3D打印的具可调节机械和细胞粘附特性的海藻酸钠/明胶复合水凝胶调节肿瘤球体

作者：余颖康

可调节生物打印墨水能够创建广泛的生理模拟三维模型，使体外研究能够更准确地模拟体内条件，但调节生物墨水使其同时具有生物可打印性和仿生性仍然是一个关键的挑战。肿瘤微环境（TEM）是一个高度动态的系统，而传统的二维细胞培养技术，重建TME并促进肿瘤球体（MCTS）的体外形成非常困难。最近，来自加拿大麦吉尔大学Joseph M. Kinsella教授团队研发了一种由明胶和海藻酸组成的工程复合水凝胶，可通过改变这两种成分的初始浓度来调整3D打印的乳腺肿瘤模型的机械性和生物特性，成功实现了在体外肿瘤球体（MCTS）的形成，从而表明该类水凝胶可用于创建高通量、低成本和高重复性的三维疾病模型。（图1）。

Figure 1. Schematic depicting the generation of the composite gels, bioprinting process, and subsequent generation of MCTS of breast cancer cells in bioprinted alginate/gelatin hydrogels.

研究者对复合水凝胶前聚体的生物可打印性进行了分析，发现AxGy（AxGy：x%海藻酸和y% 明胶）的所有组合物都可以使用相应的最小压力在不同的初始时间点进行打印(图2b)。在所有AxGy水凝胶前聚体中，对于具有相同(x + y)值的样品，它们的凝胶化曲线具有相似性。图（2c-k）表明所有打印样品都具有稳定性，有足够的屈服应力来支撑结构。大多数打印丝状体表面粗糙度在10%左右，其中以A1G5为最差(18.4%)，A5G5为最佳(6.5%)。不同水凝胶的表观杨氏模量（E）可根据海藻酸的浓度进行调整，其可调节范围为5.46 ~ 22.88 kPa，而明胶对E的影响不明显（图3）。

Figure 2. Printability of hydrogel precursors. (a) CAD of printed mesh model (unit: mm). (b)shows printing windows of precursors with different alginate and gelatin concentrations. Each round panel inside the plot represents one type of AxGy precursors. The numbers on the perimeter of the panel represent the time of gelling (min) before the printing. The color bar indicates the minimum pressure required to extrude the material using a G27 conical nozzle at RT. (c-k) demonstrate cuboid mesh models printed of AxGy. The time of gelling, extrusion pressure, and normalized roughness are shown for eachprinted mesh. Scale bar is 1 mm. (l) scatter plot of minimum extrusion pressure versus yield stress. The solid red line is the upper bound defined by equation (3), the solid green line and dashed green line are lower bounds defined by equation (4) and (5). Blue dashed line represents a linear regression, with the estimated equation and goodness of the fitting. (m) shows the geometric parameters of a Gauge 27 conical nozzle. (n) shows the explicit formulas of the boundary conditions.

Figure 3. Apparent Young’s Modulus measured 24 hours after crosslinking by micro-indentation. Plotted with the concentrations of gelatin and alginate on vertical and horizontal axes, and color bar represents the values of apparent Young s modulus. Asterisks (*) represent a significant difference between two groups, calculated by pooling all the data for the different gelatin concentrations, with P < 0.05, n=10. ns means non-significant difference.

一般来说，较软的水凝胶更容易诱发MCTS。所有的AxGy样品在培养第7天开始诱导MCTS的形成，直到第28天实验停止，结果发现A1G7凝胶在培养14天后促进中、大MCTS的形成(图4 (a, c))，而A3G7在培养14天后产生中MCTS, 28天后才形成大MCTS(图4 (b, d))。

Figure 4. Confocal images of bioprinted A1G7 and A3G7 disks and quantitative analysis of MCTS in a 28-dayperiod. Row (a) and (b) show the morphological MCTS variation by time in A1G7and A3G7, respectively. Magnification ×10. Images (c) shows the volume of each spheroid in a representative A1G7 sample during 28 days of culture, with categories of small (15,000–200,000 μm3), medium (200,000–700,000 μm3), andlarge (>700,000 μm3) MCTS presented in black, red and blue color. (d) showsthe same data for A3G7, with the same thresholds in categorization. Box plotgraphs were plotted using a box limit of 25th and 75th percentiles with aminimum-maximum whisker’s range.

为了评价MCTSs在A1G7或A3G7中的状态，研究者对其进行了活死分析（图5）。发现细胞在A1G7和A3G7水凝胶中均表现出较高的活力，并随着时间的推移，A1G7中细胞和MCTSs的增殖率较高(图5 a)，而A3G7中MCTSs的增殖率与第0天相同。进一步，使用高倍三维重建考察了A1G7和A3G7在培养21天后形成的MCTS的分布、体积和形态(图6)，发现与A3G7相比，A1G7可以产生较大的MCTS(图6 a, b)。

Figure 5. MDA-MB-231 cellviability during 28 days of culture within A1G7 or A3G7 hydrogels. (a) the viability of single cells as well as MCTS was determined each 7 days and normalized against day 0. Data presented as Mean ±SD, n≥3. Confocal images of live (green) and dead (red) MCTS in A1G7 (b) and A3G7 (c). Magnification ×4, scale bar 500 μm.

Figure 6. 3D reconstructionof MCTS showing the representative morphologies and sizes of MCTS formed inA1G7 (a, b) and A3G7 (c, d) hydrogels after 21 days of culture. A zoom in ofthe MCTS is presented in b) and d), displaying the actin organization in the spheroids. Magnification 20, scale bar 50  μm.

本研究由加拿大麦吉尔大学Joseph M. Kinsella教授团队完成，并于2019年8月12日在线发表于Biofabrication。

论文信息：
Tao Jiang‡, Jose G. Munguia-Lopez‡, Kevin Gu, Maeva M. Bavoux, Salvador Flores-Torres, Jacqueline Kort-Mascort, Joel Grant, Sanahan Vijayakumar, Antonio De Leon-Rodriguez, Allen J. Ehrlicher, Joseph M. Kinsella*. Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth. Biofabrication 2019, DOI:10.1088/1758-5090/ab3a5c.