Name: bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation
Uploaded: 2018-07-02T14:30:00.000+00:00
Duration: 16 min 20 s
Description: Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

陶江感谢中国奖学金委员会 (201403170354) 和麦吉尔工程博士奖 (90025) 为他们提供奖学金。何塞 g. Munguia-洛佩兹感谢 CONACYT (250279, 290936 和 291168) 和 FRQNT (258421) 为他们的奖学金资助。萨尔瓦多 CONACYT 感谢他们的奖学金资助 (751540)。约瑟夫 m. 金塞拉感谢国家科学和工程研究理事会, 加拿大创新基金会, 汤森-Lamarre 家庭基金会, 麦吉尔大学为他们提供资金。我们要感谢艾伦 Ehrlicher 允许我们使用他的流变仪, 丹 Nicolau 允许我们使用他的共焦显微镜, Morag 公园让我们访问荧光标记的细胞线。

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作者没有什么可透露的。

如果污染 (生物或化学) 发生在过程中的任何一点上, 就会危及细胞的结构。通常, 生物污染是在三天的文化后, 作为一种颜色变化的文化媒体或 bioprinted 结构。因此, 灭菌 (物理和化学消毒) 是所有细胞相关过程的关键步骤。值得注意的是, 热处理明胶改变了其凝胶性质, 这使得它在我们进行的试验中更慢。因此, 我们通过紫外线照射对海藻酸盐和明胶的能量进行杀菌。由于 UV 光的穿透能力非?...

虽然2D 细胞培养在癌症研究中得到广泛应用, 但由于细胞以单层格式生长, 营养和氧气浓度均匀, 所以存在局限性。这些文化缺乏在原生肿瘤微环境中存在的重要细胞细胞和细胞基质相互作用。因此, 这些模型不太概括的生理条件, 导致异常细胞行为, 包括非自然形态, 不规则的受体组织, 膜极化, 和异常的基因表达, 除其他情况1,2,3,4。另一方面, 3D 细胞培养, 细胞被扩展在一个体积空间作为聚合体, 球体, 或 organoids, 提供了一种替代技术, 以创造更准确的体内环境, 以研究基本细胞生物学和生理学。3D 细胞培养模型还可以鼓励细胞 ECM 相互作用, 这是本机1,4,5的重要生理特性。新兴的3D 生物打印技术为构建模仿异类的模型提供了可能性。
3D 生物打印是从快速原型, 并使制造的3D 显微组织, 可以模仿一些复杂的活体标本6,7。目前的生物打印方法包括喷墨、挤出和激光辅助印刷8。其中, 挤出方法可以在不同的初始位置精确定位各种不同类型的材料, 从而在印制矩阵中控制异质性。因此, 它是制造多类型细胞或矩阵的异质体模型的最佳方法。挤压生物打印已成功地用于建立耳形支架9, 血管结构10,11,12, 皮肤组织13, 导致高印刷保真度和细胞可行性。该技术还具有多种材料选择, 有能力储存与已知密度的细胞的材料, 高重现性14,15,16,17.天然和合成水凝胶经常被用作3D 生物打印的 bioinks, 因为它们的生物相容性, 生物活性, 和他们的亲水性网络, 可以工程结构类似 ECM7,18 ,19,20,21,22,23。水凝胶也很有利, 因为它们可以包括细胞的粘接部位、结构元素、养分和气体的渗透性, 以及促进细胞发育的适当机械特性24。例如, 胶原水凝胶提供整合素锚固点, 细胞可以用来连接到基质。凝胶, 变性胶原蛋白, 保留类似的细胞黏附部位。相比之下, 海藻酸盐是 bioinert, 但通过形成 crosslinks 与价离子25,26,27,28提供机械完整性。
在这项工作中, 我们开发了复合水凝胶作为 bioink, 由海藻酸盐和明胶组成, 与原生肿瘤基质的显微结构相似。乳腺癌细胞和成纤维细胞被嵌入在水凝胶中,通过挤压的 bioprinter 印刷, 以创建模拟体内微环境的3D 模型。设计的3D 环境允许癌细胞形成多细胞肿瘤球体 (MCTS), 具有很高的生存能力, 长时间的细胞培养 (> 30 天)。该协议展示了合成复合水凝胶的方法, 表征了材料的显微组织和适印性, 生物打印细胞异构模型, 观察 MCTS 的形成。这些方法可以应用于其他 bioinks 挤压生物打印, 以及不同的设计的异构组织模型的潜在应用, 在药物筛选, 细胞迁移检测, 和研究, 重点在基本细胞生理功能。

