具有特定生物和機(jī)械性能的3D功能性組織結(jié)構(gòu)在再生醫(yī)學(xué)和組織工程領(lǐng)域相當(dāng)重要。但是，高度組織化、功能性的3D組織的發(fā)展仍面臨一個(gè)未解決的挑戰(zhàn)。在體外重現(xiàn)包含多種細(xì)胞和細(xì)胞外基質(zhì)（ECM）的3D多級結(jié)構(gòu)是一個(gè)相當(dāng)不容易的任務(wù)。在這樣的背景下，生物打印作為一種有潛力制備仿生3D組織結(jié)構(gòu)的新技術(shù)已經(jīng)出現(xiàn)。生物打印可以根據(jù)需要實(shí)現(xiàn)多細(xì)胞結(jié)構(gòu)的精確定位。以往研究者們已經(jīng)將這種技術(shù)應(yīng)用于生物需要，例如打印軟骨或多細(xì)胞結(jié)構(gòu)，且在打印分辨率和細(xì)胞需求方面都取得了不錯(cuò)的結(jié)果。盡管在3D生物打印方面有著不錯(cuò)的進(jìn)展，但對不同細(xì)胞的精確定位和轉(zhuǎn)換以及尋求合適的生物材料仍是主要的挑戰(zhàn)之一。

基于此，哈佛醫(yī)學(xué)院的Ali Khademhosseini教授和羅馬大學(xué)的Mariella Dentini教授合作團(tuán)隊(duì)提出了一種新的生物打印范例，他們將3D生物打印和微流控芯片相結(jié)合，將打印分辨率和打印效率提升到一個(gè)新的水平，同時(shí)該方法還具有以下優(yōu)勢：

1）能夠同時(shí)打印多種材料；

2）可制備具有活性的載細(xì)胞3D結(jié)構(gòu)；

3）使用可以誘導(dǎo)細(xì)胞擴(kuò)散和遷移的生物墨水；

4）能夠在生物打印的支架上播種另一種細(xì)胞。首先，該團(tuán)隊(duì)選擇使用海藻酸鹽和GelMA混合的低粘度生物墨水進(jìn)行生物打印，選用GelMA是因?yàn)槠淇纱龠M(jìn)細(xì)胞粘附和細(xì)胞遷移。低粘度的GelMA對細(xì)胞的封裝效果更接近于天然基質(zhì)，但低粘度GelMA很難作為生物墨水進(jìn)行打印，在固化前易出現(xiàn)擴(kuò)散現(xiàn)象。而海藻酸鹽作為混合組分之一，可通過離子交聯(lián)起到固定結(jié)構(gòu)的作用。該生物打印技術(shù)的示意圖如（圖1 a-c）所示，先通過物理交聯(lián)得到打印模板，再通過UV共價(jià)交聯(lián)得到穩(wěn)定的3D組織結(jié)構(gòu)（圖1 g-h）。

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.

為了證明制備多組分或多細(xì)胞組織結(jié)構(gòu)的可行性，研究人員將一個(gè)微流控體系與生物打印相結(jié)合，可快速制備不同材料構(gòu)成的仿生組織結(jié)構(gòu)（圖2 a）。將“Y”型通道的微流控芯片與同軸針頭鏈接，分別注入不同墨水，用紅色和綠色熒光顆粒標(biāo)記。通過選擇不同的墨水打印，研究者們可實(shí)現(xiàn)連續(xù)打印不同材料，得到包含不同墨水在不同層上的異質(zhì)結(jié)構(gòu)（圖2 b-e）?；蛘?，當(dāng)兩種墨水同時(shí)擠出將得到多相纖維組成的3D結(jié)構(gòu)（圖2 d，f-i）。

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.

為證明細(xì)胞可在生物打印結(jié)構(gòu)中遷移，該團(tuán)隊(duì)使用人臍靜脈內(nèi)皮細(xì)胞（HUVECs）  打印10層結(jié)構(gòu)，在培養(yǎng)10天后在共聚焦顯微鏡下觀察。同時(shí)通過DAPI、F-actin、CD31染色也可觀察到在培養(yǎng)10天后，細(xì)胞多位于3D支架外層，證明細(xì)胞在培養(yǎng)過程中發(fā)生遷移（圖3 a-h）。最后，研究人員為證明該結(jié)構(gòu)可作為體外支架用于心臟組織工程，將原代心肌細(xì)胞種于該結(jié)構(gòu)上，培養(yǎng)2天后，發(fā)現(xiàn)心肌細(xì)胞在結(jié)構(gòu)4個(gè)區(qū)域顯示同步搏動(dòng)。

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. 

本研究由哈佛醫(yī)學(xué)院的AliKhademhosseini教授和羅馬大學(xué)的Mariella Dentini教授合作團(tuán)隊(duì)完成，于2015年11月26日發(fā)表于Advanced Materials。

論文信息：

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.

論文鏈接：

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