Name: growing cells in bioreactors
Uploaded: 2023-04-30T06:32:43.000Z
Duration: 9 min 40 s
Channel: JoVE

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生物反应器也称为发酵罐，是为细胞培养提供适宜生长环境的细胞培养系统。在细胞培养中，常用的生物反应器主要有两种类型：分批式反应器和连续搅拌釜式反应器。分批式反应器是一种封闭的容器系统，包含细胞培养所需的所有组分；而连续搅拌釜式反应器则是一种开放系统，组分可主动流入和流出，从而实现废物的去除和营养物质的补充。为了最大限度地促进细胞生长并提高细胞密度，两种系统均可控制多个反应器参数，包括温度、压力、溶解氧和pH值。本视频将介绍分批式和连续式反应器的基本原理，并演示在实验室中使用这些反应器的具体操作步骤。
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最简单的生物反应器类型是分批式反应器，它是一个封闭系统，内含细胞培养基和一定数量的细胞。随着细胞生长，它们消耗营养物质并排出代谢废物，这些废物会在反应器中逐渐积累。另一种常用的反应器是连续搅拌釜式反应器，属于开放系统，其中营养物质持续流入，细胞和细胞代谢废物则持续流出。这种方法可通过清除废物和补充营养物质来调控细胞密度。在使用上述任一系统之前，通常需用蒸汽对组件进行灭菌&#160;，以减少污染风险。当反应器完全组装后，将无菌培养基加入主反应容器，并接种初始培养物。在运行过程中，通过搅拌桨控制混合与供氧，以维持均匀的溶液环境，确保细胞获得足够的营养物质和氧气用于生长。此外，发酵罐系统通常配备探针，用于监测温度、溶解氧和pH等参数，以确保细胞生长处于最佳条件。在分批培养过程中，细胞群体经历典型的生长阶段：首先为延滞期，细胞适应环境，生长缓慢；随后进入对数期，细胞以指数速率分裂，直至营养物质耗尽或有毒副产物达到临界水平；最终生长速度减缓，细胞进入稳定期，此时可从培养物中收获目标产物。目标产物可能由细胞分泌并直接从培养液中收集，也可能保留在细胞内，需通过细胞裂解方式提取。收获完成后，反应器需经过清洗和灭菌处理，为下一批细胞培养做准备。
连续搅拌釜式反应器在初始阶段表现出类似的生长模式&#160;，并在稳态运行时达到恒定的细胞密度。然而，该细胞密度取决于稀释速率，即进料与出料速率除以反应器体积。因此，当稀释速率接近1时，细胞密度会下降。在考虑生物反应器构型时，需注意分批式反应器因其结构简单、成本较低而被广泛使用，但其细胞密度受限；连续式反应器则可提高细胞密度。然而，在高细胞密度下可能发生细胞聚集，从而影响最佳生长状态。此外，长时间的发酵过程可能增加培养物污染的风险。
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在介绍了间歇式反应器和连续式反应器之后，让我们来考察这两种反应器的操作流程，首先从间歇式反应器开始。在使用间歇式反应器之前，需将细胞培养基加入一个洁净的反应容器中。容器装填完毕后，用去离子水冲洗各组件并进行组装。这些组件包括顶板、用于取样培养物的收获管、用于维持培养物均匀性和培养基氧合的搅拌桨，以及向培养物中通入气体的气体分布器。接下来安装溶氧探头，用于测量容器中溶解氧的含量，同时安装用于添加酸或碱以调节pH的进料管路，以及经过校准的pH探头。当所有组件安装完毕后，将组装好的反应器通过高压灭菌器进行灭菌。随后将容器安装到发酵罐底座上，并将pH探头和溶氧探头连接至计算机。此外，气体分布器连接至气体转子流量计，用于测量气体流速。此时启动搅拌桨，并调节气体流量、温度和容器搅拌速度，直至达到所需参数。接着用酒精对接种口进行灭菌，然后将种子培养物注入反应器容器中。在运行过程中，于选定的时间点采集细胞培养样品，根据细胞密度测量结果绘制生长曲线。当细胞密度达到预期水平时，停止培养，并通过一系列过滤步骤从容器中收获细胞。剩余的容器内容物则使用漂白剂或其他抗菌物质进行处理。
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与分批式反应器类似，连续搅拌釜式反应器（有时称为恒化器）在组装前也需要将其各个部件用去离子水彻底冲洗。清洗完成后，将搅拌装置与驱动轴部件组装在一起，并向反应器容器中加入去离子水，以提高高压灭菌过程中的灭菌效果。随后对溶解氧、pH 和温度探头进行校准并安装。容器完全组装后，使用高压灭菌器对反应器进行灭菌。灭菌完成后，将容器置于加热套中以控制其温度，并连接已校准的探头。然后连接培养基大瓶和带有无菌滤器的空气供应系统，再开启气流。通过松开大瓶的管路夹，使无菌培养基流入系统，启动基础发酵程序。当培养基流入正在运行的反应器时，先用酒精对接种口进行消毒，然后向容器中加入1毫升的种子培养物。在运行过程中，于选定的时间点采集培养样品以测定细胞密度。这些数据可用于构建生长曲线。当培养物达到稳态（即细胞密度保持恒定）时，即实现最佳生长。
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在了解了间歇式反应器和连续搅拌釜式反应器之后，让我们来看一下这项技术的一些实际应用。啤酒酿造通常在间歇式反应器系统中进行。将一组基本原料——包括纯化水、麦芽大麦、啤酒花和酵母——加入反应器中。麦芽大麦作为富含糖分的营养源，可促进酵母的发酵过程；酵母消耗糖分后，将酒精作为代谢废物产生。当混合物达到目标酒精浓度，或按规定的时长完成发酵后，即可进行过滤、包装，并静置陈化以实现碳酸化，最终产品便可投入市场。标准反应器也可根据特定用途进行定制。例如，可通过专用反应器实现持续搅拌，从而增强细胞与营养物质的分布，提高组织支架中的细胞存活率。同时，可对支架施加机械刺激，以促进细胞外基质的生成，并主动引导细胞的生长与分化。由此获得的支架具有更优的生理和力学性能，因而适用于植入应用。
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您刚刚观看了 JoVE 关于间歇式反应器和连续搅拌反应器的视频。现在，您应该了解间歇式反应器和连续搅拌釜式生物反应器的工作原理，以及这些系统在生物工程领域的应用。感谢观看。
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批次式与连续式生物反应器

