Name: encapsulated cell technology for the delivery of biologics to the mouse eye
Uploaded: 2020-03-30T13:00:00
Description: Watch this Scientific Journal Video about Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye at JoVE.com

这项研究部分得到了国家卫生研究院（R01EY019320）、退伍军人事务部（RX000444和BX003050）和南卡罗来纳州智能国家基金会授予B.R.的赠款的支持。

所有实验均根据《动物在眼科和视力研究中使用ARVO声明》进行，并经南卡罗来纳医科大学动物护理和使用委员会根据协议ID00399批准。
1. 细胞培养
<ol><li>根据公布的协议14、15，,15生成人类视网膜色素上皮细胞（ARPE-19）细胞系，稳稳地表达选择的基因。</li>
	<li>维持Dulbecco改良鹰中（DMEM）中的细胞，辅以10%的胎儿牛血清（FBS）。</li...

<ol>
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Effects of formulations and process on morphology and particle size.</a> Journal of Microencapsulation. 23 (1), 69-78 (2006).</li><li>Gryshkov, 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=Process+engineering+of+high+voltage+alginate+encapsulation+of+mesenchymal+stem+cells.">Process engineering of high voltage alginate encapsulation of mesenchymal stem cells.</a> Materials Science and Engineering: C. 36, 77-83 (2014).</li><li>Thanos, C. G., 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=Sustained+secretion+of+ciliary+neurotrophic+factor+to+the+vitreous,+using+the+encapsulated+cell+therapy-based+NT-501+intraocular+device.">Sustained secretion of ciliary neurotrophic factor to the vitreous, using the encapsulated cell therapy-based NT-501 intraocular device.</a> Tissue Engineering. 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S., 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=Retinal+Pigment+Epithelium+Is+Protected+Against+Apoptosis+by+&#945;B-Crystallin.">Retinal Pigment Epithelium Is Protected Against Apoptosis by &#945;B-Crystallin.</a> Investigative Ophthalmology &amp; Visual Science. 43 (11), 3575-3582 (2002).</li><li>Chiu, K., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreous+injection+for+establishing+ocular+diseases+model.">Intravitreous injection for establishing ocular diseases model.</a> Journal of Visualized Experiments. (8), 313 (2007).</li><li>Jove Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.+Anesthesia+Induction+and+Maintenance.">Lab Animal Research. Anesthesia Induction and Maintenance.</a> Journal of Visualized Experiments. , (2019).</li><li>Holz, F. G., 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=Efficacy+and+Safety+of+Lampalizumab+for+Geographic+Atrophy+Due+to+Age-Related+Macular+Degeneration:+Chroma+and+Spectri+Phase+3+Randomized+Clinical+Trials.">Efficacy and Safety of Lampalizumab for Geographic Atrophy Due to Age-Related Macular Degeneration: Chroma and Spectri Phase 3 Randomized Clinical Trials.</a> JAMA Ophthalmology. 136 (6), 666-677 (2018).</li><li>Kassa, E., Ciulla, T. A., Hussain, R. M., Dugel, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+inhibition+as+a+therapeutic+strategy+in+retinal+disorders.">Complement inhibition as a therapeutic strategy in retinal disorders.</a> Expert Opinion in Biological Therapy. 19 (4), 335-342 (2019).</li><li>Cashman, S. M., Ramo, K., Kumar-Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Non+Membrane-Targeted+Human+Soluble+CD59+Attenuates+Choroidal+Neovascularization+in+a+Model+of+Age+Related+Macular+Degeneration.">A Non Membrane-Targeted Human Soluble CD59 Attenuates Choroidal Neovascularization in a Model of Age Related Macular Degeneration.</a> PLoS ONE. 6 (4), e19078 (2011).</li><li>Vincent, 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=Generation+of+combination+PDGF+/+VEGF-antagonist+ECT+devices.">Generation of combination PDGF / VEGF-antagonist ECT devices.</a> Investigative Ophthalmology &amp; Visual Science. 54, 3290 (2013).</li><li>Zhang, K., 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=Ciliary+neurotrophic+factor+delivered+by+encapsulated+cell+intraocular+implants+for+treatment+of+geographic+atrophy+in+age-related+macular+degeneration.">Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration.</a> Proc Natl Acad Sci USA. 108 (15), 6241-6245 (2011).</li><li>Chew, E. 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=Ciliary+neurotrophic+factor+for+macular+telangiectasia+type+2:+results+from+a+phase+1+safety+trial.">Ciliary neurotrophic factor for macular telangiectasia type 2: results from a phase 1 safety trial.</a> American Journal of Ophthalmology. 159 (4), 659-666 (2015).</li><li>Birch, D. G., 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=Randomized+trial+of+ciliary+neurotrophic+factor+delivered+by+encapsulated+cell+intraocular+implants+for+retinitis+pigmentosa.">Randomized trial of ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for retinitis pigmentosa.</a> American Journal of Ophthalmology. 156 (2), 283-292 (2013).</li></ol>

这种细胞封装技术相对快速且易于执行;但是，必须牢记某些点，以获得准确的下游结果。细胞应在培养物中保存，然后封装，并适当保持汇合。如果可能，应在适当的通风罩中进行封装，并调节气流。气流太强会影响胶囊的形成，特别是在长期实验中。无菌器具和溶液对胶囊内细胞的长期维护至关重要。
目前，活死染色被用作确定胶囊内细胞生存能力的确认工具。每个胶囊?...

