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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(xué)研究的原代細胞的分離和收集。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(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(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。
眼組織消化手冊
| 物種 | 詳細來源 | 細胞名稱 | 消化用酶 | 培養(yǎng)基 | 參考文獻 |
| 牛 | 牛 | Pericyte | Collagenase: 0.2% | DMEM | Bryan, B. and D'Amore, P.: Pericyte Isolation and USe is Endothelial/Pericyte Coculture Models, Meth Enzymol 443, 315, 2008 |
| 牛 | 牛 | Microvascular endothelial | Collagenase/Dispase: 0.1% | MEM | Bowman, P., Betz, A., and Goldstein, G.: Primary Culture of Microvascular Endothelial Cells From Bovine Retina, In Vitro 18 (7), 626, 1982 |
| 雞 | 小雞, 胚胎, 6 天 | Retinal cells | Trypsin: 0.005% | DMEM/F12 | Jacob Vanessa, Rothermel Andrae, Wolf Peter, Layer Paul G: Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions, Cell Death Differ 180, 159-68, 2005 |
| Deoxyribonuclease I: 0.005% | |||||
| 雞 | 小雞 胚胎 | Corneal epithelia | Collagenase: 0.08% | HBSS | Reenstra Wende R, Orlow Daniel L, Svoboda Kathy K H: ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent, Inv Ophthal Visual Sci 43, 3181-9, 2002 |
| Trypsin: 0.08% | |||||
| 雞 | 小雞 胚胎 | Retinal | Trypsin: 0.6% | DMEM | Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999 |
| (包括大鼠) | |||||
| 雞 | 小雞, 胚胎, 10-14 天 | Flat, retina | Trypsin: 0.1% | Tyrode's solution, CMF | Moyer, M., Bullrich, F., and Sheffield, J.: Emergence of Flat Cells From Glia in Stationary Cultures of Embryonic Chick Neural Retina, In Vitro Cell Dev Biol 26, 1073, 1990 |
| 魚類 | 魚, 斑馬魚 | Retinal | Papain: 10 u/ml | L-15 | Diekmann, H., Kalbhen, P. and Fischer, D.: Characterization of Optic Nerve Regeneration using Transgenic Zebrafish., Front Cell Neurosci 9, 118, 2015 |
| 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, 18-68 歲 | Corneal endothelial | Collagenase: 0.2% | DMEM/F12 | Li Wei, Sabater Alfonso L, Chen Ying-Ting, Hayashida Yasutaka, Chen Szu-Yu, He Hua, Tseng Scheffer C G: A novel method of isolation, preservation, and expansion of human corneal endothelial cells, Inv Ophthal Visual Sci 48, 614-20, 2007 |
| Neutral Protease: 1.0% | |||||
| 人類 | 人類 | Corneal limbal epithelial sheet | Neutral Protease: 5% | see reference | Espana EM, Romano AC, Kawakita T, Di Pascuale M, Smiddy R, and Tseng SC:Novel enzymatic isolation of an entire viable human limbal epithelial sheet, Inv Ophthal Visual Sci 44(10), 4275, 2003 |
| 人類 | 人類, 5-65 歲 | Retinal pigment epithelial (RPE) | Trypsin: 0.25% | HBSS | Von Recum, H., Okano, T., Kim, S, and Bernstein, P.: Maintenance of Retinoid Metabolism in Human Retinal Pigment Epithelium Cell Culture, Exp Eye Res 69, 97, 1999 |
| 猴 | 獼猴和狒狒 | Retinal | Papain: 20-40 u/ml | Ames' solution | Han ,Y., Jacoby, R. and Wu, S.: Morphological and Electrophysiological Properties of Dissociated Primate Retinal Cells., Brain Res 875, 175, 2000 |
| Deoxyribonuclease I: 400 u/ml | |||||
