas, S., Mackay, A. and Rajan, P.: A Novel Composition for the Culture of Human Adipose Stem Cells which Includes Complement C3.,Cytotechnology 62, 389-402, 2010
人類	人類, 40-65 year	Adult human adipose stem cells	Collagenase Type 2: 0.075%	DMEM	Sun, N., Panetta, N., Gupta, D., Wilson, K., Lee, A., Jia, F., Hu, S., Cherry, A., Robbins, R., Longaker, M. and Wu, J.: Feeder-Free Derivation of Induced Pluripotent Stem Cells from Adult Human Adipose Stem Cells., Proc Natl Acad Sci U S A 106, 15720, 2009
人類	人類	Filum terminale neural progenitor	Trypsin: see reference	DMEM/F-12	Varghese, M., Olstorn, H., Berg-Johnsen, J., Moe, M., Murrell, W. and Langmoen, I.:Isolation of Human Multipotent Neural Progenitors from Adult Filum Terminale., Stem Cells Dev 18, 603, 2009
人類	人類	skin and keloid progenitor cells	Collagenase Type 1: 0.4%	PBS	Zhang, Q., Yamaza, T., Kelly, AP, Shi, S., Wang, S., Brown, J., Wang, L., French, S., Shi, S. and Le, A.: Tumor-like Stem Cells Derived from Human Keloid are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis., PLoS ONE 4, e7798, 2009
			Neutral Protease: 0.3%
人類	人類, 男性	Spermatogonial stem cells	Collagenase: 1%	DMEM	Kossack, N., Meneses, J., Shefi, S., Nguyen, H., Chavez, S., Nicholas, C., Gromoll, J., Turek, P. and Reijo-Pera, R.: Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells., Stem Cells , , 2008
			Deoxyribonuclease I: 0.22%
			Trypsin: 0.4%
人類	人類	Muscle derived multiprogenitor cells	Collagenase Type 2: 0.05%	DMEM	Nesti, L., Jackson, W., Shanti, R., Koehler, S., Aragon, A., Bailey, J., Sracic, M., Freedman, B., Giuliani, J. and Tuan, R.: Differentiation Potential of Multipotent Progenitor Cells Derived from War-Traumatized Muscle Tissue., J Bone Joint Surg Am 90, 2390, 2008
人類	人類	Adipose derived stem cells	Collagenase Type 1: 0.075%	DMEM	Jeong, J.: Adipose Stem Cells as a Clinically Available and Effective Source of Adult Stem Cell Therapy, Int J Stem Cells 1, 43, 2008
人類	人類	Hepatic stem cells and heptoblasts	Collagenase Type 4: 0.014-0.06%	various	Wauthier, E., Schmelzer, E., Turner, W., Zhang, L., LeCluyse, E., Ruiz, J., Turner, R., Furth, M., Kubota, H., Lozoya, O., Barbier, C., McClelland, R., Yao, H., Moss, N., Bruce, A., Ludlow, J. and Reid, L.: Hepatic Stem Cells and Hepatoblasts: Identification, Isolation, and Ex Vivo Maintenance., Methods Cell Biol 86, 137, 2008
人類	人類	Umbilical mesenchymal stem	Collagenase: 0.1%	DMEM	Secco, M., Zucconi, E., Vieira, N., Fogaca, L.,Cerqueira, A., Carvalho, M., Jazedje, T., Okamoto, O., Muotri, A. and Zatz, M.: Multipotent Stem Cells from Umbilical Cord: Cord is Richer Than Blood!, Stem Cells 26, 146, 2008
人類	人類	Stem Cells	Collagenase: 280 u/ml	D-PBS	Pilgaard, L., Lund, P., Rasmussen, J., Fink, T. and Zachar, V.: Comparative Analysis of Highly Defined Proteases for the Isolation of Adipose Tissue-Derived Stem Cells., Regen Med3, 705-15, 2008
人類	人類	Bone marrow derived MSC	Trypsin: 0.05%	DMEM	Welter, J., Solchaga, L.and Penick, K.: Simplification of Aggregate Culture of Human Mesenchymal Stem Cells as a Chondrogenic Screening Assay, Biotechniques 42, 732, 2007
