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首页> 外文期刊>RSC Advances >Preservation of biomacromolecular composition and ultrastructure of a decellularized cornea using a perfusion bioreactor
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Preservation of biomacromolecular composition and ultrastructure of a decellularized cornea using a perfusion bioreactor

机译：使用灌注生物反应器保存生物分子组合物和脱细胞角膜的超微结构

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An attempt has been made to formulate a new method of corneal decellularization using a direct perfusion system through the cornea. Here, we compared the direct perfusion method to some commonly used decellularization strategies including chemical methods; non-ionic detergent TRITON X-100 and ionic detergent; sodium dodecyl sulphate (SDS) based orbital shaker method and physical methods of liquid nitrogen and freeze-thaw to decellularize a goat cornea. Histochemical evaluation and biochemical estimation highlighted that liquid nitrogen, freeze-thaw and TRITON-based orbital shaker methods resulted in incomplete removal of resident cells from the native cornea. On the contrary, direct perfusion of the cornea using TRITON and SDS completely removed all the cells from the cornea while preserving the ultrastructure of the extracellular matrix at a steady flow rate of 10 mu l min (1). Raman and ATR-FTIR spectra indicated the relative abundance of the alpha-helical conformation of collagen type I in the perfused cornea while a beta-sheet conformation was predominantly observed in other treatment methods. FACS was used to determine the cell death modality in different methods of decellularization. In the direct perfusion system, 13.1% higher apoptotic cells, the preferred route of cell death, were observed in the cornea compared to orbital shaker-based methods. Further, feasibility studies conducted for 7 days to investigate the recellularization potential of the perfused decellularized matrix demonstrated a well attached viable population of seeded corneal stromal cells. In summary, we demonstrated that the direct perfusion method for decellularization of a cornea using 0.1% TRITON detergent at 10 mu l min(-1) is an optimal strategy for efficiently removing the resident corneal cells while maintaining the ultrastructure of the corneal matrix intact and therefore could serve as an excellent source for corneal transplantation.
机译：已经尝试使用直接灌注系统通过角膜制定一种新的角膜脱细胞化方法。在这里，我们将直接灌注方法与一些常用的脱细胞化策略进行比较;非离子洗涤剂Triton X-100和离子洗涤剂;十二烷基硫酸钠（SDS）基于氧化物振荡器的方法和液氮物理方法，脱脂 - 脱羊角切口。组织化学评估和生化估计突出显示液氮，冻融和基于Triton的轨道振动器方法导致来自天然角膜的常规细胞不完全去除。相反，使用Triton和SDS直接灌注角膜，并且SDS完全从角膜中除去所有细胞，同时以10μm1min（1）的稳定流速保持细胞外基质的超微结构。拉曼和ATR-FTIR光谱表明，在灌注角膜中胶原型I的α-螺旋构象的相对丰度，而在其他治疗方法中主要观察到β-片状构象。 FACS用于确定不同脱细胞化方法中的细胞死亡方式。在直接灌注系统中，与基于轨道振荡器的方法相比，在角膜中观察到凋亡细胞，凋亡细胞，细胞死亡的优选途径。此外，进行7天进行的可行性研究探讨灌注的脱细胞化基质的转速潜力，显示出良好的接种角膜基质细胞群。总之，我们证明，使用0.1％Triton洗涤剂在10μlmin（-1）的基角膜脱果优化的直接灌注方法是有效地除去常驻角膜细胞的最佳策略，同时保持角膜基质的超微结构完好无损因此可以作为角膜移植的优秀来源。

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  • 来源
    《RSC Advances》 |2016年第3期|共16页
  • 作者

    Nara Sharda; Chameettachal Shibu; Midha Swati; Murab Sumit; Ghosh Sourabh;

  • 作者单位

    Indian Inst Technol Dept Text Technol New Delhi 110016 India;

    Indian Inst Technol Dept Text Technol New Delhi 110016 India;

    Indian Inst Technol Dept Text Technol New Delhi 110016 India;

    Indian Inst Technol Dept Text Technol New Delhi 110016 India;

    Indian Inst Technol Dept Text Technol New Delhi 110016 India;

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