笔者想承认"诱导多能干细胞的行为控制的组织工程支架材料"NSERC发现格兰特402462。

开始之前协议的注释:纤维蛋白原是血源性蛋白，从而适当的安全训练之前，必须处理完成。此协议需要2天才能完成所需的干文化，所以时间适当，以确保它们的播种准备。在计算多少纤维蛋白原权衡，三个35毫米培养皿菜TRIS缓冲液（TBS，pH值7.4），含有110-130毫克的纤维蛋白原溶解于3毫升的TBS将足以产生1 24孔板含400在每口井的微升纤维支架。 1。第一天:纤维蛋白原的解决方案和隔夜透析<ol><li>冻干纤维蛋白原的冰箱，让它坐了20分钟，以允许它来室温。 </li><li>加入3毫升的Tris缓冲液（TBS）每35毫米培养皿。每个板的纤维蛋白原产量2至3毫升的纤维蛋白原溶液浓度从12到20mg / mL和纤维蛋白原总额可以根据这些近似计算。 </li><li>称取约100-130毫克纤维蛋白原（Fg）和TBS电视台目前表面上洒在每一道菜。等待5分钟的纤维蛋白原开始进入解决方案。盖上盖培养皿。 </li><li>孵育培养皿菜在37°C的2个小时，使纤维蛋白原完全进入TBS的解决方案。 </li><li>湿透析管（7000兆瓦切断），用TBS。透析钳倍底端油管钳年底关闭。 </li><li>吸取纤维蛋白原的解决方案，从菜肴到透析管。倍多高端和透析钳钳关闭。 </li><li>成的TBS 4公升的容器中放置透析管含有纤维蛋白原的解决方案。混合溶液搅拌盘设置为低转速（100转）。 </li><li>隔夜透析溶液，搅拌至少12小时板。 TBS的解决方案并不需要通过这段时间内改变。 </li></OL&gt; 2。第2天:聚合纤维蛋白支架和干细胞播种到支架这个过程中描述的所有步骤应执行这些支架，将干细胞培养接种在无菌组织培养引擎盖。 <ol><li>删除到一个适当大小的锥形管（15毫升或50毫升）从透析管和地方的纤维蛋白原的解决方案。 </li><li> 5.0微米的注射器过滤除去溶液中大量杂质的过滤纤维蛋白原的解决方案。根据解决方案的体积，使用多个过滤器可能需要处理的整个解决方案。 </li><li>无菌过滤器的过滤用0.22微米到50毫升锥形管的纤维蛋白原的解决方案。使用适当的大小吸管，确定过滤纤维蛋白原的解决方案，以备后用做计算时的音量。根据解决方案的体积，可能需要使用多个过滤器处理耳鼻喉科愤怒的解决方案。 </li><li>测量用分光光度计在280 nm的纤维蛋白原溶液的吸光度。一个50的稀释因素往往是必要的情况下取得1吸光度。 </li><li>纤维蛋白原解决方案，使用下列公式计算:[毫克/毫升血浆纤维蛋白原浓度的蛋白质目前] =（280×稀释因子）÷1.55，其中1.55是在一个280人纤维蛋白原13消光系数。接下来计算所需的TBS稀释浓度为11.1毫克/毫升的初始音量根据纤维蛋白原的解决方案。终浓度的蛋白质纤维蛋白支架将聚合后的10毫克/毫升。 </li><li>获取无菌凝血酶和氯化钙解决方案。首先，添加15μL的凝血酶溶液（浓度为40 U / ml的 - 在脚手架终浓度将是2个U / mL）的右侧第一排的24孔板中，每口井。接下来，添加15μL50毫米钙CHLoride解决方案，以每口井的左侧。最后，添加270μL的纤维蛋白原溶液的板块。摇板从侧面一边摇晃后到前的解决方案，以确保混合。 </li><li>让行包含聚合混合物在室温下5分钟。棚架变得更加不透明，为他们聚合。 </li><li>重复步骤2.6和2.7，直到所需数量的纤维蛋白支架已聚合。 </li><li>最后5分钟的潜伏期后，放在里面的37℃培养箱板，棚架，以便完全聚合。 </li><li>经过一个小时的潜伏期是完整的，下一步就是种子胚体的脚手架。使用20μLpipettemen，选择个别胚体中的纤维蛋白支架的中心地点。用显微镜检查，每口井包含一个单一的胚体。 </li><li>添加5μL凝血酶溶液（浓度为40 U / ml的）和5μL50毫米氯化钙纤维蛋白原的解决方案，以每孔90μL每个胚体顶部的解决方案。该解决方案应形成泡沫封装胚体。 </li><li>现浇板早在37°Ç孵化器增加一个小时，以确保聚合。 </li><li>孵化后，合适的细胞培养基添加1毫升，每口井的地方板成37°C间孵化器。 </li></ol> 3。代表结果 图1显示了个人以及包含的三维纤维支架文化系统种植胚体。 图2A侧视示意图显示鼠标诱导多能干细胞培养小鼠胚胎成纤维细胞饲养层的代表图像。然后，这些细胞被诱导形成胚体，含有神经祖细胞的集合体，用8天的维甲酸治疗协议（ 图2B）previouslŸ描述14，而图3显示的IPS衍生的胚体经过3天的文化内的三维纤维蛋白支架的外观。已取得了类似的结果，以前用小鼠胚胎干细胞7。此外，这种方法已被用于文化IPS-衍生的胚体制作使用不同的协议，涉及维甲酸和purmorphamine的15三维纤维支架内，展示这个文化系统的多功能性。 <img alt="图1。" src="/files/ftp_upload/3641/3641fig1.jpg" /> 图1。示意图显示的个人以及三维纤维支架种子含胚体的侧视图。 <img alt="图2。" src="/files/ftp_upload/3641/3641fig2.jpg" /> 图2。小鼠诱导多能干细胞（iPS）细胞培养和分化 。这些细胞分化成neur人的表型，iPS细胞悬浮培养产生细胞的集合体称为胚体（EBS）。这些胚，然后用维甲酸诱导神经细胞分化和本议定书以前用来诱导胚胎干细胞的神经分化。一）未分化的小鼠iPS细胞集落培养对小鼠胚胎成纤维细胞饲养层。 b）从小鼠iPS细胞的悬浮培养的胚体维甲酸治疗后第8天，诱导神经细胞分化。比例尺是100微米。 <img alt="图3。" src="/files/ftp_upload/3641/3641fig3.jpg" /> 图3示例结果的小鼠iPS胚状体的三维纤维支架内经过3天的文化机构。细胞迁移和分化的纤维蛋白支架内已经开始。比例尺是500微米。

