Name: characterization of a novel human organotypic retinal culture technique
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作者要感谢慷慨的眼组织捐赠者和新西兰国家眼科银行团队的支持。这项工作得到了莫里斯和菲利斯·佩克尔信托基金和奥克兰医学研究基金会（1117015）的项目赠款的财政支持。IDR 的董事职位由布坎南慈善基金会支持。CK的奖学金由新西兰光学师教育与研究基金协会（CC36812）提供，HHL的奖学金由布坎南慈善基金会提供。

人体捐赠眼杯是在角膜切除后从新西兰国家眼库获得的，并经北B健康和残疾伦理委员会（NTX/06/19/CPD/AM07）批准。
注：该培养物应在 II 类生物安全柜中进行，以确保无菌组织培养条件。组织必须在24小时验尸后培养，以避免严重的视网膜完整性损失。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62046/62046fig02.jpg"> 图2：显示收?...

<ol>
	<li>Kolb, H., Fernandez, E., Nelson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facts+and+Figures+Concerning+the+Human+Retina.">Facts and Figures Concerning the Human Retina.</a> Webvision-The Organization of the Retina and Visual System. , (2005).</li><li>Gemenetzi, M., De Salvo, G., Lotery, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+serous+chorioretinopathy:+an+update+on+pathogenesis+and+treatment.">Central serous chorioretinopathy: an update on pathogenesis and treatment.</a> Eye. 24 (12), 1743-1756 (2010).</li><li>Buschini, E., Piras, A., Nuzzi, R., Vercelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+related+macular+degeneration+and+drusen:+neuroinflammation+in+the+retina.">Age related macular degeneration and drusen: neuroinflammation in the retina.</a> Progress in Neurobiology. 95 (1), 14-25 (2011).</li><li>Chen, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+regarding+developmental+mechanisms+in+diabetic+retinopathy+in+Taiwan.">Current concepts regarding developmental mechanisms in diabetic retinopathy in Taiwan.</a> Biomedicine. 6 (2), (2016).</li><li>Kaaja, R., Loukovaara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+retinopathy+in+type+1+diabetic+women+during+pregnancy.">Progression of retinopathy in type 1 diabetic women during pregnancy.</a> Current Diabetes Reviews. 3 (2), 85-93 (2007).</li><li>Azizzadeh Pormehr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organotypic+retinal+flat-mount+culture+(HORFC)+as+a+model+for+retinitis+pigmentosa11.">Human organotypic retinal flat-mount culture (HORFC) as a model for retinitis pigmentosa11.</a> Journal of Cellular Biochemistry. 119 (8), 6775-6783 (2018).</li><li>Fernandez-Bueno, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+modifications+in+organotypic+culture+of+human+neuroretina.">Time course modifications in organotypic culture of human neuroretina.</a> Experimental Eye Research. 104, 26-38 (2012).</li><li>Niyadurupola, N., Sidaway, P., Osborne, A., Broadway, D. C., Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+human+organotypic+retinal+cultures+(HORCs)+to+study+retinal+neurodegeneration.">The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration.</a> British Journal of Ophthalmology. 95 (5), 720-726 (2011).</li><li>Osborne, A., Hopes, M., Wright, P., Broadway, D. C., Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organotypic+retinal+cultures+(HORCs)+as+a+chronic+experimental+model+for+investigation+of+retinal+ganglion+cell+degeneration.">Human organotypic retinal cultures (HORCs) as a chronic experimental model for investigation of retinal ganglion cell degeneration.</a> Experimental Eye Research. 143, 28-38 (2016).</li><li>Caffe, A., Visser, H., Jansen, H., Sanyal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histotypic+differentiation+of+neonatal+mouse+retina+in+organ+culture.">Histotypic differentiation of neonatal mouse retina in organ culture.</a> Current Eye Research. 8 (10), 1083-1092 (1989).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> Journal of Neuroscience Methods. 173 (1), 47-58 (2008).</li><li>Liu, L., Cheng, S. -. H., Jiang, L. -. Z., Hansmann, G., Layer, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pigmented+epithelium+sustains+cell+growth+and+tissue+differentiation+of+chicken+retinal+explants+in+vitro.">The pigmented epithelium sustains cell growth and tissue differentiation of chicken retinal explants in vitro.</a> Experimental Eye Research. 46 (5), 801-812 (1988).</li><li>Kuo, C., Green, C. R., Rupenthal, I. D., Mugisho, O. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin43+hemichannel+block+protects+against+retinal+pigment+epithelial+cell+barrier+breakdown.">Connexin43 hemichannel block protects against retinal pigment epithelial cell barrier breakdown.</a> Acta Diabetologica. 57 (1), 13-22 (2020).</li><li>Mugisho, O. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome+pathway+is+amplified+and+perpetuated+in+an+autocrine+manner+through+connexin43+hemichannel+mediated+ATP+release.">The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release.