原代细胞分离中，针对眼睛组织涉及到视网膜内皮、脑内皮细胞、视网膜细胞和角膜上皮 、米勒细胞等等的解离通常会使用酶，如何选择合适的酶来完成对应脑组织的解离，以及原代细胞采集的常规操作， 艾美捷针对眼睛相关的组织分类解离分种属、不同的细胞、酶组合推荐，推荐浓度、培养基以及参考文献，详细汇总指南：
本worthington 眼睛组织分离指南描述了标准实验室程序； 为建立细胞分离方案提供了一种合乎逻辑的实验方法； 并列出了许多组织特异性参考资料，以帮助开发有效的方法。更多其他组织的解离指南，点击查看：各组织分离指南。
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| 种属 | 种属详情 | 细胞名称 | 酶组合推荐，含推荐浓度 | 培养基 | 参考文献 |
| 牛 | Bovine | 视网膜内皮 / Retinal endothelial | Collagenase Type 4: 0.05%； Deoxyribonuclease I: 0.02%； Pronase: 0.02% |
DMEM | Terlizzi, V., Kolibabka, M., Burgess, J., Hammes, H. and Harmsen, M.: The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2., Stem Cells 36, 240-251, 2018 |
| 牛 | Bovine | 周细胞 / 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 | Bovine | 微血管内皮 / 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 |
| 鸡 | Chick, embryo, 6 day | 视网膜细胞 / Retinal cells | Trypsin: 0.005%； Deoxyribonuclease I: 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 |
| 鸡 | Chick embryo | 角膜上皮 / Corneal epithelia | Collagenase: 0.08%； Trypsin: 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 |
| 鸡 | Chick embryo (also rat) | 视网膜 / Retinal | Trypsin: 0.6% | DMEM | Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999 |
| 鸡 | Chick, embryo, 10-14 day | 平坦，视网膜 / 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 |
| 鱼 | Fish, zebrafish | 视网膜 / 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 |
| 鱼 | Goldfish | 视网膜双极 / Retinal Bipolar | Papain: 30 u/ml | HEPES | Graffe, M., Zenisek, D. and Taraska, J.: A Marginal Band of Microtubules Transports and Organizes Mitochondria in Retinal Bipolar Synaptic Terminals., J Gen Physiol 146, 109-17, 2015 |
| 人 | Human | 角膜基质细胞角质细胞 / Corneal stromal keratocytes | Collagenase Type 1: 0.1% | DMEM/F-12 | Yusoff,B, Riau, A., Yam, G., Halim, N. and Mehta, J.: Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy.,Cells Vol. 11,, , , 2022 |
| 人 | Human | 米勒细胞 / Muller glia | PDS kit: per instructions | DMEM | Xu, N., Chen, Y., Dean, K., Lu, X., Liu, X., Wang, W., Dean, D., Kaplan, H., Gao, L., Dong, F. and Liu, Y.: Sphere-Induced Rejuvenation of Swine and Human Muller Glia Is Primarily Caused by Telomere Elongation., Stem Cells 35, 1579-1591, 2017 |
| 人 | Human | 角膜基质细胞干细胞 / Corneal stroma stem | Collagenase Type 1: 0.1% | DMEM | Sidney, L., Branch, M., Dua, H. and Hopkinson, A.: Effect of Culture Medium on Propagation and Phenotype of Corneal Stroma-Derived Stem Cells., Cytotherapy 17, 1706-22, 2015 |
| 人 | Human | 角膜基质细胞干细胞 / Corneal stromal stem | Neutral Protease: 1.2 u/ml； Collagenase: 0.1% |
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 |
| 人 | Human, 18-68 yr | 角膜内皮 / Corneal endothelial | Collagenase: 0.2%； Neutral Protease: 1.0% |
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 |
| 人 | Human | 角膜缘上皮片 / 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 | Human, 5-65 years | 视网膜色素上皮 (RPE) / 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 | Macaques and baboon | 视网膜 / Retinal | Papain: 20-40 u/ml； Deoxyribonuclease I: 400 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 |