| 猴 | 猴, 獼猴, 青少年 | Conjunctival lymphocytes | Collagenase Type 1: 0.02% | RPMI 1640 | Whittum-Hudson JA, Taylor HR: Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection, Infect Immun 57, 2977, 1989 |
| 小鼠 | 小鼠, 成年的 | Retinal photoreceptor | PDS kit: with modifications | EBSS | Feodorova, Y., Koch, M., Bultman, S., Michalakis, S. and Solovei, I.: Quick and Reliable Method for Retina Dissociation and Separation of Rod Photoreceptor Perikarya from Adult Mice., MethodsX 2, 39, 2015 |
| 小鼠 | 小鼠, 4-8 天 | Photoreceptors | PDS kit: per instructions | Neurobasal | Balmer, J., Zulliger, R., Roberti, S. and Enzmann, V.: Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways., Int J Mol Sci 16, 15086-103, 2015 |
| 小鼠 | 小鼠, 胚胎 和 P0蛋白 | Retinal | Papain: 8 u/ml | DPBS | Dvoriantchikova, G., Perea-Martinez, I., Pappas, S., Barry, A., Danek, D., Dvoriantchikova, X., Pelaez, D. and Ivanov, D.: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina., PLoS ONE 10, e0131054, 2015 |
| Deoxyribonuclease I: 124 u/ml | |||||
| 小鼠 | 小鼠 | Vitreous cells | Collagenase: 0.1% | DMEM | Iqbal, N., Xu, L., Devitt, C. and Skapek, S.: Isolation and Characterization of Mammalian Cells Expressing the Arf Promoter During Eye Development.,Biotechniques 56, 239-49, 2014 |
| Hyaluronidase: 0.03% | |||||
| 小鼠 | 小鼠, 6 周 | Retinal | PDS kit: per instructions | DMEM | Singh, M., Issa, P, Butler, R., Martin, C., Lipinski, D., Sekaran, S., Barnard, A. and Maclaren, R.: Reversal of End-Stage Retinal Degeneration and Restoration of Visual Bunction by Photoreceptor Transplantation., Proc Natl Acad Sci U S A , , 2013 |
| 小鼠 | 小鼠 | Retinal ganglion | CLSPA: 240 u/ml | Ames | Schmidt, T. and Kofuji, P.: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells., J Vis Exp 47, 2367, 2011 |
| Hyaluronidase: 0.2% | |||||
| 小鼠 | 小鼠, 成年的 | Cilary epithelial | PDS kit: per instructions | EBSS | Gualdoni, S., Baron, M., Lakowski, J., Decembrini, S., Pearson, R., Ali, R. and Sowden, J.: Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells., Curr Protoc Stem Cell Biol Chapter 1, Unit 1H.4, 2011 |
| 小鼠 | 小鼠, 出生后一天的 | Retinal progenitor | Collagenase: 0.1% | HBSS | Jiang, C. Klassen, H., Zhang, X. and Young, M.: Laser Injury Promotes Migration and Integration of Retinal Progenitor Cells into Host Retina., Mol Vis 16, 983, 2010 |
| 小鼠 | 小鼠 | Retinal | Collagenase: 0.1% | RPMI | Amadi-Obi, A., Yu, C., Liu, X., Mahdi, R., Clarke, G., Nussenblatt, R, Gery, I., Lee, Y. and Egwuagu, C.: TH17 Cells Contribute to Uveitis and Scleritis and are Expanded by IL-2 and Inhibited by IL-27/STAT1., Nat Med 13, 711, 2007 |
| Deoxyribonuclease I: 0.001% | |||||
| 小鼠 | 小鼠, 成年的 | Retinal | Papain: 50 u/ml | DMEM/F-12 | Jadhav AP, Cho SH and Cepko CL: Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property, Proc Natl Acad Sci U S A 103, 18998, 2006 |
