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

<div class="chapter_text" id="script_0">在体外培养的部分或全部器官往往是为了准确地模型组织和器官功能的各种测试条件。整个器官文化可以涉及细胞、切除器官或细胞的移除, 以利用本机器官结构。其次是 recellularization 与新的细胞。使用专门的生物反应器, 往往被纳入 recellularization 过程中, 以模拟组织生长的身体。本视频将介绍整个器官组织培养背后的基本原理, 并在实验室中演示其程序。</div><div class="chapter_text" id="script_1">这一过程始于收获一个捐赠器官。在这个例子中, 我们展示了一只猴子的捐献者肺。通过一个叫做洗涤剂灌注的过程, 隔离的器官通过一系列的洗涤来系统地净化其原生细胞的数量。导致无菌脱细胞的器官基质。接下来的组织基质是 recellularized 使用特定的细胞类型, 如建立的干细胞线。细胞也可以由接受工程组织的人捐赠。称为自体细胞移植。这减少了排斥并增强了器官的生物相容性。或者, 可以从不同的捐赠者那里使用细胞。被称为同种异体移植。如果有足够数量的细胞不能从潜在的接受者那里获取, 这可能需要被追求。一旦细胞被播种在器官上, 组织生物反应器就被用来刺激细胞增殖和组织的直接生长。这些反应堆的目的是动态培养器官和模仿在体内发现的当地环境。例如, 该器官可以连接到蠕动泵, 以模拟血流。现在, 你已经了解了器官文化的原理, 让我们来看看一个例子, 涉及整个器官培养的捐赠肺部。</div><div class="chapter_text" id="script_2">开始供体肺被安置在解剖盘和肺动脉空心通过介绍一个女性口连接器入开放洞。第二个女性口连接器, 然后插入到气管开口。磷酸缓冲盐或 PBS, 含有30单位每毫升肝素和五微克每毫升硝普钠, 然后灌输, 以促进血管的扩大和从肺部被困的空气清除。该溶液被自然反冲排出, 并在盖住套管之前重复两次, 以便在肺部保留溶液以溶解残余的血液。然后两个心房被撕裂, 气管口插管帽被移除, 以利于液体引流。灌注是继续与 PBS, 肝素和硝普钠溶液, 直到尽可能多的血液被排除在肺血管。开始 decellurization, 肺部膨胀, 并渗透到去离子水。在五动脉和血管冲洗后, 肺部被从水中移除并浸入一种叫做海卫一的洗涤剂中, 以去除细胞, 同时最小程度地影响器官基质。在四摄氏度的夜间孵化之前, 肺部被海卫一溶液充气两次。随着新的去离子水, 肺部会被洗涤五次。接下来, 肺部被淹没在2% 钠胆酸溶液中, 然后再洗几次, 用水和缓冲液, 以方便细胞和去除细胞碎片。当完全清洁的器官是储存在无菌 PBS 溶液在四摄氏度, 直到使用。</div><div class="chapter_text" id="script_3">为了模拟肺部的自然行为, 可以使用一个专门的生物反应器, 就像这里所示。首先, 反应器的主室充满了培养基, 这已被平衡到5% 的二氧化碳大气中。然后器官被安装。一旦连接的盖子是安全的, 所有的空气被删除, 从油管使用注射器。然后将生物反应器移到组织培养箱中平衡。接下来, 肺部的通风大约每两分钟一次, 呼吸一次。介质通过血管循环, 通过蠕动泵, 大约每分钟10毫升, 总共30分钟。</div><div class="chapter_text" id="script_4">对于气道播种, 肺是充气的, 其中含有骨髓间充质干细胞的悬浮细胞。recellularize 肺泡, 这是负责的氧气和二氧化碳, 气体交换发生在肺部。然后在夜间孵育肺部, 使细胞附着在细胞基质上。在夜间孵化后, 通气是重新的, 细胞可以在器官基质中生长数天。接下来的血管播种是通过使用蠕动泵逐渐引入内皮细胞来完成的, 以启动小血管的 recellularization。然后静态培养几个小时, 以促进细胞精确器官的发育。培养基再次循环, 细胞被培养一周, 以促进生长和附着在动态条件下。一旦组织生长是完整的组织学进行, 以确认的连接和生长的骨髓间充质干细胞和内皮细胞, 在血管和呼吸道的器官。组织学显示在基质支架和小血管血管中, 骨髓间充质干细胞和内皮细胞附着在肺泡内。创造出原生肺组织的外观。</div><div class="chapter_text" id="script_5">现在, 你已经了解了整个器官的文化, 让我们来看看这项技术的一些实际应用以外的主要重点是再生医学和器官置换。整个器官的培养也可以用来作为一种方法来测试药剂或药物传递装置。例如, 在这项研究中, 胚胎小鼠甲状腺被说明, 培养, 并作为一个器官模型来观察实验药物是如何通过器官和组织运输的。这一模拟最终可能导致更现实的表示药物如何在体内转移到一个器官。最后, 整个器官组织培养可用于研究各种条件下的组织行为。例如, 从牛尾巴上采集椎间盘, 研究椎间盘退变的可能机制。采用专门设计的生物反应器, 在圆盘上诱发机械负荷, 以便更好地了解这些负载如何影响退化。</div><div class="chapter_text" id="script_6">你刚刚看了朱庇特的整个器官组织培养的视频。你现在应该了解如何在体外培养整个器官, 以及如何在生物工程领域应用这种技术。谢谢收看</div>

整体器官组织培养

整个器官可以使用专门的生物反应器来培养体外, 目的是修复或替换整个器官. 这种方法使用的捐赠器官被剥离所有细胞, 留下三维结构, 然后重新与新的细胞。这段视频展示了整个肺部器官的培养, 并展示了一个动态的文化, 模仿身体的机械刺激, 是需要诱导本组织的性质。

Whole organs can be cultured ex vivo using specialized bioreactors&#160;with the goal of repairing or replacing entire organs. This method uses a donor ...

