作者感谢UVM尸检服务对人类肺部采购和Robert Pouliot博士对整体解剖技术的贡献。这些研究得到了R01 HL127144-01（DJW）的支持。

所有动物研究均按照佛蒙特大学（UVM）的IACUC进行。所有人类肺均从UVM尸检服务中获得，并根据UVM的IRB指南进行相关研究。
注意:猪和人肺的去细胞化先前已由我们的组7,8,9,10,21描述。简而言之，使用蠕动泵用一系列 2 L 洗涤剂和酶溶液对气道和脉管系统进行顺序灌注，使整个肺叶脱细胞:0.1% Triton-X 100、2% 脱氧胆酸钠、1 M 氯化钠、30 μg/mL 脱氧核糖核酸酶/1.3 mM MgSO 4/2 mM CaCl2、0.1% 过氧乙酸/4% 乙醇， 和去离子水清洗。确认有效去细胞化的标准方法包括通过凝胶电泳测定脱细胞肺内<50 ng / mg残留的双链DNA，以及通过苏木精和伊红（H&E）染色9,21进行核染色。
1. 设置
<ol><li>收集解剖程序所需的所有必要设备，包括玻璃砂锅，两对手术镊子，一对镊子和一对手术剪刀，并在使用前高压灭菌。</li>
	<li>获取一部分肺，将其放入玻璃砂锅中，并定向，以便可以清楚地看到气道的上端。</li>
	<li>确定脉管系统的近端，并保持其完整，直到后续步骤。脉管系统的末端应清晰可见，并且颜色完全不透明。</li>
	<li>使用一把镊子和手术剪刀，去除可能衬在肺外部的任何胸膜并丢弃。</li>
</ol>2. 暴露气道
<ol><li>使用手术剪刀的铺展技术，轻轻地工作以暴露额外的气道。<ol><li>找到最大的气道，其直径通常约为 2-4 厘米。识别气道的另一种方法是通过观察软骨环，可以通过视觉或通过触诊组织 来 检测。</li>
		<li>使用一对镊子，触诊气道的长度，以确定看不见的气道的位置，深度约为 1 英寸。 注意:气道衬有软骨环，其特征是比其他肺组织更硬。因此，发现和触诊看不见的气道应该相对简单。</li>
		<li>将手术剪刀平行于气道，将闭合的尖端插入直接围绕看不见的气道的组织中。</li>
		<li>慢慢打开手术剪刀，轻轻拉开周围的膜。随后，取下手术剪刀，避免切割任何组织。</li>
		<li>在整个解剖过程中间歇性地重复此过程以继续暴露气道。</li>
	</ol></li>
	<li>使用手术剪刀，在分支点切割气道并沿任一分支独立解剖。 注意:分支点是一个气道分裂成两个独立气道的位置。</li>
	<li>一旦确信完整的末端仍然可识别并易于定位以进行进一步解剖，气道的切断区域就会被切断。</li>
	<li>将气道的切断区域放入相应的管中。切断区域的大小将根据样品而有所不同，但通常长度在 1-5 厘米之间。宽度根据沿气道树的相对位置而变化，远端区域的宽度比更近端的区域保持较小的宽度。</li>
</ol>3. 暴露和切除脉管系统区域
<ol><li>对脉管系统施加温和的压力，然后慢慢远离气道。让脉管系统稍微伸展，并使用手术剪刀进一步将脉管系统与气道分开。 注意:压力过大会撕裂脉管系统。如果脉管系统撕裂，只需将该部分脉管系统放入相应的标记管中并识别其完整端。</li>
	<li>当维管树中的分支点暴露时，使用手术剪刀和镊子暴露脉管系统的更多下部区域。<ol><li>首先将手术剪刀的闭合尖端插入分支点下方和两个相应的脉管系统区域之间。</li>
		<li>慢慢打开剪刀，将下面的组织分开。</li>
		<li>间歇性地使用一把镊子去除使用手术剪刀散开的组织，以及直接围绕脉管系统的任何其他组织。</li>
	</ol></li>
	<li>当脉管系统覆盖气道区域或对夹层操作中的任何步骤变得繁琐时，在分支点切割脉管系统，然后沿任一分支独立进一步解剖。</li>
	<li>一旦确信完整的末端将保持可识别并易于定位以进行进一步解剖，脉管系统的切断区域。</li>
</ol>4.识别和切除肺泡组织
<ol><li>使用一对镊子或镊子，捏住然后轻轻撕下牙槽组织的小区域。<ol><li>定位不在气道或脉管系统的直接附近的组织区域。</li>
		<li>使用镊子捏一小块似乎没有任何脉管系统或气道的组织区域。</li>
		<li>从肺部撕裂组织的受压区域。</li>
	</ol></li>
	<li>观察切除的组织区域并确认它是否是肺泡组织。 注意:肺泡组织存在于整个肺部，因此可以并且应该在整个解剖过程中将其去除。任何不容易识别为主要肺泡、脉管系统或气道的组织都应归类为散装组织，并放置在相应的标记管中。</li>
</ol>

区域脱细胞肺组织分离以研究细胞间和细胞间 ECM 相互作用

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作者均无任何利益冲突。

来自人类和其他物种的脱细胞组织经常被用作生物材料，用于研究离体培养模型中的ECM组成以及细胞-ECM相互作用，包括3D水凝胶12,13。与其他器官类似，脱细胞肺以前已被用于确定健康肺与患病（即肺气肿和IPF）肺中的ECM组成差异，并且越来越多地用作研究ECM动力学和细胞 - ECM相互作用的水凝胶7,8,9,10,11,14,15,16,1...

