Name: lung decellularization and recellularization
Uploaded: 2023-04-30T06:33:07.000Z
Duration: 8 min 45 s
Channel: JoVE

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体外培养部分或完整器官常用于在各种测试条件下精确模拟组织和器官的功能。完整器官培养可能涉及对切除器官进行去细胞化&#160;或去除细胞&#160;，以利用器官的天然结构。随后通过重新接种新细胞实现再细胞化。在再细胞化过程中，通常会使用专用的生物反应器&#160;来模拟体内组织的生长环境&#160;。本视频将介绍完整器官组织培养的基本原理，并演示实验室中的操作流程。
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该过程始于获取供体器官。在此示例中，我们展示了一只猴子的供体肺。通过一种称为去垢剂灌注的方法，利用一系列冲洗步骤系统性地清除离体器官原有的细胞成分，从而获得无菌的无细胞器官基质。接下来，使用特定类型的细胞对组织基质进行再细胞化，例如已建立的干细胞系。这些细胞也可来自接受该工程化组织的个体，称为自体细胞移植。这种方法可降低排斥反应，并提高器官的生物相容性。或者，也可使用来自其他供体的细胞，称为同种异体移植。当无法从潜在受体获取足够数量的细胞时，可能需要采用此方法。细胞接种到器官上后，利用组织生物反应器刺激细胞增殖并引导组织生长。这些反应器旨在对器官进行动态培养，以模拟体内原有的微环境。例如，可将器官连接至蠕动泵，以模拟血流。现在您已了解器官培养的基本原理，接下来让我们来看一个具体操作示例——供体肺的全器官培养过程。
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首先，将供体肺置于解剖托盘中，通过将一个雌性鲁尔接头插入开放的腔内来插管肺动脉。第二个雌性鲁尔接头&#160;然后插入气管切口。磷酸盐缓冲液（PBS）&#160;含30单位/毫升肝素和5微克/毫升硝普钠&#160;随后注入溶液以促进血管扩张，并清除肺内滞留的空气。溶液通过自然回缩排出&#160;并重复操作两次，以使溶液保留在肺部，溶解残留的血液。随后，切开双侧心房，并取下气管卢尔接头导管帽&#160;以促进液体引流。继续用PBS、肝素和硝普钠溶液进行灌注&#160;直到尽可能多地清除肺血管系统中的血液。开始脱细胞处理时，将肺组织充气并浸透去离子水。经过五次动脉和血管冲洗后，将肺组织从水中取出，并浸入一种名为Triton的去垢剂中&#160;以尽量减少对器官基质的影响，用Triton溶液再次充盈肺部两次&#160;在4摄氏度下孵育过夜前，肺组织用新鲜去离子水再清洗五次。孵育后，将肺组织浸入2%脱氧胆酸钠溶液中，随后再多次清洗。&#160;用水和缓冲液进行脱细胞处理并清除细胞碎片。当器官完全清洁后，将其保存在无菌的PBS溶液中，于4摄氏度下直至使用。
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为了模拟肺部的自然行为，可以使用一种专用的生物反应器，如此处所示。首先，将反应器的主腔室填充已与5%二氧化碳气氛平衡的培养基。然后安装器官。连接完毕后，盖上盖子，并使用注射器从管路中完全抽出所有空气&#160;。随后将生物反应器转移至组织培养孵育箱中进行平衡。接下来，以大约每两分钟一次完整呼吸的频率对肺进行通气，同时通过蠕动泵以大约10毫升/分钟&#160;的速度使培养基在血管系统中循环，总时长为30分钟。
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对于气道接种，需用含有骨髓来源间充质干细胞的细胞悬液使肺组织膨胀&#160;，以实现肺泡的再细胞化，肺泡是肺内进行氧气与二氧化碳气体交换的部位。随后将肺组织过夜孵育&#160;，以使细胞附着于脱细胞基质上。过夜孵育后，重新启动通气，并在器官基质中继续培养数天，以促进细胞生长。接下来，通过蠕动泵&#160;逐步引入内皮细胞完成血管接种，以启动小血管&#160;的再细胞化，随后静态培养数小时&#160;，以促进形成结构准确的细胞化器官。再次循环培养基，并在动态条件下继续培养一周，以促进细胞生长和附着。当组织生长完成后，进行组织学分析&#160;，以确认间充质干细胞和内皮细胞&#160;在器官血管系统和气道中的附着与生长情况。组织学结果显示，间充质干细胞和内皮细胞已附着于基质支架内的肺泡以及小血管结构上，呈现出类似天然肺组织的形态。
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现在您已经了解了全器官培养技术，接下来让我们看看该技术在再生医学和器官替代这一主要应用领域之外的一些实际应用。全器官培养还可用于测试药物制剂或药物递送装置。例如，在这项研究中，研究人员将小鼠胚胎甲状腺组织取出并进行培养，将其作为器官模型，用以观察实验性药物制剂在器官和组织中的转运过程。这种模拟最终可能更真实地反映药物在体内器官中的转移方式。此外，全器官组织培养还可用于研究组织在不同条件下的行为特征。例如，研究人员从牛尾中获取椎间盘组织，以探究椎间盘退变的可能机制。通过使用专门设计的生物反应器&#160;对椎间盘施加机械负荷，从而更深入地了解这些负荷如何影响退变过程。
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您刚刚观看了 JoVE 关于全器官组织培养的视频。现在您应该了解如何在体外培养整个器官，以及该技术在生物工程领域的应用。感谢观看。
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全器官组织培养

