支持这项工作是由卵巢癌研究基金赠款LT/UIC/01.2011]，UIC的临床和转化科学，UIC的癌症中心，国家卫生部授予C06RR15482研究院的中心。 通过AAALAC认证的规定下批准的动物护理和使用委员会在UIC动物保健协议的准则和法规的规定进行了动物实验。这个项目的动物被安置在伊利诺伊州芝加哥大学的生物资源实验室（BRL）阻隔室。的BRL有一个充分的兽医人员，监控动物每天至少两次，并提供动物保健咨询。

1。卵巢解剖和组织隔离<ol><li>准备的海藻酸钠0.5％（W / V）的解决方案，准备从该协议的修改后的版本，描述以前5，使用无菌PBS和加热到37 ° C。混合倒置或摇晃，但不旋涡。绘制成1 ml注射器，海藻酸钠与25 G针，并保持在37 ° C。 </li><li> 10mm的无菌氯化钙溶液准备组织封装后交联海藻酸钠和温暖至37 ° C。 </li><li>准备1毫升培养液（αMEM+ 5个单位的青霉素和5微克/ ml链霉素），每口井在每个实验24孔板。包括各种药物，多肽，在培养液中或病毒。器官封装前进行转染，2000年与脂质体和质粒DNA四个小时孵化组织在37 ° C </li><li>解剖动物的器官，删除任何脂肪或残留组织，轻轻一拉，卵巢周围的滑囊膜，切成四大块平分的器官，卵巢或输卵管。动物安乐死的CO 2窒息颈椎脱位。对于输卵管，轻轻地解开组织拉除连接膜和扩展，在相反的方向之前，使用切割钳管的两端。 </li><li>放置到一个单一的海藻酸钠液滴（〜1-3μL），消毒的网状纤维。 </li><li>用无菌镊子，海藻酸钠液滴和中心的液滴，使用产钳或消毒针头移动到一块的器官。 </li><li>反转的雾滴含有网状纤维，用无菌镊子抓，把它倒过来，让重力力成悬滴海藻酸钠器官件（类器官）。 </li><li>塔上的一个10厘米培养皿含有10MM 氯化钙溶液，使溶液放入菜边缘的镊子。 </li><li>孵育2分钟，取出用无菌含有毛孔释放到培养基中额外的10毫米氯化钙2，转让的勺子。多达4个海藻酸凝胶可以被转移到一个含有培养基中板24井的单井。文化部规定的时间内。 </li></ol> 2。海藻酸钠降解和细胞集合<ol><li>海藻酸钙凝胶的增溶，孵化中的褐藻胶裂解酶（3.4毫克/毫升）溶解在37 ° C为30分钟或直到凝胶完全溶解在莱博维茨的L - 15媒体的文化。 </li><li>删除从褐藻胶裂解酶的组织。 </li><li>收集的OSE，孵化Leibovitz的L - 15的1小时8媒体与500单位的胶原酶类器官。涡在2个脉冲（2秒涡和2秒休息），中等功率为1分钟1秒脉冲（1秒涡和1秒休息1分钟）。让器官件落入新鲜无菌的离心管底部的离心管和移液器上清。在3000转颗粒卵巢表面上皮离心3分钟。 </li><li>收集的输卵管上皮，切管纵向和地方组织的DMEM培养液的塑料与5个单位青霉素和5微克/ ml链霉素和4％（V / V）FBS的。 3-5天，细胞就会“爬”出来的输卵管间质和组织培养plastic9增长。 </li></ol> 3。固定石蜡切片封装机关文化海藻酸钠<ol><li>收集在选定的时间点的培养基从封装珠海藻酸钠。 </li><li> Recrosslink海藻酸钠凝胶变硬两分钟，放入无菌10mm 的氯化钙溶液文化。 </li><li>修复器官在含有2％多聚甲醛50毫米蔗糖，10毫米氯化钙2和50毫米的钠cacodylate的海藻酸钠封装，以确保凝胶保持牢固和完全封装在固定的组织。 </li><li>组织处理，嵌入到整个石蜡，用切片机产生5微米的部分节海藻酸钠 - 封装器官培养片。 </li><li>海藻酸钠将被解散，并从幻灯片中删除，如果执行由于热量为基础的抗原修复免疫组织化学。海藻酸钠仍将站在苏木精和曙红染色，伊红染色下。 </li></ol> 4。