			Papain: 0.0025%
人類	人類, 8-12 year	Tendon stem/progenitor	Collagenase Type 1: 0.3%	DMEM	Bi Yanming, Ehirchiou Driss, Kilts Tina M, Inkson Colette A, Embree Mildred C, Sonoyama Wataru, Li Li, Leet Arabella I, Seo Byoung-Moo, Zhang Li, Shi Songtao, Young Marian F:Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche, Nat Med 13, 1219-27, 2007
			Neutral Protease: 0.4%
人類	人類, 男性	Adipose derived adult stem cells	Collagenase Type 1: 0.1%	DMEM	Lei, L., Liao, W., Sheng, P., Fu, M., He, A. and Huang, G.: Biological Character of Human Adipose-Derived Adult Stem Cells and Influence of Donor Age on Cell Replication in Culture.,Sci China C Life Sci 50, 320, 2007
人類	人類	Pancreatic cancer stem cells	Collagenase Type 4: 200 u/ml	medium 199	Li, C., Heidt, D., Dalerba, P., Burant, C., Zhang, L., Adsay, V., Wicha, M., Clarke, M. and Simeone, D.: Identification of Pancreatic Cancer Stem Cells., Cancer Res 67, 1030, 2007
人類	人類, 成年人	Adipose-derived adult stem	Collagenase Type 1: 0.1%	DMEM/F-12 Ham's	Mitchell James B, McIntosh Kevin, Zvonic Sanjin, Garrett Sara, Floyd Z Elizabeth, Kloster Amy, Di Halvorsen Yuan, Storms Robert W, Goh Brian, Kilroy Gail, Wu Xiying, Gimble Jeffrey M: Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers, Stem Cells 24, 376-85, 2006
人類	人類	Mesenchymal stem	Collagenase Type 1: 0.075%	DMEM	Kern Susanne, Eichler Hermann, Stoeve Johannes, Kluter Harald, Bieback Karen:Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue, Stem Cells 24, 1294-301, 2006
人類	人類	Placental mesenchymal stem	Collagenase Type 2: 270 u/ml	MEM	Portmann-Lanz CBettina, Schoeberlein Andreina, Huber Alexander, Sager Ruth, 男性k Antoine, Holzgreve Wolfgang, Surbek DanielV: Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration, Am J Obstet Gynecol194, 664-73, 2006
			Neutral Protease: 2.4 u/ml
人類	人類	Stromal stem cells	Collagenase Type 1: 0.2%	HBSS	Boquest Andrew C, Shahdadfar Aboulghassem, Brinchmann Jan E, Collas Philippe:Isolation of stromal stem cells from human adipose tissue, Methods Mol Biol 325, 35-46, 2006
人類	人類, 女性	Adipose derived adult stem cells	Collagenase Type 1: 0.1%	DMEM/F-12	Guilak, F., Lott, K., Awad, H., Cao, Q., Hicok, K., Fermor, B. and Gimble, J.: Clonal Analysis of the Differentiation Potential of Human Adipose-Derived Adult Stem Cells., J Cell Physiol206, 229, 2006
人類	人類	Adult stem cells	Collagenase Type 1: 0.1%	PBS	Devireddy, R., Thirumala, S. and Gimble, J.: Cellular Response of Adipose Derived Passage-4 Adult Stem Cells to Freezing Stress., J Biomech Eng 127, 1081, 2005
人類	人類, 女性	Adipose derived adult stem cells	Collagenase Type 1: 0.1%	DMEM-Ham's F-12	Aust, L., Devlin, B., Foster, S., Halvorsen, Y., Hicok, K., du Laney, T., Sen, A., Willingmyre, G. and Gimble, J.: Yield of Human Adipose-Derived Adult Stem Cells from Liposuction Aspirates., Cytometry 6, 7-14, 2004
人類	人類, 成年人, 男性	Human epidermal keratinocyte stem cells	Neutral Protease: 0.5%	DMEM	Papini S, Cecchetti D, Campani D, Fitzgerald W, Grivel J, Chen S, Margolis L, Revolla R.: Isolation and Clonal Analysis of Human Epidermal Keratinocyte Stem Cells in Long-term Culture, Stem Cells 21, 481, 2003