| 猴 | Monkey, cynomolgus, young adult | 结膜淋巴细胞 / 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 | Mouse, 2-4 week | 米勒细胞 / Muller | PDS kit: per instructions | DMEM | Liu, X., Tang, L. and Liu Y.: Mouse Muller Cell Isolation and Culture., Bio-protocol 7, , 2017 |
| 小鼠 | Mouse | 视网膜 / Retinal | Papain: 20 u/ml； Deoxyribonuclease I: 0.005% |
PBS | Ortin-Martinez, A., Tsai, E., Nickerson, P., Bergeret, M., Lu, Y., Smiley, S., Comanita, L. and Wallace, V.: A Reinterpretation of Cell Transplantation: GFP Transfer From Donor to Host Photoreceptors., Stem Cells 35, 932-939, 2017 |
| 小鼠 | Mouse | 视网膜 / Retinal | Papain: 20 u/ml； Deoxyribonuclease I: 0.005% |
HBSS | Wohl, S., Jorstad, N., Levine, E. and Reh, T.: Muller Glial microRNAs are Required for the Maintenance of Glial Homeostasis and Retinal Architecture., Nat Commun 8, 1603, 2017 |
| 小鼠 | Mouse, C57BL/6 | 视网膜星形胶质细胞 / Retinal astrocytes | Papain: 16.5 u/ml； Deoxyribonuclease I: 0.004% |
HBSS | Blandford, S. and Baldridge, W.: The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca(2+)]i., Biomed Res Int 2016, 8178162, 2016 |
| 小鼠 | Mouse, adult | 视网膜感光器 / 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 | Mouse, 4-8 day | 光感受器细胞 / 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 | Mouse, embryonic and P0 | 视网膜 / Retinal | Papain: 8 u/ml； Deoxyribonuclease I: 124 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 |
| 小鼠 | Mouse | 玻璃体细胞 / Vitreous cells | Collagenase: 0.1%； Hyaluronidase: 0.03% |
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 |
| 小鼠 | Mouse, 6 week | 视网膜 / 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 | Mouse | 视网膜神经节 / Retinal ganglion | CLSPA: 240 u/ml； Hyaluronidase: 0.2% |
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 |
| 小鼠 | Mouse, adult | 纤毛上皮 / 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 | Mouse, 4-5 week | 透镜纤维 / Lens Fiber | Collagenase Type 4: 0.125%； Protease: 0.5% |
PBS | Ebihara, L., Tong, J., Vertel, B., White, T. and Chen, T.: Properties of Connexin 46 Hemichannels in Dissociated Lens Fiber Cells., Inv Ophthal Visual Sci 52, 882-9, 2011 |
| 小鼠 | Mouse, postnatal day 1 | 视网膜祖细胞 / 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 | Mouse, 4-8 week | 角膜上皮 / Corneal epithelial | Neutral Protease: 1.5%； Trypsin: 0.25% |
DMEM/F12 | Kobayashi, T., Yoshioka, R., Shiraishi, A. and Ohashi, Y.: New Technique for Culturing Corneal Epithelial Cells of Normal Mice., Mol Vis 15, 1589-93, 2009 |
| 小鼠 | Mouse | 视网膜 / Retinal | Collagenase: 0.1%； Deoxyribonuclease I: 0.001% |
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 |
| 小鼠 | mouse, adult | 视网膜 / 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 | Mouse, adult | 基质细胞 / Stromal | Trypsin: 0.05%； Collagenase: 78 u/ml； Hyaluronidase: 38 u/ml |
DMEM/F-12 | Yoshida, S., Shimmura, S., Nagoshi, N., Fukuda, K., Matsuzaki, Y., Okano, H. and Tsubota, K.: Isolation of Multipotent Neural Crest-Derived Stem Cells from the Adult Mouse Cornea., Stem Cells 24, 2714-22, 2006 |
| 小鼠 | Mouse, 2-5 month | 小鼠视网膜和双极 / Mouse retinal and bipolar | Papain: 20 u/ml； Deoxyribonuclease I: 200 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 |
| 小鼠 | Mouse, 4 week | 视网膜内皮细胞 / 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 |