| 小鼠 | 小鼠, 2-5 個月 | Mouse retinal and bipolar | Papain: 20 u/ml | HBSS | Maxeiner Stephan, Dedek Karin, Janssen-Bienhold Ulrike, Ammermuller Josef, Brune Hendrik, Kirsch Taryn, Pieper Mario, Degen Joachim, Kruger Olaf, Willecke Klaus, Weiler Reto: Deletion of connexin45 in mouse retinal neurons disrupts the rod/cone signaling pathway between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission, J Neurosci 25, 566-76, 2005 |
| Deoxyribonuclease I: 200 u/ml | |||||
| 小鼠 | 小鼠, 4 周 | Retinal endothelial cells | Collagenase Type 1: 0.1% | DMEM | Su X, Sorenson CM, and Sheibani N.: Isolation and characterization of murine retinal endothelial cells, Mol Vis 9, 171, 2003 |
| 豬 | 豬, 5-60 公斤 | Retinal pigment epithelial cells | Collagenase: 2% | DMEM | Wiencke, A., Kiilgaard, J., Nicolini, J., Bundgaard, M., Ropke, C., and La Cour, M.: Growth of Cultured Porcine Retinal Pigment Epithelial Cells, Acta Opthalmol Scand 81(2), 170, 2003 |
| 兔 | 兔, New Zealand, Adult | Corneal Endothelial | Collagenase Type 2: 0.2% | HBSS | Lai, J., Cheng, H. and Ma, D.: Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering, PLoS ONE 10, 1371, 2015 |
| 兔 | 兔 | Corneal keratocytes | Hyaluronidase: 0.05% | DMEM | Hao, M., Flynn, K., Nien-Shy, C., Jester, B., Winkler, M., Brown, D., La Schiazza, O., Bille, J., Jester, J.: In Vivo Non Linear Optical (NLO) Imaging in Live Rabbit Eyes Using the Heidelbert Two-Photon Laser Opthalmoscope, Exp Eye Res 91, 308, 2010 |
| Collagenase: 0.2% | |||||
| 兔 | 兔, 新西蘭 | Corneal keratocytes | Trypsin: 0.25% | PBS | Stramer Brian M, Kwok Michael G K, Farthing-Nayak Pamela J, Jung Jae-Chang, Fini M Elizabeth, Nayak Ramesh C: Monoclonal antibody (3G5)-defined ganglioside: cell surface marker of corneal keratocytes, Inv Ophthal Visual Sci 45, 807-12, 2004 |
| Collagenase: 0.5% | |||||
| 兔 | 兔, 成年的 | Retinal neurons | Papain: 26 u/ml | DMEM | Brockway LM, Zhou Z-H, Bubien JK, Jovov B, Benos DJ, Keyser KT: Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits, Am J Physiol Cell Physiol283, C126-34, 2002 |
| 兔 | 兔, 新西蘭, 成年的, 雄性, 白化變種, | Epithelial | Trypsin: 0.25% | HBSS/DMEM | Johnson-Muller, B., and Gross, J.: Regulation of Corneal Collagenase Production: Epithelial-Stromal Cell Interactions, Proc Natl Acad Sci U S A 75 (9), 4417, 1978 |
| 2 公斤 | |||||
| 大鼠 | SD大鼠,6-8 周 | Retinal | PDS kit: per instructions | EBSS | Vigneswara, V., Esmaeili, M., Deer, L., Berry, M., Logan, A. and Ahmed, Z.: Eye Drop Delivery of Pigment Epithelium-Derived Factor-34 Promotes Retinal Ganglion Cell Neuroprotection and Axon Regeneration., Mol Cell Neurosci 68, 212-21, 2015 |
| 大鼠 | 大鼠, SD | Retinal ganglion | PDS kit: with modifications | Neurobasal | Tan, C., Andrews, M., Kwok, J., Heintz, T., Gumy, L., Fassler, R. and Fawcett, J.:Kindlin-1 Enhances Axon Growth on Inhibitory Chondroitin Sulfate Proteoglycans and Promotes Sensory Axon Regeneration., J Neurosci 32, 7325, 2012 |
| 大鼠 | SD大鼠, 180-220克 | Retinal ganglion | Papain: 0.2% | Neurobasal | Ma, J., Yu, W., Wang, Y., Cao, G., Cai, S., Chen, X., Yan, N., Yuan, Y., Zeng, H., Fleenor, D., Liu, X. and Pang, I.: Neuroprotective Effects of C-type Natriuretic Peptide on Rat Retinal Ganglion Cells., Inv Ophthal Visual Sci 51, 3544-53, 2010 |
