Prospective Application of Tannic Acid in Acetaminophen (APAP)-Induced Acute Liver Failure.

This study investigated the effect of tannic acid (TA), a natural plant-derived polyphenol, on hepatocyte viability and function, focusing on both hepatoprotective and hepatocurative aspects within liver failure models. In an in vitro prevention model, the TA-containing group exhibited 1.5-fold and 59-fold higher relative cell viability and albumin synthesis, respectively, in injured mature hepatocytes (MHs) and 1.14-fold and 1.10-fold higher values in injured small hepatocytes (SHs), compared with the TA-free group. In the in vitro curative model, the TA-containing group exhibited 3.25-fold and 113-fold higher relative cell viability and albumin synthesis, respectively, in injured MHs and 0.36-fold and 3.55-fold higher values in injured SHs, compared with the TA-free group. In the in vivo disease model, the administration of 300 µL of 1 µg/mL TA significantly mitigated acute liver failure damage and post-APAP toxicity in mice. This was evident in serum analysis, where the levels of alanine transaminase, aspartate aminotransferase, and total bilirubin notably decreased, in agreement with histological observations. The study findings reveal that TA can enhance hepatic function at specific additive concentrations. Furthermore, even when injured by APAP, hepatocytes could revert to their preinjury state after additional TA supplementation. Additionally, pretreating hepatocytes with TA can alleviate subsequent damage. Thus, TA holds clinical potential in the treatment of APAP-induced liver failure.

Monitoring Cultured Rat Hepatocytes Using RNA-Seq In Vitro.

Compared to other techniques, RNA sequencing (RNA-Seq) has the advantage of having details of the expression abundance of all transcripts in a single run. In this study, we used RNA-Seq to monitor the maturity and dynamic characteristics of in vitro hepatocyte cultures. Hepatocytes, including mature hepatocytes and small hepatocytes, were analyzed in vitro using RNA-Seq and quantitative polymerase chain reaction (qPCR). The results demonstrated that the gene expression profiles measured by RNA-Seq showed a similar trend to the expression profiles measured by qPCR, and can be used to infer the success of in vitro hepatocyte cultures. The results of the differential analysis, which compared mature hepatocytes against small hepatocytes, revealed 836 downregulated and 137 upregulated genes. In addition, the success of the hepatocyte cultures could be explained by the gene list screened from the adopted gene enrichment test. In summary, we demonstrated that RNA-Seq could become an effective method for monitoring the whole transcriptome of hepatocyte cultures and provide a more comprehensive list of factors related to the differentiation of small hepatocytes into mature hepatocytes. This monitoring system not only shows high potential in medical applications but may also be a novel method for the clinical diagnosis of liver-related diseases.

Determination of hematocrit by voltage-induced hemolysis on a disposable electrochemical sensing strip.

Besides being a crucial parameter for surgery and clinical diagnosis, hematocrit tends to affect the analytical results of point-of-care analytical devices. Therefore, an accurate and quick method for measuring hematocrit was developed on the basis of screen-printed carbon electrodes (SPCE). An impulse DC voltage of 3.0 V was imposed on ferricyanide-coated SPCE to induce hemolysis, and the released hemoglobin reduced the ferricyanide, generating a higher oxidation current for estimating hematocrit. Hematocrit ranging from 10 to 70% can be determined in 5 s by linear sweep voltammetry (r(2) = 0.9907) or 0.8 s by 3 V of potential step voltammetry (r(2) = 0.9833).

Fabrication of a decellularized liver matrix-based hepatic patch for the repair of CCl4-induced liver injury.

This article primarily introduces a new treatment for liver fibrosis/cirrhosis. We developed a hepatic patch by combining decellularized liver matrix (DLM) with the hepatocyte growth factor (HGF)/heparin-complex and evaluated its restorative efficacy. In vitro prophylactic results, the HGF/heparin-DLM patches effectively mitigated CCl4-induced hepatocyte toxicity and restored the cytotoxicity levels to the baseline levels by day 5. Furthermore, these patches restored albumin synthesis of injured hepatocytes to more than 70% of the normal levels within 5 days. In vitro therapeutic results, the urea synthesis of the injured hepatocytes reached 91% of the normal levels after 10 days of culture, indicating successful restoration of hepatic function by the HGF/heparin-DLM patches in both prophylactic and therapeutic models. In vivo results, HGF/heparin-DLM patches attached to the liver and gut exhibited a significant decrease in collagen content (4.44 times and 2.77 times, respectively) and an increase in glycogen content (1.19 times and 1.12 times, respectively) compared to the fibrosis group after 1 week, separately. In summary, liver function was restored and inflammation was inhibited through the combined effects of DLM and the HGF/heparin-complex in fibrotic liver. The newly designed hepatic patch holds promise for both in vitro and in vivo regeneration therapy and preventive health care for liver tissue engineering.

