Name: 3d cell growth on engineered matrices
Uploaded: 2023-04-30T06:32:19.000Z
Duration: 9 min 35 s
Channel: JoVE

<div class="chapter_text" id="script_0">Histotypic 组织培养可以使细胞在三维度中生长, 从而创造出体外组织形态, 紧密模仿逼真的组织功能, 可作为组织修复的可行结构。这些文化是典型的三维结构组成的单一细胞类型的高密度增长。三维结构, 也称为脚手架, 模仿自然细胞外基质。根据所使用的细胞类型, 脚手架可以设计为一个特定的应用, 通常作为一个模板的生物仿生组织。这段视频将介绍 histotypic 组织培养的基本原理, 一个细胞分离的程序, 和一个多孔的丝绸组织支架的制作和 cellularization 模拟心脏组织。</div><div class="chapter_text" id="script_1">所有的组织包括两个基本成分, 细胞外基质和组织特异细胞的栖息。细胞外基质是一种结构蛋白网络, 它为细胞创造了一个三维的环境, 它的细胞是用来重述组织的自然生理过程的。目前, 一种用于组织模型的常用技术是二维组织培养, 将细胞分配到平坦的基底上, 并允许形成薄膜。一般来说, 这种方法是不可靠的维持在体内表型, 器官特异功能, 细胞或细胞, 以基底的上下文相互作用。Histotypic 组织培养通过为细胞生长提供一个3D 的支架来缓解这些局限性, 从而形成一个密度更高的细胞网络, 更紧密地模仿当地细胞的形态, 并促进现实的细胞间网络的发展和沟通途径。各种3D 聚合物网络, 包括水凝胶和纺丝垫, 提供了在三维度培养组织特异细胞的简便方法。为了填充这些支架, 细胞必须隔离。在这个视频中使用的主要细胞从活体组织中获取, 这是切碎, 然后在酶溶液中消化, 将靶细胞从细胞外基质中分离出来。一旦细胞被隔离, 就有两种技术用来播种脚手架。液滴技术包括移在支架上以一个缓慢而恒定的速率来解决细胞的问题。第二, 或细胞悬浮技术, 淹没支架在细胞悬浮。通常情况下, 支架和悬挂被动摇, 以鼓励细胞迁移到矩阵。这两种技术都能产生高细胞密度的生物工程结构。下面的过程将涉及隔离心脏细胞和细胞悬浮技术, 以创建一个心脏细胞特异性支架, 因为它将保留本机心脏组织形态学。</div><div class="chapter_text" id="script_2">开始从捐献者组织中分离细胞的过程, 首先要确保工作区域和解剖器械被消毒。然后, 在生物安全柜的工作表面放置一个无菌的褶皱。将不育的手术器械放在褶皱上, 不接触它们, 然后打开一个不育的数20手术刀刀片。在安乐标本后, 用控告浸泡过的纱布消毒手术区域。准备好后, 确保样品的安全, 并开始手术, 分离出感兴趣的组织。在这种情况下, 它将是心脏。一旦切除, 将组织放在含有 PBS 葡萄糖的培养皿中的冰上。取出任何残留的血液或结缔组织, 然后将组织转移到培养皿中, 用新鲜的 PBS 葡萄糖。然后, 用无菌的微剪刀和镊子, 仔细地将组织样本切成大约1立方毫米的碎片。使用吸管, 将工件和缓冲器转移到锥形管上。然后, 除去所有的10毫升的缓冲。加入7毫升胶原酶溶液, 然后在37摄氏度的7分钟内摇动混合物。然后, 轻轻吸管10次, 打破了组织件。让这些碎片沉淀, 然后抽吸液体并丢弃它。接下来, 重复消化, 轻轻吸管的解决方案, 打破了组织件。在这些碎片解决后, 抽出上清液并将其收集在一个单独的50毫升锥形管中。然后, 加入10毫升的停止溶液, 每一个含有上清的锥形管停止消化。</div><div class="chapter_text" id="script_3">现在, 主要细胞已被隔离, 让我们制造一个多孔的丝绸组织脚手架。首先, 将30毫升的丝绸溶液倒入模具中。然后, 在溶液中均匀地分散60克被筛的氯化钠。然后, 让丝绸在室温下聚合48小时不受干扰。然后, 将模具放置在一个60度的烤箱中1小时, 以确定交联和蒸发任何剩余的液体。一旦聚合, 浸泡在一个烧杯的蒸馏水48小时, 以水蛭出盐的模具。然后, 从模具中取出支架, 用5毫米的活检穿孔切割小圆盘。将光盘修剪到2毫米的高度, 最后, 用2毫米的活检穿孔去除每个棋子的中心, 以创建一个圆环。最后, 在湿循环中压下支架20分钟。</div><div class="chapter_text" id="script_4">随着脚手架的准备, 让我们开始细胞播种过程。首先, 将一个无菌支架放在96井板上。然后, 加入细胞培养基浸泡在37摄氏度的组织培养孵化器, 以平衡他们至少30分钟。继孵育后, 吸取多余的培养基, 然后将分离的原细胞悬浮液添加到支架上。接下来, 把盘子放回孵化器, 然后过夜, 让细胞附着在支架上。在第二天早晨, 仔细地吸取非附着细胞和更换200升的新鲜细胞培养基每井。所得到的脚手架是一个多孔结构, 具有高的细胞密度, 准备使用。</div><div class="chapter_text" id="script_5">现在你已经学会了如何进行 histotypic 组织培养, 让我们来看看这些材料的一些实际应用。Histotypic 组织培养可以创建模拟原生组织的细胞微环境, 因此能够为研究单个细胞类型的细胞行为提供一个合适的模型。例如, 在支架上的3D 纤维, 更精确地模仿在体内发现的干细胞, 可以用多能干细胞进行播种, 以筛选生物线索, 并确定它们对干细胞分化的影响。这项工作最终可能会更深入地了解干细胞是如何分化的, 并能为组织工程应用中增强细胞分化和再生提供一些见解。就像动态的文化一样, 机械调节也可以通过使3D 组织支架承受自然组织在体内可能体验到的各种机械载荷来增强。通过在细胞生长过程中施加压缩和双向载荷, 改变细胞形态和胞外基质, 以反映这些机械载荷。这导致了一个预先准备好的生物工程组织支架与细胞结构类似的原生组织, 使其适合在可能遇到类似机械力的地区植入。最后, 工程组织结构也可用于替代或修复组织缺损。为了做到这一点, 组织支架必须首先带血管, 从而允许血液自由地通过该结构的运动。一旦血管化, 支架可以转移并植入组织缺损, 开始修复。成功的嫁接可以通过组织学证实, 这表明组织结构是否完全修复了受损区域。</div><div class="chapter_text" id="script_6">你刚刚看了朱庇特的 histotypic 组织文化的介绍。你现在应该了解如何简单的3D 结构的准备, 如何分离和种子的主要细胞在脚手架上, 以及这些文化在生物工程领域的各种应用。谢谢收看</div>

