3D-printing Enabled Micro-assembly of Microfluidic Electroporation System for 3D Tissue Engineering

Abstract

Electro-transfection is an essential workhorse tool for regulating cellular responses and engineering cellular materials in tissue engineering. However, existing approaches, including microfluidic platforms and bench top methods, are only able to study monolayer cell suspensions in vitro, and are incapable of clinical translation within in vivo tissue microenvironment.Knowledge regarding the three-dimensional (3D) electric field distribution and mass transport in a tissue microenvironment is lacking. However, building a 3D electro-transfection system that is compatible with 3D cell culture for mimicking in vivo tissue microenvironment is challenging, due to the substantial difficulties in control of 3D electric field distribution as well as the cellular growth. To address such challenges, we introduce a novel 3D micro-assembling strategy assisted by 3D printing, which enables the molding of 3D microstructures as LEGO® parts from 3D-printed molds. The molded PDMS LEGO  bricks are then assembled into a 3D-cell culture chamber interconnected with vertical and horizontal perfusion microchannels as a 3D channel network. Such 3D perfusion microchannel network is unattainable by direct 3D printing or other microfabrication approaches, which can facilitate the high-efficient exchange of nutrition and waste for 3D cell growth.Four flat electrodes are mounted into the 3D culture chamber via a 3D-printed holder and controlled by a programmable power sequencer for multi-directional electric frequency scanning (3D -electro-transfection). This multi-directional scanning not only can create transient pores all over the cell membrane, but also can generate local oscillation for enhancing mass transport and improving cell transfection efficiency. As a proof-of-concept, we electro-delivered pAcGFP1-C1 vector to 3D cultured HeLa cells within peptide hydrogel scaffolding. The expressed GFP level from transfected HeLa cells reflects the transfection efficiency. We found two key parameters including electric field strength and plasmid concentration playing more important roles than manipulating pulse duration and duty cycles. The results showed an effective transfection efficiency of ~15% with ~85% cell viability, which is a 3-fold increase compared to the conventional benchtop 3D cell transfection. This 3D -electrotransfection system was further used for genetically editing 3D-cultured Hek-293 cells via direct delivery of CRISPR/Cas9 plasmid which showed successful transfection with GFP expressed in the cytoplasm as the reporter. The 3D-printing enabled micro-assembly allows facile creation of novel 3D culture system for electro-transfection which can be employed for versatile gene delivery and cellular engineering, as well as building in-vivo like tissue models for fundamentally studying cellular regulatory mechanisms. the orientation of cells to the electric fields and so on. [16][17] Thus, the clinical in vivo gene delivery faces tremendous problems. 3,18 Although the in vitro cellular spheroid model is often applied to study t...

Emerging microfluidic chips on medical application

“…，⼜称芯⽚实验室(Lab-on-a Chip)或者⽣物芯⽚。微流控技术 [2] 由微 机电加⼯系统(Micro-Electro-Mechanical System，MEMS)发展⽽来，它是⼀种在微⽶级微管 [3] 中精确操纵 [4] 微量流体的技术⼿段，具有将⽣物、化学等实验室微缩到⼀个⼏平⽅厘⽶芯⽚中的基本功能(样品的制备、 分离、反应、检测等)。随着材料科学的发展，作为微流控芯⽚载体的材料也层出不穷，从硅、玻璃到纸基 [5] 、 ⽔凝胶 [6] 以及各类聚合物和纳⽶材料 [7,8] 。与此同时，微流控芯⽚的制备技术也蓬勃发展如丝⽹印刷，喷墨打 印、3D 打印 [9]， [10] 等，⼀些⾼精尖的加⼯技术如⻜秒激光加⼯技术、双光⼦ 3D 打印技术也为⾼精密度的微流 控芯⽚的制作提供了更多可能性。 微流控芯⽚有着微型化、⾼灵敏度、⾼集成、⾼通量、反应快、检测时间短等技术优势，在⽣物医学研 究，合成分析 [11] ，司法鉴定等众多领域有着⼴泛的应⽤(可穿戴微流控设备 [12] 、体外医疗诊断 [13] 、仿⽣⽪肤 [14] 组织器官 [15] ，⽣化 [16] 与环境分析、单细胞分析 [17] 、核酸分析、药物筛选递送 [18] )。迄今为⽌，微型化、集成 化和智能化已经成为现代科技⼿段的⼀个重要趋势，在这样的⼤环境下微流控芯⽚的发展⼗分迅速，它的分 类⽅法丰富多样，可根据不同的分析检测⽅法可以将微流控芯⽚分为电化学检测法 [19] 、光谱分析法(核磁共 振 [20] 、化学发光分析法 [21] )、⾮标记检测法 [22] ，分离富集 [8] 等。 微流控芯⽚是医学领域新⼀代床旁诊断(Point of care testing,POCT) [23,24] 主流技术。POCT 可直接在被检 者身边提供快捷有效的⽣化指标，现场指导⽤药，使检测、诊断、治疗成为⼀个连续过程，对于疾病的早期 发现和治疗具有突破性的意义。基于微流控芯⽚技术的 POCT 仪器发展趋势是⼩型化、操作便捷，直接输⼊ 体液样本 [25] ，即可迅速得到诊断结果 [26] ，由医⽣指导⽤药。⽬前，市场上有多种即时诊断⽅法，仅有简单地 流动测试⼯作没有流体管理技术，⽽当测试复杂性增加时，微流控技术是⼀⻔不可或缺的技术。在治疗肿瘤 癌症的医疗领域⽅⾯，微流控技术同样⼤放异彩，如何使药物特异性杀死肿瘤细胞⽽不伤害正常细胞，这⼀ 直是个业界难题，代尔夫特理⼯⼤学 Jan H. van Esch 的课题组 [27] 将微流控设备中搭载⼀种具有双重功能的新 ⽉形微凝胶，研究发现这些微凝胶对肺癌细胞有⾼亲和、⾼选择性，可以达到肺癌化疗的效果并且减轻副作 ⽤对⼈体的伤害。微流控细胞/组织/器官操作芯⽚是哺乳动物细胞以及微环境操作最重要的技术平台，渴望 应⽤于代替各类动物模型，模拟⼈体内环境(⼈体、组织芯⽚ [28] )，⽤于药物筛查递送，研究药物毒理和药 理作⽤，⼈体耐药性 [29] [30] . (b)基于激光打印技术制备 3D 纳⽶等离⼦体的示意图 [31] .…”

Study on the Preparation and Test of Hydrodynamic Focusing Microfluidic Chip Fabricated by Liquid Crystal Display Mask Photo-Curing Method

“…For example, the development cost of mold for micro injection molding or hot embossing usually ranges from US $10000 to US $100000 and the development cycle usually takes one to several months, while the LIGA technology relies on expensive equipment and complex operation process, which is especially unfriendly for the fabrication of microfluidic chips in small and medium quantities [7], [8]. Additive manufacturing technology [9], [10] has the advantages of cost effective and flexibility, which is an ideal method for microfluidic chip preparation, especially in the case of small fabrication quantity. Some miniaturized microfluidic chips have been fabricated by additive manufacturing technology, such as functional structure, microvascular networks and scaffolds.…”