Whole Organ Tissue Culture

整个器官可以进行培养 离体 使用专用生物反应器&#160;旨在修复或替换整个器官。该方法利用供体器官，去除其所有细胞，仅保留三维结构，&#160;随后用新细胞重新填充。本视频展示了肺器官的全器官培养&#160;并说明需要一种模拟体内机械刺激的动态培养环境，才能诱导出天然组织的特性。

整个器官可以进行培养&#160;离体&#160;使用专用生物反应器&#160;旨在修复或替换整个器官。该方法利用供体器官，将其所有细胞去除，仅保留三维结构，&#160;随后用新细胞重新填充。本视频展示了肺器官的全器官培养&#160;并表明需要一种能够模拟体内机械刺激的动态培养环境，以诱导出天然组织的特性。

完整的器官可以在体外利用特制的生物反应器进行培养，目的是修复或替换整个器官。该方法使用供体……

部分或完整器官的体外培养常用于在各种测试条件下精确模拟组织和器官的功能。全器官培养可能涉及切除器官后的去细胞化，或去除细胞以利用器官原有的结构，随后用新细胞进行再细胞化。再细胞化过程通常结合使用专用的生物反应器，以模拟体内组织的生长环境。本视频将介绍全器官组织培养的基本原理，并在实验室中演示该操作流程。该过程始于获取供体器官。在此示例中，我们展示了一只猴子的供体肺。通过一种称为去污剂灌注的方法，对分离的器官进行一系列洗涤，系统性地清除其原有的细胞群体，从而获得无菌的无细胞器官基质。接下来，使用特定类型的细胞（例如已建立的干细胞系）对组织基质进行再细胞化。这些细胞也可来自接受该工程化组织的个体，称为自体细胞移植。这可以减轻排斥反应，并提高器官的生物相容性。 alternatively，可使用来自不同供体的细胞，称为同种异体移植。如果无法从潜在受体身上获取足够数量的细胞，则可能需要采用此方法。当细胞接种到器官上后，利用组织生物反应器刺激细胞增殖并引导组织生长。这些反应器旨在动态培养器官，并模拟体内原有的微环境。例如，可将器官连接至蠕动泵以模拟血液流动。现在您已了解器官培养的基本原理，接下来让我们来看一个涉及供体肺全器官培养的示例操作流程。首先，将供体肺置于解剖托盘中，通过将一个雌性鲁尔接头插入开放的腔内来插管肺动脉。随后将第二个雌性鲁尔接头插入气管开口。然后灌注含有每毫升30单位肝素和每毫升5微克硝普钠的磷酸盐缓冲盐水（PBS），以促进血管扩张并清除肺内滞留的空气。液体依靠肺组织的自然回缩力排出，并重复此过程两次，之后封闭插管以使溶液保留在肺内，溶解任何残留的血液。接着切开左右心房，并移除气管鲁尔插管的帽盖，以促进液体引流。继续使用PBS、肝素和硝普钠溶液进行灌注，直至尽可能清除肺血管系统中的血液。开始脱细胞处理时，用去离子水充盈并渗透肺组织。经过五次动脉和血管冲洗后，将肺从水中取出并浸入一种名为Triton的去垢剂中，以去除细胞成分，同时最大程度保留器官基质。再用Triton溶液对肺组织进行两次充盈，随后在4°C条件下孵育过夜。孵育结束后，用新鲜的去离子水再冲洗五次。接下来，将肺浸入2%脱氧胆酸钠溶液中，然后用清水和缓冲液进一步多次冲洗，以促进脱细胞过程及细胞碎片的清除。当器官完全净化后，将其保存于4°C的无菌PBS溶液中，直至使用。为了模拟肺部的自然行为，可以使用一种专用的生物反应器，例如此处所示的装置。首先，将反应器的主腔室填充已与5%二氧化碳环境平衡的培养基。然后安装器官。连接完毕后，盖上盖子，并使用注射器通过管路抽出所有空气。随后将生物反应器转移至组织培养孵育箱中进行平衡。接下来，以大约每两分钟一次完整呼吸的频率对肺部进行通气，同时通过蠕动泵以约10毫升/分钟的速度使培养基在血管系统中循环，持续30分钟。对于气道接种，需用含有骨髓来源间充质干细胞的细胞悬液对肺部进行灌注充气，以实现肺泡的再细胞化，肺泡是肺内进行氧气与二氧化碳气体交换的部位。随后将肺组织过夜孵育，使细胞能够附着于脱细胞基质上。过夜孵育后，重新启动通气，使细胞在器官基质中生长数天。接下来，通过蠕动泵逐步引入内皮细胞完成血管接种，以启动小血管的再细胞化，之后静态培养数小时，以促进形成具有准确细胞结构的器官。再次循环培养基，并在动态条件下继续培养一周，以促进细胞生长和附着。当组织生长完成后，进行组织学分析，以确认间充质干细胞和内皮细胞在器官血管系统和气道中的附着与生长情况。组织学结果显示，间充质干细胞和内皮细胞已成功附着于基质支架内的肺泡以及小血管结构上，呈现出类似天然肺组织的形态。现在您已经了解了器官整体培养技术，接下来让我们看看该技术在再生医学和器官替代以外领域的一些实际应用。器官整体培养还可用于测试药物制剂或药物递送装置。例如，在这项研究中，研究人员将小鼠胚胎的甲状腺组织分离并进行外植体培养，作为器官模型，用以观察实验性药物制剂在器官和组织中的转运过程。这种模拟实验最终可能更真实地反映药物在活体器官内的转移情况。此外，器官组织整体培养还可用于研究组织在不同条件下的生物学行为。例如，研究人员从牛尾中获取椎间盘组织，以探讨椎间盘退变的可能机制。通过使用专门设计的生物反应器对椎间盘施加机械负荷，以深入理解这些力学负荷对退变过程的影响。您刚刚观看了 JoVE 关于全器官组织培养的视频。现在您应该了解如何在体外培养整个器官，以及该技术在生物工程领域的应用。感谢观看。

