From 3D cell culture to organs-on-chips

Abstract

3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.

Figure 1. Microengineering technologies used to construct 3D culture systems and organs-on-chips

(a) Photolithography is a core microfabrication technique used to transfer microscale patterns to photosensitive materials by selective exposure to optical radiation. A silicon wafer is spin coated with a thin uniform film of a photosensitive material (photoresist), which is then aligned and brought in close contact with a photomask that typically consists of a transparent glass plate covered with a pattern defined by opaque chrome layers; the microscale pattern desired by the user is generated with computer-assisted design software. This is followed by exposure of the photoresist to high-intensity ultraviolet (UV) light through the photomask, which protects some regions of the photoresist from UV and exposes others based on the design of the pattern. UV-exposed areas become soluble in a developer solution and dissolve away during the following step, called development, thus leaving the desired microscale pattern etched into the photoresist. (b) Soft lithography involves fabrication of elastomeric stamps using a replica-molding technique in which liquid prepolymer of PDMS is cast against the bas-relief pattern of photoresist produced by photolithography (a) to generate a PDMS substrate that replicates the 3D topography of the original master. In microcontact printing, the PDMS stamp is inked with protein solution, dried, and brought in conformal contact with a surface for a period ranging from 30s to several minutes. Upon removal of the stamp, a pattern is generated on the surface that is defined by the raised bas-relief structure of the stamp, and hence precisely recreates the microscale pattern of the original master. (c) Microfluidic devices are typically created by bonding a PDMS substrate containing microchannel features created by replica molding with a blank PDMS slab. In these microdevices, two fluids (red and green) flowing through independent inlets meet at a Y-junction and enter a straight microchannel in which they flow in adjacent, laminar streams without mixing (arrows indicate flow direction; scale bar, 500 µm). Reproduced from [84] with permission.

Figure 2. Microengineered organs-on-chips

(a) A microfluidic kidney epithelium model composed of a multi-layered microdevice that incorporates stacked layers of PDMS microchannels and a PDMS well separated by a porous polyester membrane. The 3D architecture of this microsystem provides physiologically relevant culture environments for polarized kidney epithelial cells, and enables precise control of fluid flows, selective exposure of the apical and basal sides of the cells to fluid shear, hormones, and chemical gradients, and collection of samples from both sides of the polarized tissue. (b) A microengineered liver-on-a-chip reconstitutes hepatic microarchitecture. The functional unit of this microsystem consists of a central liver cell culture chamber and a surrounding nutrient flow channel separated by microfabricated barrier structures patterned with a set of narrow (2 µm in width) microchannels that mimic the highly permeable endothelial barrier between hepatocytes and the liver sinusoid. This biomimetic device closely approximates transport of nutrients and waste products in the liver sinusoid and provides more favorable environments for the maintenance of primary liver cells in a differentiated state (scale bar, 50 µm). (c) Heterotypic interactions between tumor cells and endothelial cells are studied in a microfluidic device that permits co-culture of these cells in two separate microchannels connected via scaffold channels filled with 3D collagen gels (pink channels in the diagram). This microfluidic system is used to model the tumor microenvironment and gain better understanding of important disease processes such as angiogenesis and cancer cell invasion during cancer progression. Reproduced from [50], [55], [60, 61] with permission.

Figure 3. A human breathing lung-on-a-chip

A microengineered model of the alveolar-capillary interface within a clear flexible microfluidic chip about the size of a computer memory stick (top right visualized on the microscope under fluorescence illumination; bottom right shows scanning electron micrographic view) that reconstitutes the cellular, biochemical and mechanical functions of the living human lung. The critical tissue-tissue interface of the alveolus (top left) is replicated in this bioinspired microdevice by co-culturing human alveolar epithelial cells and pulmonary capillary endothelial cells on the opposite sides of a thin, flexible, porous, ECM-coated PDMS membrane (bottom left). To accomplish mechanical actuation that mimics physiological breathing movements, air pressure in two hollow side chambers microfabricated within the device is decreased and increased in a cyclic manner by using a small vacuum pump; this causes the membrane and attached human cell layers to cyclically stretch and relax under physiological mechanical strain. The lung epithelial cells are cultured at an air-liquid interface, and culture medium is flowed through the lower microchannel containing the capillary cell layer to mimic blood flow through lung microvasculature. This system effectively mimics the entire human inflammatory response when pathogens or inflammatory cytokines are placed in the air channel and human neutrophils are flowed through the capillary channel; the device also can be used to study absorption and toxic effects of airborne particles, chemicals or drugs. Reproduced from [71] with permission.

Figure 4. The human-on-a-chip concept

Biomimetic microsystems representing different organs can be integrated into a single microdevice and linked by a microfluidic circulatory system in a physiologically relevant manner to model a complex, dynamic process of drug absorption, distribution, metabolism and excretion, and to more reliably evaluate drug efficacy and toxicity. As shown in this example, an integrated system of microengineered organ mimics (lung, heart, gut, liver, kidney and bone) can be used to study absorption of inhaled aerosol drugs (red) from the lung to microcirculation, as well as measure their cardiotoxicity (e.g. changes in heart contractility or conduction), transport and clearance in the kidney, metabolism in the liver and immune cell contributions to these responses. Drug substances (blue) also can be introduced into the gut compartment to investigate interplay between orally administered drugs and molecular transporters and metabolizing enzymes expressed in the various organs.