RESUMEN

The provided dataset describes the transcriptomic profile of human liver spheroid co-cultures consisting of a human hepatoma cell line (C3A/HepG2 cells) and an immortalized activated human hepatic stellate cell line (LX-2 cells) upon exposure to total parenteral nutrition. High-throughput RNA sequencing was performed using DNBSEQTM sequencing technology. Following the quality check and filtering of raw sequence reads, the clean reads were aligned to the reference human genome and used to determine differential gene expression. Raw and processed data are deposited in the Gene Expression Omnibus with accession number GSE264357. These data could serve further mechanistic studies on parenteral nutrition-induced liver injury and support translational research on intestinal failure-associated liver disease occurring in individuals receiving total parenteral nutrition.

RESUMEN

Parenteral nutrition (PN) is typically administered to individuals with gastrointestinal dysfunction, a contraindication for enteral feeding, and a need for nutritional therapy. When PN is the only energy source in patients, it is defined as total parenteral nutrition (TPN). TPN is a life-saving approach for different patient populations, both in infants and adults. However, despite numerous benefits, TPN can cause adverse effects, including metabolic disorders and liver injury. TPN-associated liver injury, known as intestinal failure-associated liver disease (IFALD), represents a significant problem affecting up to 90% of individuals receiving TPN. IFALD pathogenesis is complex, depending on the TPN components as well as on the patient's medical conditions. Despite numerous animal studies and clinical observations, the molecular mechanisms driving IFALD remain largely unknown. The present study was set up to elucidate the mechanisms underlying IFALD. For this purpose, human liver spheroid co-cultures were treated with a TPN mixture, followed by RNA sequencing analysis. Subsequently, following exposure to TPN and its single nutritional components, several key events of liver injury, including mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, apoptosis, and lipid accumulation (steatosis), were studied using various techniques. It was found that prolonged exposure to TPN substantially changes the transcriptome profile of liver spheroids and affects multiple metabolic and signaling pathways contributing to liver injury. Moreover, TPN and its main components, especially lipid emulsion, induce changes in all key events measured and trigger steatosis.

Asunto(s)

RESUMEN

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.

Asunto(s)

RESUMEN

Nanobiomaterials, or NBMs, have been used in medicine and bioimaging for decades, with wide-reaching applications ranging from their uses as carriers of genes and drugs, to acting as sensors and probes. When developing nanomedicine products, it is vitally important to evaluate their safety, ensuring that both biocompatibility and efficacy are achieved so their applications in these areas can be safe and effective. When discussing the safety of nanomedicine in general terms, it is foolish to make generalised statements due to the vast array of different manufactured nanomaterials, formulated from a multitude of different materials, in many shapes and sizes; therefore, NBM pre-clinical screening can be a significant challenge. Outside of their distribution in the various tissues, organs and cells in the body, a key area of interest is the impact of NBMs on the liver. A considerable issue for researchers today is accurately predicting human-specific liver toxicity prior to clinical trials, with hepatotoxicity not only the most cited reasons for withdrawal of approved drugs, but also a primary cause of attrition in pre-launched drug candidates. To date, no simple solution to adequately predict these adverse effects exists prior to entering human experimentation. The limitations of the current pre-clinical toolkit are believed to be one of the main reasons for this, with questions being raised on the relevance of animal models in pre-clinical assessment, and over the ability of conventional, simplified in vitro cell-based assays to adequately assess new drug candidates or NBMs. Common 2D cell cultures are unable to adequately represent the functions of 3D tissues and their complex cell-cell and cell-matrix interactions, as well as differences found in diffusion and transport conditions. Therefore, testing NBM toxicity in conventional 2D models may not be an accurate reflection of the actual toxicity these materials impart on the body. One such method of overcoming these issues is the use of 3D cultures, such as cell spheroids, to more accurately assess NBM-tissue interaction. In this study, we introduce a 3D hepatocellular carcinoma model cultured from HepG2 cells to assess both the cytotoxicity and viability observed following treatment with a variety of NBMs, namely a nanostructured lipid carrier (in the specific technical name = LipImage™ 815), a gold nanoparticle (AuNP) and a panel of polymeric (in the specific technical name = PACA) NBMs. This model is also in compliance with the 3Rs policy of reduction, refinement and replacement in animal experimentation [1], and meets the critical need for more advanced in vitro models for pre-clinical nanotoxicity assessment. Pipeline for the pre-clinical assessment of NBMs in liver spheroid model.

Asunto(s)

RESUMEN

The liver is the most important metabolic hub of endo and xenobiotic compounds. Pre-clinical studies using rodents to evaluate the toxicity of new drugs and cosmetics may produce inconclusive results for predicting clinical outcomes in humans, moreover being banned in the European Union. Human liver modeling using primary hepatocytes presents low reproducibility due to batch-to-batch variability, while iPSC-derived hepatocytes in monolayer cultures (2D) show reduced cellular functionality. Here we review the current status of the two most robust in vitro approaches in improving hepatocyte phenotype and metabolism while mimicking the hepatic physiological microenvironment: organoids and liver-on-chip. Both technologies are reviewed in design and manufacturing techniques, following cellular composition and functionality. Furthermore, drug screening and liver diseases modeling efficiencies are summarized. Finally, organoid and liver-on-chip technologies are compared regarding advantages and limitations, aiming to guide the selection of appropriate models for translational research and the development of such technologies.

RESUMEN

Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 µm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

Asunto(s)

RESUMEN

Generating microliver tissues to recapitulate hepatic function is of increasing importance in tissue engineering and drug screening. But the limited availability of primary hepatocytes and the marked loss of phenotype hinders their application. Human induced hepatocytes (hiHeps) generated by direct reprogramming can address the shortage of primary hepatocytes to make personalized drug prediction possible. Here, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Transducing human fetal and adult fibroblasts with low vector dosage generated human induced hepatocyte-like cells (hiHeps) displaying characteristics of mature hepatocytes and capable of drug metabolism. To mimic the physiologic liver microenvironment and improve hepatocyte function, we prepared 3D scaffold-free microliver spheroids using hiHeps and human liver nonparenchymal cells through self-assembly without exogenous scaffolds. We then introduced the microliver spheroids into a two-organ microfluidic system to examine interactions between hepatocytes and tumor cells. The hiHeps-derived spheroids metabolized the prodrug capecitabine into the active metabolite 5-fluorouracil and induced toxicity in downstream tumor spheroids. Our results demonstrate that hiHeps can be used to make microliver spheroids and combined with a microfluidic system for drug evaluation. Our work could make it possible to use patient-specific hepatocyte-like cells to predict drug efficacy and side effects in various organs from the same patient.

Asunto(s)