Three-dimensional (3D) culture via micropattern arrays to generate cellular spheroids seems a promising in vitro biomimetic system for liver tissue engineering applications, such as drug screening. Recently, organ-derived decellularized extracellular matrix emerges as arguably the most biomimetic bioink. Herein, decellularized liver matrix (DLM)-derived micropattern array chips were developed to fabricate size-controllable and arrangement-orderly HepG2 spheroids for drug screening. The porcine DLM was obtained by the removal of cellular components and then ground into powder, followed by enzymolysis. DLM as a coating substrate was compared with collagen type I (Col I) and Matrigel in terms of biological performance for enhancing cell adhesion, proliferation, and functions. Subsequently, we used poly­(dimethylsiloxane) (PDMS) to adsorb DLM as the bioink to fabricate micropattern array chips. The optimal shape and size of micropattern were determined by evaluating the morphology, viability, and functions of HepG2 3D cellular aggregates. In addition, drug-susceptibility testing (paclitaxel, doxorubicin HCl, and disulfiram) was performed on this novel platform. The DLM provided the tissue-specific microenvironment that provided suitable supports for HepG2 cells, compared to Col I and Matrigel. A circular micropattern with a diameter of 100 μm was the optimal processing parameter to rapidly fabricate large-scale, size-controllable, and arrangement-orderly HepG2 cellular aggregates with 3D spheroid’s shape and high cell viability. Drug screening testing showed that the effect of a drug could be directly demonstrated on-chip by confocal microscopy measuring the viability of spheroids. We provide a novel platform for the large-scale generation of HepG2 spheroids with uniform size and arrangement, thus bringing convenience, reducing error, and increasing reproducibility for a rapid drug discovery by fluorescence quantitative analysis. This methodology may be possible to apply in advancing personalized medicine and drug discovery.

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

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Hydrogels based on poly-(2-hydroxyethyl methacrylate) (pHEMA) have been widely used as biomaterials in tissue engineering due to their biocompatibility, hydrophilicity, and low friction coefficient. The terminal sterilization of hydrogels is a critical step in clinical applications. However, regulations and standardization for the sterilization of hydrogels based on pHEMA are still lacking. In this study, we explored six sterilization methods on pHEMA-based materials (A1: pHEMA, A2: pHEMA copolymerizes with acrylic acid, and A3: pHEMA copolymerizes with acrylic acid and further coordinated with iron ions), such as gamma irradiation, 75% ethanol, ultraviolet (UV), ethylene oxide (EtO), and autoclaving with or without deionized water (autoclaving-H 2 O or autoclaving-dry). Combining results from the multifaceted approaches with assessment, pHEMA-based hydrogels can be completely sterilized via the autoclaving-H 2 O method analyzed by sterilized testing. The physicochemical properties and cell behavior of sterilized hydrogels were not influenced by this sterilization approach, validated by Fourier transform infrared (FT-IR) spectroscopy and tensile tests. The pHEMA-based hydrogel sterilized by the autoclaving-H 2 O method also had no effect on the cell behavior evaluated by in vitro cytotoxicity experiments and caused no evident inflammatory reaction in tissue in vivo implantation experiments. However, it was also found that there were still some defects in the A2 and A3 groups as biomaterials possibly because of an inappropriate proportion of formulations or raw material used in exploring sterilization methods. These findings have implications for the improvement and clinical application of pHEMA-based hydrogels.