</a> Biochimica et Biophysica Acta (BBA)-General Subjects. 1862 (3), 385-393 (2018).</li><li>Mugisho, O. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal+pro-inflammatory+cytokines+in+non-obese+diabetic+mice:+Modelling+signs+of+diabetic+retinopathy.">Intravitreal pro-inflammatory cytokines in non-obese diabetic mice: Modelling signs of diabetic retinopathy.</a> PLoS ONE. 13 (8), 0202156 (2018).</li><li>. R&amp;D Systems, I Available from: <a target="_blank" href="https://www.rndsystems.com/protocol-types/luminex">https://www.rndsystems.com/protocol-types/luminex</a> (2020)</li><li>Nakazawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+glial+reactions+and+photoreceptor+degeneration+after+retinal+detachment+in+mice+deficient+in+glial+fibrillary+acidic+protein+and+vimentin.">Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin.</a> Investigative Ophthalmology, Visual Science. 48 (6), 2760-2768 (2007).</li><li>Okada, M., Matsumura, M., Ogino, N., Honda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cells+in+detached+human+retina+express+glial+fibrillary+acidic+protein+and+vimentin.">M&#252;ller cells in detached human retina express glial fibrillary acidic protein and vimentin.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 228 (5), 467-474 (1990).</li><li>Murali, A., Ramlogan-Steel, C. A., Andrzejewski, S., Steel, J. C., Layton, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+explant+culture:+A+platform+to+investigate+human+neuro-retina.">Retinal explant culture: A platform to investigate human neuro-retina.</a> Clinical &amp; Experimental Ophthalmology. 47 (2), 274-285 (2019).</li><li>Koleva-Georgieva, D. N., Sivkova, N. P., Terzieva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+inflammatory+cytokines+IL-1beta,+IL-6,+TNF-alpha+and+VEGF+have+influence+on+the+development+of+diabetic+retinopathy.">Serum inflammatory cytokines IL-1beta, IL-6, TNF-alpha and VEGF have influence on the development of diabetic retinopathy.</a> Folia Medica (Plovdiv). 53 (2), 44-50 (2011).</li><li>Lee, J. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+type+2+diabetes+mellitus+patients+with+diabetic+retinopathy.">Cytokine profile of peripheral blood in type 2 diabetes mellitus patients with diabetic retinopathy.</a> Annals of Clinical &amp; Laboratory Science. 38 (4), 361-367 (2008).</li><li>Chorostowska-Wynimko, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiomodulatory+activity+of+sera+from+type+2+diabetic+patients+with+background+retinopathy.">In vitro angiomodulatory activity of sera from type 2 diabetic patients with background retinopathy.</a> Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society. 56, 65-70 (2005).</li><li>Khalifa, R. A., Khalef, N., Moemen, L. A., Labib, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+interleukin+12+(IL-12),+interferon-inducible+protein+10+(IP-10)+and+Interleukin+18+(IL-18)+in+the+angiogenic+activity+of+diabetic+retinopathy.">The role interleukin 12 (IL-12), interferon-inducible protein 10 (IP-10) and Interleukin 18 (IL-18) in the angiogenic activity of diabetic retinopathy.</a> Research Journal of Medicine and Medical Sciences. 4, 510-514 (2009).</li><li>Zhou, J., Wang, S., Xia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+intravitreal+inflammatory+cytokines+and+angiogenic+factors+in+proliferative+diabetic+retinopathy.">Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy.</a> Current Eye Research. 37 (5), 416-420 (2012).</li><li>Song, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+intravitreous+interleukin-18+correlated+to+vascular+endothelial+growth+factor+in+patients+with+active+proliferative+diabetic+retinopathy.">Increased intravitreous interleukin-18 correlated to vascular endothelial growth factor in patients with active proliferative diabetic retinopathy.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 252 (8), 1229-1234 (2014).</li><li>Louie, H. H., Shome, A., Kuo, C. Y. J., Rupenthal, I. D., Green, C. R., Mugisho, O. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin43+hemichannel+block+inhibits+NLRP3+inflammasome+activation+in+a+human+retinal+explant+model+of+diabetic+retinopathy.">Connexin43 hemichannel block inhibits NLRP3 inflammasome activation in a human retinal explant model of diabetic retinopathy.</a> Experimental Eye Research. , (2020).</li></ol>

HORC是目前临床前视网膜研究中最临床可翻译的模型。与体外细胞培养模型相比，HORC可以通过保留动态视网膜细胞类型及其与神经元、血管和细胞外环境的连接，更好地在原地代表人类视网膜的解剖学。与动物模型相比，HORC在研究人类视网膜疾病的病理生理学和设计药物治疗方面更有利，因为物种间的变化，如不同的视网膜细胞类型以及不同的光受体密度和比例，尽管动物模型<...