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作者没有透露。

上面详细介绍该协议产生的多能干细胞培养小鼠胚胎和诱导多能干细胞的三维纤维支架，专门提供了一个方法。这三维生物材料为基础的文化系统，更准确地模仿体内发现的干细胞小生境，作为一个结果，它可以用来筛选生物的线索，以确定对干细胞分化6的影响。我们的观察表明，干胚体细胞在体外培养2周保持完全降解之前，播种时，这些支架。为了进一步提高这些支架的稳定，...

所需设备: 天平 pH计 组织文化的孵化器（37℃，5％CO 2） 搅拌板 分光光度计 无菌组织培养罩 Tris缓冲液（pH值7.4）（需要4升，加上​​足够的溶解纤维蛋白原） 50毫米的氯化钙溶液 无菌锥形管（15或50毫升） 35毫米培养皿 透析管（7,000兆瓦截止） 透析剪辑 5.0微米的针头式过滤器 单独包装的无菌0.22微米针头式过滤器 注射器 24孔组织培养板<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>纤维蛋白原（人） </td><td> calbioc下摆</td><td> 341578 </td><td></td></tr><tr><td>凝血酶（人） </td><td>西格玛</td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

干细胞被发现在三维微环境中自然产生的体内 ，它通常被称为干细胞小生境1。三维生物材料支架内培养干细胞提供了一种方法，准确地模仿这些微环境，提供文化，用聚苯乙烯以及更换工程的组织2的方法比传统的2D方法的优势。虽然已为大多数细胞培养实验中使用的二维组织培养聚苯乙烯，三维生物材料支架可以更密切地复制在体内环境中，使更准确的细胞极性建立和拥有生化和机械性能类似于软组织的微环境。 3一个多种自然派生和合成生物材料支架已被用于支持干细胞生长的3D环境调查。虽然合成支架材料可以合成有一个更大的ŕ安格机械和化学性能，往往有更大的重复性，通常由天然生物​​材料在细胞外基质含有细胞粘附的结合位点，并随时支持细胞培养的结果发现蛋白质和多糖。纤维蛋白支架，通过聚合获得的血浆蛋白纤维蛋白原生产，已被广泛研究了各种组织工程的应用， 在体外和体内 4。这种支架可以修改使用多种方法纳入控释系统提供治疗的因素5。以前的工作表明，这种支架可以用来成功地培养胚胎干细胞，并基于此脚手架的文化系统，可用于筛选内6,7种子的干细胞分化的各种生长因子的影响。 该协议的细节过程polymerizinĞ纤维支架纤维蛋白原使用凝血酶酶活性的解决方案。这个过程需要2天才能完成，包括纤维蛋白原的解决方案，以消除，抑制聚合的柠檬酸隔夜透析的步骤。这些详细的方法依赖于胚胎和诱导多能干细胞培养确定为最佳的纤维蛋白原浓度。其他团体作进一步调查的细胞类型和应用范围广泛的纤维蛋白支架-证明了这种方法的8-12的多功能性。

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

这项工作的具体编制三维纤维支架plutipotent干细胞培养和分化。这种支架可用于筛选各种生物化合物对干细胞的行为的影响，以及修改为包含药物输送系统。

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Research

JoVE Journal

Bioengineering

25.1K 次观看.  University of Victoria. 这项工作的具体编制三维纤维支架plutipotent干细胞培养和分化。这种支架可用于筛选各种生物化合物对干细胞的行为的影响，以及修改为包含药物输送系统。

视频: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

科研

生物工程

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

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 ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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 ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...