Cells on chips
Abstract
Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world.
微系统通过将模拟复杂生物化学和细胞外基质几何形状的表面与调节流体和可溶性因子传输的微流体通道相结合，为细胞生长和刺激的空间和时间控制创造了新的机会。与生物分析微系统的进一步整合产生了用于细胞和组织的基本生物学见解的多功能平台，以及具有生物化学，生物医学和环境功能的基于细胞的传感器。高度集成的微器件为基础生物医学和药物研究带来了巨大希望，强大的便携式床边护理设备可用于发达国家和发展中国家的临床环境。Introduction
In their normal environment, cells are subject to multiple cues that vary in time and space, including gradients of cytokines and secreted proteins from neighboring cells, biochemical and mechanical interactions with the extracellular matrix (ECM), and direct cell–cell contacts (Box 1). Microfabricated systems can present cells with these cues in a controllable and reproducible fashion that cannot easily be achieved by standard tissue culture, and can be used to link cell culture with integrated analytical devices that can probe the biochemical processes that govern cell behavior. Some cell-based microsystems simply represent miniaturized versions of conventional laboratory techniques, whereas others exploit the advantages of small length scales and low Reynolds numbers 1, such as favorable scaling of electrical fields and the ability to create well-controlled laminar flows. In this Review, we discuss the application of microtechnology to cell biology and describe methods for cell culture, regulation of extracellular cues, cell fractionations and biochemical analysis on a micrometer scale (Fig. 1). Emphasis is placed on microsystems aimed at gaining biological insight, as well as on efforts to realize increasing cell-handling integration and biochemical analysis levels on chips.
在正常环境中，细胞受到多种时间和空间变化的线索的影响，包括细胞因子和邻近细胞分泌的蛋白质的梯度，与细胞外基质（ECM）的生化和机械相互作用，以及直接的细胞-细胞接触（方框1））。微制造系统可以以可控且可再现的方式呈现具有这些提示的细胞，这些方式不能通过标准组织培养容易
地实现，并且可以用于将细胞培养物与可以探测控制细胞行为的生化过程的集成分析装置联系起来。一些基于细胞的微系统仅代表传统实验室技术的小型化版本，而其他微型系统利用小长度尺度和低雷诺数1的优点，例如有利的电场缩放和产生良好控制的层流的能力。在本综述中，我们讨论了微技术在细胞生物学中的应用，并描述了细胞培养方法，细胞外线索的调节，细胞分级和微观尺度的生化分析（图1）。重点放在旨在获得生物学洞察力的微系统，以及实现增加芯片上的细胞处理整合和生化分析水平的努力。
We believe these devices will become increasingly implemented in applied and basic biomedical research, mainly because soft lithography 2 has put microfluidics within the reach of biology-focused academic laboratories. Elastomeric materials used in soft lithography, typically poly(dimethylsiloxane) (PDMS), are relatively easy to fabricate, and are compatible with most biological assays. Devices that are based on microfabrication of silicon and glass require access to advanced cleanroom facilities similar to those used for microelectronics. This typically involves higher cost, but has unique advantages for specialized applications, such as electrophoresis in glass devices.
我们相信这些设备将越来越多地应用于应用和基础生物医学研究，主要是因为软光刻技术2已将微流体技术置于以生物学为重点的学术实验室的范围内。用于软光刻的弹性材料，通常是聚（二甲基硅氧烷）（PDMS），相对容易制造，并且与大多数生物测定相容。基于硅和玻璃微加工的设备需要使用
类似于微电子设备的高级洁净室设施。这通常涉及更高的成本，但对于专门的应用具有独特的优势，例如玻璃装置中的电泳。
Much cell-based microsystem research takes place under a ‘lab-on-a-chip’ or ‘micro-total-analysis-system’ (µTAS) framework that seeks to create microsystems incorporating several steps of an assay into a single system 3–5. Integrated microfluidic devices perform rapid and reproducible measurements on small sample volumes while eliminating the need for labor-intensive and potentially error-prone laboratory manipulations. Thus, microfluidics allows experiments to be carried out
that cannot be performed simply by miniaturizing and mechanizing conventional laboratory procedures using robotics and microplates. For example, in cell-based studies, the transition from 384- to 1,536-well plates is proving challenging, largely because edge effects and uncontrolled evaporation from very small wells result in poorly defined culture conditions. Conventional handling of very small fluidic volumes is difficult, and subject to both variability and high fixed losses. The fabrication of many copies of an analytic device, small reagent volumes, and diminished labour associated with use of automated microfabricated devices should make them highly cost effective. Moreover, the small footprint and low power consumption of integrated systems creates opportunities
for portable, point-of-care devices that can perform analyses hitherto possible only in the research or clinical laboratory. Devices such as these with sophisticated diagnostic capabilities are likely to become important in the personalization of medical care.