人類	人類	Umbilical vein mesenchymal stem cells	Collagenase: 1%	PBS	Covas DT, Siufi JL C, Silva AR L, Orellana MD: Isolation and culture of umbilical vein mesenchymal stem cells, Braz J Med Biol Res 36, 1179-83, 2003
人類	人類, 7-8 歲	Stem cells Human exfoliated deciduous teeth	Collagenase Type 1: 0.3%	see reference	Miura, M., Gronthos, S, Zhao, M, Lu, B, Fisher, L, Robey, P, and Shi, S.: SHED: Stem Cells from Human Exfoliated Deciduous Teeth, Proc Natl Acad Sci U S A 100, 5807, 2003
			Neutral Protease: 0.4%
人類	人類, 胎兒	Epithelial progenitor	Collagenase: 0.03%	DMEM	Malhi, H., Irani, A., Gagandeep, S. and Gupta, S.: Isolation of Human Progenitor Liver Epithelial Cells with Extensive Replication Capacity and Differentiation into Mature Hepatocytes., J Cell Sci 115, 2679, 2002
人類	人類, 成年人	Human skin mast cells	Collagenase Type 2: 0.15%	HBSS	Kambe, N, Kambe, M, Kochan, J, and Schwartz ,L.: Human Skin-derived Mast Cells Can Proliferate While Retaining Their Characteristic Functional and Protease Phenotypes, Blood 97, 2045, 2001
			Hyaluronidase: 0.07%
			Deoxyribonuclease I: 0.03%
人類	人類	Central nervous system stem	Collagenase: 0.1%	HBSS	Uchida, N., Buck, D., He, D., Reitsma, M., Masek, M., Phan, T., Tsukamoto, A., Gage, F. and Weissman, I.: Direct Isolation of Human Central Nervous System Stem Cells., Proc Natl Acad Sci U S A 97, 14720, 2000
			Hyaluronidase: 0.1%
人類	人類	Embryonic stem	Neutral Protease: 1%	DMEM	Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts, Science 282, 1145-7, 1998
			Collagenase Type 4: 0.1%
人類	人類	Muscle-derived stem cells	Trypsin: 0.25%	DMEM/F12	Alessandri Giulio, Pagano Stefano, Bez Alessandra, Benetti Anna, Pozzi Stefano, Iannolo Gioacchin, Baronio Manuela, Invernici Gloria, Caruso Arnaldo, Muneretto Claudio, Bisleri Gianluigi, Parati Eugenio: Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages, Lancet 364, 1872-83,
猴	猴	Embryonic stem	Collagenase Type 4: 0.08%	DMEM	Chen, S., Revolla, R., Papini, S., Michelini, M., Fitzgerald, W., Zimmerberg, J.,and Margolis, L.: Multilineage Differentiation of Rhesus Monkey Embryonic Stem cells in Three-dimensional Culture Systems, Stem Cells 21(3), 281, 2003
猴	Baboons, 1歲, 7歲, 14 歲	Primate spermatogonial	Collagenase Type 2: 0.1%	DMEM	Nagano, M., McCarrey, J., and Brinster, R.: Primate Spermatogonial Stem Cells Colonize Mouse Testes, Biol Reprod 64, 1409, 2001
			Trypsin: 0.05%
			Deoxyribonuclease I: 0.1%
小鼠	小鼠, 3-4 周	Cardiac progenitor	Collagenase Type 1: 0.1%	DMEM	Wang, H., Chen, H., Feng, B., Wang, X., He, X., Hu, R., Yin, M., Wang, W., Fu, W. and Xu, Z.: Isolation and Characterization of a Sca-1+/CD31- Progenitor Cell Lineage Derived From Mouse Heart Tissue., BMC Biotechnol 14, 75, 2014
小鼠	小鼠, P4	Enteric neural crest progenitors	Neutral Protease: 0.5%	DMEM/F12	Hotta, R., Stamp, L., Foong, J., McConnell, S., Bergner, A., Anderson, R., Enomoto, H., Newgreen, D., Obermayr, F., Furness, J. and Young, H.: Transplanted Progenitors Generate Functional Enteric Neurons in the Postnatal Colon., J Clin Invest 123, 1182, 2013
