3D打印的具可調(diào)節(jié)機(jī)械和細(xì)胞粘附特性的海藻酸鈉/明膠復(fù)合水凝膠調(diào)節(jié)腫瘤球體

作者：余穎康

可調(diào)節(jié)生物打印墨水能夠創(chuàng)建廣泛的生理模擬三維模型，使體外研究能夠更準(zhǔn)確地模擬體內(nèi)條件，但調(diào)節(jié)生物墨水使其同時(shí)具有生物可打印性和仿生性仍然是一個(gè)關(guān)鍵的挑戰(zhàn)。腫瘤微環(huán)境（TEM）是一個(gè)高度動態(tài)的系統(tǒng)，而傳統(tǒng)的二維細(xì)胞培養(yǎng)技術(shù)，重建TME并促進(jìn)腫瘤球體（MCTS）的體外形成非常困難。最近，來自加拿大麥吉爾大學(xué)Joseph M. Kinsella教授團(tuán)隊(duì)研發(fā)了一種由明膠和海藻酸組成的工程復(fù)合水凝膠，可通過改變這兩種成分的初始濃度來調(diào)整3D打印的乳腺腫瘤模型的機(jī)械性和生物特性，成功實(shí)現(xiàn)了在體外腫瘤球體（MCTS）的形成，從而表明該類水凝膠可用于創(chuàng)建高通量、低成本和高重復(fù)性的三維疾病模型。（圖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.

研究者對復(fù)合水凝膠前聚體的生物可打印性進(jìn)行了分析，發(fā)現(xiàn)AxGy（AxGy：x%海藻酸和y% 明膠）的所有組合物都可以使用相應(yīng)的最小壓力在不同的初始時(shí)間點(diǎn)進(jìn)行打印(圖2b)。在所有AxGy水凝膠前聚體中，對于具有相同(x + y)值的樣品，它們的凝膠化曲線具有相似性。圖（2c-k）表明所有打印樣品都具有穩(wěn)定性，有足夠的屈服應(yīng)力來支撐結(jié)構(gòu)。大多數(shù)打印絲狀體表面粗糙度在10%左右，其中以A1G5為最差(18.4%)，A5G5為最佳(6.5%)。不同水凝膠的表觀楊氏模量（E）可根據(jù)海藻酸的濃度進(jìn)行調(diào)整，其可調(diào)節(jié)范圍為5.46 ~ 22.88 kPa，而明膠對E的影響不明顯（圖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.

一般來說，較軟的水凝膠更容易誘發(fā)MCTS。所有的AxGy樣品在培養(yǎng)第7天開始誘導(dǎo)MCTS的形成，直到第28天實(shí)驗(yàn)停止，結(jié)果發(fā)現(xiàn)A1G7凝膠在培養(yǎng)14天后促進(jìn)中、大MCTS的形成(圖4 (a, c))，而A3G7在培養(yǎng)14天后產(chǎn)生中MCTS, 28天后才形成大MCTS(圖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.

為了評價(jià)MCTSs在A1G7或A3G7中的狀態(tài)，研究者對其進(jìn)行了活死分析（圖5）。發(fā)現(xiàn)細(xì)胞在A1G7和A3G7水凝膠中均表現(xiàn)出較高的活力，并隨著時(shí)間的推移，A1G7中細(xì)胞和MCTSs的增殖率較高(圖5 a)，而A3G7中MCTSs的增殖率與第0天相同。進(jìn)一步，使用高倍三維重建考察了A1G7和A3G7在培養(yǎng)21天后形成的MCTS的分布、體積和形態(tài)(圖6)，發(fā)現(xiàn)與A3G7相比，A1G7可以產(chǎn)生較大的MCTS(圖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.

本研究由加拿大麥吉爾大學(xué)Joseph M. Kinsella教授團(tuán)隊(duì)完成，并于2019年8月12日在線發(fā)表于Biofabrication。

論文信息：
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.