3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions

The cell spheroid technology, which greatly enhances cell-cell interactions, has gained significant attention in the development of in vitro liver models. However, existing cell spheroid technologies still have limitations in improving hepatocyte-extracellular matrix (ECM) interaction, which have a significant impact on hepatic function. In this study, we have developed a novel bioprinting technology for decellularized ECM (dECM)-incorporated hepatocyte spheroids that could enhance both cell-cell and -ECM interactions simultaneously. To provide a biomimetic environment, a porcine liver dECM-based cell bio-ink was developed, and a spheroid printing process using this bio-ink was established. As a result, we precisely printed the dECM-incorporated hepatocyte spheroids with a diameter of approximately 160–220 μm using primary mouse hepatocyte (PMHs). The dECM materials were uniformly distributed within the bio-printed spheroids, and even after more than 2 weeks of culture, the spheroids maintained their spherical shape and high viability. The incorporation of dECM also significantly improved the hepatic function of hepatocyte spheroids. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed approximately 4.3- and 2.5-fold increased levels of albumin and urea secretion, respectively, and a 2.0-fold increase in CYP enzyme activity. These characteristics were also reflected in the hepatic gene expression levels of ALB, HNF4A, CPS1, and others. Furthermore, the dECM-incorporated hepatocyte spheroids exhibited up to a 1.8-fold enhanced drug responsiveness to representative hepatotoxic drugs such as acetaminophen, celecoxib, and amiodarone. Based on these results, it can be concluded that the dECM-incorporated spheroid printing technology has great potential for the development of highly functional in vitro liver tissue models for drug toxicity assessment

Inflammatory Responses Are Not Sufficient to Cause Delayed Neuronal Death in ATP-Induced Acute Brain Injury

BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+) and Iba-1(+) cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+) cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic

Effect of detergent type on the performance of liver decellularizedextracellular matrix-based bio-inks

Decellularized extracellular matrix-based bio-inks (dECM bio-inks) for bioprinting technology have recently gained attention owing to their excellent ability to confer tissue-specific functions and 3D-printing capability. Although decellularization has led to a major advancement in bio-ink development, the effects of detergent type, the most important factor in decellularization, are still unclear. In this study, the effects of various detergent types on bio-ink performance were investigated. Porcine liver-derived dECM bio-inks prepared using widely used detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton X-100 (TX), and TX with ammonium hydroxide (TXA), were characterized in detail. SDS and SDC severely damaged glycosaminoglycan and elastin proteins, TX showed the lowest rate of decellularization, and TXA-based dECM bio-ink possessed the highest ECM content among all bio-inks. Differences in biochemical composition directly affected bio-ink performance, with TXA-dECM bio-ink showing the best performance with respect to gelation kinetics, intermolecular bonding, mechanical properties, and 2D/3D printability. More importantly, cytocompatibility tests using primary mouse hepatocytes also showed that the TXA-dECM bio-ink improved albumin secretion and cytochrome P450 activity by approximately 2.12- and 1.67-fold, respectively, compared with the observed values for other bio-inks. Our results indicate that the detergent type has a great influence on dECM damage and that the higher the dECM content, the better the performance of the bio-ink for 3D bioprinting

RNA-Guided Genome Editing in Drosophila with the Purified Cas9 Protein

We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay.

Extent of linkage disequilibrium and effective population size of Korean Yorkshire swine

Objective We aimed to characterize linkage disequilibrium (LD) and effective population size (Ne) in a Korean Yorkshire population using genomic data from thousands of individuals. Methods We genotyped 2,470 Yorkshire individuals from four major Grand-Grand-Parent farms in Korea using the Illumina PorcineSNP60 version2 BeadChip, which covers >61,565 single nucleotide polymorphisms (SNPs) located across all chromosomes and mitochondria. We estimated the expected LD and inferred current Ne as well as ancestral Ne. Results We identified 61,565 SNP from autosomes, mitochondria, and sex chromosomes and characterized the LD of the Yorkshire population, which was relatively high between closely linked markers (>0.55 at 50 kb) and declined with increasing genetic distance. The current Ne of this Korean Yorkshire population was 122.87 (106.90; 138.84), while the historical Ne of Yorkshire pigs suggests that the ancestor Ne has decreased by 99.6% over the last 10,000 generations. Conclusion To maintain genetic diversity of a domesticated animal population, we must carefully consider appropriate breed management methods to avoid inbreeding. Although attenuated selection can affect short-term genetic gain, it is essential for maintaining the long-term genetic variability of the Korean Yorkshire population. Continuous and long-term monitoring would also be needed to maintain the pig population to avoid an unintended reduction of Ne. The best way to preserve a sustainable population is to maintain a sufficient Ne