Self-Healing Hydrogel Containing Decellularized Liver Matrix and Endothelial Cell-Covered Hepatocyte Spheroids for Rescue of Injured Hepatocytes.

Liver fibrosis occurs in many chronic liver diseases, while severe fibrosis can lead to liver failure. A chitosan-phenol based self-healing hydrogel (CP) integrated with decellularized liver matrix (DLM) is proposed in this study as a 3D gel matrix to carry hepatocytes for possible therapy of liver fibrosis. To mimic the physiological liver microenvironment, DLM is extracted from pigs and mixed with CP hydrogel to generate DLM-CP self-healing hydrogel. Hepatocyte spheroids coated with endothelial cells (ECs) are fabricated using a customized method and embedded in the hydrogel. Hepatocytes injured by exposure to CCl4-containing medium are used as the in vitro toxin-mediated liver fibrosis model, where the EC-covered hepatocyte spheroids embedded in the hydrogel are co-cultured with the injured hepatocytes. The urea synthesis of the injured hepatocytes reaches 91% of the normal level after 7 days of co-culture, indicating that the hepatic function of injured hepatocytes is rescued by the hybrid spheroid-laden DLM-CP hydrogel. Moreover, the relative lactate dehydrogenase activity of the injured hepatocytes is decreased 49% by the hybrid spheroid-laden DLM-CP hydrogel after 7 days of co-culture, suggesting reduced damage in the injured hepatocytes. The combination of hepatocyte/EC hybrid spheroids and DLM-CP hydrogel presents a promising therapeutic strategy for hepatic fibrosis.

Development of a 3D porous chitosan/gelatin liver scaffold for a bioartificial liver device.

Functional artificial livers (FALs), with embedded hepatocytes that perform the functions of a normal liver, have been developed during the past decades. It is important to note that the liver scaffold, which is a biologically functional core of bioartificial livers, plays a vital role in the bio-cartridge within a bioartificial liver. In this study, a three-dimensional (3D) liver scaffold for in vitro cultures was fabricated by freeze-drying a chitosan/gelatin (CG) solution. A CG scaffold has advantages such as (i) inexpensive and easy-to-make; (ii) easy to fabricate with varying compressive modulus by changing the concentration of glutaraldehyde; (iii) non-cytotoxicity; and (iv) porous structure is similar to extracellular matrix (ECM), thus facilitating hepatocyte adhesion and proliferation. The results revealed that the compressive modulus and maintainability of a CG scaffold was correlated to the increase in glutaraldehyde. Furthermore, hepatocyte viability and hepatic functions showed the best performances with a 0.61% glutaraldehyde-CG scaffold. This CG scaffold not only had higher hepatocyte biocompatibility and mechanical strength, but also maintained hepatic functions and viability in vitro cultures; especially, the mechanical properties of 0.61% glutaraldehyde-CG scaffold were very similar to those in normal liver. The CG scaffold as a liver scaffold may have high potential for further bioartificial liver design in the near future.

Decellularized liver matrix as substrates for rescue of acute hepatocytes toxicity.