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整体器官组织培养

lung decellularization and recellularization

Whole organs can be cultured ex vivo using specialized bioreactors to support repair or replacement of entire organs. In this approach, a donor organ is first stripped of all cells. What remains is the three-dimensional structure of the organ, which can then be repopulated with new cells.

The lung is the example shown here. The method uses a dynamic culture system that provides mechanical stimulation, similar to what the organ experiences in the body. This type of culture helps induce native tissue properties in the developing lung tissue.

Lung Decellularization and Recellularization

lung-decellularization-and-recellularization

overview of biomaterials

Biomaterials in Medicine and Engineering

Biomaterials are materials designed to interact well with biological organisms or molecules. They may come from an organism, be produced by an organism, or be made as a synthetic polymer. Because of these properties, engineers use biomaterials in medicine and other biological applications. These materials are used in tissue engineering, biosensing, and drug delivery. Tissue engineering uses materials to support the growth or repair of tissues. Biosensing uses materials to detect biological...

collagen hydrogels

Porcine Collagen for Tissue Engineering

Collagen is a useful biomaterial with uses that go beyond the lab. It has also found commercial use in photography. In tissue engineering, collagen can be turned into hydrogels that give engineered tissue support and structure. This page follows the path from porcine skin to a collagen hydrogel. Porcine skin is used as the source of collagen. The material is then prepared for use in tissue engineering applications. The video also shows several applications of collagen and the methods used to...

electrospinning of silk biomaterials

Silk Cocoon Processing for Fiber Mats

Silk cocoons can be processed into a soluble material that is useful for making biomaterials. Silk fibers have been used for fabrics and threads for centuries, but turning them into a versatile pre-polymer solution is a newer technique. That solution can then be shaped into materials with controllable mechanical properties. The process begins with silk worm cocoons. The silk is solubilized, or dissolved, so it can be handled as a liquid-like solution. The video then shows how that silk...

overview of biomem devices

BioMEMs in Diagnostics and Miniaturization

Bio-microelectromechanical systems, or BioMEMs, are microscale devices used in diagnostic settings. They work with very small sample and reagent volumes in vivo and in vitro, which means inside a living body or outside it in a lab. These compact systems support bioengineering research and help make diagnostic tools more efficient. BioMEMs can carry out functions such as filtration, sensing, and synthesis on a tiny scale. Using less material can lower costs and improve sensitivity. The...

microfabrication via photolithography

Photolithography for Silicon Wafer Patterning

Photolithography is a common microfabrication method used to pattern a silicon wafer. It uses light to transfer a design onto the wafer surface. This process helps form the basis for many BioMEM devices. The page also shows how photolithography is carried out in a cleanroom. Cleanroom steps are important because they support careful fabrication of small, detailed features. The video ends by introducing applications of this process in BioMEM device production.