histotypic 组织培养

虽然二维组织培养在一段时间内很常见, 但细胞在三维的文化中表现得更逼真, 更接近于本土组织。 该视频介绍 histotypic 组织培养, 其中一个细胞线的生长和繁殖是在一个工程的三维矩阵, 以达到高的细胞密度。 在这里, 我们展示了从捐赠组织收集细胞, 其次是细胞培养的工程建设。

Histotypic 组织培养

Although two-dimensional tissue culture has been common for some time, cells behave more realistically in a three-dimensional culture, and more closely ...

组织型组织培养允许细胞在三维空间中生长，从而产生与实际组织功能非常相似的体外组织形态，可用作组织修复的活构建体。这些培养物通常是三维结构，由高密度生长的单一细胞类型组成。三维结构，也称为支架，模仿天然细胞外基质。根据所使用的细胞类型，支架可以针对特定应用进行设计，通常用作仿生组织的模板。本视频将介绍组织型组织培养的基础知识、细胞分离程序以及多孔丝组织支架的制造和细胞化以模拟心脏组织。所有组织都由两个基本成分组成，即细胞外基质和栖息在其中的组织特异性细胞。细胞外基质是一个结构蛋白网络，为细胞创造了一个三维环境，其中的细胞旨在概括组织的天然生理过程。目前，用于组织建模的常用技术是二维组织培养，其中细胞被分配到平坦的基质上并使其形成薄膜。一般来说，这种方法对于维持体内表型、器官特异性功能以及细胞间或细胞间底物环境相互作用并不可靠。组织型组织培养通过为细胞生长提供 3D 支架来缓解这些限制，从而产生更接近天然细胞形态的致密细胞网络，并促进逼真的细胞间网络和通信途径的开发。各种 3D 聚合物网络，包括水凝胶和静电纺丝垫，为在三维空间中培养组织特异性细胞提供了便捷的方法。为了填充这些支架，必须分离细胞。本视频中使用的原代细胞是从活组织中收获的，活组织被切碎，然后在酶溶液中消化，以将靶细胞与细胞外基质分离。分离细胞后，有两种技术可用于接种支架。液滴技术包括以缓慢且恒定的速率将细胞溶液移液到支架上。第二种，或细胞悬液技术，将支架浸入细胞悬液中。通常，摇动支架和悬液以促进细胞迁移到基质中。这两种技术都产生了具有高细胞密度的生物工程构建体。以下程序将涉及心肌细胞的分离和细胞悬液技术，以创建心肌细胞特异性支架，因为它将保留天然心脏组织形态。要开始从供体组织中分离细胞的过程，首先要确保工作区域和解剖器械已消毒。然后，将无菌窗帘放在生物安全柜的工作表面上。将无菌手术器械放在窗帘上，不要触摸它们，然后打开无菌的 20 号手术刀刀片。对标本实施安乐死后，用 betadine 浸泡的纱布垫对手术区域进行消毒。准备好后，固定样品并开始外科手术以分离感兴趣的组织。在这种情况下，它将是心脏。切除后，将组织放在含有 PBS 葡萄糖的培养皿中的冰上。去除任何残留的血液或结缔组织，然后将组织转移到装有新鲜 PBS 葡萄糖的培养皿中。然后，使用无菌微剪刀和镊子，小心地将组织样本切成大约 1 立方毫米的小块。使用移液管，将碎片和缓冲液转移到锥形管中。然后，除 10 mL 缓冲液外，除 10 mL 外，其余全部取出。加入 7 毫升胶原酶溶液，然后在 37 摄氏度下摇晃混合物 7 分钟。然后，轻轻移液 10 次以打碎组织块。让碎片沉淀，然后吸出液体并丢弃。接下来，重复消化并轻轻吸取溶液以打碎组织碎片。碎片沉淀后，吸出上清液并将其收集在单独的 50 毫升锥形管中。然后，向每个含有上清液的锥形管中加入 10 毫升 STOP 溶液以停止消化。现在原代细胞已经被分离出来，让我们制作一个多孔丝组织支架。首先，将 30 毫升蚕丝溶液倒入模具中。