More and more scholars regard the lesion of liver cirrhosis as a series of progressive clinical stages. Besides, liver cirrhosis is a late stage of scarring (fibrosis) of the liver, and has long been a common cause of death for the global adult population, thus, the treatment of liver cirrhosis is a key point investigated in the biomedical field. Here, we propose a novel hypothesis; if decellularized liver matrix (DLM) is possible injected into the injurant-induced fiberized liver via the hepatic portal vein to thoroughly repair the liver, it may be an effective therapeutic method for liver fibrosis or even liver cirrhosis. This study mixed rat DLM with gelatin-hydroxyphenylpropionic acid (Glt-HPA) to form a three-dimensional structure to simulate the in vivo liver environment, and cultured the primary rat hepatocytes in it. Afterward, the hepatocytes were treated using D-galactosamine (GaIN), CHCl3 , and CCl4 -containing medium to simulate the toxin-mediated liver fibrosis in vitro. Finally, they were cultured in a DLM-containing medium to observe the viability and functions of the damaged hepatocytes, and the hypothesis of this research was proved, meaning that Glt-HPA-DLM acting on damaged hepatocytes may repair them. Results have shown that Glt-HPA-DLM was effective for hepatocytes culture and repaired injured hepatocytes from GaIN, CHCl3 , and CCl4 (albumin synthesis was increased by 219, 108, and 12%, respectively, whereas relative lactate dehydrogenase activity was reduced by 38, 68, and 67%, after 5 days of culture, separately). This research shows promising effects against hepatic fibrosis and may have potential for liver cirrhosis in vivo.

Angiogenic potential of co-spheroids of neural stem cells and endothelial cells in injectable gelatin-based hydrogel.

Appropriate crosstalk between neural stem cells (NSCs) and endothelial cells (ECs) is essential for establishment of the neurovascular network and neuroregeneration in the central nervous system (CNS) in vivo. However, platforms used to study the interaction of NSCs and ECs in three-dimensional (3D) environment are still rare. Here, we employed the chitosan-based substrates to rapidly generate the 3D NSC/EC co-spheroids in vitro, and then analyzed their crosstalk in the co-spheroids. By the analysis of gene and protein expression, NSCs in the NSC/EC co-spheroids displayed greater differentiation potential than the regular 2D co-culture on plastic dish. We also encapsulated the NSC/EC co-spheroids into chitosan- or gelatin-based hydrogels to further support the long-term growth of cell spheroids in a 3D environment. We observed that NSC/EC co-spheroids exhibited greater viability in the gelatin-based hydrogel, and even formed tube-like structures from the surface of the co-spheroids after FGF2 induction, indicating the increased angiogenic potential of ECs in the NSC/EC co-spheroids embedded in the FGF2-containing gelatin-based hydrogel. Finally, we demonstrated the injectability and printability of NSC/EC co-spheroids encapsulated in the gelatin-based hydrogel, revealing the possibility of using NSC/EC co-spheroids to build the biomimetic neurovascular constructs in the future.

Label-free and reagentless capacitive aptasensor for thrombin.

This investigation develops a label-free and reagentless aptasensor, based on a capacitive transducer with simple face-to-face electrode pairs. The electrode pairs of the transducer are composed of a gold electrode and an indium tin oxide film with micrometer separation with a double-side polyethylene terephthalate tape. Aptamers and 1-dodecanethiol are modified to form a self-assembled monolayer (SAM) on the gold electrode surfaces, and function as bio-recognition elements and preventers of non-specific protein binding, respectively. Electrochemical characterization results indicate that the SAM also forms an effective insulating layer, which is sufficient for capacitive sensing. The feasibility of the capacitive biosensor is validated using thrombin as a model analyte. The ultra-small value changes of capacitance originating from thrombin binding with the aptamers modified on the biosensor were measured with a home-made capacitance measuring circuit based on switched capacitor (SC) technology. The developed biosensor has detection limits of 1 pM and 10 pM of thrombin in phosphate buffered saline and mimic serum solution, respectively. The linear range for thrombin detection in human serum solution is from 10 pM to 1 µM, with a regression coefficient of 0.98. Additionally, the proposed aptasensor does not have significant levels of non-specific binding of bovine serum albumin and human serum albumin. Accordingly, the combination of SC and SAM bringing capacitive transduction at the forefront of ultrasensitive label-free and reagentless biosensing devices, particularly for point-of-care clinical analysis, which adopts small numbers of biological samples with low analyte concentrations.

Direct Photometric Assay for Copper Chlorophyll Adulterants in Edible Oil by the Aid of an Ultraviolet-Photobleaching Pretreatment.