许多基于细胞的微系统研究都是在“芯片实验室”或“微全分析系统”（μTAS）框架下进行的，该框架旨在创建将多个测定步骤合并到一个系统中的微系统3-5。集成的微流体设备可在小样本量上执行快速且可重复的测量，同时无需劳动密集型且可能容易出错的实验室操作。因此，微流体允许进行实验，这些实验不能简单地通过使用机器人和微孔板小型化和机械化传统的实验室程序来进行。例如，在基于细胞的研究中，从384孔板到1,536孔板的转变证明是具有挑战性的，主要是因为边缘效应和非常小的井的不受控制的蒸发导致不明确的培养条件。常规处理非常小的流体体积是困难的，并且受可变性和高固定损失的影响。制造分析装置的许多副本，小试剂量和与使用自动微制造装置相关的减少的劳动力应该使它们具有高成本效益。此外，集成系统的小尺寸和低功耗为便携式即时护理设备创造了机会，这些设备可以仅在研究或临床实验室中进行迄今为止可能的分析。具有复杂诊断功能的这些设备可能在医疗保健的个性化方面变得重要。
Many of the promises of µTAS have yet to be realized: integration and packaging of several functionalities into a single system is proving to be a complex task (Fig. 1), and many cell-based microsystems available today are still in the proof-of-concept phase. Typical unit operations (for exa
mple, growth, treatment, selection, lysis, separation and analysis) have been demonstrated (Fig. 2), but robust approaches to fabrication, integration and packaging (such as communication with the macroenvironment) remain major areas of research.
μTAS的许多承诺尚未实现：将多个功能集成和封装到单个系统中被证明是一项复杂的任务（图1），现在许多基于单元的微系统仍然可以证明- 概念阶段。已经证明了典型的单元操作（例如，生长，处理，选择，裂解，分离和分析）（图2），但是制造，集成和包装的强大方法（例如与宏观环境的通信）仍然是研究的主要领域。
Box 1 | Cell physiology, phenotype and fate are regulated by cell-autonomous processes and extracellular signaling molecules.方框1 | 细胞生理学，表型和命运受细胞自主过程和细胞外信号分子的调节。
Soluble signalling molecules include hormones, cytokines and growth factors produced by local or distant cells (giving rise to paracrine and endocrine signals, respectively), and even by the receiving cell itself (autocrine signals). Insoluble signalling molecules include components of the ECM and membrane-bound proteins on neighbouring cells. Cells sense most extracellular signals (including proteins, peptides and carbohydrates) via transmembrane receptors that activate complex biochemic
al cascades of kinases, proteases, adaptor proteins, transcription factors and so on, which together act to regulate cell physiology. At the same time, cells alter their surrounding environment by making or destroying ECM or soluble factors and by exerting mechanical force 99, 100.
可溶性信号分子包括由局部或远端细胞产生的激素，细胞因子和生长因子（分别产生旁分泌和内分泌信号），甚至由接收细胞本身产生（自分泌信号）。不溶性信号分子包括ECM的组分和相邻细胞上的膜结合蛋白。细胞通过跨膜受体感知大多数细胞外信号（包括蛋白质，肽和碳水化合物），这些受体激活复杂的生物化学级联激酶，蛋白酶，衔接蛋白，转录因子等，它们共同起到调节细胞生理作用的作用。同时，细胞通过制造或破坏ECM或可溶性因子并通过施加机械力99,100来改变其周围环境。
In animals, cells typically reside in environments with very specific three-dimensional (3D) features. Cells are sensitive to the presence of neighbouring cells of similar or different type and often make long-lasting mechanical and biochemical connections to them. In columnar epithelia, for example, identical cells lined up side by side assemble junctions with neighbours to form continuous impermeable sheets. These sheets are polarized such that cells interact with their surroundings in very different ways on the lumenal and basolateral surfaces. In addition, epithelia usually establish a close relationship with specific types of immune cell. In these epithelia, both homotypic and heterotypic cell–cell interactions are essential to maintain cell and tissue function.
