笔者要感谢东轩佳博士为技术援助。在这篇文章中出现的一些图像在与美国普林斯顿大学分子生物学系，马康斯坦伯格博士合作。笔者还要感谢部和国防部前列腺癌研究计划（赠款PC - 030482 PC - 991552）的慷慨支持美国国立癌症研究所/美国国立卫生研究院（批R01CA118755）。

1。单细胞悬液的制备<ol><li>于是单层贴壁细胞培养应增长到90％汇合，应当用PBS漂洗两次。排水良好后，加入2毫升，0.05％胰蛋白酶1毫米EDTA（100毫米板块），并在37 ° C，直到细胞分离。停止加入2 MLS的完全培养基，轻轻地用5毫升吸管磨碎的混合物，直至细胞悬浮液中的胰蛋白酶。细胞转移到15毫升锥形管。 </li><li>添加在室温为5分钟40μL10 mg / ml的DNA酶股票和孵化。涡短暂离心5分钟，在200的XG。 </li><li>弃上清，用1毫升完整的组织培养基颗粒。重复，然后在2 MLS的完整的组织培养液中的悬浮细胞。 </li><li>计数细胞，使用hemacytometer，或自动细胞计数，调整浓度为2.5 × 10 6细胞/ ml 。对于本演示一个BioRad公司TC10自动细胞计数器使用。 </li></ol> 2。形成悬滴。 <ol><li>删除从60毫米组织培养皿并将其放置在底部的菜5毫升的PBS的盖子。这将作为一个水化室。 </li><li>倒置的盖子，并用20μL移液器存款10μL滴上的盖子底部。确保，滴放置足够的距离，以不碰。这是可能的地方至少有20％菜滴。 </li><li>反转到的PBS填充的底部室和孵化在37 ° C / 5％的CO 2 / 95％湿度盖子，监察滴每天孵育直到细胞表或聚集已经形成。一个立体显微镜可用于评估汇总形成。 </li><li>一旦表的形式，他们可以转移到圆底玻璃振动筛瓶含有3 MLS和完全培养基在摇晃水浴孵育37 ° C和5％，直到球体形成的CO 2。 </li></ol> 3。注释<ol><li>根据细胞的大小，浓度可能需要向上或向下调整。 </li><li>可染细胞与膜插层荧光染料前悬滴形成。 </li><li>两种不同的细胞类型可以不同的染色和前悬滴的形成1:1的比率（或其他）混合。这些可以成为癌症和基质细胞，胚胎组织或细胞基因工程有特定的粘合剂签名。 </li><li>用0.05％胰酶/ 2毫米的钙，以维持钙粘蛋白功能，细胞可以脱离组织培养皿。 </li><li>根据实验，表的大小可以衡量，或聚合，可用于机械试验或生化或分子分析。 </li></ol> 4。代表性的成果： 表“或”球体的形成通常发生在24小时内，但可能需要更长的时间取决于细胞类型。图1代表未经治疗的前列腺癌的MAT - LyLu（MLL）细胞在悬滴文化（图1A）或25微米MEK抑制剂PD98059处理的细胞培养18小时后拍摄的图像。请注意，在标记的细胞表压实MEKi的治疗效果。压实是可以量化的测量大小为10-20（或更多）挂滴和治疗组之间的平均规模比较。我们通常使用阈值每一个图像，然后将图像转换为二进制模式ImageJ软件分析图像，于是我们申请颗粒分析，以揭示总像素的图像区域。这可以很容易地转换为平方微米。图2表示了未处理和治疗MEKi MLL基因细胞的大小比较。如前所述，MEKi治疗，大大降低了学生的t检验（P &lt;0.0001）相比，纸张尺寸。对于这种比较，分别测定了10个未处理和处理过的细胞聚集。图3表示一个鸡胚肝悬滴培养24小时，2天在摇床浴后形成的球体。 悬滴检测，也可以修改，以包括一个以上的细胞类型，揭示空间定位通过。例如，可以是孤立的，鸡胚肝细胞，用荧光膜插层染料染色，用鸡胚胎的心脏细胞已用不同的荧光标记染色混合的。图，而不是剩余混合4A显示，肝脏和心脏细胞往往从一个“排序”，并采取一个领域内的一个领域配置，其中心脏细胞，肝细胞弥漫。此配置可通过细胞间粘附在肝脏和心脏细胞之间的差异解释。这个概念是编纂差速贴壁的假说已被用来解释细胞为胰岛细胞组织 5和细胞之间的细胞群相同的I排序排序两栖动物 3和硬骨鱼类 4胚胎的胚层组织之间的行为，ñ每一个方面比其他为钙粘蛋白（6和图4B）同一类型的表达水平。这个结果显得尤为重要示范工程在细胞群之间的附着力的简单的差异如何，可能会导致器官结构的产生，例如，胰岛5。 <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="图1" /> 图1。挂18小时，无论是在下降的情况下（A）或存在文化细胞的图像（二）的MEK抑制剂PD98059 25微米。使用尼康的Eclipse TE 300 epifluorescence显微镜和Photometrics CoolSnap ES数码相机捕获图像。 <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="图2" /> 图2。MEKi诱导大鼠前列腺癌的MLL细胞压实的量化。悬滴文化的未经处理和MEKi处理的MLL细胞培养18小时，届时总的大小是决定如上所述。骨料粒径的统计分析是由学生的t -检验。星号代表在骨料粒径之间的未经处理和MEKi处理的细胞和建议，MEKi治疗效果显着性差异明显变小，更紧凑的聚合性（P &lt;0.0001）。 <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="图3" /> 图3。挂18小时，然后在下拉文化的孵化器/惊天48小时洗澡孵化鸡胚肝细胞形成一个球体。使用尼康SMZ800立体显微镜和索尼黑色CCD相机拍摄图像。 <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="图4" /> 图4。悬滴文化，揭示出不同类型的细胞之间的行为进行排序。在A组中，鸡胚肝细胞PKH - 2插层膜染料染色和混合PKH - 26染色小鸡胚胎的心脏细胞的比例相等。在B组中，两个N - cadherin的转染L细胞克隆（LN4和LN2a），在2.4:1的比例在其表面表达的N - CAD，也沾上插层染料的荧光膜PKH - 2和PKH - 26 （Sigma - Aldrich公司），混合比例相等和悬滴7培养。文化在18小时后，细胞表转移到摇瓶和孵育48小时。总量在2％多聚甲醛固定在磷酸缓冲液和一个BioRad公司MRC600激光扫描共聚焦显微镜系统连接到尼康Optiphot - 2显微镜观看。 （一）从绿色和红色通道的光学部分收集和硅谷Graphix公司深红VGX工作站，配备了生命图像体素查看超成像软件用于分配两个通道假彩色，并合并图像，揭示了产生的解剖配置。共焦光学部分（这里是一个伪彩色黄色）通过聚合证明心脏细胞的包围伪蓝色的肝细胞 （8 ）。 （二）共焦光学部分通过N - cadherin的高表达细胞表达水平较低的N - cadherin的（红色）（6）（绿色）的总展示包络。