			Collagenase animal free: 0.05%
小鼠	小鼠	Epithelial Stem	Trypsin: 0.25%	HBSS	Jensen, U., Ghazizadeh, S. and Owens, D.: Isolation and Characterization of Cutaneous Epithelial Stem Cells., Methods Mol Biol 989, 61-9, 2013
			Collagenase Type 1: 0.4%
小鼠	小鼠, 10 周	Synovial mesenchymal	Collagenase: 0.1%	DMEM	Futami, I., Ishijima, M., Kaneko, H., Tsuji, K., Ichikawa-Tomikawa, N., Sadatsuki, R., Muneta, T., Arikawa-Hirasawa, E., Sekiya, I. and Kaneko, K.: Isolation and Characterization of Multipotential Mesenchymal Cells from the Mouse Synovium., PLoS ONE7, e45517, 2012
			Deoxyribonuclease I: 0.005%
小鼠	小鼠, 6-8 周	Neural stem cells	Papain: 0.01%	DMEM/F12	Bracko, O., Singer, T., Aigner, S., Knobloch, M., Winner, B., Ray, J., Clemenson, G., Suh, H., Couillard-Despres, S., Aigner, L., Gage, F. and Jessberger, S.: Gene Expression Profiling of Neural Stem Cells and their Neuronal Progeny Reveals IGF2 as a Regulator of Adult Hippocampal Neurogenesis., J Neurosci 32, 3376-87, 2012
			Neutral Protease: 0.1%
			Deoxyribonuclease I: 0.01%
小鼠	小鼠, 8-10 周	Adipose derived stem	Collagenase Type 1: 0.025%	HBSS	Sugii, S., Kida, Y., Berggren, W. and Evans, R.: Feeder-Dependent and Feeder-Independent iPS Cell Derivation from Human and Mouse Adipose Stem Cells., Nat Protoc 6, 346, 2011
小鼠	小鼠	Spleen, bone marrow endothelial	Collagenase Type 4: 0.3-1.0%	PBS	Shi, C., Jia, T., Mendez-Ferrer, S., Hohl, T., Serbina, N., Lipuma, L., Leiner, I., Li, M., Frenette, P. and Pamer, E.: Bone Marrow Mesenchymal Stem and Progenitor Cells Induce Monocyte Emigration in Response to Circulating Toll-Like Receptor Ligands., Immunity 34, 590, 2011
			Deoxyribonuclease I: 20 u/ml
小鼠	小鼠, 8-10 周	Lung mesenchymal stem	Collagenase Type 2: 0.2%	HBSS	Chow, K., Jun, D., Helm, K., Wagner, D. and Majka, S.: Isolation & Characterization of Hoechst(low) CD45(negative) Mouse Lung Mesenchymal Stem Cells., J Vis Exp 56, e3159, 2011
小鼠	小鼠, 新生的	Dental Pulp Stem Cells	Collagenase Type 4: 0.2%	PBS	Janebodin, K., Horst, O., Ieronimakis, N., Balasundaram, G., Reesukumal, K., Pratumvinit, B. and Reyes, M.: Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice., PLoS ONE 6, e27526, 2011
			Neutral Protease: 1.2 u/ml
小鼠	小鼠	Stem and progenitor	Collagenase Type 2: 0.2%	HBSS	Han, J., Koh, Y., Moon, H., Ryoo, H., Cho, C., Kim, I. and Koh, G.: Adipose Tissue is an Extramedullary Reservoir for Functional Hematopoietic Stem and Progenitor Cells., Blood 115, 957, 2010
小鼠	小鼠, 6-8 周	Bone marrow mesenchymal stem	Collagenase Type 1: 0.25%	RPMI 1640	Xu, S., De Becker, A., Van Camp, B., Vanderkerken, K. and Van Riet, I.: An Improved Harvest and In Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells., J Biomed Biotechnol 2010, 105940, 2010
小鼠	小鼠, 成年人	Adult neural stem	Trypsin: 0.05%	DMEM	Deleyrolle, L. and Reynolds, B.: Isolation, Expansion, and Differentiation of Adult Mammalian Neural Stem and Progenitor Cells using the Neurosphere Assay., Methods Mol Biol 549, 91, 2009