Batch and Continuous Bioreactors

生物反应器用于大量培养生物体，从而实现目标产物的大规模生产。这些反应器可以是分批式反应器，其中包含细胞生长所需的所有组分；也可以是连续式反应器，具有进样和出样端口，可添加新鲜培养基并去除细胞代谢废物。
本视频介绍了间歇式反应器和连续式反应器&#160;，并演示了在实验室中利用生物反应器培养细菌的方法。最后，本视频还探讨了这些反应器在生物工程领域中的应用，例如生产蛋白质治疗药物甚至啤酒。

生物反应器用于大量培养生物体，从而实现目标产物的大规模生产。这些反应器可以是分批式反应器，其中包含细胞生长所需的所有组分；也可以是连续式反应器，具有进样和出样端口，可添加新鲜培养基并去除细胞代谢废物。
本视频介绍了间歇式反应器和连续式反应器&#160;，并演示了如何在实验室中利用生物反应器培养细菌。最后，本视频还探讨了这些反应器在生物工程领域中的应用，例如生产蛋白质治疗药物甚至啤酒。

生物反应器用于大规模培养生物体，从而实现目标产物的大量生产。这些反应器可以是……

生物反应器也称为发酵罐，是一种为细胞培养提供适宜生长环境的细胞培养系统。在细胞培养中，常用的生物反应器有两种类型：分批式反应器和连续搅拌釜式反应器。分批式反应器是一种封闭的容器系统，包含细胞培养所需的所有组分；而连续搅拌釜式反应器则是一种开放系统，组分可主动流入和流出，从而实现废物的去除和营养物质的补充。为了最大限度地促进细胞生长并提高细胞密度，可对这两种系统的多个反应器参数进行控制，包括温度、压力、溶解氧和pH值。本视频将介绍分批式和连续式反应器的基本原理，并演示在实验室中使用这些反应器的具体操作步骤。最简单的生物反应器类型是分批式反应器，它是一个封闭系统，内含细胞培养基和一定数量的细胞。随着细胞生长，它们消耗营养物质并排出代谢废物，这些废物会在反应器中逐渐积累。另一种常用的反应器是连续搅拌釜式反应器，属于开放系统，其中营养物质持续流入，细胞及其代谢废物则不断流出。这种方式可通过清除废物和补充营养物质来调控细胞密度。在使用上述任一系统之前，所有组件通常需通过蒸汽灭菌，以防止污染。当反应器完全组装后，将无菌的培养基加入主反应容器，并接种起始培养物。在运行过程中，通过搅拌桨控制混合与供氧，以维持均匀的溶液环境，确保细胞获得足够的营养物质和氧气用于生长。此外，发酵罐系统通常配备探针，用于监测温度、溶解氧和pH等参数，以确保细胞生长处于最佳条件。在分批培养过程中，细胞群体经历典型的生长阶段：首先，细胞进入迟缓期，此时细胞生长缓慢，正在适应其环境；随后进入对数生长期，细胞以指数速率分裂，直至营养物质耗尽或有毒副产物达到临界水平；最终，生长速度减慢，细胞进入稳定期，此时可从培养物中收获目标产物。产物可能由细胞分泌并直接从培养液中收集，也可能保留在细胞内，需通过细胞裂解来提取。收获完成后，反应器需经过清洗和灭菌，为下一轮细胞培养做准备。连续搅拌釜式反应器在初始阶段表现出相似的生长模式，并在稳态运行时达到恒定的细胞密度。然而，该细胞密度取决于稀释速率，即进料和出料速率除以反应器体积。因此，当稀释速率接近1时，细胞密度会下降。在考虑生物反应器构型时，需注意分批式反应器由于其结构简单且成本较低而常被使用，但其细胞密度受限。连续式反应器能够提高细胞密度，但在高细胞密度下可能发生聚集现象，从而阻碍最佳生长。此外，较长的发酵时间可能增加培养物污染的风险。在介绍了间歇式反应器和连续式反应器之后，我们接下来将分别考察两者的操作流程，首先从间歇式反应器开始。在使用间歇式反应器之前，需向一个洁净的反应容器中加入细胞培养基。容器注满后，用去离子水冲洗各组件并进行组装。这些组件包括顶板、用于取样培养物的收获管、用于维持培养液均匀性和培养基氧合的搅拌桨，以及向培养液中通入气体的气体分布器。随后安装溶氧探头以测量容器内溶解氧含量，安装进料管路以加入酸或碱来调控pH，并安装经过校准的pH探头。当所有组件安装完毕后，将组装好的反应器通过高压灭菌器进行灭菌。然后将容器安装到发酵罐底座上，并将pH探头和溶氧探头连接至计算机。