肺部疾病，包括慢性阻塞性肺病（COPD），特发性肺纤维化（IPF）和囊性纤维化（CF），目前仍未治愈1,2,3,4。肺移植通常是晚期患者的唯一选择，但由于提供合适的供体肺以及移植后的急性和慢性排斥反应，这仍然是一个有限的选择3,5,6。因此，迫切需要新的治疗策略。呼吸生物工程中一种有前途的方法是应用由去细胞化的天然肺组织制备的组织来源支架。由于无细胞全肺支架保留了天然细胞外基质（ECM）组成和生物活性的大部分复杂性，因此它们已被深入研究用于全器官工程，并作为研究肺部疾病机制的改进模型7,8,9,10。同时，人们越来越有兴趣利用来自不同器官（包括肺）的脱细胞组织作为水凝胶和其他底物来研究类器官和其他组织培养模型中的细胞-细胞和细胞-ECM相互作用11,12,13,14,15,16,17.这些提供了比市售底物（例如来自肿瘤来源的基质胶）更相关的模型。然而，目前关于人肺源性水凝胶的信息相对有限。我们之前已经描述了来自脱细胞猪肺的水凝胶，并表征了它们的机械和材料特性，并证明了它们作为细胞培养模型的实用性18,19。最近的一份报告详细介绍了源自脱细胞正常和患病（COPD，IPF）人肺的水凝胶的初始机械和粘弹性表征20。我们还提供了表征去细胞正常肺和COPD人肺糖胺聚糖含量的初始数据，以及它们在研究细胞 - 细胞和细胞 - ECM相互作用中的适用性11。
这些例子说明了将脱细胞人肺ECM用于调查目的的力量。然而，肺是一个复杂的器官，其结构和功能在肺的不同区域各不相同，包括ECM组成和其他特性，如硬度21,22。因此，研究肺部各个区域的ECM是有意义的，包括气管和大气道，中型和小型气道和肺泡，以及大，中和小血管。为此，我们开发了一种可靠且可重复的方法，用于解剖脱细胞的人肺和猪肺，并随后分离出每个解剖区域。这使得可以对正常肺和患病肺中的区域蛋白质含量进行详细的差异分析21。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Curved, S/S, Blunt, Serrated - 130mm</td>
			<td>CellPath</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Iris Forceps</td>
			<td>Fine Science Tools</td>
			<td>11373-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Glass Casserole Dish</td>
			<td>Cole-Parmer</td>
			<td>3175-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and isolation of region-specific decellularized lung tissue

肺移植通常是严重肺部疾病晚期患者的唯一选择，但由于合适的供体肺的供应以及移植后的急性和慢性排斥反应，这都是有限的。确定用于替代患病肺的新型生物工程方法对于提高患者生存率和避免与当前移植方法相关的并发症至关重要。另一种方法是使用缺乏细胞成分的去细胞化全肺，这些成分通常是急性和慢性排斥反应的原因。由于肺是一个如此复杂的器官，因此检查特定区域的细胞外基质成分（包括脉管系统，气道和肺泡组织）是有意义的。这种方法的目的是建立简单且可重复的方法，研究人员可以通过这些方法从完全脱细胞的肺中解剖和分离区域特异性组织。目前的方案是为猪和人类的肺设计的，但也可以应用于其他物种。对于该方案，指定了组织的四个区域:气道，脉管系统，肺泡和大量肺组织。与传统的批量分析方法相比，该程序允许获取更准确地代表去细胞化肺组织内容物的组织样本。

该协议的总体原理图如图1所示。一旦掌握，脱细胞肺组织的区域解剖很容易重现。确定每个切断组织样本的分类对于解剖程序的成功至关重要。血管组织比气道更具弹性，因此使用镊子拉伸组织通常是特定样本是脉管系统还是气道的有力指标。通常，血管组织平行于气道（图2A）。血管组织也往往比气道组织（图2C）更白和不透?...

Watch this Scientific Journal Video about Dissection and Isolation of Region-Specific Decellularized Lung Tissue at JoVE.com

作者聚焦:区域特异性脱细胞肺组织的解剖和分离  

这里介绍的是用于分离区域脱细胞肺组织的方案。该协议为研究细胞外基质和细胞-基质相互作用的复杂性提供了强大的工具。

区域特异性去细胞化肺组织的解剖和分离

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

脱细胞器官不断用于各种组织工程应用。然而，这些研究中的大多数都考虑了整个器官，而不是单个解剖区域。我们开发了这种方法来观察去细胞化人肺的单个解剖区域，以便为下游应用提供更精确的模型系统。在开发模型系统时，重要的是要考虑不同的设计方面，以便您的系统最好地概括正常的生物学。对于肺，这包括环境应力、循环机械应力和弹性等因素，这些因素在肺的独立区域之间可能有所不同。利用这种方法，我们已经能够证明来自健康和患病肺的单个解剖肺区域的细胞外基质具有不同的蛋白质组学特征。这使我们能够进一步了解肺部疾病，并有可能提出新的治疗途径。为了继续这项研究，我们的实验室目前正在开发来自健康和患病肺细胞外基质的三维水凝胶。这些水凝胶使我们能够进行体外类器官建模，以阐明细胞外基质相互作用对相应细胞行为的作用。

dissection-and-isolation-of-region-specific-decellularized-lung-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

5.1K 次观看.  University of Vermont. 脱细胞器官不断用于各种组织工程应用。然而，这些研究中的大多数都考虑了整个器官，而不是单个解剖区域。我们开发了这种方法来观察去细胞化人肺的单个解剖区域，以便为下游应用提供更精确的模型系统。在开发模型系统时，重要的是要考虑不同的设计方面，以便您的系统最好地概括正常的生物学。对于肺，这包括环境应力、循环机械应力和弹性等因素，这些因?...

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Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

科研

生物工程

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...