代表性的成果： 三维器官培养的卵巢和输卵管的一个例子是如图1所示。卵巢可切成不同大小的块，和OSE的增殖和迁移到修复的表面，用手术刀切开伤员。在这个过程中，保持正确的方向的OSE相对底层的基质，与封装卵巢的OSE。正如图2所示，削减了一半的卵巢OSE，更彻底封装卵巢相比，卵巢切成宿舍或八分。卵巢创面修复在整个卵巢的情况下，可以研究表面，并可能阐明如何排卵和表面修复诱导卵巢表面改造。 实验的终点取决于所需的信息。使用石蜡切片组织结构，细胞形态，和特定的细胞生长率很容易监测。例如，卵巢表面上皮单层观察7天（图3A），而另外5微克/毫升胰岛素7天诱导卵巢表面上皮细胞过度增殖和形成的多层次培养后在无血清αMEM细胞（图3B）。溴脱氧尿嘧啶核苷加入终浓度在10微米24小时之前，以固定的文化，可以测量细胞的增殖（插图3A和3B）。在无血清αMEM保持文化，约3-5％的OSE进行期间24小时的标签的时期（插图3A）的增殖，而比例较大的颗粒细胞增殖。除了增加胰岛素文化传媒增殖的OSE总OSE，约30％（插图3B）的百分比。 cDNA转染（图3C），可以通过海藻酸钙凝胶与标准的显微镜观察绿色荧光蛋白（GFP）的免疫。虽然卵巢表面上皮细胞和初级卵泡的生长和分析提供海藻酸钠机关文化，应该指出的是，次级卵泡成熟，无法生存在这个文化体系（图3D）。个别卵泡成熟海藻酸钠水凝胶矩阵内的文化已在别处 10 。 从整个器官或从特定的细胞类型，用酶降解（图4），可以分析蛋白质或RNA的改变。治疗胶原酶培养卵巢organoids叶完好（图4C），同时允许在得到治疗后的细胞沉淀丰富的OSE底层基质，虽然一些基质细胞也隔离（图4D）。 <img alt="图1" src="/files/ftp_upload/2804/2804fig1.jpg" /> 图1小鼠卵巢三维培养系统，用于传播伤人后的OSE 。一）在显微镜下，布尔萨和周围的脂肪垫被拆除。二）完整的卵巢（海外）输卵管管（T）和脂肪垫（F）的。三）卵巢布尔萨和脂肪垫去除（左）和uncoiled输卵管（右）。四）卵巢或输卵管organoids放入海藻酸钠液滴。五）被投进一个氯化钙溶液交 ​​联形成凝胶的海藻酸钠海藻酸钠包裹organoids。 F）的海藻酸钠封装卵巢类器官。 G）的海藻酸钠封装输卵管类器官。 H）褐藻胶封装organoids在培养基中培养。 <img alt="图2" src="/files/ftp_upload/2804/2804fig2.jpg" /> 图2。卵巢表面细胞角蛋白8测量面积再生。卵巢organoids封装由被削减到八分，季度，半，在BSA的培养基中培养，并用细胞角蛋白8（CK8）抗体的免疫分析测定后的OSE。 <img alt="图3" src="/files/ftp_upload/2804/2804fig3.jpg" /> 图3。海藻酸钠文化系统允许通过生长因子的基因转染到的OSE，但不支持快速增长和成熟卵泡。一）卵巢类器官，7天CK8的表达，这标志着的OSE分析的基础培养基中培养。插图，串行部分BrdU掺入染色。 B）的培养基中加入胰岛素，导致乳腺增生的OSE。插图，串行部分BrdU掺入染色。三）卵巢类器官表达绿色荧光蛋白基因转染。四）海藻酸钠文化系统支持的原始卵泡（红色箭头）的增长，但尚未成熟的次级卵泡（黑色箭头）。 <img alt="图4" src="/files/ftp_upload/2804/2804fig4.jpg" /> 图4。OSE中可以分离分析以下文化。 A）类器官培养中等至2周。二）封装类器官被视为与褐藻胶裂解酶的不完整的类器官。卵巢与CK8染色标记的OSE。三）单独的OSE从底层基质胶原酶治疗类器官。剩余的一块组织与CK8染色切除卵巢表面突出。四）治疗后收集细胞丰富的OSE CK8（红色）染色。 counterstained用DAPI标记细胞核的细胞。

