JoVE Science Education
Bioengineering
需要订阅 JoVE 才能查看此.
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。
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
Histotypic tissue culture allows cells to be grown in three dimensions, thereby creating in-vitro tissue morphologies that closely mimic realistic tissue function, which can be used as viable constructs for tissue repair. These cultures are typically three dimensional structures consisting of a single cell type grown in high density. The three dimensional structure, also known as the scaffold, mimics the natural extracellular matrix. Depending on the cell type used, scaffolds can be designed for a specific application and typically act as a template for bio-mimetic tissue. This video will introduce the fundamentals of histotypic tissue cultures, a procedure for the isolation of cells, and the fabrication and cellularization of a porous silk tissue scaffold to mimic cardiac tissue.
All tissues consist of two fundamental components, the extracellular matrix and the tissue-specific cells that inhabit it. The extracellular matrix is a network of structural proteins that create a three dimensional environment for cells to occupy, and the cells within it are meant to recapitulate the native physiological processes of the tissue. Currently, a common technique utilized to model tissue is two dimensional tissue culture, where cells are dispensed onto a flat substrate and allowed to form a thin film. In general, this method is not reliable for maintaining an in vivo phenotype, organ-specific functions, and cell to cell or cell to substrate contextual interactions. Histotypic tissue culture alleviates those limitations by providing a 3D scaffold for cells to grow on, resulting in a dense network of cells that more closely mimics native cell morphologies and facilitates the development of realistic intercellular networks and communication pathways. A variety of 3D polymer networks, including hydrogels and electrospun silk mats, offer convenient ways to culture tissue-specific cells in three dimensions. In order to populate these scaffolds, cells must be isolated. Primary cells used in this video are harvested from live tissue, which is minced and then digested in an enzyme solution to separate the target cells from the extracellular matrix. Once the cells are isolated, there are two techniques used to seed the scaffolds. The droplet technique involves pipetting a solution of cells onto the scaffold at a slow and constant rate. The second, or cell suspension technique, submerges the scaffold in a cell suspension. Often, the scaffold and suspension are shaken to encourage cell migration into the matrix. Both techniques result in bio-engineered constructs with high cell densities. The following procedures will involve the isolation of cardiac cells and the cell suspension technique to create a cardiac cell-specific scaffold, as it will retain the native heart tissue morphology.
To begin the process of isolating cells from donor tissue, start by ensuring that the work area and dissection instruments are sterilized. Then, place a sterile drape on the work surface in the bio-safety cabinet. Place the sterile surgical instruments onto the drape without touching them, and then open a sterile number 20 scalpel blade. After euthanizing the specimen, sterilize the surgical area with a betadine-soaked gauze pad. When ready, secure the sample and begin the surgical procedure to isolate the tissue of interest. In this case, it will be the heart. Once excised, place the tissue on ice in the Petri dish containing PBS glucose. Remove any residual blood or connective tissue, and then transfer the tissue to a Petri dish with fresh PBS glucose. Then, using sterile micro-scissors and forceps, carefully mince the tissue samples into roughly 1 cubic millimeter pieces. Using a pipette, transfer the pieces and buffer to a conical tube. Then, remove all but 10 milliliters of buffer. Add 7 milliliters of collagenase solution, and then shake the mixture at 37 degrees Celsius for 7 minutes. Then, gently pipette 10 times to break up the tissue pieces. Allow the pieces to settle, and then aspirate the liquid and discard it. Next, repeat the digestion and gently pipette the solution to break up the tissue pieces. After the pieces have settled, draw off the supernatant and collect it in a separate 50 milliliter conical tube. Then, add 10 milliliters of STOP solution to each conical tube containing supernatant to stop the digestion.
