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Worthington组织消化指南
引言
组织消化酶主要用于组织培养和细胞生物学研究的原代细胞的分离和收集。尽管这些消化酶已经普遍应用多年，但是对其组织消化和细胞收集的作用机制却没有全面的理解。 由此导致的结果便是选择一种消化方法大多是随机选择或基于过去的经验，而不是在*理解消化原理的基础上选择的消化方案，以及如何根据具体实验目的修正消化方案以获得更好的实验结果。
细胞分离程序的目的是zui大程度的获得功能活性的单个细胞，而影响结果的原因有很多，包括（不限于）以下：
1、组织类型
2、物种来源
3、动物年龄
4、遗传改造情况（如knockouts）
5、所用的消化培养液
6、所用的酶
7、消化酶中的杂质
8、所用消化酶的工作浓度
9、温度
10、孵育时间
通常前四个原因没办法选择，而剩下的原因一般由经验决定以获取zui适实验结果。
通过查阅文献来选择*组织消化酶和zui适消化条件的研究人员通常被互相矛盾的实验数据搞得一头雾水。这些巨大差异来源于组织胞外基质的复杂而动态的特性，以及过去常用的粗制、未提纯的组织分离酶。另外，组织胞外基质由各种蛋白、糖蛋白、脂类和糖脂组成，这些成分的组成比例在不同物种、不同组织、甚至不同发育（年龄）之间存在明显的差异，这些因素都极大的影响了消化方案的制定。目前通常使用的粗制酶，如链霉蛋白酶、NF1:250、以及含有几种其他不同浓度蛋白酶的胶原酶、以及各种多糖酶、核酶和脂酶。
本指南总结了目前常用组织消化酶的zui适消化和细胞收集知识和方法，描述了标准的实验步骤， 以便于制定一个*的细胞分离方案，同时列举了一些相关的参考文献。
下表列出多种组织的适宜消化酶（含参考文献，以组织类型和物种分类，详见链接）
脂肪组织 | 肾上腺组织 | 骨组织 | 脑组织 |
软骨组织 | 结肠组织 | 内皮组织 | 上皮组织 |
眼 | 心脏 | 肠 | 肾 |
肝 | 肺 | 淋巴结 | 乳腺 |
其它组织 | 其他组织 | 肌组织 | 神经组织 |
胰腺 | 腮腺 | 垂体 | 前列腺 |
生殖 | 动物鳞片 | 皮肤 | 脾脏 |
干细胞 | 胸腺 | 甲状腺/甲状旁腺 | 扁桃体 |
肿瘤 |
|
|
|
注：使用Worthington公司消化酶的文献不限于上表链接中所列，一般而言，Worthington公司的组织消化酶可用于大多组织类型的消化。
肠组织消化手册
| 物种 | 详细来源 | 细胞名称 | 消化用酶 | 培养基 | 参考文献 |
| 犬 | 狗, 成年的 | Intestinal L-cells | Collagenase Type 1: 75 u/ml | HBSS | Damholt AB, Buchan AM, Kofod H: Glucagon-like-peptide-1 secretion from canine L-cells is increased by glucose-dependent-insulinotropic peptide but unaffected by glucose,Endocrinology 139, 2085-91, 1998 |
| 人类 | 人类 | Lamina propria lymphocytes | Collagenase: 20 u/ml | Medium | Ebert, Ellen.: CD2 Activation of Human Lamina Propria Lymphocytes Reduces CD3 Responsiveness., Immunology 117, 71-7, 2006 |
| 人类 | 人类 | Lamina propria mononuclear cells | Collagenase: 0.02% | HBSS | Kanai Takanori, Totsuka Teruji, Uraushihara Koji, Makita Shin, Nakamura Tetsuya, Koganei Kazutaka, Fukushima Tsuneo, Akiba Hisaya, Yagita Hideo, Okumura Ko, Machida Utako, Iwai Hideyuki, Azuma Miyuki, Chen Lieping, Watanabe Mamoru: Blockade of B7-H1 suppresses the development of chronic intestinal inflammation, J Immunol 171, 4156-63, 2003 |
| 人类 | 人类 | Intestinal epithelial | Collagenase Type 4: 72.5 u/ml | HBSS | Fahlgren, A., Hammarstrom, S., Danielsson, A. and Hammarstrom, M.: Increased Expression of Antimicrobial Peptides and Lysozyme in Colonic Epithelial Cells of Patients with Ulcerative Colitis., Clin Exp Immunol 131, 90, 2003 |
| 人类 | 人类 | Mucosal mononuclear cells | Collagenase Type 3: 0.01% | RPMI | Stallmach A, Schaffer F, Hoffmann S, Weber S, Muller-Molaian I, Schneider T, Kohne G, Ecker KW, Feifel G, Zeitz M: Increased state of activation of CD4 positive T cells and elevated interferon gamma production in pouchitis, Gut 43, 499-505, 1998 |
