一个培养人关节软骨细胞的三维系统在滑液
Summary
一个3D系统培养人关节软骨在滑液的高层次描述。滑液反映关节软骨的最自然的微环境，并可以很容易地获得和存储。因此，该系统可用于软骨再生研究和筛选疗法治疗关节炎。
Abstract
软骨破坏的骨性关节炎的病理特征是中央在美国的事业，一个残疾的领先。在成人软骨没有非常有效的再生，在体内，因此，骨关节炎导致不可逆转的软骨丢失及伴有慢性疼痛和行动不便 1,2 。软骨组织工程提供了有希望的潜在的再生和恢复组织功能。这种技术通常涉及到播种天然或人工合成的支架和培养软骨细胞产生的三维构造过了一段时间，在一个平衡的介质中的工程目标的生物化学和生物力学成熟的组织 ，可以在体内3-6成缺损部位移植。软骨组织工程的成功实现，为软骨细胞的生长和外基质沉积的最佳条件是必不可少的。
在本机的关节腔，在北极的软骨ular表面的骨沐浴在滑液。这清晰和粘性流体股骨头关节软骨提供营养，并包含 7,8软骨细胞代谢的重要生长因子，细胞因子和酶。此外，滑液有利于低摩擦的软骨表面之间的运动，主要是通过分泌两个重要组成部分， 透明质酸和 lubricin 9 10。与此相反，组织工程化软骨是最常见的人工培养基培养。虽然这些媒体很可能能够为研究软骨细胞代谢提供更明确的条件，滑液最准确地反映关节软骨细胞，其中居住的自然环境
事实上，滑液容易获取和存储的优势，往往可以定期身体补充。一些团体补充滑液在培养基中的人类，牛，兔和狗的彗星hondrocytes，但主要用于滑液（低于20％）11-25只低的水平。虽然鸡，马和人类的软骨细胞已在滑液的百分比较高的中等培养，这些文化系统二维26-28。在这里，我们目前在3D系统培养人关节软骨细胞的方法，我们与滑液的高（可达100％）的比例超过21天的期限。这样做，我们克服了滑液的高粘度提出的一个主要障碍。该系统提供学习人体软骨在滑液在3D设置，可进一步与其他两个重要因素（氧张力和机械负荷）29,30，软骨构成的自然环境，模仿自然的环境相结合的可能性软骨生长。此外，该系统也可用于化验滑液对软骨细胞的活动，并为发展中国家提供了一个平台软骨再生技术和关节炎的治疗方案。
Protocol
一个培养人关节软骨细胞的三维系统在滑液在这项工作中，我们人体关节软骨细胞封装在使用修改后的制造商建议的封装协议（ 龙沙 ，和31）的海藻酸钠珠。使用这些三维结构，我们已经发展为培养细胞中含有人体滑液的不同比例的中等文化制度和评估软骨基因表达的这些三维结构。 1。准备人关节软骨细胞三维（3D）封装（HAC） 解冻在3…
Discussion
在这份报告中，我们开发了一个人关节软骨细胞培养的方法，允许在一个中型的3D环境，它包含人体滑液的高浓度。滑液的主要成分构成的关节腔，关节软骨细胞所在的自然环境之一。然而，滑液的粘度一直是立体培养软骨细胞的长期的重大挑战。要克服的挑战，保持甚至养分分布在三维构造在粘稠的环境，并防止聚集，我们将不断的运动，温和的摇摆下的软骨结构。为了提高结构的软骨结构完?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们想感谢提供帮助滑液存储和离心罗宾奈（塔夫茨医学中心），智也内村和Dana凯恩斯（塔夫茨大学）。这项工作是由NIH（1R01AR059106 – 01A1为LZ）
Materials
Table of specific reagents and equipment:
| Name of the Reagent | Company | Catalogue Number | Comments |
|---|---|---|---|
| Alginate (Alginic Acid sodium salt) | Sigma | A2158-250G | 2.4% solution stored at 40°C |
| Calcium Chloride Dihydrate, Granular | J.T. Baker | A19339 | |
| Chondrogenic Growth media | Lonza | CC-3156 (base media) | |
| CC-4409 (supplement) | |||
| Chondrogenic Differentiation Media | Lonza | CC-3226 (base media) | |
| CC-4408 (supplement) | |||
| Human articular chondrocytes | Lonza | CC-2550 | |
| Dapi (4′,6-Diamidino-2-phenylindole dihydrochloride) | Sigma-Aldrich | D9542 | |
| RNeasy mini kit (for RNA extraction) | Qiagen | 74104 | |
| PCR reagents: SYBR-green | Quanta | 95053-500 | |
| 12 ml syringe | Tyco-Kendall-Monoject | 512852 | |
| 22-Gague Hypodermic Needle | Tyco-Kendall-Monoject | 8881 | |
| Microscope | Olympus | IX71 | |
| Platform rocker | Thermoscientific thermolyne | Vari-mix | |
| Primers sequences | |||
| Collagen IIa-forward | 5′-TTC ATC CCA CCC TCT CAC AGT-3′ | ||
| Collagen IIa-reverse | 5′-CCTCTGCCTTGACCCGAA-3′ | ||
| MMP13-forward | 5′-TGT GCC CTT CTT CAC ACA GAC ACT-3′ | ||
| MMP13-reverse | 5′-GAG AGC AGA CTT TGA GTC ATT GCC-3′ | ||
| Caspase 3-forward | 5′-TCA TTA TTC AGG CCT GCC GTG GTA-3′ | ||
| Caspase 3-reverse | 5′-TGG ATG AAC CAG GAG CCA TCC TTT -3′ |
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Tags
3D SystemCulturingHuman Articular ChondrocytesSynovial FluidCartilage DestructionOsteoarthritisDisabilityCartilage RegenerationChronic PainImmobilityScaffoldsBalanced MediumIn Vivo TransplantationChondrocyte GrowthMatrix DepositionNative Joint CavitySynovial FluidAvascular Articular CartilageGrowth FactorsCytokinesEnzymesChondrocyte MetabolismHyaluronanLubricinArtificial Media
Cite This Article
Brand, J. A., McAlindon, T. E., Zeng, L. A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid. J. Vis. Exp. (59), e3587, doi:10.3791/3587 (2012).