在动物中，细胞通常存在于具有非常特定的三维（3D）特征的环境中。细胞对相似或不同类型的相邻细胞的存在敏感，并且通常与它们形成持久的机械和生物化学连接。例如，在柱状上皮细胞中，并排排列的相同细胞与邻居组装
连接以形成连续的不可渗透的片。这些薄片被极化，使得细胞在腔和基底外侧表面上以非常不同的方式与它们的周围环境相互作用。此外，上皮细胞通常与特定类型的免疫细胞建立密切关系。在这些上皮细胞中，同型和异型细胞- 细胞相互作用对于维持细胞和组织功能是必需的。
Most in vitro experiments with adherent human cells are performed in two-dimensional (2D) cultures in which cells are plated onto plastic surfaces treated to stimulate cell binding. Depending on their type, cells either grow directly on the plastic, secrete ECM components that coat the plastic to facilitate cell adhesion, or require pre-coating of the plastic with ECM. Standard 2D culture conditions are poor mimics of the cellular environment in an animal: soluble growth factors are present at abnormally high concentrations, 3D cues are largely absent, oxygen tension is too high and cell–cell interactions are rarely organized. Attempts have been made to overcome these problems using organ culture and various laboratory-scale bioreactors, but microsystems provide a much more effective means of controlling cell environment in vitro. Particularly promising are various artificial organ systems in which multiple cell types are grown together under conditions that mimic normal 3D
environmental and circulatory cues.
粘附人细胞的大多数体外实验在二维（2D）培养中进行，其中将细胞接种到处理以刺激细胞结合的塑料表面上。根据其类型，细胞可直接在塑料上生长，分泌涂覆塑料的ECM组分以促进细胞粘附，或需要用ECM预涂塑料。标准的2D培养条件是动物细胞环境的差模拟：可溶性生长因子以异常高的浓度存在，3D提示基本上不存在，氧张力太高并且细胞- 细胞相互作用很少组织。已经尝试使用器官培养和各种实验室规模的生物反应器来克服这些问题，但是微系统提供了更有效的体外控制细胞环境的手段。特别有希望的是各种人工器官系统，其中多种细胞类型在模拟正常3D环境和循环线索的条件下一起生长。
Figure 1 | Tissue organization, culture and analysis in microsystems.Advanced tissue organization and culture can be performed in microsystems by integrating homogeneous and heterogeneous cell ensembles, 3D scaffolds to guide cell growth, and microfluidic systems for transport of nutrients and other soluble factors. Soluble factors — for example, cytokines for cell stimulation — can be presented to the cells in precisely defined spatial and temporal patterns using integrated microfluidic systems. Microsystems technology can also fractionate heterogeneous cell populations into homogeneous populations, including single-cell selection, so different cell types can be analysed separately. Microsystems can incorporate numerous techniques for the analysis of the biochemical reactions in cells, including image-based analysis and techniques for gene and protein analysis of cell lysates. This makes microtechnology an excellent tool in cell-based applications and in the fundamental study of cell biology. As indicated by the yellow arrows, the different microfluidic components can be connected with each other to form an integrated system, realizing multiple functionalities on a single chip. However, this integration is challenging with respect to fluidic and sample matching between the different components, not least because of the difficulty in simultaneously packaging fluidic, optical, electronic and biological components into a single system.
图1 |微系统中的组织组织，培养和分析。通过整合同源和异质细胞集合，3D支架以指导细胞生长，以及用于运输营养素和其他可溶
性因子的微流体系统，可以在微系统中进行先进的组织组织和培养。可溶性因子- 例如，用于细胞刺激的细胞因子- 可以使用集成的微流体系统以精确限定的空间和时间模式呈现给细胞。微系统技术还可以将异质细胞分成均质体，包括单细胞选择，因此可以分别分析不同的细胞类型。Microsystems可以结合多种技术分析细胞中的生化反应，包括基于图像的分析和细胞裂解物的基因和蛋白质分析技术。这使得微技术成为基于细胞的应用和细胞生物学基础研究的优秀工具。如黄箭头所示，不同的微流体组件可彼此连接以形成集成系统，从而在单个芯片上实现多种功能。然而，这种集成在不同组件之间的流体和样品匹配方面具有挑战性，尤其是因为难以将流体，光学，电子和生物组件同时包装到单个系统中。
细胞芯片
Microfabricated cell cultures
微纤维化细胞培养物
Culturing cells in vitro is one of the cornerstones of modern biology. Nevertheless, even for intensively studied tissues, many of the factors that induce or stabilize differentiated phenotypes are poorly understood and difficult to mimic in vitro 6. One approach to increase control over cell–cell and soluble cues typical of in vivo cell environments is to combine microfabrication of 3D ECM structures and microfluidic networks that transport soluble factors such as nutrients and oxygen. Micr
ofluidics has the additional advantage of being capable of creating mechanical strain, through shear, in the physiological range.