细胞疗法代表了在医学上广泛应用的革命性生物技术。最近，它们已成功应用于神经退行性疾病、眼疾和癌症的治疗。细胞疗法涵盖从细胞替代到药物输送的广泛领域，该协议侧重于后者。可生物降解的藻酸盐微胶囊（MC）作为一种输送系统已经显示出有效性，并且正在生物医学领域得到广泛应用。Alginate 由于其简单的凝胶工艺、生物降解性、优,异的生物相容性以及1、2、323、44在体内条件下的稳定性，一直用于微封装。
电喷法作为一种微封装技术，已成功利用藻酸盐（基聚合物）和聚l-ornithine（二次涂层聚合物）来封装肽和蛋白质。两种聚合物都是自然发现的，并用于其生物相容性,5，6，7。,7然而，在基于细胞的疗法的主要挑战是抑制宿主免疫系统，以避免免疫抑制药物引起的副作用。藻酸盐微胶囊的渗透性被认为是细胞封装的合适特性，它允许将营养物质、废物和治疗因子扩散到细胞中，同时将其从宿主免疫反应88、9、109,10中屏蔽。
在眼睛中，封装细胞被用于临床试验，以持续传递生物制剂（即生长因子11，12,12和生长因子拮抗剂13）用于治疗视网膜炎色素瘤或年龄相关的黄斑变性。其他目标，如补充抑制剂14目前正在临床前环境中探索。

<table>
	<tbody>
		<tr>
			<td>3 mL Syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 1&#34; Blunt needle</td>
			<td>SAI Infusion technology</td>
			<td>B30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt, from brown algae</td>
			<td>Sigma</td>
			<td>A0682</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulfate Ophthalmolic solution (1%)</td>
			<td>Akorn</td>
			<td>NDC 17478-215-15</td>
			<td>for pupil dilation</td>
		</tr>
		<tr>
			<td>BD 1 mL Syringe 26 G x 3/8 (0.45 mm x 10 mm)</td>
			<td>Becton, Dickinson and Company</td>
			<td>DG518105 500029609 REF 309625</td>
			<td>to generate the guide hole</td>
		</tr>
		<tr>
			<td>Calcium chloride, Anhydrous, granular</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal Tears</td>
			<td>Alcon</td>
			<td>NDC 0078-0429-47</td>
			<td>to lubricate the eyes during anesthesia</td>
		</tr>
		<tr>
			<td>Goniotaire: Hypromellose (2.5%) Ophthalmolic Demulcent Solution (Sterile)</td>
			<td>Altaire Pharmaceuticals Inc.</td>
			<td>NDC 59390-182-13</td>
			<td>to lubricate the eyes during anesthesia</td>
		</tr>
		<tr>
			<td>Hamilton Needle/syringe Tip: 27 Gauge, Small Hub RN NDL, custum length (12mm), point style 3, 6/PK</td>
			<td>Hamilton</td>
			<td>7803-01</td>
			<td>for intravitreal delivery of capsules</td>
		</tr>
		<tr>
			<td>Hamilton Syringe: 2.5 &#181;L, Model 62 RN SYR, NDL Sold Separately</td>
			<td>Hamilton</td>
			<td>7632-01</td>
			<td>for intravitreal delivery of capsules</td>
		</tr>
		<tr>
			<td>HEPES buffer, 1M</td>
			<td>Fisher Bioreagents</td>
			<td>BP299100</td>
			<td></td>
		</tr>
		<tr>
			<td>High voltage generator</td>
			<td>ESD EMC Technology</td>
			<td>ES813-D20</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Thermofisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ornithine hydrochloride, 99%</td>
			<td>Alfa Aesar</td>
			<td>A12111</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmolic Ointment</td>
			<td>SANDOZ</td>
			<td>NDC 61314-631-36</td>
			<td>antibiotic to prevent infection after intravitreal injection</td>
		</tr>
		<tr>
			<td>Phenolephrine Hydrochloride Ophthalmolic Solution (2.5%)</td>
			<td>Akorn</td>
			<td>NDC 17478-201-15</td>
			<td>for pupil dilation</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filters, 0.2 um</td>
			<td>VWR</td>
			<td>28143-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>GRASEBY</td>
			<td>MS16A</td>
			<td></td>
		</tr>
	</tbody>
</table>

encapsulated cell technology for the delivery of biologics to the mouse eye

目前许多正在开发治疗后视点疾病的治疗方法都是生物制剂。这些药物需要经常施用，通常通过静脉注射。表达选择的生物学的封装细胞正在成为当地蛋白质生产和释放的工具（例如，通过长期药物输送）。此外，封装系统利用可渗透材料，允许将营养物质、废物和治疗因素扩散到细胞中和细胞外。这发生在遮蔽宿主免疫反应的细胞时，避免了对宿主免疫系统的抑制。该协议将藻酸盐作为微封装中的聚合物与电喷法一起描述为一种微封装技术。ARPE-19细胞是一种自发产生的人类RPE细胞系，由于其终身功能，已用于长期细胞治疗实验，并在这里用于胶囊的封装和向小鼠眼睛的传递。手稿总结了细胞微封装、质量控制和眼部传递的步骤。

