State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

Abstract

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Fig. 1. Microenvironmental factors affecting cell behavior

Numerous aspects of the microenvironment that change spatially and temporally may affect how accurately a 3D model reflects cellular behavior in vivo. Conversely, cells (center) can actively modify their local microenvironment. Reprinted with permission from Yamada and Cukierman (2007).

Fig. 2. Schematic representation of the situation at the cell culture media/biological tissue interface of a tissue engineering construct

From the interface, substance A is distributed via diffusion and eliminated throughout the biological tissue.

Fig. 3. Cellular cross talk via soluble factors in a 2D system

(A) Cells in a monolayer secrete and internalize signaling factor. The messenger is distributed in the supernatant. (B) Simulated distribution of the concentration of a signaling molecule in the supernatant. The molecule is secreted and eliminated along the x-axis by the cells. qS indicates secretion, qI internalization. The formation of a gradient at the cell/supernatant interface is possible.

Fig. 4. 3D liver cell cultivation techniques

(A) Polarized hepatocytes in collagen sandwich cultures (reprinted with permission from Tuschl, 2009). (B) 3D membrane bioreactors with perfusion system (reprinted with permission from Zeilinger et al., 2011) (C) Stirrer bioreactor for 3D aggregates (reprinted with permission from Tostoes et al., 2012). (D) Hanging drop liver organoids (reprinted with permission from Mueller et al., 2013). (E) 3D polystyrene scaffold culture (sc-scaffold, grey arrow indicates microvilli) (reprinted with permission from Bokhari, 2007). (F) A microfluidic device ensuring perfusion in multi-well plate format (reprinted with permission from Domansky, 2010).

Fig. 5. Schematic representation of 3D in vitro models of the nervous system

The first 3D in vitro models used rodent acute or organotypic brain slices. These are still a major model for electrophysiological studies (A). Alternative approaches use co-culture models consisting of, e.g., a layer of neurons directly on top of a glial cell layer (B). Transwell culture models are used, e.g., for in vitro blood-brain barrier models by growing endothelial cells on a perforated membrane above a glial cell layer. Neurons may be attached to the bottom compartment (C). Re-aggregating brain cell culture models are formed by spontaneous re-aggregation of dissociated rodent brain cells under continuous shaking (D). Floating neurospheres can be generated from neural stem cells/neural progenitor cells, kept in low adherence plastic plates (E). Secondary 3D cultures are produced by plating neurospheres onto a planar adhesive substrate, which facilitates outgrowth of radial glia and migrating neurons (F). Engineered neural tissue (ENT) is generated by growing highly concentrated suspensions of stem cells on a membrane floating at the air-liquid interphase. This tissue is polarized (e.g., astrocytes at the bottom), consists of several subtypes of neurons and shows rosettes as neural tube-like structures (G).

Fig. 6. Schematic representation of 3D in vitro models of lung

Many co-cultures mimicking the alveolar barrier are described in the literature (A). The most common approach is to use permeable membrane inserts in a two chamber system with epithelial cells grown on the upper side and endothelial cells on the opposite (B). Design of the triple cell co-culture composed of epithelial cells with macrophages on top and dendritic cells on the opposite of the insert membrane. (C) The microfabricated lung device uses compartmentalized polydimethylsiloxane (PDMS) microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with extracellular matrix. The device recreates physiological breathing movements by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier (reprinted with permission from Huh et al., 2010).

Fig. 7. In monolayer culture, cellular morphology differs from in vivo morphology

Upper picture: Fibroblasts in monolayer culture on lamella observed by scanning electron microscopy (SEM): population sub-confluence. Cells have rather starry shapes. Length of the line 10 μm (Image: A. Minondo, L’Oréal); lower pictures: human fibroblasts project a dendritic network of extensions in collagen matrices but not on collagen-coated coverslips. Fibroblasts were incubated for 5 h on collagen-coated surfaces (A) or in collagen matrices (D). At the end of the incubations, samples were fixed and stained for actin. Reprinted with permission from Grinnell et al. (2003).

Fig. 8. Reconstructing human skin

Normal fibroblasts and keratinocytes are isolated from, e.g., human foreskin and expanded in monolayer culture. Fibroblasts are embedded into a collagen matrix (step 1) and the collagen is allowed to contract (growth medium). Keratinocytes are seeded on top and medium is changed to differentiation medium (step 2). Finally the culture is lifted to the air-medium interface (step 3). Total culture duration is about 14 days. For the reconstruction of human epidermis step 1 is omitted and keratinocytes are seeded on a supporting membrane.

Fig. 9

Histology of a reconstructed epidermis, EpiSkin^™

Fig. 10. Matrix effects on 3D RHS

Both 3D organization and cell-derived matrix components are required for formation of 3D matrix adhesions (triple label localization of α5 integrin (green, A), paxillin (red, B), and fibronectin blue, (B)) in RHS. The yellow color (A) represents the merged labels of α5 in green and paxillin in red. Triple merged images for the indicated matrices are shown in (C) to (F). Note that complete triple co-localization (white) is observed only with in vivo-like, cell-derived 3D matrix (C) or mouse tissue-derived 3D matrix (F) in 3D matrix adhesions (arrows). Reprinted with permission from Cukierman et al. (2001).