提高2D和3D皮肤<em>体外</em>模型使用大分子拥挤
Summary
我们提出了一个协议，以获得富含胞外基质蛋白的细胞来源的基质，使用大分子沟渠扫污机（MMC）。此外，我们提出该结合MMC在三维器官皮肤共培养产生，从而降低了培养时间，同时保持结构的成熟的协议。
Abstract
胶原的糖蛋白家族代表在人体内的主要结构蛋白，并且是在现代组织工程中使用的生物材料的重要组成部分。一个技术瓶颈是胶原的体外沉积，因为它是相当慢，导致结缔组织和随后的组织凝聚力亚最佳的形成，特别是在皮肤的模型。在这里，我们描述了包括加入差分大小蔗糖共聚物的皮肤培养物，以产生高分子拥挤（MMC），其导致胶原沉积的急剧增强的方法。具体地，真皮成纤维细胞相比，控制沉积下的MMC胶原I / IV / VII和纤连蛋白的一个显著量。
该协议还描述了一种方法，以decellularize拥挤的细胞层，暴露显著量细胞外基质（ECM）的该被保留的培养表面上由immunocy证明组化。使用干扰反射显微镜研究总矩阵的质量和分布格局。变化的组成和形态的有趣的是，成纤维细胞，角质形成细胞和共培养生产细胞衍生的基质（CDM）的。 CDM可作为“生物支架”进行二次细胞接种，其中当前使用的涂层或支架的，典型地从异种动物来源的，是可以避免的，由此实现更临床相关的应用程序移动。
此外，该协议描述中一个三维器官皮肤共培养模型这是足以增强的ECM沉积于真皮 – 表皮交界处（DEJ），特别是，胶原VII，主要部件的浸没阶段MMC卡的应用锚固纤维。电子显微镜确认为与对照相比锚固在使用MMC的发展，培养原纤维的存在。这是因为显著锚原纤维系绳真皮表皮，因此，具有预成形的成熟DEJ可以以接枝稳定性和整体的伤口愈合方面受益皮肤移植接受者。此外，培养时间第5周冷凝至3周，以获得成熟的构建物，使用MMC时，降低了成本。
Introduction
皮肤形成通过防止水分流失和病原体进入的保护性屏障。它是由三大部分组成;基质丰富的真皮1，分层表皮2,3,4在它的上面，并在5,6之间的真皮表皮交界处。真皮是由主要的胶原蛋白和弹力纤维，并用稀疏的7成纤维细胞填充。与此相反，细胞丰富表皮是由角化细胞的多层组成。最内层的角质形成细胞是增殖性并提供新的基底细胞，其更新和替换末端分化的角质形成细胞是不断移动到皮肤的最外层，并失去了核和细胞质材料，导致其经历角化层脱屑。
真皮 – 表皮交界，特定类型的基底膜，是互连矩阵分子，其tethe组成的复杂结构RS表皮到真皮。真皮的胶原蛋白I的纤维交织胶原VII锚定它们锚定到IV型胶原丰富致密板纤维。反过来锚固丝（5层粘连蛋白，胶原十七和整合）与基底角朊细胞的半桥粒连接致密板。基底角质形成细胞（基底层）有能力的增殖和更新，以及分化和分层，以形成基底层层;棘层，颗粒层，以及最后的角质层，它代表了皮肤的接触面与环境。从基底层的角化层的途中 ，角化切换角蛋白的表达模式和最后经历细胞凋亡和包住自己在角化信封，共价由转谷氨酰胺酶活性的交联的特定蛋白质的船体。
重建皮肤及其体外层，包括的复杂结构真皮 – 表皮连接和真皮细胞外基质，并以模拟角化过程中，具有长好奇科学家和生物工程作为一个具有挑战性的任务。已经出现了皮肤组织工程显著的进步，例如，皮肤细胞从病人活检成功提取和皮肤器官培养物使用源自患者的皮肤细胞8的生成。然而，未解决的问题保持由皮肤细胞有关的细胞外基质蛋白的分泌差本身并导致次优的皮肤模型。此外，所需要的时间，以产生使用当前的协议三维器官皮肤共培养四到八周之间变化，这可能潜在地与大分子沟渠扫污机的掺入缩短一个时间段。减少培养时间可以节省试剂成本，减少细胞衰老的发生率，降低了患者的等待时间应该产品在临床中使用。
t“的>高分子拥挤（MMC）涉及引入特定大分子到培养基中，以产生排除体积效应。这些影响酶促反应速率，包括原胶原的蛋白水解切割这是标准的水性培养条件9-13下缓慢。在MMC中，酶反应被加速，而不相比不拥挤的对照10增加试剂导致14,15的量，以原胶原裂解，I型胶原在拥挤的培养物分子的量增加的情况下，由于原胶原胶原的转换允许胶原蛋白的形成组件，具有MMC 48小时培养成纤维细胞产生显著更多的胶原蛋白我相比，监测四个星期11,16,17拥挤的成纤维细胞培养的。除了对影响ECM的形成，稳定和重塑酶活性，MMC还效应已显示直接提升和调制胶原纤维的形成18,19。我们在这里提出的方法，以增强由皮肤细胞的胞外基质（ECM）的生产，特别是真皮成纤维细胞和表皮角质形成细胞。此外，我们表明，在单层培养下的MMC中产生的富集的ECM可脱细胞并用作纯细胞衍生的基质（CDM）的。
我们使用非传统的方式来可视化和充分理解皮肤细胞培养MMC沉积在细胞外基质。干扰反射镜通常用于研究细胞 – 基质相互作用或细胞至玻璃的接触点。这种技术是在我们的系统中使用，以查看基质的沉积在玻璃表面上的总金额。干扰反射显微镜加上荧光免疫染色，以获得在细胞外基质组合物和图案方面最量的信息，在存在和不存在的MMC。
Organotypic皮共培养是在皮肤上的体外在三维上下文建模经典方法。而二维共培养可能提供显著的信息，它转换该数据和应用回体内环境，这是固有的三维结构时是有限的。皮肤角质形成细胞，尤其是偏振和含有心尖和基底段这对于稳态和细胞粘附是必不可少的。此外，典型的基底层蛋白在角化细胞的基底层之上，如角蛋白1，角蛋白10和聚丝蛋白的表达仅在分层和角化细胞的终末分化的过程中存在。由于终末分化是典型的单层培养几乎不存在，基底层蛋白的表达通常不会在这个培养体系来实现的。因此，器官型培养开始淹没在培养基中，但随后提升到空气 – 液体界面驱动keratinocytË分化。这导致分层的标记，甚至角化和表皮生理学的总体上更好的反射的表达。虽然其他团体先前生成的器官皮肤共培养成功，建立了功能真皮表皮交界地带一直是一个问题。这里，我们提出用于培养器官皮肤共培养具有增强的基底膜，在稠时间帧，在不损害这些构建体的成熟的新方法。这将提供皮肤类似物在体外模型，皮肤生物学研究和筛选试验的品种。
Protocol
Representative Results
Discussion
Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time wh…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.
Materials
| Ascorbic acid | Wako | 013-12061 | Cell culture media additive |
| Cell culture inserts (6-well format) | Greiner Bio-One | 657610 | Organotypic cultures |
| Citrate solution | Dako | S2369 | DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10) |
| Collagen (rat tail) | Corning | 354236 | Organotypic cultures |
| CnT-57 | CELLnTEC | CnT-57 | Cell culture media |
| DAB Substrate + chromogen kit | Dako | K3468 | Histology |
