Janani G, Kumar Shivanshi, Mandal Biman B
Biomaterial and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India.
Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India.
ACS Biomater Sci Eng. 2019 Nov 11;5(11):6137-6151. doi: 10.1021/acsbiomaterials.9b01162. Epub 2019 Oct 7.
Urokinase plasminogen activator (uPA) has been extensively used as a thrombolytic drug in cases of myocardial infarction, thromboembolism, and ischemic brain stroke. Media optimization and high-density perfusion culture are the decisive factors that facilitate enhanced urokinase production in a conditioned medium. In this study, we have aimed for a high-density perfusion culture of HT1080, a human fibrosarcoma cell line, by formulating optimal media for enhanced urokinase productivity. Four scaffold variants were fabricated from silk fibroin and microfibers of (BM) and (AA) and physico-chemically characterized. Field emission scanning electron microscopy studies revealed a heterogeneous distribution of pores with interconnected networks supporting cell infiltration, attachment, and long-term viability. AA-based fiber-reinforced scaffolds (ASAF) demonstrated superior mechanical strength, integral stability, and increased cell proliferation as compared to pure silk scaffolds. Media formulation was accomplished by limiting serum concentration (2% FBS) and supplementing with 20 μg/mL arginine and 20 ng/mL TGF-β1 to retain the stationary phase of cells and augment the urokinase production. A perfusion bioreactor culture of HT1080-laden scaffolds in the presence of formulated media was performed for improving the production of urokinase, with a maximum activity of 432 U/L. Also, gene expression analysis revealed that the individual silk scaffolds have different effects on regulating the expression of plasminogen activator urokinase and plasminogen activator urokinase receptor. In brief, our results suggest that a perfusion bioreactor culture of HT1080-laden ASAF scaffolds in formulated media promotes an increased urokinase production, such that it can be further used as a novel 3D matrix platform for industrial production of the lifesaving uPA drug.
尿激酶型纤溶酶原激活剂（uPA）已被广泛用作治疗心肌梗死、血栓栓塞和缺血性脑卒中等病症的溶栓药物。培养基优化和高密度灌注培养是促进条件培养基中尿激酶产量提高的决定性因素。在本研究中，我们旨在通过配制优化培养基以提高尿激酶生产力，对人纤维肉瘤细胞系HT1080进行高密度灌注培养。用丝素蛋白以及（BM）和（AA）的微纤维制备了四种支架变体，并对其进行了物理化学表征。场发射扫描电子显微镜研究显示，孔隙呈异质分布，具有相互连接的网络，支持细胞浸润、附着和长期存活。与纯丝支架相比，基于AA的纤维增强支架（ASAF）表现出卓越的机械强度、整体稳定性和更高的细胞增殖能力。通过限制血清浓度（2%胎牛血清）并补充20μg/mL精氨酸和20ng/mL转化生长因子-β1来完成培养基配方，以维持细胞的静止期并增加尿激酶的产量。在配制培养基存在的情况下，对负载HT1080的支架进行灌注生物反应器培养，以提高尿激酶的产量，最大活性为432U/L。此外，基因表达分析表明，单个丝支架对调节纤溶酶原激活剂尿激酶和纤溶酶原激活剂尿激酶受体的表达有不同影响。简而言之，我们的结果表明，在配制培养基中对负载HT1080的ASAF支架进行灌注生物反应器培养可促进尿激酶产量增加，因此它可进一步用作生产救命药物uPA的新型3D基质平台。