| 大鼠 | Fishe大鼠, 成年的 | Retinal | PDS kit: per instructions | MEM | Suzuki, T., Mandai, M., Akimoto, M., Yoshimura, N. and Takahashi, M.: The Simultaneous Treatment of MMP-2 Stimulants in Retinal Transplantation Enhances Grafted Cell Migration into the Host Retina., Stem Cells 24, 2406, 2006 |
| 大鼠 | 大鼠, 暗條紋, 3-4 周 | Ciliary-derived eye | PDS kit: with modifications | DMEM/F12 | Akagi T, Mandai M, Ooto S, Hirami Y, Osakada F, Kageyama R, Yoshimura N, Takahashi M.: Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue, Inv Ophthal Visual Sci 45, 4570, 2004 |
| 大鼠 | 大鼠, 胚胎 17 天 | Retinal | Trypsin: 0.6% | DMEM | Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999 |
| 大鼠 | 大鼠, SD | Retina | Papain: 120 U | HBSS, PBS | Jing, S., Wen, D., Yu, Y., Holst, P., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., et al.: GDNF-Induced Activation of the Ret Protein Tyrosine Kinase is Mediated by GDNFR-a, a Novel Receptor for GDNF, Cell 85, 1113, 1996 |
| 大鼠 | 大鼠, 4-14 week | Retinal pigment epithelial cells | Collagenase Type 1: 65 u/ml | CF Hanks with EDTA | Wang N, Koutz CA, and Anderson RE.: A method for the isolation of retinal pigment epithelial cells from adult rats, Inv Ophthal Visual Sci 34(1), 101, 1993 |
| Hyaluronidase: 220 u/ml | |||||
| 大鼠 | 大鼠, 6-8 天 | Retinal pigment epithelial | Neutral Protease: 2% | DMEM | Chang CW, Roque RS, Defoe DM, and Caldwell RB.: An improved method for isolation and culture of pigment epithelial cells from rat retina, Curr Eye Res 10(11), 1081, 1991 |
| 大鼠 | 大鼠, 長伊萬斯, 1-15 天 | Neurons, visual cortex | Papain: 20 u/ml | BSS | Huettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats, J Neurosci 6, 3044, 1986 |
| (see reference) | |||||
| 大鼠 | 大鼠, SD, 小狗 | Retina | Trypsin: 0.25% | Ham's F-12 | Sarthy PV, Curtis BM, and Catterall WA.: Retrograde Labeling, Enrichment, and Characerization of Retinal Ganglion Cells from the Neonatal Rat, J Neurosci 3 (12), 2532, 1983 |
| 蠑螈 | 蠑螈 | Neurons | Papain: 14 u/ml | Ringers | Clarke, R, Wang, J. and Townes-Anderson, E.: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro., J Vis Exp 19, 911, 2008 |
| 蠑螈 | 蠑螈, 18-25 厘米 | Retina | Papain: 14 u/ml | Saline | Townes-Anderson, E., MacLeish, P., and Raviola, E.: Rod Cells Dissociated from Mature Salamander Retina: Ultrastruture and Uptake of Horseradish Peroxidase, J Cell Biol 100, 175, 1985 |
| 蠑螈 | 蠑螈 (A. tigrinum) | Photoreceptors, retina | Papain: 0.05% | (see reference) | Bader, C., MacLeish, P., and Schwartz, E.: Responses to Light of Solitary Rod Photoreceptors Isolated From Tiger Salamander Retina, Proc Natl Acad Sci U S A 75, 3507, 1978 |
| 龜 | 龜(紅耳龜) | Retinal | Papain: 0.1% (13.5 u/mg) | Kreb's Ringer | Lam, D.: Biosynthesis of Acetylcholine in Turtle Photoreceptors, Proc Natl Acad Sci U S A 69, 1987, 1972 |
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