<table><tbody><tr><td>Sodium alginate</td><td>FMC BioPolymer</td><td>CAS-No: 9005-38-3</td><td>Protanal LF 10/60 FT</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td>Type B gelatin from bovine skin</td></tr><tr><td>Dubelcco&#039;s phosphate buffered saline (DPBS 1X)</td><td>Gibco</td><td>LS14190136</td><td>1&amp;times;, w/o calcium, w/o magnesium</td></tr><tr><td>Magnetic hotplate</td><td>Corning&amp;nbsp;</td><td>N/A</td><td>Stirrer/hot plate model PC-420</td></tr><tr><td>50 mL centrifuge tubes</td><td>Corning</td><td>352098</td><td>Falcon&amp;reg; 50mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile</td></tr><tr><td>Centrifuge</td><td>GMI</td><td>N/A</td><td>Sorvall RT6000D, GMI, USA</td></tr><tr><td>Calcium chloride anhydrous</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>MilliQ water</td><td>Millipore</td><td>N/A</td><td></td></tr><tr><td>Millipore 0.22 &amp;micro;m filters</td><td>Millipore</td><td>SLGS033SB</td><td>Millex-GS Syringe Filter Unit, 0.22&amp;nbsp;&amp;micro;m, mixed cellulose esters, 33&amp;nbsp;mm, ethylene oxide sterilized</td></tr><tr><td>Oscillation rheometer MCR 302</td><td>Anton Paar</td><td>N/A</td><td></td></tr><tr><td>Rheometer measuring tool CP25</td><td>Anton Paar</td><td>79038</td><td>Conical plate geometry for rheometer</td></tr><tr><td>RheoCompass</td><td>Anton Paar</td><td>N/A</td><td>Software controlling rheometer MCR 302</td></tr><tr><td>Scanning electron microscope</td><td>Hitachi</td><td>N/A</td><td>SEM, Hitachi SU-3500 Variable Pressure</td></tr><tr><td>Paraformaldehyde, 96%, extra pure</td><td>Acros Organics</td><td>416785000</td><td></td></tr><tr><td>Dulbecco modified eagle medium (DMEM)</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>Antibiotic/Antimycotic solution (100X) stabilized</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Wisent Bioproducts</td><td>080-150</td><td></td></tr><tr><td>Cell culture T-75 flasks</td><td>Sigma-Aldrich</td><td>CLS430641</td><td>75 cm&lt;sup&gt;2&lt;/sup&gt; TC-Treated surface treatment</td></tr><tr><td>3D bioprinter BioScaffolder 3.1</td><td>GeSiM</td><td>N/A</td><td></td></tr><tr><td>GeSim software</td><td>GeSiM</td><td>N/A</td><td>Software controlling BioScaffolder 3.1</td></tr><tr><td>10cc cartridge UV resist</td><td>EFD Nordson</td><td>7012126</td><td></td></tr><tr><td>End cap</td><td>EFD Nordson</td><td>7014472</td><td></td></tr><tr><td>Tip cap</td><td>EFD Nordson</td><td>7014469</td><td></td></tr><tr><td>Piston&amp;nbsp;</td><td>EFD Nordson</td><td>7012182</td><td></td></tr><tr><td>Stainless nozzle G25</td><td>EFD Nordson</td><td>7018345</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>N/A</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td>Bioreagent, for molecular biology</td></tr><tr><td>Costar 6-well plates&amp;nbsp;</td><td>Corning</td><td>3516</td><td>TC-Treated Multiple Well Plates, Individually Wrapped, Sterile&amp;nbsp;</td></tr><tr><td>Confocal spinning disk inverted microscope</td><td>Olympus Life Science</td><td>N/A</td><td>Olympus IX83</td></tr><tr><td>MTS assay kit</td><td>Promega</td><td>G3582</td><td>CellTiter 96&amp;reg; AQueous&amp;nbsp;One Solution Cell Proliferation Assay&amp;nbsp;</td></tr><tr><td>Live/Dead viability cytotoxicity kit</td><td>Molecular Probes,ThermoFisher Scientific</td><td>L3224</td><td></td></tr><tr><td>Trypsin 0.25/EDTA 1X</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Corning 96-well plate</td><td>Corning</td><td>3595</td><td>Clear Flat Bottom Polystyrene TC-Treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile</td></tr><tr><td>Autoclave Tuttnauer</td><td>Heidolph Brinkmann</td><td>N/A</td><td>Heidolph Tuttnauer 2540E Autoclave Sterilizer Electronic Model with 4 Stainless Steel Trays, 23L Capacity</td></tr><tr><td>Trypan blue</td><td>Invitrogen&amp;nbsp;</td><td>T10282</td><td>0.4% solution</td></tr><tr><td>Ethanol</td><td>Commercial Alcohols</td><td>P016EA95</td><td>Greenfield Speciality Alcohols</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Panasonic</td><td>N/A</td><td>MCO 19AIC-PA</td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>SP Scientific</td><td>N/A</td><td>Virtis Sentry 2.0</td></tr><tr><td>SolidWorks</td><td>Dassault Systems</td><td>N/A</td><td>A CAD software used to build demostrative propeller-like model</td></tr><tr><td>MATLAB</td><td>The MathWorks</td><td>N/A</td><td>A programming software used to generate G-code for BioScaffolder 3.1</td></tr></tbody></table>

bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation

使用常规的二维 (2D) 细胞培养法, 生长永生化癌细胞株不概括本机肿瘤微环境的细胞、生物化学和生物物理异质性。这些挑战可以克服, 通过使用生物打印技术建立异构的三维 (3D) 肿瘤模型, 即不同类型的细胞嵌入。海藻酸盐和明胶是生物打印中使用的两种最常见的生物材料, 其生物相容性、仿生学和力学性能。通过结合这两种聚合物, 我们取得了 bioprintable 复合水凝胶与原生肿瘤基质的显微结构相似。通过流变学研究了复合水凝胶的适印性, 得到了最佳的印刷窗口。将乳腺癌细胞和成纤维细胞嵌入水凝胶中, 并印成3D 模型, 模拟体内微环境。bioprinted 异构模型为长期细胞培养 (> 30 天) 提供了很高的生存能力, 并促进了乳腺癌细胞的自组装成多细胞肿瘤球体 (MCTS)。我们观察了肿瘤相关成纤维细胞 (咖啡馆) 与 MCTS 在该模型中的迁移和相互作用。利用 bioprinted 细胞培养平台作为共培养系统, 为研究肿瘤发生对基质成分的依赖性提供了独特的工具。该技术具有吞吐量高、成本低、重现性高的特点, 也为常规细胞单层培养和动物肿瘤模型的研究提供了一种可替代的模型。

温度扫描显示 A3G7 前驱体在25°c 和37°c 上的明显差异。前体是液体在37°c 和有一个复杂的粘度 1938.1, 84.0 mPa x s, 这是验证由一个更大的 g "超过 g"。随着温度的降低, 前驱体经过物理凝胶, 由于明胶分子自发物理纠缠成三螺旋形成29,30。g ' 和 g ' 增加和汇合在30.6 °c, 表明溶胶-凝胶转折。模数继续增加, 温度降低, 达到 468.5 34.2 pa g ' ?...

Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

我们开发了一种异种乳癌模型, 由永生化的肿瘤和成纤维细胞组成的 bioprintable 海藻酸盐/明胶 bioink。该模型概括体内肿瘤微环境, 促进多细胞肿瘤球体的形成, 对肿瘤的发生机制有深入的认识。

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

bioprintable-alginategelatin-hydrogel-3d-vitro-model-systems-induce

Research

JoVE Journal

Bioengineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

18.5K 次观看.  McGill University Montreal. 我们开发了一种异种乳癌模型, 由永生化的肿瘤和成纤维细胞组成的 bioprintable 海藻酸盐/明胶 bioink。该模型概括体内肿瘤微环境, 促进多细胞肿瘤球体的形成, 对肿瘤的发生机制有深入的认识。

视频: Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫与感染

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

在 PEG 水凝胶中制备肿瘤球状体后进行 3D 封装

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

作者聚焦:胶质母细胞瘤微环境研究的体外水凝胶模型

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

摘要

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一个简单的悬滴细胞生成三维球体文化协议

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

3D Microtissues 用于可注射再生疗法和高通量药物筛选

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

将3D多细胞乳房球体直接生物打印到内皮网络

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用

聚乙二醇水凝胶中的肿瘤球状体制造和封装用于研究球状体-基质相互作用