体外培养细胞是现代生物学的基石之一。然而，即使对于深入研究的组织，许多诱导或稳定分化表型的因素也很难理解并且难以在体外模拟 6.增加对体内细胞环境典型的细胞- 细胞和可溶性线索的控制的一种方法是结合3D ECM结构的微制造和传输可溶性因子如营养素和氧气的微流体网络。微流体的另一个优点是能够在生理范围内通过剪切产生机械应变。
Cells and the extracellular matrix
细胞和细胞外基质
Microfabrication integrating micropatterning techniques with advanced surface chemistry makes it possible to reproducibly engineer cell micro-environment at cellular resolution. A large variety of surface-patterning techniques are available, including standard photolithography liftoff techniques, photoreactive chemistry and, increasingly, techniques based on soft lithography (microcontact printing and fluidic patterning) 7. Surface patterning of micrometre-sized features allows micrometrescale control over cell–ECM interactions and can be used to generate ensembles of cells with defined geometry. Lamination, moulding and photo-polymerization techniques all allow fabricatio
n of 3D scaffolds with feature sizes in the lower micrometre range, including microstructured scaffolds made of biodegradable materials 8.
微细加工将微图案化技术与先进的表面化学相结合，可以在细胞分辨率下重复设计细胞微环境。有多种表面图案化技术可供选择，包括标准光刻剥离技术，光反应化学以及越来越多的基于软光刻（微接触印刷和流体图案化）的技术.7。微米尺寸特征的表面图案化允许对细胞进行微米级控制- ECM交互并可用于生成具有已定义几何的单元集合。层压，模塑和光聚合技术都可以制造出具有较低微米范围特征尺寸的3D支架，包括由可生物降解材料制成的微结构支架8。
The precise control of the cellular environment that has been made possible by microtechnology provides new opportunities for understanding biochemical and mechanical processes responsible for changes in behaviour such as the effects of cell shape on the anchorage-dependence of cell growth 9,10. For example, by altering the spacing of a grid of cell-adhesive islands it is possible to control the extent of cell spreading, while keeping the cell–ECM contact area constant 10 (Fig. 3a). Human capillary endothelial cells confined to closely spaced islands undergo apoptosis, whereas cells that can spread freely survive and proliferate normally 10. Adhesive ECM patches can also be designed so that the locations of focal adhesions (integrin-mediated links between the ECM and actin cytoskeleton) result in the same overall cell shape, but with a different underlying cytoskeletal organi
zation (Fig. 3b). By allowing cells to spread and proliferate on these adhesive patches the orientation of the cell division axis can be controlled 11. Similar regulation of the division axis by the ECM is likely to be important for tissue morphogenesis and other developmental processes.
通过微技术实现的细胞环境的精确控制为理解负责行为变化的生化和机械过程提供了新的机会，例如细胞形状对细胞生长的锚定依赖性的影响9,10。例如，通过改变细胞粘附岛网格的间距，可以控制细胞扩散的程度，同时保持细胞-ECM接触面积恒定10（图3a）。限制在间隔紧密的岛上的人毛细血管内皮细胞经历细胞凋亡，而能够自由扩散的细胞可以正常存活和增殖10.粘附性ECM贴片也可以设计成使得粘着斑的位置（整合素介导的ECM和肌动蛋白细胞骨架之间的连接）导致相同的整体细胞形状，但具有不同的潜在细胞骨架组织（图3b）。通过使细胞在这些粘性贴剂上扩散和增殖，可以控制细胞分裂轴的方向11. ECM对分裂轴的类似调节可能对组织形态发生和其他发育过程很重要。
The force exerted on the ECM by cells can be measured in several ways. A particularly powerful method involves measuring
the deflection of arrays of micrometre-sized vertical elastomer posts (Fig. 3c). When tested with smooth muscle cells, forces acting in the plane of the substrate are in the range of 100 nN, and appear to scale with the area covered by focal adhesions 12. Compared with conventional methods t
hat rely on substrate distortion, the elastomer-post technique has the advantage of greater accuracy and manipulability: the mechanical properties of a surface can be varied by changing post geometries without altering surface chemistry 12.
