Strategic Replication of the Hepatic Zonation In Vitro Employing a Biomimetic Approach.

The varied functions of the liver are dependent on the metabolic heterogeneity exhibited by the hepatocytes within the liver lobule spanning the porto-central axis. This complex phenomenon plays an important role in maintaining the physiological homeostasis of the liver. Standard in vitro culture models fail to mimic this spatial heterogeneity of hepatocytes, assuming a homogeneous population of cells, which leads to inaccurate translation of results. Here, we demonstrate the development of an in vitro model of hepatic zonation by mimicking the microarchitecture of the liver using a 3D printed mini bioreactor and decellularized liver matrix to provide the native microenvironmental cues. There was a differential expression of hypoxic and metabolic markers across the developed mini bioreactor, showing the establishment of gradients of oxygen, Wnt/ß-catenin pathway, and other metabolic pathways. The model also showed the establishment of zone-dependent toxicity on treatment with acetaminophen. The developed model would thus be a promising avenue in the field of tissue engineering for understanding the liver physiology and pathophysiology and for drug screening to evaluate the potential of new pharmaceutical interventions.

Galactose Tethered Decellularized Liver Matrix: Toward a Biomimetic and Biofunctional Matrix for Liver Tissue Engineering.

The major challenge in liver tissue engineering is the replication of the microenvironment and microarchitecture of the liver tissue at the nanoscale. Decellularized liver matrix (DLM) provides an ideal material for scaffold preparation, as it retains the relevant structural and biochemical composition. However, the loss of bioactive factors during decellularization needs to be taken into account when using DLM and should be supplemented accordingly for an expected outcome. This study reports on the modification of DLM by the addition of galactose residues using a two-step thiol-ene-mediated photoclick chemistry for the coupling of galactose moieties to the DLM. Modification with galactose enhanced the function of hepatocytes and provides many advantages over currently used DLM and DLM-based materials. The galactose modified DLM enhanced the initial HepG2 cell adhesion to the substrate with changes in dynamics over time such as spheroid formation and further migration on the matrix. Our observation is that the galactose ligand decoration can also enhance the liver-specific metabolism of HepG2 compared to unmodified DLM. Galactosylated DLM also showed a better establishment of cellular polarity which also contributes to the function of HepG2 cells. Together our results demonstrate the advantages of adding galactose residues to currently available biomaterials, which makes this approach an attractive method for ECM-based liver tissue engineering.

Influence of Liver Extracellular Matrix in Predicting Drug-Induced Liver Injury: An Alternate Paradigm.

In vitro drug-induced liver injury (DILI) models are promising tools for drug development to predict adverse events during clinical usage. However, the currently available DILI models are not specific or not able to predict the injury accurately. This is believed to be mainly because of failure to conserve the hepatocyte phenotype, lack of longevity, and difficulty in maintaining the tissue-specific microenvironment. In this study, we have assessed the potential of decellularized liver extracellular matrix (DLM) in retaining the hepatic cellular phenotype and functionality in the presence of a tissue-specific microenvironment along with its role in influencing the effect of the drug on hepatic cells. We show that DLM helps maintain the phenotype of the hepatic cell line HepG2, a well-known cell line for secretion of human proteins that is easily available. Also, the DLM enhanced the expression of a metabolic marker carbamoyl phosphate synthetase I (CPS1), a regulator of urea cycle, and bile salt export pump (BSEP), a marker of hepatocyte polarity. We further validated the DLM for its influence on the sensitivity of cells toward different classes of drugs. Interestingly, the coculture model, in the presence of endothelial cells and stellate cells, exhibited a higher sensitivity for both acetaminophen and trovafloxacin, a toxic compound that does not show any toxicity on preclinical screening. Thus, our results demonstrate for the first time that a multicellular combination along with DLM can be a potential and reliable DILI model to screen multiple drugs.

Organoid Platform in Preclinical Investigation of Personalized Immunotherapy Efficacy in Appendiceal Cancer: Feasibility Study.

