This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1，创建PNIPAAM涂层板的<ol><li>解散2.6克PNIPAAM的2毫升60％甲苯/ 40％正己烷溶液。 </li><li>加热到60℃，将混合物搅拌10分钟，直到PNIPAAM溶解。 </li><li>切滤纸为60毫米直径的圆和地点纸张布氏漏斗。 </li><li>过滤该溶液，通过用布氏漏斗放入预先称重的玻璃烧杯（不使用塑料，如己烷将熔化的塑料）。 </li><li>将烧杯中，内容为钟真空（24磅）的O / N（16小时）。注：直到残留物用异丙醇反应，它会氧化所以一定要确保它不与氧气接触。 </li><li>称量烧杯建立PNIPAAM的重量。 </li><li>添加异丙醇的PNIPAAM，创造一个各占50％w / w的解决方案。 </li><li>放置2毫升在UV光下的溶液中的组织培养板的表面上，并且涂层为5分钟的。 </li><li>用2毫升温的PBS洗板两次使用的细胞培养之前。 </li></ol> 2，创建细胞片的注：，或通过如这里描述的涂覆的组织培养表面与热响应性聚合物可以使用许多不同的方法来创建的初级细胞对靶器官的细胞片层。预涂温敏板是由一些厂商也提供。 注：该协议是使用35毫米的菜文化。简言之，将细胞首先在37℃下进行至少24小时，在合流建立相邻单元之间的横向连接。释放细胞片材，板材受到温度低于32℃。细胞片，然后转移到含有新生血管容许水凝胶与血管内皮细胞的强碱纤维状基质。 <ol><li>分离的细胞群。注意：此方法是依赖于个体的推导过程和细胞类型。大鼠主动脉平滑肌万亩SCLE细胞（RASMC）被用于本实例。这些是主要的平滑肌细胞从大鼠的腹主动脉分离。 </li><li>用2毫升温的PBS洗细胞。 </li><li>加入3 ml胰蛋白酶（或其他切割/解离溶液）向细胞5分钟。 </li><li>通过加入3毫升含10％胎牛血清（FBS）的培养基中，或磷酸盐缓冲液（PBS）的抑制的胰蛋白酶。 </li><li>收集细胞在锥形管和计数等分。 </li><li>旋转细胞以1000rpm（228 XG）5分钟。 </li><li>吸出上清液并将细胞重新悬浮在生长培养基（SmGM2加子弹试剂盒培养基用于RASMC）。 </li><li>将含有细胞的培养基上35毫米热敏制版 - PNIPAAm的涂层板，其浓度将达到100％汇合。注意：对于RASMCs该号码被确定为100000细胞/ cm 2。然而，由于细胞的传递过程中的损失，120是VA的％<html>略被使用。 </li><li>放入培养箱中，在37℃CO / N。注意：维持细胞在37℃下维持细胞粘附到板是重要的。 </li></ol> 3，准备基础性矩阵注意：各种三维纤维基质可用于层中的微妙细胞片层之间强的纤维基体。一些例子包括：明胶海绵，生物玻璃，天然脱细胞物质26或nanospun材料27,28在这些研究中所用的猪膀胱基质层（UBM）的慷慨从我们的合作者，Badylak医生提供29。 <ol><li>在使用之前，确定矩阵特征包括缺乏细胞含量如果脱细胞基质的情况下，27,28细胞特异性存活率，和空隙空间22中</li><li>切断已预先灭菌的基质成所需的尺寸和形状。注：在这里，打孔用于切割4毫米直径的圆。 </li></ol>ve_title“&gt; 4。播种内皮细胞成新生血管宽容的水凝胶注意：内皮细胞可从各种来源，包括从干细胞或祖细胞分化而得到。这里，的HuVECs被使用。 <ol><li>使用任何允许的水凝胶（血纤蛋白，胶原蛋白凝胶），只要交联时间短得足以使细胞保持活力。注意：在此，透明质酸（HA）的基于凝胶的交联与二硫键被使用。 </li><li>准备医管局水凝胶按照公司的协议。 </li><li>收集内皮细胞和成分散使用1X胰蛋白酶单细胞溶液。注意：的Accutase或细胞解离缓冲液也可用于单细胞分散。 </li><li>用大豆胰蛋白酶抑制剂等量或在PBS中的10％FBS，收集该溶液/细胞入15ml锥形管中（如果该细胞不接触到血清接触是很重要的）去激活胰蛋白酶的酶。 </li><li> ç指望。2-15的细胞，并计算出所需要的补丁尺寸的体积（先前量化）。注意：对于4毫米的补丁，这里200万内皮细胞使用。 </li><li>提取200万细胞，并放入一个新的15 mL锥形管。 </li><li>转速为（228 XG）5分钟。 </li><li>吸出上清液，留下细胞在锥形管的颗粒。 </li><li>混合HA和明胶液体物料在1:1比值。然后加入80％总体积的成锥形管含有沉淀。 </li><li>重悬内皮细胞在1:1的HA /明胶混合</li><li>将悬浮的细胞在HA /明胶混合物制成从步骤2中的碱纤维基体。 </li><li>加入所需的交联剂的总体积的五分之一（20％）的</li><li>孵育1小时，在37℃。 </li></ol> 5，隔离单元表的<ol><li>除去含有从培养箱和地方的细胞35毫米PNIPAAM处理板中的细胞培养罩在RT。 </li><li>从细胞中迅速吸出介质，并加入2毫升6％正常明胶已被加热到37℃。 </li><li>而明胶仍然温暖时，将金属晶格入明胶，淹没它的正常明胶（电影1）的表面之下。 </li><li>放置5-7分钟，整个板在冰上，使明胶硬化。 </li><li> 7分钟后，用刮勺从板的侧面仔细分离明胶边缘，然后用钳子抬起金属晶格从板注：6％的明胶，和细胞片应该解除与晶格。 </li><li>将电池板的菜，并放置在基体的纤维基质的水凝胶组合的顶部，仔细设置在构建体的上方的格子。注：电池板的顶面仍然会在榜首的位置。 </li><li>加入2毫升温介质（37℃）。 </li><li>孵育O 2 / N使细胞的薄片粘附到水凝胶的表面上。 </li><li>除去叔后的溶液升温（约1小时），或在第二天，他的金属晶格。 </li></ol>