细胞施加在ECM上的力可以用几种方法测量。一种特别有效的方法涉及测量微米尺寸垂直弹性体柱阵列的偏转（图3c）。当用平滑肌细胞测试时，作用在基底平面中的力在100nN的范围内，并且看起来与焦点粘附所覆盖的区域成比例。与依赖于基底变形的常规方法相比，弹性体柱技术具有更高精度和可操作性的优点：通过改变柱几何形状而不改变表面化学12，可以改变表面的机械性能。
Figure 2| Microsystems enabling cell-based assays from cell culture to biochemical analysis. A collection of microsystems enabling cell-based assays, covering all the steps from cell culture, through selection and treatment, to biochemical analysis. a, Image showing six bioreactors that can operate in parallel on a single chip. Each reactor can be used to monitor the growth of extremely small numbers of cells. (Image reproduced, with permission, from ref. 20.) b, Microfluidic cell-culture array with integrated concentration gradient generator (left). Image of concentration gradient across ten columns when loaded with blue and yellow dye. (Image reproduced, with permission, from ref. 33.) c, Two different laminar streams exposing two sides of a single cell to different conditions 34. d, Perfusion over a single hydrodynamically trapped cell. Switching of the perfused media can occur in ~100 ms. (Image reproduced, with permission, from ref. 38.) e, Single-cell dielectrophoresis (DEP) trap, consisting of four electroplated electrodes (left). Fluorescent image of a trapped cell (indicated by blue arrow; right). The cell has been loaded with calcein through the microfluidic system. (Image reproduced, with permission, from ref. 46.) f, Fluorescent image of light path at the detection zone in a micro flow cytometer with integrated waveguides and lenses. (Image reproduced, with permission, from ref. 53.) g, Scanning electron micrograph of a mechanical lysis device with sharp knife-like protrusions. (Image reproduced, with permission, from ref. 55.) h, Schematic of electrical lysis device with integrated microelectrodes. (Image reproduced, with permission, from ref. 56.) i, Isoelectric focu
sing of cell organelles from whole-cell lysate. The mitochondria focuses in a band at pI between 4 and 5. (Image reproduced, with permission, from ref. 62.) j, Two-dimensional separation of
four model proteins. Isoelectric focusing (top) followed by SDS gel electrophoresis. (Image reproduced, with permission, from ref. 64.) k, Schematic of immunoassay performed using microbeads as solid support in a microfluidic system. (Image adapted, with permission, from ref. 69.) l, Schematic of a hollow cantilever-based mass sensor for analyte detection. (Image adapted, with permission, from ref. 74.)
图2 |微系统使细胞培养从细胞培养到生化分析成为可能。一系列微系统，可实现基于细胞的分析，涵盖从细胞培养，选择和到生化分析的所有步骤。a，图像显示了六个可在单个芯片上并行运行的生物反应器。每个反应器可用于监测极少量细胞的生长。（图片转载，经许可，参考文献20）.b，具有集成浓度梯度发生器的微流体细胞培养阵列（左）。当加载蓝和黄染料时，十列浓度梯度的图像。（图片转载，经许可，参见参考文献33.）c，两个不同的层流将单个细胞的两侧暴露于不同的条件34. d，在单个流体动力学捕获的细胞上灌注。灌注介质的切换可以在~100ms内发生。（图片转载，经许可，参见参考文献38.）e，单细胞介电电泳（DEP）捕获器，由四个电镀电极组成（左）。被困细胞的荧光图像（用蓝箭头表示；右）。细胞已通过微流体系统装载钙黄绿素。（图片转载，经许可，参考文献46.）f，微流式细胞仪检测区光路的荧光图像，带有集成的波导和镜头。（经许可，
转载自参考文献53的图像。）g，具有锋利的刀状突起的机械裂解装置的扫描电子显微照片。（经许可，转载自参考文献55的图像。）h，具有集成微电极的电解冻装置的示意图。（图片转载，经许可，参考文献56.）i，从全细胞裂解液中细胞器的等电聚焦。线粒体聚焦在pI在4和5之间的条带中。（图片转载，经许可，参考文献62）.j，四种模型蛋白质的二维分离。等电聚焦（上图），然后进行SDS凝胶电泳。（经许可，参考文献64）再现图像。）k，使用微珠作为微流体系统中的固体支持物进行的免疫测定的示意图。（经许可，图片改编自参考文献69.）l，用于分析物检测的基于空心悬臂的质量传感器的示意图。（图片改编，经许可，参考文献74.）
Liver-cell culture肝细胞培养
In vitro culture of liver cells has received particular attention in biotechnology as many drugs fail in clinical studies either because they damage the liver directly or because liver metabolites are toxic 13. The study of hepatotoxicity would be greatly facilitated by the availability of in vitro culture systems that mimic real liver conditions. However, the development of liver-cell cultures as biosensors for drug toxicity faces challenges because of the difficulty in maintaining the differentiated phenotypes.