小鼠	小鼠, 6-8 周	Prostate epithelial/stem	Collagenase Type 2: 0.5%	HBSS	Burger, P., Gupta, R., Xiong, X., Ontiveros, C., Salm, S., Moscali, D. and Wilson, E.:High Aldehyde Dehydrogenase Activity: A Novel Functional Marker of Murine Prostate Stem/Progenitor Cells., Stem Cells 27, 2220-8, 2009
			Trypsin: 0.05%
小鼠	小鼠	Adipose tissue-derived stem	Collagenase Type 1: 0.075%	Modified Eagles	Cho, K., Park, H., Park, H., Jung, J., Jeon, S., Kim, Y. and Roh, H.: IFATS Collection: Immunomodulatory Effects of Adipose Tissue-Derived Stem Cells in an Allergic Rhinitis Mouse Model., Stem Cells 27, 259-65, 2009
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	Neural progenitor cell	Papain: 10 ul/ml	PBS	Hutton, S. and Pevny, L.: Isolation, Culture, and Differentiation of Progenitor Cells from the Central Nervous System., Cold Spring Harb. Protoc. 11, 5077, 2008
小鼠	小鼠, 3-6 周	Ear mesenchymal stem	Collagenase Type 1: 0.2%	DMEM/F12	Staszkiewicz, J., Gimble, J., Manuel, J. and Gawronska-Kozak, B.: IFATS Collection: Stem Cell Antigen-1-Positive Ear Mesenchymal Stem Cells Display Enhanced Adipogenic Potential.,Stem Cells 26, 2666, 2008
小鼠	小鼠, 胚胎	HES-BC cells	Trypsin: 0.05%	DMEM	Lu Shi-Jiang, Feng Qiang, Caballero Sergio, Chen Yu, Moore Malcolm A, Grant Maria B, Lanza Robert: Generation of functional hemangioblasts from human embryonic stem cells,Nat Methods 4, 501-9, 2007
小鼠	小鼠, 6-8 周	Tendon stem/progenitor	Collagenase Type 1: 0.3%	DMEM	Bi Yanming, Ehirchiou Driss, Kilts Tina M, Inkson Colette A, Embree Mildred C, Sonoyama Wataru, Li Li, Leet Arabella I, Seo Byoung-Moo, Zhang Li, Shi Songtao, Young Marian F:Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche, Nat Med 13, 1219-27, 2007
			Neutral Protease: 0.4%
小鼠	小鼠	Liver epithelial progenitor cells	Collagenase Type 4: 0.1%	DMEM	Li Wen-Lin, Su Juan, Yao Yu-Cheng, Tao Xin-Rong, Yan Yong-Bi, Yu Hong-Yu, Wang Xin-Min, Li Jian-Xiu, Yang Yong-Ji, Lau Joseph T Y, Hu Yi-Ping: Isolation and characterization of bipotent liver progenitor cells from adult mouse, Stem Cells 24, 322-32, 2006
			Deoxyribonuclease I: 0.05%
小鼠	小鼠, 6 周	Adipose mesenchymal stem	Collagenase: 0.2%	PBS	Di Rocco Giuliana, Iachininoto Maria Grazia, Tritarelli Alessandra, Straino Stefania, Zacheo Antonella, Germani Antonia, Crea Filippo, Capogrossi Maurizio C: Myogenic potential of adipose-tissue-derived cells, J Cell Sci 119, 2945-52, 2006
小鼠	小鼠	Neural stem cells	Papain: see reference	DMEM/F-12	Meletis, K., Wirta, V., Hede, S., Nister, M., Lundeberg, J. abd Frisen, J.: p53 Suppresses the Self-Renewal of Adult Neural Stem Cells., Development 133, 363, 2006
小鼠	小鼠, 7天	Cerebellar stem cells	Papain: 10u/ml	Dulbecco's PBS	Lee, A, Kessler, J, Read, T, Kaiser, C, Corbeil, D, Huttner ,W, Johnson, J, Wechsler-Reya, R.: Isolation of Neural Stem Cells from the Postnatal Cerebellum, Nat Neurosci 8, 723, 2005
			Deoxyribonuclease I: 250 u/ml