Now that the primary cells have been isolated, let's fabricate a porous silk tissue scaffold. To begin, pour 30 milliliters of the silk solution into a mold. Next, scatter 60 grams of sieved sodium chloride evenly over the solution. Then, allow the silk to polymerize undisturbed at room temperature for 48 hours. Then, place the mold in a 60 degree oven for 1 hour to finalize cross-linking and evaporate any remaining liquid. Once polymerized, immerse the mold in a beaker of distilled water for 48 hours to leech out the salt. Then, remove the scaffold from the mold and cut small discs with a 5 millimeter biopsy punch. Trim the discs to a height of 2 millimeters, and finally, remove the centers of each piece with a 2 millimeter biopsy punch to create a ring. Lastly, autoclave the scaffolds in a wet cycle for 20 minutes.
With the scaffold prepared, let's begin the cell seeding process. First, place one sterile scaffold per well in a 96 well plate. Then, add cell culture medium to immerse the scaffolds and incubate at 37 degrees Celsius in a tissue culture incubator to equilibrate them for at least 30 minutes. Following incubation, aspirate the excess medium and then add the isolated primary cell suspension to the scaffolds. Next, return the plate to the incubator and leave overnight for the cells to attach to the scaffolds. On the following morning, carefully aspirate the non-attached cells and replace with 200 microliters of fresh cell culture medium per well. The resulting scaffold is a porous construct with a high cell density ready to be used.
Now that you have learned how to perform histotypic tissue culture, let's look at some practical applications of these materials. Histotypic tissue culture can create cellular micro-environments that mimic native tissues, and as a result are able to provide a suitable model for the study of cellular behavior concerning a single cell type. For example, 3D fiber in scaffolds, which more accurately mimic the stem cell niche found in vivo, can be seeded with pluripotent stem cells to screen for biological cues and determine their effects on stem cell differentiation. This work may ultimately provide a greater understanding of how stem cells differentiate and may offer insights into enhancing cell differentiation and regeneration for tissue engineering applications. Like dynamic cultures, mechanical conditioning can also enhance the 3D tissue scaffold by subjecting it to various mechanical loads that natural tissue may experience in vivo. By applying compression and biaxial loads during cell growth, the cell morphology and extracellular matrix is altered to reflect those mechanical loads. This results in a preconditioned bio-engineered tissue scaffold with a cellular structure resembling native tissue, making it ideal for implantation in areas that may experience similar mechanical forces. Finally, engineered tissue constructs may also be used to replace or repair tissue defects. In order to achieve this, the tissue scaffold must be first vascularized, thereby allowing blood to freely move through the construct. Once vascularized, the scaffold can be transferred and implanted into the tissue defect to initiate repair. Successful grafting can later be confirmed through histology, which reveals whether or not the tissue construct completely repaired the damaged area.
You've just watched Jove's introduction to histotypic tissue culture. You should now understand how simple 3D structures are prepared, how primary cells are isolated and seeded onto a scaffold, and the various applications of these cultures in the bio-engineering field. Thanks for watching.
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。
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。
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。
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。
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。
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。
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
跳至...
0:06
Overview
0:56
Principles of Histotypic Tissue Culture
3:12
Isolation of Cells from Donor Tissue
5:08
Preparation of Tissue Scaffold
6:11
3D Culture Growth on Scaffold
7:13
Applications
9:12
Summary
此集合中的视频:
Now Playing
Bioengineering
11.5K Views
Bioengineering
74.0K Views
Bioengineering
53.1K Views
Bioengineering
8.9K Views
Bioengineering
15.9K Views
Bioengineering
11.0K Views
Bioengineering
10.9K Views
Bioengineering
18.5K Views
Bioengineering
9.9K Views
Bioengineering
49.2K Views
Bioengineering
11.8K Views
Bioengineering
15.9K Views
Bioengineering
13.1K Views
Bioengineering
12.8K Views
Bioengineering
14.0K Views
ISSN 2578-2614
版权所属 © 2025 MyJoVE 公司版权所有,本公司不涉及任何医疗业务和医疗服务。
我们使用 cookie 来增强您在我们网站上的体验。
继续使用我们的网站或单击“继续”,即表示您同意接受我们的 cookie。