soft lithography

Photolithography for Microfluidic Device Making

Photolithography and soft lithography are used to make BioMEM devices such as microfluidic channels. A microscale pattern is copied by curing an elastomeric polymer over a 3D structure. The result is a polymer piece that can serve as part of a biological device. These polymer structures are then used to build a range of tools. Some are microfluidic channels for biosensing applications. Others are microscale bioreactors that let scientists visualize micro-colonies. The process also shows how...

overview of bioprocess engineering

Bioreactors and Protein Production in Bioprocessing

Bioprocessing uses living organisms to make a desired target product. In many cases, that product is a protein made in a bioreactor, which is a controlled vessel for growing cells. This process often relies on genetically engineered organisms. Engineers design the system so the cells can produce proteins at large scale. That approach is central to making biotherapeutics, which are drugs made from biological materials. Biotherapeutics are important medicines for complex diseases such as...

synthetic biology

Engineering Cells to Produce Proteins

Synthetic biology is a bioengineering method for programming cells to make useful products. In many cases, the goal is to genetically modify an organism so it can produce large amounts of a protein. That protein may already be made by the cell, or it may come from a newly inserted DNA sequence. The process depends on changing an organism’s genetic material. This is done with transformation or transfection, two common ways to introduce DNA into cells. The video also shows how this work is...

batch and continuous bioreactors

Growing Cells in Bioreactors

Bioreactors are used to grow organisms in large volumes. This makes it possible to produce large amounts of a target product. In laboratory and bioengineering settings, they provide a controlled place for cell growth and product formation. The two main reactor types are batch reactors and continuous reactors. A batch reactor contains all of the materials needed for cell growth at the start. A continuous reactor has inlet and outlet ports, so fresh growth media can be added while cell waste is...

overview of biosensing

How Biosensors Detect Molecules

Biosensors detect biological molecules and chemical contaminants by combining biology with physical measurement. They can identify targets such as proteins or cells, as well as non-biological molecules like chemicals or contaminants. This makes biosensing a useful field for studying and monitoring many kinds of samples. These devices rely on different signal types to find the target molecule. Some biosensors use electrical signals, while others depend on optical, electrochemical, or mechanical...

electrochemical biosensing

Glucose Sensors and Cancer Detection

Electrochemical biosensors detect target molecules by measuring an oxidation-reduction event. That signal changes when a molecule binds to the sensor surface. These sensors helped shape modern biosensing after the first glucose biosensor was invented. The glucose biosensor is a key example of this technology. It shows how electrochemical sensing can track a biological target through a measurable electrical change. The same basic idea also helps explain how electrochemical biosensors can be...

optical biosensing

Detecting Molecules with Light

Optical biosensors detect target molecules with light. They monitor binding by reading an optical signal from the sample. This makes them useful for seeing when a target molecule is present. Some optical biosensors use a label molecule. The label creates a measurable signal, such as fluorescence. Other optical biosensors are label-free and track changes in optical properties, such as refractive index, as binding occurs. This page introduces both label and label-free optical biosensors. It...

overview of tissue engineering

Building Artificial Tissue with Biomaterials

Tissue engineering uses biomaterials, specific cells, and growth factors to build artificial tissue. These engineered tissue constructs are designed for tissue repair and, in some cases, organ replacement. The field focuses on how to make tissue work like natural tissue. It begins with a scaffold, which is a structure that supports cell growth. Cells are then added to the scaffold, and growth factors help encourage proliferation, or cell multiplication. The video also outlines common methods...

histotypic tissue culture

3D Cell Growth on Engineered Matrices

Histotypic tissue culture uses an engineered three-dimensional matrix to grow and propagate cells. Compared with common two-dimensional tissue culture, cells in a 3D setting behave more realistically and better mimic native tissue. The method is useful when high cell density is needed. The process begins by harvesting cells from donor tissue. Those cells are then placed onto an engineered construct for culture. This setup supports growth in a three-dimensional space instead of a flat surface.

whole organ tissue culture

Whole organs can be cultured ex vivo using specialized bioreactors to support repair or replacement of entire organs. In this approach, a donor organ is first stripped of all cells. What remains is the three-dimensional structure of the organ, which can then be repopulated with new cells. The lung is the example shown here. The method uses a dynamic culture system that provides mechanical stimulation, similar to what the organ experiences in the body. This type of culture helps induce native...

bioengineering

该系列涵盖了核心生物工程概念, 包括生物材料的生产、histotypic 和整个器官组织培养、生物技术, 以及 bioMEMs 和传感的复杂系统级领域。

Learn how donor lungs are decellularized, repopulated with new cells, and grown in bioreactors that provide dynamic mechanical stimulation.

Lung Decellularization and Recellularization

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