通常使用部分或整个器官的体外培养物来准确模拟各种测试条件下的组织和器官功能。全器官培养可以涉及切除器官的脱细胞或去除细胞，以利用天然器官结构。然后是新细胞的再细胞化。专用生物反应器的使用通常被纳入再细胞化过程，以模拟体内的组织生长。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过程。这个过程从收获供体器官开始。在这个例子中，我们展示了一只猴子的供体肺。通过称为去污剂灌注的过程，分离的器官通过一系列洗涤系统地清除其天然细胞群，从而产生无菌的脱细胞器官基质。接下来，使用特定细胞类型（例如已建立的干细胞系）对组织基质进行再细胞化。接受工程组织的人也可以捐献细胞，称为自体细胞移植。这减轻了排斥反应并增强了器官的生物相容性。或者，可以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中收获足够数量的细胞，则可能需要进行此作。一旦细胞接种到器官上，组织生物反应器就用于刺激细胞增殖和指导组织生长。这些反应器旨在动态培养器官并模拟体内发现的天然环境。例如，可以将器官连接到蠕动泵以模拟血流。现在您已经了解了器官培养的原理，让我们看一个涉及供体肺整个器官培养的示例程序。首先，将供体肺放置在解剖盘中，并通过将母鲁尔连接器引入开放腔来插管肺动脉。然后将第二个母鲁尔接头插入气管开口。然后滴注含有每毫升 30 单位肝素和每毫升 5 微克硝普钠的磷酸盐缓冲盐水或 PBS，以促进血管扩张和清除肺部滞留的空气。溶液通过自然后坐力排出，并在盖上套管之前再重复两次，以将溶液保持在肺部以溶解任何残留的血液。然后，撕裂两个心房并移除气管鲁尔套管帽，以促进液体引流。继续使用 PBS、肝素和硝普钠溶液进行灌注，直到从肺血管系统中去除尽可能多的血液。为了开始脱细胞，肺部充气并渗透到去离子水中。经过五次动脉和血管清洗后，将肺从水中移出，并浸入一种叫做 Triton™ 的洗涤剂中，以去除细胞，同时对器官基质的影响最小。用 Triton 溶液再给肺充气两次，然后在 4 摄氏度下孵育过夜。孵育后，用新鲜的去离子水再洗涤肺部五次。接下来，将肺浸没在 2% 脱氧胆酸钠溶液中，然后用水和缓冲溶液再洗涤数次，以促进脱细胞和去除细胞碎片。完全清洁后，将器官储存在 4 摄氏度的无菌 PBS 溶液中直至使用。为了模拟肺部的自然行为，可以使用专门的生物反应器，如此处所示。首先，反应器的主腔室中装满已平衡至 5% 二氧化碳气氛的培养基。然后安装管风琴。连接后，盖上盖子，并使用注射器从管道中去除所有空气。然后将生物反应器移至组织培养箱中以达到平衡。接下来，大约每两分钟进行一次肺部通气，培养基通过蠕动泵以每分钟约 10 毫升的速度在脉管系统中循环，总共 30 分钟。对于气道接种，用含有骨髓来源的间充质干细胞的细胞悬液对肺进行充气。使肺泡再细胞化，肺泡负责肺部发生的氧气和二氧化碳气体交换。然后将肺孵育过夜，以使细胞附着在脱细胞基质上。孵育过夜后，重新开始通气，让细胞在器官基质中生长数天。接下来，通过使用蠕动泵逐渐引入内皮细胞来完成血管接种，以启动小血管的再细胞化，然后静态培养数小时，以促进细胞精确器官的发育。培养基再次循环，并将细胞培养一周，以促进动态条件下的生长和附着。一旦组织生长完成，进行组织学检查，以确认间充质干细胞和内皮细胞在器官的脉管系统和气道上的附着和生长。组织学显示间充质干细胞和内皮细胞附着在基质支架内的肺泡和小血管上，从而形成天然肺组织的外观。现在您已经了解了全器官培养，让我们来看看这项技术在再生医学和器官置换主要关注点之外的一些实际应用。全器官培养也可用作测试药物制剂或药物输送装置的一种方式。例如，在这项研究中，胚胎小鼠甲状腺被移植、培养并用作器官模型，以观察实验药物如何通过器官和组织运输。这种模拟最终可能会更真实地表示药物如何在体内器官内转移。最后，全器官组织培养可用于研究组织在各种条件下的行为。例如，从牛尾巴上采集椎间盘，以研究椎间盘退化的可能机制。采用专门设计的生物反应器在圆盘上诱导机械负载，以便更好地了解这些负载如何影响退化。您刚刚观看了 Jove 关于全器官组织培养的视频。您现在应该了解如何在体外培养整个器官，以及如何将这项技术应用于生物工程领域。感谢观看。

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生物工程

Whole Organ Tissue Culture

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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