| 猪 | Porcine, adult | 米勒细胞 / Muller glia | PDS kit: per instructions | DMEM | Xu, N., Chen, Y., Dean, K., Lu, X., Liu, X., Wang, W., Dean, D., Kaplan, H., Gao, L., Dong, F. and Liu, Y.: Sphere-Induced Rejuvenation of Swine and Human Muller Glia Is Primarily Caused by Telomere Elongation., Stem Cells 35, 1579-1591, 2017 |
| 猪 | Porcine, 5-60 kg | 视网膜色素上皮细胞 / 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 | Rabbit, New Zealand, Adult | 角膜内皮 / 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 | Rabbit | 角膜角质细胞 / Corneal keratocytes | Hyaluronidase: 0.05%； Collagenase: 0.2% |
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 |
| 兔 | Rabbit, New Zealand | 角膜角质细胞 / Corneal keratocytes | Trypsin: 0.25%； Collagenase: 0.5% |
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 |
| 兔 | Rabbit, adult | 视网膜神经元 / 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 Physiol 283, C126-34, 2002 |
| 兔 | Rabbit, New Zealand, adult, male, albino, 2 kg | 上皮 / 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 |
| 大鼠 | Adult, female, 170-200 g | 视网膜 / Retinal | PDS kit: with modifications | EBSS | Mead, B. and Tomarev, S.: Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms., Stem Cells Transl Med 6, 1273-1285, 2017 |
| 大鼠 | Rat, Wister, 3-30 wk | 视网膜 / Retinal | Deoxyribonuclease I: 0.005%； Papain: 30 u/ml |
EBSS | Cameron, M., Kekesi, O., Morley, J., Tapson, J., Breen, P., Van Schaik, A and Buskila, Y.: Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue., PLoS ONE 11, e0155468, 2016 |
| 大鼠 | Rat, SD, 6-8 week | 视网膜 / 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 | Rat, SD | 视网膜神经节 / 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 | Rat, SD, 180-220g | 视网膜神经节 / 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 | Rat, Fisher, adult | 视网膜 / 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 | Rat, Dark Agouti, 3-4 week | 睫状源性眼 / 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 | Rat, embryonic 17 day | 视网膜 / Retinal | Trypsin: 0.6% | DMEM | Seigel, G.: The Golden Age of Retinal Cell Culture, Mol Vis 5, 4, 1999 |
| 大鼠 | Rat, SD | 视网膜 / 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 | Rat, 4-14 week | 视网膜色素上皮细胞 / Retinal pigment epithelial cells | Collagenase Type 1: 65 u/ml； Hyaluronidase: 220 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 |
| 大鼠 | Rat, 6-8 day | 视网膜色素上皮 / 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 | Rat, Long Evans, 1-15 days | 神经元，视觉皮层 / Neurons, visual cortex | Papain: 20 u/ml | BSS (see reference) | Huettner, J., and Baughman, R.: Primary Culture of Identified Neurons From the Visual Cortex of Postnatal Rats, J Neurosci 6, 3044, 1986 |
| 大鼠 | Rat, SD, pups | 视网膜 / 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 |
| 蝾螈 | Salamander | 神经元 / 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 |
| 蝾螈 | Salamander, 18-25 cm | 视网膜 / 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 |
| 蝾螈 | Salamander (A. tigrinum) | 光感受器细胞，视网膜 / 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 |
| 龟 | Turtle (Pseudemys scripta elegans) | 视网膜 / 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 |