| Deoxyribonuclease I: 0.01% | |||||
| Soybean Trypsin Inhibitor: 0.01% | |||||
| 小鼠 | 小鼠 | Lamina propria mononuclear cells | Collagenase Type 3: 400 u/ml | HBSS | Seo, S., Kuffa, P., Kitamoto, S., Nagao-Kitamoto, H., Rousseau, J., Kim, Y., Nunez, G. and Kamada, N.: Intestinal Macrophages Arising from CCR2(+) Monocytes Control Pathogen Infection by Activating Innate Lymphoid Cells., Nat Commun 6, 8010, 2015 |
| Deoxyribonuclease I: 0.001% | |||||
| 小鼠 | 小鼠 | Lymphocytes | Collagenase Type 1: 100 u/ml | RPMI-1640 | Beura, L., Anderson, K., Schenkel, J., Locquiao, J., Fraser, K., Vezys, V., Pepper, M. and Masopust, D.: Lymphocytic Choriomeningitis Virus Persistence Promotes Effector-Like Memory Differentiation and Enhances Mucosal T cell Distribution., J Leukoc Biol 97, 217, 2015 |
| 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 | colon tumor organoids | Collagenase Type 4: 200 u/ml | DMEM | Xue, X. and Shah, Y.: In Vitro Organoid Culture of Primary Mouse Colon Tumors., J Vis Exp , e50210, 2013 |
| Neutral Protease: 0.01% | |||||
| 小鼠 | 小鼠 | Intestinal organoids | Collagenase Type 1: 800 u/ml | DMEM | Barthel, E., Speer, A., Levin, D., Sala, F., Hou, X., Torashima, Y., Wigfall, C. and Grikscheit, T.: Tissue Engineering of the Intestine in a Murine Model., J Vis Exp 70, e4279, 2012 |
| Neutral Protease: 0.013% | |||||
| 小鼠 | 小鼠 | Dendritic, macrophages | Collagenase: 0.15% | HBSS | Geem, D., Medina-Contreras, O., Kim, W., Huang, C. and Denning T.: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine., J Vis Exp 63, e4040, 2012 |
| Deoxyribonuclease I: 0.004% | |||||
| 小鼠 | 小鼠, 新生的及成年的 | Myenteric plexus | Collagenase Type 4: 0.025-0.1% | HBSS | Joseph, N., He, S., Quintana, E., Kim, Y., Nunez, G. and Morrison, S.: Enteric Glia are Multipotent in Culture but Primarily Form Glia in the Adult Rodent Gut., J Clin Invest 121, 3398, 2011 |
| Papain: 10 u/ml | |||||
| Deoxyribonuclease I: 100 u/ml | |||||
| 小鼠 | 小鼠 | Lamina propria lymphocytes | Collagenase Type 1: 0.3% | RPMI 1640 | Ito, Y., Kanai, T., Totsuka, T., Okamoto, R., Tsuchiya, K., Nemoto, Y., Yoshioka, A., Tomita, T., Nagaishi, T., Sakamoto, N., Sakanishi, T., Okumura, K., Yagita, H. and Watanabe, M.:Blockade of NKG2D Signaling Prevents the Development of Murine CD4+ T Cell-Mediated Colitis., Am J Physiol Gastrointest Liver Physiol 294, G199, 2008 |
| Deoxyribonuclease I: 0.01% | |||||
| 小鼠 | 小鼠 | Intestinal mononuclear | Collagenase Type 4: 0.1% | RPMI 1640 | Forbes, E., Groschwitz, K., Abonia, J.P., Brandt, E., Cohen, E., Blanchard, C., Ahrens, R., Seidu, L., McKenzie, A., Strait, R., Finkelman, F., Foster, P., Matthaei, K., Rothenberg, M. and Hogan, S.: IL-9- and Mast Cell-Mediated Intestinal Permeability Predisposes to Oral Antigen Hypersensitivity., J Exp Med 205, 897, 2008 |