此外，气体分布器连接至气体转子流量计，用于测量气体流速。此时启动搅拌桨，并调节气体流量、温度和容器搅拌速度，直至达到所需参数。接着用酒精对接种口进行灭菌，并将种子培养物注入反应器容器中。在运行过程中，于选定的时间点采集细胞培养样品，根据细胞密度测定结果绘制生长曲线。当细胞密度达到预期水平时，停止培养，并通过一系列过滤步骤从容器中收获细胞。剩余容器内容物则使用漂白剂或其他抗菌物质进行处理。与分批式反应器类似，连续搅拌釜式反应器（有时称为恒化器）在组装前也需要将其各个部件用去离子水彻底冲洗。清洗完成后，将搅拌装置与驱动轴部件组装在一起，并向反应器容器中加入去离子水，以提高高压灭菌过程中的灭菌效果。随后对溶解氧、pH 和温度探头进行校准并安装。容器完全组装后，使用高压灭菌器对反应器进行灭菌。灭菌完成后，将容器置于加热夹套中以控制其温度，并连接已校准的探头。然后连接含无菌过滤器的培养基大瓶和空气供应系统，再开启气流。通过松开大瓶的管路夹，用无菌培养基对容器进行预灌注，然后运行基础发酵程序。当培养基流入正在运行的反应器时，先用酒精对接种口进行消毒，再向容器中加入 1 毫升种子培养物。在运行过程中，于选定的时间点采集培养样品，以测定细胞密度。这些数据可用于构建生长曲线。当培养物达到稳态（即细胞密度保持恒定）时，即实现最佳生长。在了解了间歇式反应器和连续搅拌釜式反应器之后，让我们来看一下该技术的一些实际应用。啤酒酿造通常在间歇式反应器系统中进行。将一系列基本原料（包括纯化水、麦芽大麦、啤酒花和酵母）加入反应器中。麦芽大麦富含糖类，可作为酵母的食物来源，促进发酵过程；酵母消耗糖分后，产生酒精作为代谢废物。当混合物达到目标酒精含量或按规定的时长完成发酵后，即可进行过滤、包装，并静置陈化以产生碳酸化，随后将最终产品投入市场。标准反应器也可根据特定用途进行定制。例如，可通过持续搅拌使用专用反应器增强细胞和营养物质的分布，从而提高组织支架的细胞存活率。同时，可对支架施加机械刺激，以促进细胞外基质的生成，并主动引导细胞的生长与分化。由此获得的支架具有更优的生理和机械性能，因而适用于植入应用。您刚刚观看了 JoVE 关于间歇式反应器和连续搅拌反应器的视频。现在，您应该理解间歇式反应器和连续搅拌釜式生物反应器的工作原理，以及这些系统在生物工程领域中的应用。感谢观看。

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间歇和连续生物反应器

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 removed.

This page also shows how bioreactors are used to grow bacteria in the laboratory. These systems help explain how bioengineering can produce useful products. Examples include protein therapeutics and even beer.

Growing Cells in Bioreactors

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

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

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 how batch and continuous bioreactors grow bacteria in large volumes and help produce products like protein therapeutics and beer.

Growing Cells in Bioreactors

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