<ol>
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有研究表明，在三维环境（3D）培养细胞产生不同的细胞形态和信号时相比，一个刚性的二维（2D）文化系统 9 。例如，成纤维细胞填充的胶原凝胶表明，成纤维细胞在三维形态是相当独特的2D 10,11指出的。同样，三维培养可诱发乳腺上皮细胞分化的组织特异性。已被用来区分正常和恶性细胞三维培养系统，并已表明支持转化细胞的表型正常的回归，给予适当的刺激12,13。?...

<ul><li>自动计数仪（BioRad公司TC10） </li><li> CO 2山墙盖型号3540（实验室线）摇动水浴</li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

壁刚性基板上单层培养的细胞与细胞间的凝聚力和基质细胞黏附的研究在历史上一直执行。然而，一个组织内的细胞，通常装在一个紧密排列的组织肿块，在其中的细胞建立亲密的联系，许多附近的邻居和细胞外基质成分。因此，化学环境和一个三维组织内的细胞所经历的物理力量比单层培养细胞生长经历的根本不同。这已经显示出显着影响细胞的形态和信号。已制定了几种方法生成的三维细胞培养物，包括细胞在胶原凝胶1或生物材料支架2封装。这样的方法，虽然有用，但不复述亲密直接的细胞与细胞间的粘附结构，在正常组织中发现。相反，他们更加紧密地近似的文化系统中，单个细胞松散地分散在一个3D小梁ECM产品。在这里，我们描述了一个简单的方法中，细胞被放置在悬滴培养和生理条件下培养，直到它们形成真正的3D球体中，细胞直接接触与相互之间以及与细胞外基质成分。该方法无需专门的设备，可适应包括在非常小的数量，阐明对细胞与细胞或细胞- ECM相互作用的影响可能感兴趣的任何生物制剂此外。该方法也可用于共培养两个（或更多），不同的细胞群，以澄清在指定细胞之间的空间关系中的作用的细胞 - 细胞或细胞外基质的相互作用。细胞与细胞间的凝聚力和细胞- ECM的粘附，胚胎发育，肿瘤的间质细胞相互作用恶性侵袭，促进伤口愈合，组织工程的应用研究的基石。这个简单的方法将提供一个样组织产生的生物力学性能的测量或细胞聚集在生理相关的模型的分子和生化分析的手段。

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

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

我们描述了一个简单快速的方法，生成三维组织类似的球体和其潜在的应用，量化，在细胞间的相互作用的差异。

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 ...

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

56.8K 次观看.  UMDNJ-Robert Wood Johnson Medical School. 我们描述了一个简单快速的方法，生成三维组织类似的球体和其潜在的应用，量化，在细胞间的相互作用的差异。

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

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

科研

生物工程

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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 ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D 共培养检测：用于癌症药物筛选的血管化成骨生态位

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

作者聚焦： 在成像微孔板中使用预制聚乙二醇 （PEG） 水凝胶简单建立血管化成骨骨髓生态位  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...