| 小鼠 | 小鼠, 南非乳鼠 | Enterochromaffin cells | Collagenase: 0.025% | HBSS | Kidd, M., Modlin, I., Eick, G. and Champaneria, M.: Isolation, Functional Characterization, and Transcriptome of Mastomys Ileal Enterochromaffin Cells., Am J Physiol Gastrointest Liver Physiol 291, G778-91, 2006 |
| Pronase E: 0.07% | |||||
| 小鼠 | 小鼠, 7 周 | Interstitial cells | Collagenase: 0.04% | see reference | Goto Kazunori, Matsuoka Satoshi, Noma Akinori: Two types of spontaneous depolarizations in the interstitial cells freshly prepared from the murine small intestine, J Physiol 559, 411-22, 2004 |
| Trypsin: 0.02% | |||||
| 小鼠 | 小鼠, 9-13 天 | Interstitial cells of Cajal | Collagenase Type 2: 0.13% | HBSS | Ordag Tamas, Redelman Doug, Miller Lisa J, Horvath Viktor J, Zhong Qiao, Almeida-Porada Graca, Zanjani Esmail D, Horowitz Burton, Sanders Kenton M: Purification of interstitial cells of Cajal by fluorescence-activated cell sorting, Am J Physiol Cell Physiol 286, C448-56, 2004 |
| 小鼠 | 小鼠, 0-15 天 | Interstitial cells of Cajal | Collagenase Type 2: 0.13% | Hanks | Lee Young Mee, Kim Byung Joo, Kim Hyun Jin, Yang Dong Ki, Zhu Mei Hong, Lee Kyu Pil, So Insuk, Kim Ki Whan: TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach, Am J Physiol/Gastro 284, G604-16, 2003 |
| 小鼠 | 小鼠 | Lamina propria lymphocytes | Collagenase Type 4: 300 u/ml | PBS | Wu Y, Wang X, Csencsits KL, Haddad A, Walters N, Pascual DW: M cell-targeted DNA vaccination, Proc Natl Acad Sci U S A 98, 9318-23, 2001 |
| 小鼠 | 小鼠, F14 | Intestinal mesenchymal | Collagenase Type 2: 0.03% | HBSS | Sakagami Y, Inaguma Y, Sakakura T, Nishizuka Y: Intestine-like remodeling of adult mouse glandular stomach by implanting of fetal intestinal mesenchyme, Cancer Res 44, 5845-9, 1984 |
| 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 | SD大鼠,胚胎 | Sciatic Nerve and Gut Neural Crest Stem Cells | Collagenase Type 4: 0.025% | HBSS | Bixby, S., Kruger, G., Mosher, J., Joseph, N. and Morrison, S.: Cell-Intrinsic Differences Between Stem Cells from Different Regions of the Peripheral Nervous System Regulate the Generation of Neural Diversity., Neuron 35, 643, 2002 |
| Trypsin: 0.005% | |||||
| Deoxyribonuclease I: 0.05% | |||||
| 大鼠 | 大鼠,雄性, 18-24 天 | Intestinal epithelial | Collagenase Type 1: 0.1% | DMEM | Quaroni A, Wands J, Trelstad RL, Isselbacher KJ: Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria, J Cell Biol 80, 248-65, 1979 |
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