可调水凝胶由肺细胞外基质的三维细胞培养
Summary
这是创建从肺的细胞外基质的三维细胞培养物的支架的方法。完好肺被加工成可支持细胞以三维生长的水凝胶。
Abstract
在这里，我们提出了体外肺细胞培养建立的多组分细胞培养水凝胶的方法。用健康途中从猪，大鼠，或小鼠集团肺组织开始，组织灌注并在随后的化学洗涤剂浸没以除去细胞碎片。处理之前和之后的组织的组织学比较证实除去双链DNA和α-半乳糖苷酶染色的95％以上表明大部分细胞碎片除去。脱细胞后，组织是冻干然后cryomilled成粉末。基质粉末在酸性胃蛋白酶消化溶液消化48小时，然后中和形成的预凝胶溶液中。该预凝胶溶液的凝胶化可通过温育在37℃下被诱导，并且可以立即使用以下中和或储存在4℃下长达两周。涂层可以用在未处理板c中的预凝胶溶液来形成埃尔附件。细胞可以悬浮在之前自组装，实现了3D培养的预凝胶，电镀形成的凝胶从该细胞可以通过在支架迁移，或镀在涂层的表面上。改变的策略提出可影响凝胶化温度，强度，或蛋白质片段的大小。以外的水凝胶的形成，所述水凝胶的刚度可使用京尼平可以增加。
Introduction
Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6.
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research.
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.
Protocol
Representative Results
Discussion
之一的生物学的积分方面是自组织分子成执行特定任务层次结构。 13在实验室中，自组装取决于许多因素，如盐浓度，pH和消化时间。如图所示，自组织的水凝胶形式时溶解蛋白质返回到生理温度。形成的水凝胶是能够促进在体外的细胞附着和增殖的。
从ECM生物物理信号的细胞应答不能使用的聚合物进行研究。生物物理信号组织特异性细胞应答不能使用其他组?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们要感谢史密斯菲尔德农场捐赠了完整的猪肺组织。我们也想感谢胡扬博士，唐克里斯蒂娜博士和VCU整形外科部允许我们使用他们的设备。水凝胶和组织样品进行SEM准备在解剖学和神经生物学显微镜基金的VCU部支持的，一部分是由NIH-NINDS中心核心格兰特5 P30 NS047463的资金，部分通过资金形式NIH-NCI癌症中心支援津贴P30 CA016059。在VCU纳米技术核心表征基金（NCC）的SEM图像。这项工作是由美国国家科学基金会，CMMI 1351162资助。
Materials
| Triton X-100 | Fisher Scientific | BP151-100 | Use in fume hood with eye protection and gloves. |
| Sodium Deoxycholate | Sigma-Aldrich | D6750-100g | Use with eye protection and gloves. |
| Magnesium Sulfate | Sigma-Aldrich | M7506-500g | None |
| Calcium Chloride | Sigma-Aldrich | C1016-500g | None |
| DNase | Sigma-Aldrich | D5025-150KU | None |
| HCl | Sigma-Aldrich | 258148-500ML | Use with eye protection and gloves. |
| Pepsin | Sigma-Aldrich | P6887-5G | Use in fume hood with eye protection and gloves. |
| Sodium Hydroxide | Fisher Scientific | BP359-500 | Use with eye protection and gloves. |
| Genipin | Wako Chemicals | 078-03021 | Use in fume hood with eye protection and gloves. |
| PBS 10x | Quality Biological | 119-069-151 | None |
| PBS | VWR | 45000-448 | None |
| Filter Paper | Whatman | 8519 | N/A |
| Hand pump | Fisher Scientific | 10-239-1 | N/A |
| Graduate Beaker | VitLab | 445941 | N/A |
| Cryomill | SPEX | 6700 | Use cryogloves and eye protection. |
| Lyophilizer | FTS FlexiDry | Use gloves. | |
| Rheometer | Discovery | HR-2 | Use gloves and eye protection. |
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