视网膜是一种高度专业化的眼部结构，负责将传入的光能转化为电信号，然后由大脑处理，以进行视觉感知。人类视网膜包含一系列动态的细胞类型，高度组织在一个独特的拉梅拉结构，包括两个突触和三个核层1（图1）。视网膜平衡是由神经视网膜细胞、血管、神经、结缔组织和RPE1之间的复杂连接维持的。由于复杂的视网膜解剖学和生理学，许多视网膜疾病的机制仍然缺乏了解2，3，4，5。为了更好地研究视网膜疾病，已经开发了6，7，8，9的HORC模型。与动物研究和体外培养相比，HORC模型具有优势，因为它们保留了动态细胞环境和复杂的神经血管相互作用，为临床翻译提供了良好的模型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62046/62046fig01.jpg"> 图1：人眼的后眼结构。前后视网膜层为：神经纤维层（NFL）、结节细胞层（GCL）、内丛状层（IPL）、内核层（INL）、外丛状层（OPL）、外核层（ONL）、光受体内段（IS）和光受体外层（OS）。视网膜内的细胞包括结节细胞（蓝色）、阿马林细胞（黄色）、双极细胞（红色）、水平细胞（紫色）、棒光受体（粉红色）和锥体光受体（绿色）。视网膜位于前。RPE，布鲁奇的膜，胆囊和硬膜位于视网膜的后。请注意，显示的图像只是视网膜的示意图，每个层内细胞/视网膜连接的比例可能不表示体内设置。<a href="https://www.jove.com/files/ftp_upload/62046/62046fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
先前的荷尔克协议6，7，8，9涉及使用手术颤音将视网膜从底层RPE-胆碱和硬化物中分离出来。然而，如果没有这些基础结构的支持，半透明视网膜变得脆弱，难以处理，诸如钳子等工具很容易破坏其完整性。此外，在没有RPE的情况下分离视网膜已证明会导致结核细胞凋亡和光受体退化10，11，12。因此，一个替代的HORC协议，尽量减少视网膜完整性的损失，并更好地模仿体内环境将是有用的。这在研究视网膜疾病机制时尤其重要，因为外植过程中的身体损伤可能会引入人工制品。因此，本研究的目的是开发一种新的HORC模型，其中包括RPE-巧克力和硬皮，以保护视网膜的完整性，在外植物处理和文化。
为了实现这一目标，在残余的病毒和底层的RPE-胆囊和硬化物之间"夹"出视网膜外植物。在三明治外植物中，视网膜压低，以防止视网膜分离和折叠，而坚韧、纤维状硬化的硬化物既充当结构支撑的脚手架，又充当钳子的接触点。此外，动物模型已经表明，在培养中保留RPE可以防止视网膜退化和胶质增殖，这是穆勒细胞对缺氧和炎症等危险信号的反应，10，11，12。
为了描述模型的特点，三明治视网膜外植物被染色了血氧林和Eosin（H&E），以评估解剖结构和免疫造血化学（IHC）的进行， 标记外生植物与终端脱氧核苷酸转移酶dUTP尼克结束标签（TUNEL，凋亡细胞标记），胶质纤维酸性蛋白（GFAP，视网膜炎症和穆勒细胞激活标记），和维他命，穆勒细胞完整性的标志。为了确定该模型是否可以诱导形成分子疾病迹象， 这些外生植物暴露在高血糖（HG）与亲炎细胞因子（Cyt），白细胞素介素-1+（IL-1+）和肿瘤坏死因子-α（TNF-α），一种培养环境，已被证明模仿糖尿病视网膜病变（DR）在细胞和动物疾病模型13，14，15。在DR模型中使用了Luminex检测来测量释放到培养基中的细胞因子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disposable Biopsy Punches (5 mm)</td>
			<td>Integra York PA Inc., USA</td>
			<td>21909-142</td>
			<td>Referred as surgical trephines 
			in this article</td>
		</tr>
		<tr>
			<td>Human Recombinant IL-1&#946;</td>
			<td>Peprotech, USA</td>
			<td>200-01B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Human Recombinant&#160; TNF-a</td>
			<td>Peprotech, USA</td>
			<td>300-01A</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>DAPI (1 &#181;g/mL)</td>
			<td>Sigma Aldrich, Germany</td>
			<td>#D9542</td>
			<td>Nuclear stain. Working dilution 1:1000</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Gibco, Thermofisher, Scientific Inc., USA</td>
			<td>10565018</td>
			<td>Dulbecco&#8217;s Modified Eagle Medium nutrient mixture F-12 containing a 1&#215; antibiotics and antimycotics mixture (AA, 100&#215; stock)</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools (F.S.T)</td>
			<td>14028-10</td>
			<td>Tips: Sharp-Blunt, Cutting Edge: 27mm, Length: 10cm, Alloy/Material: Stainless Steel, Serrated: No, Tip Shape: Straight</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools (F.S.T)</td>
			<td>11050-10</td>
			<td>Length:10cm, Tip shape: Straight, Tips: Serrated, Tip Width: 0.8mm, Tip Dimensions:0.8 x 0.7mm, Alloy/Materials: Stainless Steel</td>
		</tr>
		<tr>
			<td>Mouse monoclonal GFAP-Cy3</td>
			<td>Sigma Aldrich, Germany</td>
			<td>#C9205</td>
			<td>Primary antibody conjugated to Cy3. Working dilution 1:1000.</td>
		</tr>
		<tr>
			<td>Mouse monoclonal Vimentin-Cy3</td>
			<td>Sigma Aldrich, Germany</td>
			<td>#C9080</td>
			<td>Primary antibody conjugated to Cy3. Working dilution 1:50.</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal TUNEL (In Situ Cell Death Detection Kit, Fluorescein)</td>
			<td>Sigma Aldrich, Germany</td>
			<td>#11684795910</td>
			<td>Primary antibody conjugated to Fluorescein-dUTP. Working dilution 1:10 with enzyme-buffer solution.</td>
		</tr>
	</tbody>
</table>