PURPOSE: Immunotherapy efficacy data on appendiceal cancer from clinical trials does not exist, due to appendiceal cancer incidence of 0.97 per 100,000. The goal of this study was to preclinically explore the application of immunotherapy in treating appendiceal cancer in a personalized organoid model. EXPERIMENTAL DESIGN: Patient tumor organoids (PTO) were fabricated using unsorted tumor cells with and without enrichment with patient-matched immune components derived from peripheral blood leukocytes, spleen, or lymph nodes [immune-enhanced PTOs (iPTO)]. Organoids were cultured for 7 days, followed by treatment with immunotherapy (pembrolizumab, ipilimumab, nivolumab), and assessed for treatment efficacy. RESULTS: Between September 2019 and May 2021, 26 patients were enrolled in the study. Successful testing was conducted in 19 of 26 (73.1%) patients, with 13 of 19 (68.4%) and 6 of 19 (31.6%) patients having low-grade appendiceal (LGA) and high-grade appendiceal (HGA) primaries, respectively. Immunotherapy response, with increased expression of granzyme B and cleaved caspase 3 and decreased expression of CK20 and ATP activity, was exhibited in 4 of 19 (21.1%) pembrolizumab-treated and 2 of 19 (10.5%) nivolumab-treated iPTOs. Post-immunotherapy cellular viability, in responding HGA organoids to pembrolizumab, decreased to less than 15% (P < 0.05). LGA iPTO treatment responses were observed in pembrolizumab and nivolumab, with an 8%-47.4% (P < 0.05) viability compared with controls. Ipilimumab showed no efficacy in the examined cohort. CONCLUSIONS: Immunotherapy shows measurable efficacy in appendiceal cancer organoids. Information derived from immunocompetent organoids may be applied in selecting patients for clinical trial enrollment in rare diseases where preclinical models of disease are lacking.

Personalized Identification of Optimal HIPEC Perfusion Protocol in Patient-Derived Tumor Organoid Platform.

BACKGROUND: Chemotherapy dosing duration and perfusion temperature vary significantly in HIPEC protocols. This study investigates patient-derived tumor organoids as a platform to identify the most efficacious perfusion protocol in a personalized approach. PATIENTS AND METHODS: Peritoneal tumor tissue from 15 appendiceal and 8 colon cancer patients who underwent CRS/HIPEC were used for personalized organoid development. Organoids were perfused in parallel at 37 and 42 °C with low- and high-dose oxaliplatin (200 mg/m2 over 2 h vs. 460 mg/m2 over 30 min) and MMC (40 mg/3L over 2 h). Viability assays were performed and pooled for statistical analysis. RESULTS: An adequate organoid number was generated for 75% (6/8) of colon and 73% (11/15) of appendiceal patients. All 42 °C treatments displayed lower viability than 37 °C treatments. On pooled analysis, MMC and 200 mg/m2 oxaliplatin displayed no treatment difference for either appendiceal or colon organoids (19% vs. 25%, p = 0.22 and 27% vs. 31%, p = 0.55, respectively), whereas heated MMC was superior to 460 mg/m2 oxaliplatin in both primaries (19% vs. 54%, p < 0.001 and 27% vs. 53%, p = 0.002, respectively). In both appendiceal and colon tumor organoids, heated 200 mg/m2 oxaliplatin displayed increased cytotoxicity as compared with 460 mg/m2 oxaliplatin (25% vs. 54%, p < 0.001 and 31% vs. 53%, p = 0.008, respectively). CONCLUSIONS: Organoids treated with MMC or 200 mg/m2 heated oxaliplatin for 2 h displayed increased susceptibility in comparison with 30-min 460 mg/m2 oxaliplatin. Optimal perfusion protocol varies among patients, and organoid technology may offer a platform for tailoring HIPEC conditions to the individual patient level.

3D hepatic mimics - the need for a multicentric approach.

The liver is a center of metabolic activity, including the metabolism of drugs, and consequently is prone to drug-induced liver injury. Failure to detect hepatotoxicity of drugs during their development will lead to the withdrawal of the drugs during clinical trials. To avoid such clinical and economic consequences, in vitro liver models that can precisely predict the toxicity of a drug during the pre-clinical phase is necessary. This review describes the different technologies that are used to develop in vitro liver models and the different approaches aimed at mimicking different functional aspects of the liver at the fundamental level. This involves mimicking of the functional and structural units like the sinusoid, the bile canalicular system, and the acinus.

HspB5 protects mouse neural stem/progenitor cells from paraquat toxicity.

INTRODUCTION: HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress. OBJECTIVE: The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (mNSPCs) against paraquat-induced toxicity. METHODS: The recombinant human HspB5 was expressed in E.coli and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting. RESULTS: The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of mNSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively. CONCLUSION: Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the mNSPCs and confers protection against paraquat toxicity.

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity.

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn's disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.