Adulterating edible oil with copper chlorophyll derivatives (E141i) has made a substantial impact on the edible oil industry and food safety. This study demonstrates an efficient and reliable screening method to directly identify the color adulteration by the aid of a simple photobleaching pretreatment using a 365 nm ultraviolet-light-emitting diode working at a photon flux density of 480 mmol m-2 s-1 for 24 min. The content of copper chlorophyll [predominantly Cu-pyropheophytin a (Cu-py a)] can be calculated by A600, A650, and A700 with satisfactory spike recovery [97.9-103.6%; six kinds of edible oils spiked with 1 ppm of Cu-py a; n = 3 for each kind of oil; relative standard deviation (RSD) < 5%], linearity ( R2 = 0.9961 when spiking 0.1-10 ppm of Cu-py a into soybean oil standard; n = 3 for each concentration; RSD < 5%), and reproducibility (RSD < 5% for spiking 1 ppm of Cu-py a into soybean oil standard; n = 3 over 3 days). The detection limit (S/N > 5) was 0.05 ppm. The analytical results of 50 commercially available oil samples were verified by the official high-performance liquid chromatography method.

Near-IR-Absorbing Gold Nanoframes with Enhanced Physiological Stability and Improved Biocompatibility for In Vivo Biomedical Applications.

This paper describes the synthesis of near-infrared (NIR)-absorbing gold nanoframes (GNFs) and a systematic study comparing their physiological stability and biocompatibility with those of hollow Au-Ag nanoshells (GNSs), which have been used widely as photothermal agents in biomedical applications because of their localized surface plasmon resonance (LSPR) in the NIR region. The GNFs were synthesized in three steps: galvanic replacement, Au deposition, and Ag dealloying, using silver nanospheres (SNP) as the starting material. The morphology and optical properties of the GNFs were dependent on the thickness of the Au coating layer and the degree of Ag dealloying. The optimal GNF exhibited a robust spherical skeleton composed of a few thick rims, but preserved the distinctive LSPR absorbance in the NIR region-even when the Ag content within the skeleton was only 10 wt %, 4-fold lower than that of the GNSs. These GNFs displayed an attractive photothermal conversion ability and great photothermal stability, and could efficiently kill 4T1 cancer cells through light-induced heating. Moreover, the GNFs preserved their morphology and optical properties after incubation in biological media (e.g., saline, serum), whereas the GNSs were unstable under the same conditions because of rapid dissolution of the considerable silver content with the shell. Furthermore, the GNFs had good biocompatibility with normal cells (e.g., NIH-3T3 and hepatocytes; cell viability for both cells: >90%), whereas the GNSs exhibited significant dose-dependent cytotoxicity (e.g., cell viability for hepatocytes at 1.14 nM: ca. 11%), accompanied by the induction of reactive oxygen species. Finally, the GNFs displayed good biocompatibility and biosafety in an in vivo mouse model; in contrast, the accumulation of GNSs caused liver injury and inflammation. Our results suggest that GNFs have great potential to serve as stable, biocompatible NIR-light absorbers for in vivo applications, including cancer detection and combination therapy.

A selective decoy-doxorubicin complex for targeted co-delivery, STAT3 probing and synergistic anti-cancer effect.

A novel selective decoy oligodeoxynucleotide (dODN)-doxorubicin (DOX) complex is reported for cancer theranostics. It eliminates the use of a ligand or carrier for targeted delivery and disassembles into therapeutic dODN and DOX upon encountering over-activated STAT3 in cancer cells. Hence, in situ STAT3 probing and synergistic anti-cancer effect are attained at the same time.

Silicate fiber-based 3D cell culture system for anticancer drug screening.

BACKGROUND: Three-dimensional (3D) in vitro cultures can recapitulate the physiological in vivo microenvironment. 3D Modeling techniques have been investigated and applied in anticancer drug screening. MATERIALS AND METHODS: A silicate fiber scaffold was used for 3D cell cultures, and used to model the efficacy of anticancer drugs, such as mytomicin C and doxorubicin. RESULTS: A unique 3D structure was observed in 13 human tumor cell lines on scaffold, and these cells exhibited higher drug resistance than cells in two-dimensional (2D) cultures. Furthermore, the production of lactate and expression of the nuclear factor-kappa B (NF-κB)-regulated genes B cell lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) were higher in 3D cultures than in 2D cultures. CONCLUSION: These findings suggest that a 3D model using a silicate fiber scaffold can mimic features of cancer, and is also a suitable model for the evaluation of anticancer drugs in vitro.

Growth factor/heparin-immobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis.

Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering.

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes.

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 microg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 microg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 microg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.