肝细胞的体外培养在生物技术中受到特别关注，因为许多药物在临床研究中失败，因为它们直接损害
肝脏或因为肝脏代谢物有毒13。通过模拟真实肝脏条件的体外培养系统的可用性将极大地促进肝毒性的研究。然而，肝细胞培养物作为药物毒性的生物传感器的发展面临挑战，因为难以维持分化的表型。
In the liver, hepatocytes are found in a complex 3D environment in which nutrients, soluble factors and oxygen are transported through blood capillaries and bile canaliculi. Using silicon as a substrate, perfused 3D liver reactors have been fabricated on arrays of 300-µm-wide channels (capillaries) that comprise a scaffold for the ECM 14 (Fig. 3d). Seeding hepatocytes with pre-aggregated multicellular spheroids in the 3D reactor generates cultures that are viable for a long time period (~3 weeks) and that exhibit a stable differentiated phenotype. Cells in 3D liver cultures also have cell–cell contacts, such as tight junctions and desmosomes, that resemble those found in tissues in vivo 13,14.
在肝脏中，肝细胞存在于复杂的3D环境中，其中营养物，可溶性因子和氧气通过毛细血管和胆小管传输。使用硅作为基底，灌注的3D肝脏反应器已经在300μm宽的通道（毛细管）阵列上制造，其包含ECM 14的支架（图3d）。在3D反应器中用预聚集的多细胞球体接种肝细胞产生长时间（约3周）存活并且表现出稳定分化表型的培养物。3D肝脏培养物中的细胞也具有细胞- 细胞接触，例如紧密连接和桥粒，类似于体内组织中发现的那些13,14。
It has been observed that co-culture of hepatocytes with other cell types, including liver epithelial cells and Kupffer cells, prolongs the survival of cultured hepatocytes and helps maintain liver-specific properties such as albumin secretion 15. Using a micropatterned 2D co-culture system, it has also been shown that liver-specific functions increase with heterotypic cell–cell interactions. Only hepatocytes close to the heterotypic interface maintain their differentiated phenotypes in longer-term culture 6 (Fig. 3e). Relative to conventional co-culture, in which seeding densities of two cell types are varied on a planar surface, micropatterning techniques afford greatly improved control of homo- and heterotypic cell–cell interactions 16. The ability to culture cells such as liver cells in vitro and to demonstrate protein and gene expression levels similar to those found in tissue suggests that microfabricated cultures could have applications in toxicology and could also serve as model systems for in vitro analogues of organ tissue.
已经观察到肝细胞与其他细胞类型（包括肝上皮细胞和库普弗细胞）的共培养延长了培养的肝细胞的存活并且有助于维持肝特异性特性，例如白蛋白分泌15.使用微图案2D共培养系统，还表明肝脏特异性功能随着异型细胞- 细胞相互作用而增加。只有接近异型界面的肝细胞才能在长期培养中维持其分化表型6（图3e）。相对于常规共培养，其中两种细胞类型的种子密度在平面表面上变化，微图案化技术极大地改善了对同型和异型细胞- 细胞相互作用的控制16.在体外培养肝细胞等细胞的能力并且为了证明
与组织中发现的蛋白质和基因表达水平类似的蛋白质和基因表达水平表明微制造的培养物可以在毒理学中具有应用并且还可以用作器官组织的体外类似物的模型系统。
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