用于向小鼠眼睛输送生物制剂的封装细胞技术
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 维持Dulbecco改良鹰中（DMEM）中的细胞，辅以10%的胎儿牛血清（FBS）。</li…
Representative Results
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?…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 3 mL Syringe | BD | 309656 | |
| 30 G 1" Blunt needle | SAI Infusion technology | B30-100 | |
| Alginic acid sodium salt, from brown algae | Sigma | A0682 | |
| Atropine Sulfate Ophthalmolic solution (1%) | Akorn | NDC 17478-215-15 | for pupil dilation |
| BD 1 mL Syringe 26 G x 3/8 (0.45 mm x 10 mm) | Becton, Dickinson and Company | DG518105 500029609 REF 309625 | to generate the guide hole |
| Calcium chloride, Anhydrous, granular | Sigma | C1016 | |
| GenTeal Tears | Alcon | NDC 0078-0429-47 | to lubricate the eyes during anesthesia |
| Goniotaire: Hypromellose (2.5%) Ophthalmolic Demulcent Solution (Sterile) | Altaire Pharmaceuticals Inc. | NDC 59390-182-13 | to lubricate the eyes during anesthesia |
| Hamilton Needle/syringe Tip: 27 Gauge, Small Hub RN NDL, custum length (12mm), point style 3, 6/PK | Hamilton | 7803-01 | for intravitreal delivery of capsules |
| Hamilton Syringe: 2.5 µL, Model 62 RN SYR, NDL Sold Separately | Hamilton | 7632-01 | for intravitreal delivery of capsules |
| HEPES buffer, 1M | Fisher Bioreagents | BP299100 | |
| High voltage generator | ESD EMC Technology | ES813-D20 | |
| LIVE/DEAD Viability/Cytotoxicity Kit | Thermofisher Scientific | L3224 | |
| L-Ornithine hydrochloride, 99% | Alfa Aesar | A12111 | |
| Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmolic Ointment | SANDOZ | NDC 61314-631-36 | antibiotic to prevent infection after intravitreal injection |
| Phenolephrine Hydrochloride Ophthalmolic Solution (2.5%) | Akorn | NDC 17478-201-15 | for pupil dilation |
| Sodium Chloride | Sigma | S-5886 | |
| Sterile syringe filters, 0.2 um | VWR | 28143-312 | |
| Syringe pump | GRASEBY | MS16A |
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Citer Cet Article
Belhaj, M., Annamalai, B., Parsons, N., Shuler, A., Potts, J., Rohrer, B. Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye. J. Vis. Exp. (157), e60162, doi:10.3791/60162 (2020).