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有没有透露。

利用海藻酸钠水凝胶立体器官培养系统的发展代表了通用的方法，从多种生物体分析许多不同的组织类型。使用三维文化，可以扩展到组织工程和再生医学等领域，因为它提供了一个支架上的细胞能够增长11。目前，我们的实验室正处于初步研究使用人类卵巢和输卵管组织，但是，人类和非人类灵长类动物的毛囊的文化已被描述12,13。 利用海藻酸钠水凝胶的文...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> Leibovitz的L - 15培养基</td><td> Gibco公司</td><td> 11415 </td><td></td></tr><tr><td> αMEM中等</td><td> Gibco公司</td><td> 12000-022 </td><td></td></tr><tr><td>海藻酸钠</td><td> Novamatrix </td><td></td><td></td></tr><tr><td>钠cacodylate </td><td>西格玛爱秩序</td><td> C0250 </td><td></td></tr><tr><td>聚丙烯网状纤维</td><td>麦克马斯特卡尔</td><td></td><td> 45“宽辊; 0.0041”开幕</td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Gibco公司</td><td> 15070-063 </td><td></td></tr><tr><td>褐藻胶裂解酶</td><td>西格玛爱秩序</td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td>西格玛爱秩序</td><td> C9697 </td><td>从梭菌histolyticum </td></tr><tr><td>牛血清白蛋白</td><td> MP生物医学</td><td> 103700 </td><td></td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> Gibco公司</td><td></td></tr><tr><td>它的解</td><td>罗氏公司</td><td> 11074547001 </td><td>胰岛素/转/亚硒酸钠</td></tr><tr><td>细胞角蛋白8抗体</td><td>发展研究杂交瘤细胞银行</td><td> TROMA - 1 </td><td>在1:200的使用</td></tr><tr><td> DAPI </td><td>媒介实验室</td><td> H1200 </td><td></td></tr><tr><td>溴脱氧尿嘧啶核苷</td><td>西格玛</td><td> B50002 - 1G </td><td>终浓度10μM </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

卵巢癌是导致妇女因癌症死亡的第五，并已在美国的1 63％的死亡率。卵巢癌的起源的细胞类型仍然是有问题的，可能是卵巢表面上皮（OSE），或输卵管纤毛2,3远端上皮。培养原代培养的正常细胞在体外将帮助科学家模型的具体变化可能导致卵巢癌，在不同的上皮细胞，从而明确地确定起源的细胞类型。这将允许更精确的生物标志物，具有组织特异性基因的变化的动物模型，并针对本病的更好的预防策略的发展。 保持在海藻酸钠水凝胶中的正常细胞，促进细胞的体外培养中短期内其三维的背景下，允许引进的质粒DNA，RNA干扰，小分子。培养机关，从战略的削减使用手术刀，几种文化，从单一的器官可以产生，从单一的动物实验次数增加派生件。这些削减导致扩散的OSE，这是与卵巢癌的形成相关的排卵模型的各个方面。使用此方法可以培养的细胞类型，如OSE中不生长在塑料表面，并促进正常细胞过程中或在癌症形成4的最早事件进行调查。 海藻酸钠水凝胶可用于支持许多类型的组织5增长。海藻酸钠是一种线性多糖的重复单位，对β- D - mannuronic酸和α- L - guluronic酸可与钙离子交联，导致在一个温和的凝胶行动，不破坏组织6,7组成。像其他三维细胞，如基质胶文化矩阵，海藻酸钠为组织提供机械支持;但是，蛋白质不与海藻酸钠矩阵反应，并因此作为一种人工合成的细胞外基质，不会启动细胞信号5的海藻酸钠功能。海藻酸钠水凝胶漂浮在标准细胞培养液中中， 并支持在体外培养的组织生长的体系结构。 一种方法是提交到海藻酸钠水凝胶，可以在长达两个星期的文化保持的卵巢和输卵管器官件的制备，分离，嵌入。器官培养的蛋白质和RNA样品分析细胞酶的释放，也被描述。最后，主要的细胞类型增长是可能的，没有从小鼠遗传永生，并允许使用基因敲除和转基因小鼠的调查。

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

正常细胞在其三维背景的文化代表了一种替代方法研究所需的细胞转化和肿瘤的早期事件。这种方法是用来种植正常的卵巢和输卵管细胞研究在卵巢癌的形成早期事件。

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K 次观看.  University of Illinois at Chicago. 正常细胞在其三维背景的文化代表了一种替代方法研究所需的细胞转化和肿瘤的早期事件。这种方法是用来种植正常的卵巢和输卵管细胞研究在卵巢癌的形成早期事件。

视频: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

编制完整的牛尾巴器官培养的椎间盘

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术：新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造：一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...