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We have nothing to disclose.

在该协议中的关键步骤包括：涂布板面与温敏聚合物和冷却板后操纵的细胞片层。因为不同的细胞表现出不同的物理性质，如粘合性，升降时间应为每个不同的细胞类型进行了优化。本协议的第二个，也是最显著挑战性成分，集中在电池板，为组织组装方法的一个重要方面的操作。在电池板的单细胞层是相当脆弱的，并且可以很容易地撕开，如果操作用钳子。此外，当细胞片层中的地方不被保持?...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:207px;">Table of Reagents:</td>
			<td style="width:128px;"></td>
			<td style="width:140px;"></td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:140px;">Catalogue number</td>
			<td style="width:221px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcein-AM</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">C3099</td>
			<td style="width:221px;">Cell tracker / live dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Lysotracker Red</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">L7528</td>
			<td style="width:221px;">Cell tracker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Neutral Red</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:140px;">N7005</td>
			<td style="width:221px;">Visible Cell dye</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">pNIPAAM</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">412780250</td>
			<td style="width:221px;">Poly(N-isopropylacrylamide)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Toluene</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">244511-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Hexane</td>
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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

许多组织中，如成人心，都无法破坏后，以充分再生。2,3策略在组织工程中提出的创新，以协助身体恢复和修复。例如，TE方法可能能够减轻心肌梗死（MI）后心脏重塑和可能增加总的心脏功能，以接近正常的预MI水平。4与任何功能性组织，心脏组织的再生成功涉及适当的递送多种细胞类型与环境因素有利于整合植入的细胞/组织移植和生存。工程化组织应处理的多个参数包括：评价为运载工具，其对细胞存活的影响，材料强度和细胞 - 组织机构的便利可溶信号，细胞 - 细胞相互作用，并且基质材料。研究采用直接注射移植的细胞不仅无视这些基本要素。2,5,6一种组织设计结合这些成分还有待开发。这里，我们提出的使用图案化的细胞片层的分层与两种不同类型的含有靶器官的细胞类型和内皮细胞用于增强新血管形成中的“组织”生物衍生材料集成设计的例子。虽然这些研究集中在心脏样组织的产生，此组织设计可以应用于许多器官比心脏以最小的设计和材料的变化等，并意指是断开的，现成的产品用于再生疗法。该协议包含五个详细步骤。一种温度敏感聚（N -isopropylacrylamide）（PNIPAAM）用于涂层的组织培养皿。然后，特定组织细胞的涂装板/微图案的表面的表面上培养，以形成细胞片层具有很强的横向粘连。第三，基础矩阵的组织由多孔基体结合新生血管permissi创建已经凝胶和内皮细胞。最后，将细胞片从PNIPAAm的涂覆菜肴解除，并转移到基座元件，使得整个结构。

的流程图（ 图1）示出了制作的多层片的整体方法。细胞片从PNIPAAM处理板通过降低温度低于32℃的分离。然后将细胞板置于含有接种到底层的纤维基体（ 图1）在内皮细胞中的交联水凝胶的顶部。经预处理的温敏板，也可用于创建细胞片。特殊的拓扑表面使用专门的模式（ 即对齐）单元30。 基底的纤维基质可以由脱细胞天然的组织基质?...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.3K 次观看.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

视频: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

科研

生物工程

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...