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微流控生物打印异质3D组织结构

具有特定生物和机械性能的3D功能性组织结构在再生医学和组织工程领域相当重要。但是,高度组织化、功能性3D组织的发展仍面临一个未解决的挑战。在体外重现包含多种细胞和细胞外基质ECM)3D多级结构是一个相当不容易的任务。在这样的背景下,生物打印作为一种有潜力制备仿生3D组织结构的新技术已经出现。生物打印可以根据需要实现多细胞结构的精确定位。以往研究者们已经将这种技术应用于生物需要,例如打印软骨或多细胞结构,且在打印分辨率和细胞需求方面都取得了不错的结果。尽管在3D生物打印方面有着不错的进展,但对不同细胞的精确定位和转换以及寻求合适的生物材料仍是主要的挑战之一。

基于此,哈佛医学院的Ali Khademhosseini教授和罗马大学的Mariella Dentini教授合作团队提出了一种新的生物打印范例,他们将3D生物打印微流控芯片相结合,将打印分辨率和打印效率提升到一个新的水平,同时该方法还具有以下优势:

1)能够同时打印多种材料;

2)可制备具有活性的载细胞3D结构;

3)使用可以诱导细胞扩散和迁移的生物墨水;

4)能够在生物打印的支架上播种另一种细胞。首先,该团队选择使用海藻酸盐和GelMA混合的低粘度生物墨水进行生物打印,选用GelMA是因为其可促进细胞粘附和细胞迁移。低粘度的GelMA对细胞的封装效果更接近于天然基质,但低粘度GelMA很难作为生物墨水进行打印,在固化前易出现扩散现象。而海藻酸盐作为混合组分之一,可通过离子交联起到固定结构的作用。该生物打印技术的示意图如(图1 a-c)所示,先通过物理交联得到打印模板,再通过UV共价交联得到稳定的3D组织结构(图1 g-h)。

微流控3D组织结构

Figure 1. a) Schematic illustration of thelayer-by-layer-deposition-based bioprinting technique consisting of two independent crosslinking steps. b,c) The bioink contained GelMA (red dashed lines), alginate (green lines), photoinitiator, and cells in the inner needle of thecoaxial system. Simultaneously theCaCl2(blue dots) solution flows through the outer needle to induce the gelation of alginate chains. The construct wasthen UV crosslinked to solidify the GelMA prepolymer in the fi ber. d)Printability of different alginate concentrations and different  CaCl2 concentrations. GelMA concentration was kept constant (4.5% w/v). e) Viscosities of alginateand GelMA, and the combination of both at room temperature. f) The fiber diameter varied with different deposition speeds, calculated for a bioink flow rate of 5 μL min−1 . In theupper-right corner, photographs of the 1 mm thick constructs obtained for deposition speeds of 6 mm s−1 , 3 mm s−1 , and 1 mm s−1. g) Photograph of the final construct (30 layers). h) Top, lateral and 3D μCT reconstructions of the final bioprinted 3D structure.

为了证明制备多组分或多细胞组织结构的可行性,研究人员将一个微流控体系与生物打印相结合,可快速制备不同材料构成的仿生组织结构(图2 a)。将Y”型通道的微流控芯片与同轴针头链接,分别注入不同墨水,用红色和绿色荧光颗粒标记。通过选择不同的墨水打印,研究者们可实现连续打印不同材料,得到包含不同墨水在不同层上的异质结构(图2 b-e)。或者,当两种墨水同时挤出将得到多相纤维组成的3D结构(图2 d,f-i)。

“Y”型通道的微流控芯片

Figure 2. a) A microfluidic system was used to flow two separate bioinks containing red and green fluorescent beads that exited the device through a single extruder. Photograph (inset) of the coaxial needle system with a microfluidic chip with a “Y”-shaped channel. The schematic diagram and fluorescence microscopy image of cross-section view of 3D construct with b,c) alternate deposition, d,e) alternate/simultaneous deposition, and f–i) simultaneous deposition.

为证明细胞可在生物打印结构中迁移,该团队使用人脐静脉内皮细胞(HUVECs  打印10层结构,在培养10天后在共聚焦显微镜下观察。同时通过DAPIF-actinCD31染色也可观察到在培养10天后,细胞多位于3D支架外层,证明细胞在培养过程中发生迁移(图3 a-h)。最后,研究人员为证明该结构可作为体外支架用于心脏组织工程,将原代心肌细胞种于该结构上,培养2天后,发现心肌细胞在结构4个区域显示同步搏动。

微流控生物打印异质3D组织结构

Figure 3. a)Schematic of the encapsulated HUVECs migrating to outer regions of the bioprinted fibers after 10 d of culture. Confocal microscopy images with b)top view, c) cross-section view, and d) fiber junctions showing interconnected structures. Confocal microscopy images of a 1 mm thick construct that show e) transversal cross-section, f) longitudinal cross-section, g) outer surface of the complete construct. h) Top view of a single fiber immunostained for CD31(red) and DAPI (blue). i) Schematic illustration of the HUVEC structure beforeand after the cardiomyocyte seeding. j) 3D surface plot of a microscopy image of the 3D HUVEC-cardiac tissue construct after 2 d of cardiomyocytes culture.k) The beating rates of cardiomyocytes were monitored in four different zones(1, 2, 3, 4) of the construct which showed synchronous beating behavior. 

本研究由哈佛医学院的AliKhademhosseini教授和罗马大学的Mariella Dentini教授合作团队完成,于20151126日发表于Advanced Materials

论文信息:

CristinaColosi, Su Ryon Shin, Vijayan Manoharan, Solange Massa, Marco Costantini, Andrea Barbetta, Mehmet Remzi Dokmeci, Mariella Dentini,* and Ali Khademhosseini*. Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-ViscosityBioink. Adv.Mater. 2016, 28, 677–684.

论文链接:

https://www.ncbi.nlm.nih.gov/pubmed/26606883