The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

注:此协议描述了使用背压辅助机器人分配系统（ 图1A），为表面刻蚀（ 图1B）和基于挤出的生物印刷器（ 图1C）10。 1.对聚苯乙烯表面改性<ol><li>使用1毫米聚苯乙烯板聚苯乙烯以来组织培养板倾向于向上弓为中心，既破坏蚀刻和印刷的高度一致性。 注:由于聚苯乙烯片不被修改为细胞粘附，进行等离子体处理。 </li><li>氧等离子体处理。 <ol><li>首先，选择2巴的上连接到等离子体机器氧气瓶的调节器压力。然后打开并输入以下条件到机器的控制面板等离子机（分别为电力，燃气流量和时间）为150W，氧气30 SSCM，10分钟。 </li><li>将聚苯乙烯substratË进入等离子体室，封门，按下控制面板上的启动按钮。浸泡在胎牛血清氧等离子体处理的聚苯乙烯基板和洗涤三次1×磷酸盐缓冲盐水（PBS）之前在37℃孵育2小时。 </li></ol></li></ol> 2.编程模式打印<ol><li>使用该程序首先蚀刻表面用触控笔或手术刀。 <ol><li>当使用手写笔，插入直径为1.5mm针纺织品（从非常小心内部）插入到分配注射器（或5或10毫升）的嘴，直到它被卡住并固定。如果使用手术刀，选择一个圆处理解剖刀，以便它可以被紧固到打印臂的夹紧机构。 注:手写笔蚀刻图案弯曲比手术刀更好。 </li><li>当第一次尝试创建一个bioprinted安排，素描在坐标纸上所需的图案与编号轴产生XY坐标。然后我NPUT蚀刻/ bioprinted图案的坐标到电子表格软件（ 图2）。 注意:"所需的图案"可以是多种形状，如直链的，S形，或圆形的。的选择取决于所需的实验模型，如平行的线性线的MSC的分化为心肌如这里证明。散点图功能允许提出情节模式的可视化。 </li><li>打开打印/分发软件。选择"程序&gt;添加程序"，其次是"编辑&gt;添加点"来设置程序。导出x和y坐标从电子表格中获得的值成打印/分配使用软件"复制和粘贴"的功能。 </li><li>校准每次运行前，机器人的"z"的高度，以触针/打印喷嘴放置在表面上。 <ol><li>在打印/分发软件，选择"机器人"选项，点击"更改模式＆＃34;并启用"教学模式"选项。一旦选择，软件使机器人的"点动"功能。 </li><li>慢跑，首先通过选择菜单栏下面的命令机器人初始化到默认位置; "机器人&gt;梅卡初始化"，然后选择"机器人&gt;点动"。输入中以精确地放置触笔上的图案的原点所需要的"X和Y槽"的数值（单位mm）。 </li><li>接着，在"Z槽"输入的数值（毫米）放置铁笔或打印喷嘴中与表面接触，但不挠曲或缩进表面。这点被指定为"Z"的初始值。 注:各槽的深度可以很容易地使用该系统的Z高度变化。 40，80和170微米槽证明切成1毫米厚的聚苯乙烯片材（ 图4和表1）的表面上。 </li></ol></li><li>选择在P每个坐标点RINT指令， 即 "行配给的开始"来定义的第一点和打印起始，"行传递"指定的中间点和"行配给结束"的信号给机器人终止印数。 </li><li>通过从顶部菜单栏中选择后续指令控制程序的机器人:"机器人&gt;发送C＆T的数据"。 </li><li>启动蚀刻/印数，通过改变机器人的"运行"模式。通过选择这样做从顶部菜单栏中的"机器人&gt;更改模式&gt;切换运行模式"。 </li><li>通过按下绿色"启动按钮"机器人饮水机控制台上启动打印程序。 </li></ol></li></ol> 3.制备和印刷的明胶生物油墨含有细胞的<ol><li>溶解2％的明胶在最小必需培养基阿尔法培养基（αMEM）（补充有10％FBS和2％抗生素/ antimycotIC）在60℃下2小时以制备生物油墨溶液。 </li><li>预培养的红色荧光蛋白表达间充质干细胞（RFP-MSCS）至70％汇合使用αMEM/ 10％FBS10厘米组织培养皿。通过在37℃下除去培养基和涂料用1X胰蛋白酶-EDTA溶液中5分钟，释放附着的细胞成悬浮液。 </li><li>沉淀通过离心将细胞在1000×g离心5分钟，去除上清。重悬在0.5ml 1×PBS中的细胞沉淀，并使用血细胞计数器计数细胞密度。 </li><li>允许生物油墨冷却至室温后，轻轻以足够的量混合到悬浮液中的RFP-MSC的在生物油墨以实现5×10 6个细胞毫升-1的终浓度。 </li><li>倒入细胞生物油墨轴承与密封喷嘴的打印注射器。冷却加载注射器至4℃，以达到一印刷的粘度。 </li><li>放置装注射器上的自动化全自动点胶系统，并将空气压力线。取出注射头密封和连接打印喷头。 </li><li>挤出细胞化bioinks成使用0.05MPa的背压细线，以5mm /（锥形建议在圆柱形）从经由27克针/喷嘴10毫升注射器秒写入速度到聚苯乙烯膜表面，之后预编程的沉积在步骤2.1中所述图案至细胞化生物油墨放置到预刻蚀槽。 </li><li>在室温下孵育1小时后，使用倒置荧光显微镜观察之前加入10 mL生长培养基（补充有10％FBS和抗生素），孵育细胞24小时（以使细胞以感测和反应的蚀刻特征）在放大10倍。 </li></ol>