| Deep-well plate (6-well format) | Corning | 355467 | Organotypic cultures |
| ECL detection reagent | GE Healthcare Life Sciences | RPN2106 | Amersham ECL Western Blotting Detection Reagent |
| Electron microscope (TEM) | JEOL | JEM-1010 (100kV) | Transmission electron microscopy |
| Fibroblast media (FM) | High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin | ||
| Ficoll PM70 | GE Healthcare Life Sciences | 17-0310-05 | Macromolecular crowder |
| Ficoll PM400 | GE Healthcare Life Sciences | 17-0300-05 | Macromolecular crowder |
| Keratinocyte serum-free media (KSFM) | Life Technologies | 17005-042 | Cell culture media |
| Lysis buffer | ThermoFisher Scientific | 89900 | Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer. |
| Microscope | Zeiss | LSM510 | Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag). |
| Mounting media (Hydromount) | National Diagnostics | HS-106 | Fluorescent staining |
| Mounting media (Cytoseal) | ThermoFisher Scientific | 8310-16 | Histology (HRP) |
| OCT compound (Tissue Tek) | Sakura | 4583 | Embedding for cryotomy |
| Penicillin-streptomycin antibiotics | Sigma Aldrich | A5955 | Cell culture media additive |
| Primary antibodies | |||
| Anti-Collagen I antibody | Abcam | #ab6308 | |
| Anti-Collagen Type IV | Novocastra | #NCL-COLL-IV | |
| Anti-collagen VII | LH7.2 (in house) | ||
| Anti-Fibronectin antibody | Abcam | #ab2413 | |
| Reducing agent | ThermoFisher Scientific | NP0009 | Western blot |
| Sample buffer | ThermoFisher Scientific | NP0008 | Western blot |
| Secondary antibodies (Immunostaining) | |||
| 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) | Sigma Aldrich | #D9542 | |
| AlexaFluor 594 goat-anti-rabbit | ThermoFisher Scientific | #A-11037 | |
| AlexaFluor 594 goat-anti-mouse | ThermoFisher Scientific | #A-11005 | |
| AlexaFluor 488 chicken-anti-rabbit | ThermoFisher Scientific | #A-21441 | |
| AlexaFluor 488 goat-anti-mouse | ThermoFisher Scientific | #A-11001 | |
| Secondary antibodies (HRP) | Dako | Envision + System – HRP Labelled Polymer Anti-Rabbit and Anti-Mouse | undiluted |
| Sodium deoxycholate | Prodotti Chimicie Alimentari | Decellularization | |
| Stratification media | |||
| Dulbecco's Modified Eagle's Medium | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| Ham’s F12 | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 10% fetal bovine serum | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 100U/mL penicillin and 100U/mL streptomycin | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 0.4mg/mL hydrocortisone | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 5mg/mL insulin | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 1.8·10-4 M adenine | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 5mg/mL transferrin | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| 2·10- 11 M triiodothyronine | Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert. | ||
| Trypsin | Biopolis Shared Facilities | 0.125% Trypsin/Versene pH 7.0 + 0.3 | Used to trypsinize cells |
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