小鼠	小鼠, 胚胎	Embryonic fibroblast feeder cells	Collagenase Type 4: 0.1%	DMEM	Schatten Gerald, Smith Joseph, Navara Christopher, Park Jong-Hyuk, Pedersen Roger:Culture of human embryonic stem cells, Nat Methods 2, 455-63, 2005
小鼠	小鼠, 1天	Neural progenitor	PDS kit: per instructions	see reference	Seaberg, R., Smukler, S. and Van der Kooy, D.: Intrinsic Differences Distinguish Transiently Neurogenic Progenitors from Neural Stem Cells in the Early Postnatal Brain., Dev Biol 278, 71, 2005
小鼠	小鼠, 8-10 周	Liver derived stem cells	Collagenase Type 1: 0.1%	HBSS	Kotton, D., Fabian, A. and Mulligan, R.: A Novel Stem-Cell Population in Adult Liver with Potent Hematopoietic-Reconstitution Activity., Blood 106, 1574, 2005
			Neutral Protease: 2.4 u/ml
小鼠	小鼠	Bone marrow	Collagenase Type 1: 0.15%	PBS	Bertoncello, I. and Williams, B.: Hematopoietic Stem Cell Characterization by Hoechst 33342 and Rhodamine 123 Staining., Methods Mol Biol 263, 181, 2004
			Neutral Protease: 0.15%
小鼠	小鼠, 4-7 天	Pluripotent stem cells	Collagenase Type 1: 220 u/ml	MEM	Howell, J., Lee, W., Morrison, P., Zhong, J., Yoder, M. and Srour, E.: Pluripotent Stem Cells Identified in Multiple Murine Tissues., Ann N Y Acad Sci 996, 158, 2003
			Neutral Protease: 33 u/ml
小鼠	小鼠, 15 天或 3 個月	Neural stem cells	Papain: 0.1%	DMEM/F-12	Gritti, A, Bonfanti, L, Doetsch, F, Caille, I, Alvarez-Buylla, A, Lim, D, Galli, R, Verdugo J, Herrera, D, and Vescovi A.: Multipotent Neural Stem Cells Reside into the Rostral Extension and Olfactory Bulb of Adult Rodents, J Neurosci 22(2), 437, 2002
小鼠	小鼠, 胚胎	Cortical progenitors	PDS kit: per instructions	Serum free medium	Estivill-Torrus, G., Pearson, H., Van Heyningen, V., Price, D. and Rashbass, P.: Pax6 is Required to Regulate the Cell Cycle and the Rate of Progression from Symmetrical to Asymmetrical Division in Mammalian Cortical Progenitors., Development 129, 455, 2002
小鼠	小鼠, 4-8 個月	Neural subventricular zone	Trypsin: 0.13%	DMEM/F12	Gritti A, Frolichsthal-Schoeller P, Galli R, Parati E, Cova L, Pagano S, Bjornson C, and Vescovi A.: Epidermal and Fibroblast Growth Factors Behave as Mitogenic Regulators for a Single Multipotent Stem Cell-like Population from the Subventricular Region of the Adult Mouse forebrain, J Neurosci 19(9), 3287, 1999
			Hyaluronidase: 0.067%
小鼠	小鼠, 6 周	Muscle hematopoietic stem cells	Collagenase: 0.2%	DMEM	Jackson, K., and Goodell, M.: Hematopoietic Potential of Stem Cells Isolated from Murine Skeletal Muscle, Proc Natl Acad Sci U S A 96, 14482, 1999
			Trypsin: 0.1%
豬	豬, 3-4 個月	Synovial membrane and skin stem cells	Collagenase: 0.2%	DMEM	Ando, W., Kutcher, J., Krawetz, R., Sen, A., Nakamura, N., Frank, C. and Hart, D.: Clonal Analysis of Synovial Fluid Stem Cells to Characterize and Identify Stable Mesenchymal Stromal Cell/Mesenchymal Progenitor Cell Phenotypes in a Porcine Model: A Cell Source with Enhanced Commitment to the Chondrogenic Lineage., Cytotherapy 16, 776, 2014
豬	豬,雌性, 小于一歲	Adipose mesenchymal stem	Collagenase Type 1: 0.1%	DMEM	Williams, K., Picou, A., Kish, S., Giraldo, A., Godke, R. and Bondioli, K: Isolation and Characterization of Porcine Adipose Tissue-Derived Adult Stem Cells., Cells Tissues Organs188, 251, 2008