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The authors declare they have no competing financial interest.

这个过程的关键步骤是干细胞作为过程必须允许细胞沉降到该特征，打印无生物油墨扩频/出血，递送细胞无剪切应力的细胞死亡，而不是触发分化朝不希望的谱系的实际生物印刷递送。 如果预期细胞对准未能发生，则该生物油墨粘度应该评估其适合印刷。重要的是，该生物油墨允许细胞沉降到该图案化的聚合物表面上。该生物油墨聚合物的浓度，可以减少或它​​的温度升?...

单元布局的蓄意构图，可允许在体内细胞组织1模仿文化的形成。事实上，研究多种细胞类型之间的相互作用可以通过组织他们的空间位置2,3的协助。大多数图形系统依赖于表面改性方法，为推动或阻止随后的被动细胞沉积的细胞粘附。提供生物印刷在细胞分布1空间和时间的控制。除了这些功能，生物印刷已被描述为用于产生几何形状复杂的支架4在技术上简单，快速和成本有效的方法。它利用计算机设计软件，并允许引入细胞进入制造工序4。 生物印刷系统已根据自己的基础工作原理激光被分类，喷墨型或挤压型4。挤压生物印刷已被描述为是最有前途的，因为它允许一个现实的时间帧4-6内临床相关尺寸的组织构建物的制造。它是由一个细胞轴承水凝胶生物油墨的机械或背压辅挤出进行。在这里提出的方法中，被采用的背压。如所提到的，将细胞在细胞相容性生物油墨递送。这样的生物油墨应该支持细胞的递送，而不产生有害的剪切应力，并具有足够的粘度以保持在印刷迹的完整性，而不会破裂或扩散（称为"墨渗色"），7-10。 已知细胞与它们的贴壁表面的相互作用来影响细胞的行为。表面形貌可以控制细胞形状，方向11，甚至表型。尤其是，沟槽和通道的制造中已被证明诱导在多个细胞类型拉伸的，细长的形态。这种形态的通过已发现影响多能和多潜能细胞的表型。例如，当在槽对齐，间质干细胞（MSC）表现出对心肌12,13和血管平滑肌细胞分化的证据采用收缩表型在合成10,14-17。 可在聚合物表面通过许多方法来产生对准通道或沟槽的细胞，例如，深反应离子蚀刻，电子束光刻，激光直接印刷，飞秒激光，光刻和等离子干蚀刻18。这些方法通常耗时，需要复杂的设备，并且可以在所产生的图案的形状进行限制。此外，他们不与同步生物印刷图案，不允许立即细胞化。一个自动化的协调控制的运动分配系统可以遵循复杂的图案为溶液的沉积。在这里，我们展示了微型受控的运动是如何被利用来创造细胞定向通道。削尖触笔或手术刀被连接到所述打印头代替挤出注射器中，然后将设备可以蚀刻绘图软件的指导下，在聚合物表面上。该方法在图案设计提供了多功能性，并适用于在生物工程常用如聚苯乙烯，聚四氟乙烯，和聚己内酯的聚合物材料。作为后续步骤的蚀刻，细胞可以直接bioprinted到划伤凹槽。这里使用的明胶生物油墨能够既保持跟踪，并允许沉积的细胞来检测蚀刻特征。 bioprinted的刻蚀槽间充质干细胞，表现在不同的线条沿它们伸长。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Robotic Dispensing System</td><td>Janome</td><td>2300N</td><td></td></tr><tr><td>Plasma Machine</td><td>Femto Science</td><td>Covance</td><td></td></tr><tr><td>USB Microscope</td><td></td><td></td><td></td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spreadsheet</td><td>Excel</td><td>Excel</td><td></td></tr><tr><td>Printing Co-ordinate Software</td><td>Janome</td><td>JR C-Points</td><td></td></tr><tr><td>Imaging Software</td><td>National Institutes of Health (NIH)</td><td>ImageJ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stylus (Blade)</td><td>OLFA</td><td>AK-5</td><td></td></tr><tr><td>5 