接下来，将 60 克过筛的氯化钠均匀地撒在溶液上。然后，让真丝在室温下不受干扰地聚合 48 小时。然后，将模具放入 60 度的烘箱中 1 小时以完成交联并蒸发任何剩余的液体。聚合后，将模具浸入装有蒸馏水的烧杯中 48 小时以吸出盐分。然后，从模具中取出支架，用 5 毫米的活检打孔器切割小圆盘。将椎间盘修剪到 2 毫米的高度，最后，用 2 毫米的活检打孔器去除每块的中心，形成一个环。最后，在湿循环中高压灭菌支架 20 分钟。准备好支架后，让我们开始细胞接种过程。首先，在 96 孔板中每孔放置一个无菌支架。然后，添加细胞培养基浸入支架，并在 37 摄氏度下在组织培养箱中孵育至少 30 分钟。孵育后，吸出多余的培养基，然后将分离的原代细胞悬液添加到支架中。接下来，将板放回培养箱中，放置过夜，让细胞附着在支架上。第二天早上，小心吸出未贴壁的细胞，并用每孔 200 μL 的新鲜细胞培养基替换。所得支架是具有高细胞密度的多孔构建体，可随时使用。现在您已经了解了如何进行组织型组织培养，让我们看看这些材料的一些实际应用。组织型组织培养可以创造模拟天然组织的细胞微环境，因此能够为研究有关单一细胞类型的细胞行为提供合适的模型。例如，支架中的 3D 纤维更准确地模拟了体内发现的干细胞生态位，可以接种多能干细胞，以筛选生物学线索并确定它们对干细胞分化的影响。这项工作最终可能会更好地了解干细胞如何分化，并可能为增强组织工程应用的细胞分化和再生提供见解。与动态培养一样，机械调节也可以通过使 3D 组织支架承受天然组织在体内可能经历的各种机械载荷来增强 3D 组织支架。通过在细胞生长过程中施加压缩和双轴载荷，细胞形态和细胞外基质会发生变化以反映这些机械载荷。这导致预处理的生物工程组织支架具有类似于天然组织的细胞结构，使其成为植入可能受到类似机械力的区域的理想选择。最后，工程组织结构也可用于替换或修复组织缺损。为了实现这一点，必须首先使组织支架血管化，从而允许血液自由地穿过结构。一旦血管化，支架就可以转移并植入组织缺损中以开始修复。随后可以通过组织学来确认移植成功，这揭示了组织结构是否完全修复了受损区域。您刚刚观看了 Jove 对组织型组织培养的介绍。您现在应该了解如何制备简单的 3D 结构，如何分离原代细胞并将其接种到支架上，以及这些培养物在生物工程领域的各种应用。感谢观看。

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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.

3D Cell Growth on Engineered Matrices

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

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

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...

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。

Explore histotypic tissue culture, where cells are harvested from donor tissue and grown on an engineered 3D matrix to better mimic native tissue.

3D Cell Growth on Engineered Matrices

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