characterization of a novel human organotypic retinal culture technique

以前的人类组织状视网膜培养（HORC）模型已经利用了分离视网膜：然而，如果没有视网膜色素上皮-胆碱（RPE-胆碱）和硬化物的结构支持，脆弱的视网膜的完整性很容易受到损害。这项研究的目的是开发一个新的HORC模型，其中含有视网膜，RPE胆碱和硬膜，以保持视网膜的完整性，当培养视网膜外植。
沿着豪华巴士进行割礼以去除虹膜和透镜后，制作了四个深切口来平整眼罩。与以前的 HORC 协议不同，不仅用于切开视网膜，还用于切开 RPE 胆碱和硬化物。由此产生的三层外植培养为72小时。血氧林和Eosin染色（H&E）用于评估解剖结构，视网膜外植进一步的特点是免疫造血化学（IHC）的凋亡，穆勒细胞完整性和视网膜炎症。为了确认疾病诱导的可能性，外生植物暴露于高血糖（HG）和促炎细胞因子（Cyt），以模仿糖尿病视网膜病变（DR）。Luminex 磁珠检测用于测量释放到培养介质中的 DR 相关细胞因子。
H&E染色揭示了视网膜拉梅拉和紧凑的细胞核在视网膜外植与底层RPE-胆碱和硬化，而视网膜没有基础结构表现出减少厚度和严重的核损失。IHC结果显示，没有凋亡和视网膜炎症，以及保存的穆勒细胞完整性。Luminex 检测显示，与 24 小时的基线水平相比，暴露在 HG 和 Cyt 的视网膜外植体中与 DR 相关的亲炎细胞因子的分泌显著增加。
我们成功地开发并定性了一种新的HORC协议，其中视网膜完整性被保留，没有凋亡或视网膜炎症。此外，当视网膜外植物暴露在HG + Cyt中时，DR相关亲炎症生物标志物的诱导分泌表明，该模型可用于临床可翻译视网膜疾病研究。

视网膜完整性保留在这个HORC模型。 视网膜完整性保留在培养的三明治视网膜外植中，但在没有相邻结构的视网膜培养中丢失。H&E 进行了研究，以检查 72 小时后分片三明治视网膜外植的结构完整性。三明治视网膜外植显示出保存的完整性和独特的跛脚结构，从GCL到 ONL与紧凑的核在 INL 和 ONL （图4A）。然而，在同一样本的边缘发现视网膜...

