Bar Assaf, Kryukov Olga, Etzion Sharon, Cohen Smadar
The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Int J Bioprint. 2023 Jan 17;9(2):670. doi: 10.18063/ijb.v9i2.670. eCollection 2023.
In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration .
近年来，基于挤出的三维（3D）生物打印技术因其能够利用水凝胶基生物墨水组装复杂结构而被用于构建心脏补片（CP）。然而，由于生物墨水中的细胞受到剪切力作用，诱导细胞凋亡，此类心脏补片中的细胞活力较低。在此，我们研究了在生物墨水中加入经工程改造可持续递送细胞存活因子miR-199a-3p的细胞外囊泡（EVs）是否会提高心脏补片内的细胞活力。从THP-1衍生的活化巨噬细胞（MΦ）中分离出细胞外囊泡，并通过纳米颗粒跟踪分析（NTA）、低温电子显微镜（cryo-TEM）和蛋白质免疫印迹分析进行表征。在优化施加电压和脉冲后，通过电穿孔将miR-199a-3p模拟物加载到细胞外囊泡中。使用增殖标志物ki67和Aurora B激酶的免疫染色在新生大鼠心肌细胞（NRCM）单层中评估工程化细胞外囊泡的功能。为了研究工程化细胞外囊泡对3D生物打印心脏补片活力的影响，将细胞外囊泡添加到由藻酸盐-RGD、明胶和NRCM组成的生物墨水中。5天后评估3D生物打印心脏补片的代谢活性和凋亡相关的活化半胱天冬酶3的表达水平。发现电穿孔（850 V，5个脉冲）最适合miR加载；与简单孵育相比，细胞外囊泡中miR-199a-3p水平增加了五倍，加载效率为21.0%。在这些条件下，细胞外囊泡的大小和完整性得以维持。验证了NRCM对工程化细胞外囊泡的细胞摄取，因为24小时后58%的cTnT细胞内化了细胞外囊泡。与对照组相比，工程化细胞外囊泡诱导心肌细胞增殖，使cTnT细胞的细胞周期重新进入比例增加30%（Ki67），中体+细胞比例增加两倍（Aurora B）。与不含细胞外囊泡的生物墨水相比，在生物墨水中加入工程化细胞外囊泡后心脏补片的细胞活力提高了三倍。细胞外囊泡的延长效应很明显，因为5天后心脏补片的代谢活性升高，与不含细胞外囊泡的心脏补片相比，凋亡细胞更少。向生物墨水中添加负载miR-199a-3p的细胞外囊泡可提高3D打印心脏补片的活力，并有望促进它们的整合。
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