ARPE-19细胞是一种自发不朽的人类RPE细胞系，在将胶囊植入眼睛后，可以封装和长期生存。藻酸盐封装的工具如图1所示。在这项研究中，证明在藻酸盐封装后，通过明亮的场成像证实了藻酸盐胶囊中的细胞（图2A）。活死检测在胶囊内的细胞上进行，显示90%的封装后生存能力（图2B）。为了确保胶囊内细胞的长期生存能力，胶囊溶?...

Watch this Scientific Journal Video about Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye at JoVE.com

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

此处介绍的是一种协议，用于将藻酸盐作为聚合物在永生细胞的微封装中，用于长期向啮齿动物的眼睛提供生物制剂。

用于向小鼠眼睛输送生物制剂的封装细胞技术

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

该技术是将药物、蛋白质或细胞等任何内容传递到组织或动物中特定位置的一种可靠方法。您可以跟踪交付并调整内容的发布，以符合您想要的实验。它可用于治疗从伤口愈合到眼部和心脏病的许多疾病。这种方法的主要优点是能够使用藻酸盐微胶囊将治疗细胞送到目标位点，而不会引起任何免疫反应，并持续持续提供新鲜分泌的治疗药物更长时间。首先根据手稿方向培养视网膜上皮细胞。使用标准组织培养程序，当细胞达到70至80%的汇合时，通过细胞。为了封装细胞，将藻酸钠与去离子水混合，最终浓度为2%的重量，然后用0.2微米无菌注射器过滤器通过过滤净化混合物。用10毫摩尔 HEPES 缓冲盐水溶液对细胞进行试穿、离心和清洗。使用血细胞计计算细胞，将最终细胞浓度调整为每毫升藻酸溶液一百万。将藻酸盐和细胞混合物的300微升等分装入三毫升注射器中，并将其连接到注射器泵上。根据手稿指示准备凝胶浴，并放在注射器尖端下方的无菌 50 毫升烧杯中，以 7 毫米针进行沐浴喷洒距离。将电压调整为 8 千伏，将流量调整为每小时 30 毫米，以产生约 150 微米的微胶囊。将黑色接地夹连接到半浸在凝胶浴中的铜线，然后将红色阳极导线的夹子连接到针上。从每毫升的藻酸盐和细胞混合物中准备封装的细胞大约需要30分钟。形成后，用洗涤溶液洗涤微胶囊两次，并在37摄氏度和5%CO2的加湿培养箱中用10%FBS补充DMEM介质孵育。经过24小时的孵育，制备500微升微胶囊样品，在洗涤液中清洗两次，进行染色。使用 LIVE/死亡检测套件来染色以获得生存能力。为确保微胶囊大小适当，在无血清介质中稀释。用 2.5 微升汉密尔顿注射器小心吸气。缓慢地将一个微升滴滴弹射到显微镜幻灯片上，并使用直立的 Brightfield 显微镜确定微胶囊的完整性。在适当麻醉的小鼠中，涂抹眼部润滑剂以保持眼睛水分，并使用 26 量表 3/8 英寸斜面注射器针在边缘下方建立初始眼穿刺。确保以 45 度角从鼻向接近眼睛，使斜面尖端向上，以避免透镜穿孔。接下来，确保将整个针尖插入眼部以生成导孔，但要小心不要走得太远并刺穿时间视网膜。完成后，慢慢缩回针头。在微操纵器内，放置一个 27 量表钝尖端 2.5 微升汉密尔顿注射器，该注射器已充满胶囊溶液，并用初始导孔将针头向上线。将动物的头保持稳定的位置，并故意将针头转向眼睛。为确保在玻璃内有正确的针头位置，请插入注射器，直到针尖在镜头后面可视化。然后在25至30秒内缓慢注射胶囊溶液。完成后，逐渐收回钝尖注射器，以尽量减少对眼睛的伤害。如有必要，根据手稿方向调整电压和流速，调整胶囊尺寸。用布莱特菲尔德成像证实了藻酸盐细胞的封装，LIVE/死检测表明90%的细胞在封装后是可行的。通过溶解柠檬酸钠中的胶囊和重放，确保了细胞的长期生存能力。内注射是在目视检查下进行的，允许玻璃胶囊的可视化。尝试此过程时，不要将 PBS 用于任何洗涤步骤，因为 PBS 溶解形成藻酸盐微胶囊。经过开发，我们的实验室被用作一种输送系统，用于治疗心力衰竭和高血压。这项技术被认为是未来治疗不同心血管和神经系统疾病的有希望的工具。

Research

JoVE Journal

Bioengineering

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

在小型组织培养体系培养老鼠心脏瓣膜

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Intraductal Delivery to the Rabbit Mammary Gland

导管内送货到兔乳腺

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...