Watch this Scientific Journal Video about Characterization of a Novel Human Organotypic Retinal Culture Technique at JoVE.com

Characterization of a Novel Human Organotypic Retinal Culture Technique

这项研究旨在开发一种新的人体组织性视网膜培养（HORC）模型，防止在外植过程中损害视网膜的完整性。这是通过培养视网膜与过度的四肢和底层视网膜色素上皮-胆碱（RPE-胆碱）和硬膜。

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

该技术保留了所有视网膜层和细胞类型原位，使其在临床上与动物和体外模型相比更加相关。此协议的主要优点是，它最大限度地减少了组织处理过程中视网膜完整性中断，这在比较健康和患病视网膜时至关重要。视网膜疾病可以通过在特定条件下培养组织来模仿。这允许测试新的药物疗法。解剖和样品采集过程很复杂。因此，视觉演示对于帮助读者理解书面协议并成功执行程序至关重要。演示这个程序的将是来自我实验室的二年级博士生郭查里斯。准备含有杜尔贝科改性鹰中/营养混合物F-12和抗生素和抗菌混合物的培养基。首先，将500微升的介质物放到24井的盘子中，在37摄氏度和5%的二氧化碳的加湿孵化器中平衡。在外生提取之前准备好介质，避免在随后添加视网膜时脱落。要提取视网膜外植物，首先将眼罩放在培养皿上，虹膜和镜头朝上，ONH 与培养皿接触。用钳子将眼罩稳定在豪华巴士上，沿着利姆布斯的外缘进行小切口，从而分离虹膜和镜头。小心地取出虹膜和透镜，确保避免干扰视网膜。使用明亮的白光源识别 ONH，并在四个象限处向 ONH 旋转培养皿，以便于处理。小心地铺开和压平眼杯。将钳子涂抹在硬膜上，而不是视网膜上，以避免破坏视网膜的完整性。从孵化器中取出包含预制介质的板。在没有视网膜褶皱的区域，在视网膜上放置手术颤音，然后用力压入硬盘，当您穿过培养皿时，应会发出裂开的声音。旋转肌酸，以确保硬化已经完全穿透，使视网膜除植物现在从样品的其余部分分离。将钳子涂抹在硬囊上，将三明治视网膜除植物转移到培养介质中。在加湿的 5% 二氧化碳孵化器中，在 37 摄氏度下培养收集的三明治视网膜外植，持续长达 72 小时。血氧林和欧辛染色表明，三明治视网膜外植保持完整性和独特的跛脚菌结构，从GCL到 ONL，内核和外核层具有紧凑的核。在同一样本的边缘发现视网膜的完整性中断，视网膜已脱离底层RPE胆碱和硬膜，显示在 INL 和 ONL 中视网膜厚度降低和核损失。在基础条件下培养的视网膜外植中，没有发现标志着细胞凋亡的 TUNEL 阳性细胞核，即使在 72 小时后也显示出持续的细胞活力。GFAP 标签仅限于 GCL 和 IPL，没有胶质纤维化，这是病理学的标志，将被确定为 GFAP 在 ONL 中的扩展表达。维门汀从结节细胞层到外核层都得到了高度的表达，在正常的视网膜组织中可以看到，它显示了穆勒细胞的完整性。Luminex磁性检测表明，IL-18、VEGF、IL-6和IL-8水平在24小时后显著高于高葡萄糖和促炎细胞因子中三明治视网膜除菌培养物的基线。72小时后，IL-18 和 VEGF 水平仍显著提高，但在 IL-6 和 IL-8 级别中未发现显著差异。执行此程序时，留下足够的残留玻璃，这将权衡视网膜下来，使其不脱离RP胆囊，并漂浮在文化媒体。培养视网膜可以使用免疫造血化学和组织学进一步描述，而释放到培养介质中的炎症细胞因子可以使用Luminex磁性检测来测量。与动物和体外模型相比，该模型在临床上更易翻译，更适合于测试视网膜疾病新疗法的疗效。

Research

JoVE Journal

Medicine

A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation

一个新型插管困难和困难通风救援技术

We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which ...

科研

医学

A Novel Capsulorhexis Technique Using Shearing Forces with Cystotome

一种新型撕囊技术，使用剪切力与Cystotome

Purpose:
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye....

A Novel Technique of Rescuing Capsulorhexis Radial Tear-out using a Cystotome

径向的抢救囊撕式的新技术，使用一个Cystotome

Part 1 : Purpose:   To demonstrate a capsulorhexis radial tear out rescue technique using a cystotome on a virtual reality cataract surgery simulator and ...

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

简化技术<em&gt;原位</em&gt;切除角膜和视网膜组织剜人眼地球

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

详细的汗渍监控协议使用一种新的，小的，无线设备

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...