Perfusion decellularization of human and porcine lungs: Bringing the matrix to clinical scale
Perfusion decellularization of human and porcine lungs: Bringing the matrix to clinical scale
复制标题
DOI：
10.1016/j.healun.2013.10.030
复制
发表时间：
2014-03-01
影响因子：
8.9
通讯作者：
Ott, Harald C.
中科院分区：
文献类型：
--
作者：
Gilpin, Sarah Elizabeth;Guyette, Jacques P.;Ott, Harald C.
背景：器官工程是终末期器官衰竭同种异体移植的理论替代方案。全器官支架可以通过经由天然脉管系统的去污剂灌注来产生，从而产生适合于用选定的细胞类型再细胞化的无细胞基质。我们的目的是扩大这一进程，产生生物相容性支架的临床相关scale.METHODS：大鼠，猪，和人的肺脱细胞洗涤剂灌注在恒定的压力。胶原蛋白，弹性蛋白，和糖胺聚糖含量的支架通过比色测定进行定量。采用微毛细管液相色谱串联质谱法进行蛋白质组学分析。将细胞外基质（ECM）切片与人脐静脉内皮细胞（HUVEC）、小气道上皮细胞（SAEC）或肺泡上皮细胞（PAEC）一起培养，并通过延时活细胞显微镜和MTT（3[4，5-二甲基噻唑-2-基]-2，5-二苯基四唑溴化物）测定进行评价。结果：采用十二烷基硫酸钠（SDS）、脱氧胆酸钠（SDC）、3-[（3-胆酰胺丙基）二甲氨基]-1-丙磺酸钠（CHAPS）对大鼠肺进行脱细胞处理。所得的支架显示在SDS脱细胞肺中DNA的损失相当，但ECM组分的保留最大。猪（n = 10）和人（n = 7）肺需要增加SDS浓度、灌注压和时间，以实现脱细胞化（通过DNA损失确定），同时保留完整的基质组成和肺结构。人脱细胞肺的蛋白质组学分析进一步证实了ECM保存。再细胞化实验证实支架生物相容性培养时，与成熟细胞表型和支架的完整性仿生culture.CONCLUSIONS：SDS为基础的灌注脱细胞可以应用于整个猪和人的肺，以产生生物相容性器官支架保存ECM组合物和架构。(C)2014年国际心肺移植学会。All rights reserved.
BACKGROUND: Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale.METHODS: Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs.RESULTS: Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture.CONCLUSIONS: SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture. (C) 2014 International Society for Heart and Lung Transplantation. All rights reserved.