ml printing syringe</td><td>San-ei Tech</td><td>SH10LL-B</td><td></td></tr><tr><td>30 G printing needle</td><td>San-ei Tech</td><td>SH30-0.25-B</td><td></td></tr><tr><td>1 mm polystyrene sheets</td><td></td><td>Purchased locally</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen&amp;nbsp;</td><td>10270-098</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Gelatin from porcine skin, Gel strength 300, Type A</td><td>Sigma Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>41061-029</td><td></td></tr><tr><td>Antibiotc antimycotic</td><td>Sigma Aldrich</td><td>A5955-100ML</td><td></td></tr><tr><td>Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td><td>Cyagen Biosciences Incorporation</td><td>RASMX-01201</td><td></td></tr></tbody></table>

automated robotic dispensing technique for surface guidance and bioprinting of cells

这个手稿描述引进细胞指导功能接着用自动机器人分配系统对这些特征的直接递送细胞在水凝胶生物油墨。被选定的特定生物油墨，因为它使细胞对沉积物和感知功能。分配系统bioprints使用背压辅助打印头中的水凝胶bioinks活细胞。然而，通过用锋利笔或手术刀更换打印头，分配系统也可以采用以创建经过表面刻蚀地形线索。触针运动可以在X，Y的10微米的步骤和Z方向进行编程。图案化的凹槽能够定向间质干细胞，影响它们通过在对准的细长的形态与凹槽&#39;方向。图案形成可以在直线，同心圆，和正弦波使用绘图软件来设计。在随后的过程中，成纤维爆炸和间充质干细胞悬浮于2％的明胶生物油墨，用于在背压从动挤压印刷头生物印刷。然后将细胞轴承生物油墨使用用于蚀刻相同的编程坐标打印。所述bioprinted细胞能够感知和通过沿刻蚀槽的方向上其细长方向所证明反应的蚀刻特征。

代表结果表明，该反压辅助机器人分配系统可以作为一个基于挤压的生物印刷器，用于执行两个表面蚀刻和生物油墨印刷（ 图1的A）。它可用于刻蚀槽的产生到聚合物表面上，并且随后直接打印的小区轴承生物油墨的特征（ 图1 B和C）。 既蚀刻和印刷由编程坐标确定（ 图2）输入到打印/分...

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

automated-robotic-dispensing-technique-for-surface-guidance

Research

JoVE Journal

Bioengineering

6.6K 次观看.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

视频: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

暂停规定微米/亚微米尺度的纤维结构的3-D直写经全自动点胶系统

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

科研

工程学

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

生物工程

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

琼脂糖流体凝胶在凝胶过程中通过剪切处理形成，用于悬浮3D生物打印

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...