兔	兔, 新西蘭, 8-10 周	Tenocytes and tendon stem cells	Collagenase Type 1: 0.3%	DMEM	Zhang, J. and Wang, J.: Characterization of Differential Properties of Rabbit Tendon Stem Cells and Tenocytes., BMC Musculoskelet Disord 11, 10, 2010
			Neutral Protease: 0.4%
大鼠	大鼠	Mesenchymal Stem Cells	Collagenase Type 1: 0.075%	DMEM	Emre, E, Yuksel, N, Duruksu, G, Pirhan, D, Subasi, C, Erman, G and Karaoz, E:Neuroprotective Effects of Intravitreally Transplanted Adipose Tissue and Bone Marrow-Derived Mesenchymal Stem Cells in an Experimental Ocular Hypertension Model.,Cytotherapy 17, 543-59, 2015
大鼠	SD大鼠, 5 個月	Mesenchymal Stromal Cells	Collagenase Type 2: 0.075%	DMEM	Veronesi, F, Torricelli, P, Della, B, Pagani, S and Fini, M: In Vitro Mutual Interaction Between Tenocytes and Adipose-Derived Mesenchymal Stromal Cells., Cytotherapy 17, 215-23, 2015
大鼠	大鼠	Adipose Derived Stem	Collagenase Type 1: 0.1%	DMEM	Wei, X., Du, Z., Zhao, L., Feng, D., Wei, G., He, Y., Tan, J., Lee, W., Hampel, H., Dodel, R., Johnstone, B., March, K., Farlow, M. and Du, Y.: IFATS Collection: The Conditioned Media of Adipose Stromal Cells Protect Against Hypoxia-Ischemia-Induced Brain Damage In Neonatal Rats., Stem Cells 27, 478, 2009
大鼠	SD大鼠, 新生	Neutral stem cells, Schwann cells	Trypsin: 0.25%	DMEM/F12	Zeng Yuan-Shan, Ding Ying, Wu Li-Zhi, Guo Jia-Song, Li Hai-Biao, Wong Wai-Man, Wu Wu-Tian: Co-transplantation of schwann cells promotes the survival and differentiation of neural stem cells transplanted into the injured spinal cord, Dev Neurosci 27, 20-6, 2005
			Collagenase: 0.16%
大鼠	大鼠, 雄性, 200-250 克	Neural stem cells	Papain: 0.09%	EBSS	Gobbel GT, Choi SJ, Beier S, Niranjan A: Long-term c*tion of multipotential neural stem cells from adult rat subependyma, Brain Res 980, 221, 2003
			Deoxyribonuclease I: 0.1%
大鼠	大鼠, 出生后的, 成年的	Gut Neural Crest Stem Cells	Collagenase Type 4: 0.1%	HBSS	Kruger, G., Mosher, J., Bixby, S., Joseph, N., Iwashita, T. and Morrison, S.: Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness., Neuron 35, 657, 2002
			Trypsin: 0.025%
大鼠	大鼠, 成年的	Neural stem cells	Papain: 2.5 u/ml	DMEM/F-12	Palmer, T., Markakis, E., Willhoite, A., Safar, F., and Gage, F.: Fibroblast Growth Factor-2 Activates a Latent Neurogenic Program in Neural Stem Cells from Diverse Regions of the Adult CNS, J Neurosci 19, 8487, 1999
			Deoxyribonuclease I: 250 u/ml
			Neutral Protease: 1 u/ml
大鼠	大鼠	Central nervous system stem cells	Papain: 0.01%	DMEM/F-12	Ray, J. and Gage, F.: Neural Stem Cell Isolation, Characterization and Transplantation,Modern Techniques in Neuroscience Research,Johansson, H. and Windhorst, U., Springer-Verlag New York, 339, 1999
			Neutral Protease: 0.1%
大鼠	大鼠, 胚胎	Neurons and progenitor	Papain: 20 u/ml	EBSS	Maric, D., Maric, I. and Barker, J.: Buoyant Density Gradient Fractionation and Flow Cytometric Analysis of Embryonic Rat Cortical Neurons and Progenitor Cells., Methods 16, 247, 1998

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