Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.

Young’s modulus and Poisson’s ratio of a porous polymeric construct (scaffold) quantitatively describe how it supports and transmits external stresses to its surroundings. While Young’s modulus is always non-negative and highly tunable in magnitude, Poisson’s ratio can, indeed, take on negative values despite the fact that it is non-negative for virtually every naturally occurring and artificial material. In some applications, a construct having a tunable negative Poisson’s ratio (an auxetic construct) may be more suitable for supporting the external forces imposed upon it by its environment. Here, three-dimensional polyethylene glycol scaffolds with tunable negative Poisson’s ratios are fabricated. Digital micromirror device projection printing (DMD-PP) is used to print single-layer constructs composed of cellular structures (pores) with special geometries, arrangements, and deformation mechanisms. The presence of the unit-cellular structures tunes the magnitude and polarity (positive or negative) of Poisson’s ratio. Multilayer constructs are fabricated with DMD-PP by stacking the single-layer constructs with alternating layers of vertical connecting posts. The Poisson’s ratios of the single- and multilayer constructs are determined from strain experiments, which show (1) that the Poisson’s ratios of the constructs are accurately predicted by analytical deformation models and (2) that no slipping occurrs between layers in the multilayer constructs and the addition of new layers does not affect Poisson’s ratio.

This article presents a micromanufacturing method for direct projection printing of three-dimensional scaffolds for applications in the field of tissue engineering by using a digital micromirror-array device (DMD) in a layer-by-layer process. Multilayered scaffolds are microfabricated using curable materials through an ultraviolet (UV) photopolymerization process. The prepatterned UV light is projected onto the photocurable polymer solution by creating the “photomask” design with a graphic software. Poly(ethylene glycol) diacrylate is mixed with a small amount of dye (0.3wt%) to enhance the fabrication resolution of the scaffold. The DMD fabrication system is equipped with a purging mechanism to prevent the accumulation of oligomer, which could interfere with the feature resolution of previously polymerized layers. The surfaces of the predesigned multilayered scaffold are covalently conjugated with fibronectin for efficient cellular attachment. Our results show that murine marrow-derived progenitor cells successfully attached to fibronectin-modified scaffolds.

Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (~3 kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (~90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to regulate stem cell fate, and for rapidly optimizing ECM-containing scaffolds to support stem cell differentiation and tissue regeneration.

The development of biomedical scaffolds mimicking a heterogeneous cellular microenvironment for a specified regulation of cell-fates is very promising for tissue engineering. In this study, three-dimensional scaffolds with heterogeneous microstructure were developed using a DMD-PP apparatus. During the fabrication process, this apparatus can efficiently switch monomers to form microstructures with localized, different material properties; the resolution in the arrangement of material properties is comparable to the characteristic size of functional subunits in living organs, namely, a hundred microns. The effectiveness of this DMD-PP apparatus is demonstrated by a woodpile microstructure with heterogeneous fluorescence and also by a microporous cell-culturing scaffold with selected sites for protein adhesion. Cell-cultivation experiment was performed with the microporous scaffold, in which selective cell adhesion was observed.

Hydrogels (HGs) are attractive matrices for cell-based cartilage tissue regeneration given their injectability and ability to fill defects with irregular shapes. However, most HGs developed to date often lack cell scale macroporosity, which restrains the encapsulated cells, leading to delayed new extracellular matrix deposition restricted to pericellular regions. Furthermore, tissue-engineered cartilage using conventional HGs generally suffers from poor mechanical property and fails to restore the load-bearing property of articular cartilage. The goal of this study was to evaluate the potential of macroporous gelatin-based microribbon (μRB) HGs as novel 3D matrices for accelerating chondrogenesis and new cartilage formation by human mesenchymal stem cells (MSCs) in 3D with improved mechanical properties. Unlike conventional HGs, these μRB HGs are inherently macroporous and exhibit cartilage-mimicking shock-absorbing mechanical property. After 21 days of culture, MSC-seeded μRB scaffolds exhibit a 20-fold increase in compressive modulus to 225 kPa, a range that is approaching the level of native cartilage. In contrast, HGs only resulted in a modest increase in compressive modulus of 65 kPa. Compared with conventional HGs, macroporous μRB scaffolds significantly increased the total amount of neocartilage produced by MSCs in 3D, with improved interconnectivity and mechanical strength. Altogether, these results validate gelatin-based μRBs as promising scaffolds for enhancing and accelerating MSC-based cartilage regeneration and may be used to enhance cartilage regeneration using other cell types as well.

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Tissue engineering has largely focused on single tissue-type reconstruction (such as bone); however, the basic unit of healing in any clinically relevant scenario is a compound tissue type (such as bone, periosteum, and skin). Nanofibers are submicron fibrils that mimic the extracellular matrix, promoting cellular adhesion, proliferation, and migration. Stem cell manipulation on nanofiber scaffolds holds significant promise for future tissue engineering. This work represents our initial efforts to create the building blocks for composite tissue reflecting the basic unit of healing. Polycaprolactone (PCL) nanofibers were electrospun using standard techniques. Human foreskin fibroblasts, murine keratinocytes, and periosteal cells (4-mm punch biopsy) harvested from children undergoing palate repair were grown in appropriate media on PCL nanofibers. Human fat-derived mesenchymal stem cells were osteoinduced on PCL nanofibers. Cell growth was assessed with fluorescent viability staining; cocultured cells were differentiated using antibodies to fibroblast- and keratinocyte-specific surface markers. Osteoinduction was assessed with Alizarin red S. PCL nanofiber scaffolds supported robust growth of fibroblasts, keratinocytes, and periosteal cells. Cocultured periosteal cells (with fibroblasts) and keratinocytes showed improved longevity of the keratinocytes, though growth of these cell types was randomly distributed throughout the scaffold. Robust osteoinduction was noted on PCL nanofibers. Composite tissue engineering using PCL nanofiber scaffolds is possible, though the major obstacles to the trilaminar construct are maintaining an appropriate interface between the tissue types and neovascularization of the composite structure.