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Modular cardiac tissues developed both vascular and cardiac structures in vivo, provided that the host response was attenuated by omitting xenoproteins from the modules. Collagen gel modules (with Matrigel(TM)) containing cardiomyocytes (CMs) alone or CMs with surface-seeded endothelial cells (ECs; CM/EC modules) were injected into the peri-infarct zone of the heart in syngeneic Lewis rats. After 3 weeks, donor ECs developed into blood vessel-like structures that also contained erythrocytes. However, no donor CMs were found within the implant sites, presumably because host cells including macrophages and T cells infiltrated extensively into the injection sites. To lessen the host response, Matrigel was omitted from the matrix and the modules were rinsed with serum-free medium prior to implantation. Host cell infiltration was attenuated, resulting in a higher degree of vascularization with CM/EC modules than with CM modules without ECs. Most importantly, donor CMs matured into striated muscle-like structures in Matrigel-free implants., (Copyright © 2013 John Wiley & Sons, Ltd.)

Three-dimensional (3D) culture systems such as cell-laden hydrogels are superior to standard two-dimensional (2D) monolayer cultures for many drug-screening applications. However, their adoption into high-throughput screening (HTS) has been lagging, in part because of the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden prepolymer solutions into 2D well plates is a potential solution but typically requires large volumes of reagents to avoid evaporation during polymerization, which (1) increases costs, (2) makes drug penetration variable and (3) complicates imaging. Here we describe a technique to efficiently produce 3D microgels using automated liquid-handling systems and standard, nonpatterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of a ~2.5 mm diameter microwell with no concerns about evaporation or meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. The cytotoxicity of chemotherapeutics was monitored by bioluminescence and demonstrated that 3D cultures confer chemoresistance as compared with similar 2D cultures. Hence, these data demonstrate the importance of culturing cells in 3D to obtain realistic cellular responses. Overall, this system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure., (© 2014 Society for Laboratory Automation and Screening.)

Three-dimensional spheroid cultures have become increasingly popular as drug screening platforms, especially with the advent of different high throughput spheroid forming technologies. However, comparing drug efficacy across different cell types in spheroid culture can be difficult due to variations in spheroid morphologies and transport characteristics. Improving the reproducibility of compact, circular spheroids contributes to standardizing and increasing the fidelity of the desired gradient profiles in these drug screening three-dimensional tissue cultures. In this study we discuss the role that circularity and compaction has on spheroids, and demonstrate the impact methylcellulose (MethoCel) and collagen additives in the culture media can contribute to more compact and circular spheroid morphology. We demonstrate that improved spheroid formation is not a simple function of increased viscosity of the different macromolecule additives, suggesting that other macromolecular characteristics contribute to improved spheroid formation. Of the various macromolecular additives tested for hanging drop culture, MethoCel provided the most desirable spheroid formation. Additionally, the higher viscosity of MethoCel-containing media improved the ease of imaging of cellular spheroids within hanging drop cultures by reducing motion-induced image blur.

This protocol describes the fabrication of a type of micro-tissues called modules. The module approach generates uniform, scalable and vascularized tissues. The modules can be made of collagen as well as other gelable or crosslinkable materials. They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells. The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable. These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues. There are three main steps to making the modules: neutralizing the collagen and embedding cells in it, gelling the collagen in the tube and cutting the modules and coating the modules with endothelial cells.

Collagen-containing crosslinked, remodelable poloxamine derivatives were produced by introducing very short oligo(lactic acid) segments through the reaction of poloxamine with L-lactide and the later addition of unsaturated bonds by the reaction of modified poloxamine with methacryloyl chloride. Degradation studies on discs indicated a faster weight loss in comparison to the stability of lactoyl-free samples. Cell-containing modules (both HepG2 cells and two different umbilical vein smooth muscle cell (UVSMC) cell-types) were produced. Live/Dead assay showed high survival levels for both HepG2 and UVSMC cell types after crosslinking. While nondegradable modules did not change shape over time, lactoyl-poloxamine matrices showed a gradual shrinkage and size decrease and an increase in the roughness of the surface. These findings were consistent with the expected degradability of the lactoyl derivative. A UVSMC cell line (CRL-2481) embedded in a LA-poloxamine/collagen matrix showed the characteristic elongated shape at day 9. UVSMC primary cells behaved in a manner similar to that seen in collagen gels: these cells formed isolated clusters through the matrix that gradually lost viability. On tissue culture polystyrene the same cells aggregated and did not reach confluence. Modules with embedded CRL-2481 UVSMC led to a better initial adhesion of endothelial cells and a higher extent of surface coverage than seen with the UVSMC-free system. With embedded primary UVSMC, some EC attachment and formation of gap junctions was seen. The pattern was not well organized. With further improvement (and characterization), the lactoyl poloxamine derivative is potentially useful as a scaffold for modular tissue engineering, when tissue remodeling is an important consideration.