Building in vitro models for mechanistic understanding of liver regeneration in chronic liver diseases.

The liver has excellent regeneration potential and attains complete functional recovery from partial hepatectomy. The regenerative mechanisms malfunction in chronic liver diseases (CLDs), which fuels disease progression. CLDs account for 2 million deaths per year worldwide. Pathophysiological studies with clinical correlation have shown evidence of deviation of normal regenerative mechanisms and its contribution to fueling fibrosis and disease progression. However, we lack realistic in vitro models that can allow experimental manipulation for mechanistic understanding of liver regeneration in CLDs and testing of candidate drugs. In this review, we aim to provide the framework for building appropriate organotypic models for dissecting regenerative responses in CLDs, with the focus on non-alcoholic steatohepatitis (NASH). By drawing parallels with development and hepatectomy, we explain the selection of critical components such as cells, signaling, and, substrate-driven biophysical cues to build an appropriate CLD model. We highlight the organoid-based organotypic models available for NASH disease modeling, including organ-on-a-chip and 3D bioprinted models. With the focus on bioprinting as a fabrication method, we prescribe building in vitro CLD models and testing schemes for exploring the regenerative responses in the bioprinted model.

Unravelling microRNA regulation and miRNA-mRNA regulatory networks in osteogenesis driven by 3D nanotopographical cues.

Three-dimensional (3D) culturing of cells is being adopted for developing tissues for various applications such as mechanistic studies, drug testing, tissue regeneration, and animal-free meat. These approaches often involve cost-effective differentiation of stem or progenitor cells. One approach is to exploit architectural cues on a 3D substrate to drive cellular differentiation, which has been shown to be effective in various studies. Although extensive gene expression data from such studies have shown that gene expression patterns might differ, the gene regulatory networks controlling the expression of genes are rarely studied. In this study, we profiled genes and microRNAs (miRNAs) via next-generation sequencing (NGS) in human mesenchymal stem cells (hMSCs) driven toward osteogenesis via architectural cues in 3D matrices (3D conditions) and compared with cells in two-dimensional (2D) culture driven toward osteogenesis via soluble osteoinductive factors (OF conditions). The total number of differentially expressed genes was smaller in 3D compared to OF conditions. A distinct set of genes was observed under these conditions that have been shown to control osteogenic differentiation via different pathways. Small RNA sequencing revealed a core set of miRNAs to be differentially expressed under these conditions, similar to those that have been previously implicated in osteogenesis. We also observed a distinct regulation of miRNAs in these samples that can modulate gene expression, suggesting supplementary gene regulatory networks operative under different stimuli. This study provides insights into studying gene regulatory networks for identifying critical nodes to target for enhanced cellular differentiation and reveal the differences in physical and biochemical cues to drive cell fates.

Liver organoids cocultured on decellularized native liver scaffolds as a bridging therapy improves survival from liver failure in rabbits.

The present study aimed to develop viable liver organoids using decellularized native liver scaffolds and evaluate the efficacy of human liver organoid transplantation in a rabbit model of cirrhosis. Liver organoids were formed by coculture of hepatocyte-like cells derived from the human-induced pluripotent stem cells with three other cell types. Twelve 3-mo-old New Zealand White Rabbits underwent a sham operation, bile duct ligation, or biliary duct ligation followed by liver organoid transplantation. Liver organoid structure and function before and after transplantation were evaluated using histological and molecular analyses. A survival analysis using the Kaplan-Meier method was performed to determine the cumulative probability of survival according to liver organoid transplantation with significantly greater overall survival observed in rabbits that underwent liver organoid transplantation (P = 0.003, log-rank test). The short-term group had higher hepatic expression levels of ALB and CYP3A mRNA and lower expression levels of AST mRNA compared to the long-term group. The short-term group also had lower collagen deposition in liver tissues. Transplantation of human liver organoids cocultured in decellularized native liver scaffold into rabbits that had undergone bile duct ligation improved short-term survival and hepatic function. The results of the present study highlight the potential of liver organoid transplantation as a bridging therapy in liver failure; however, rejection and poor liver organoid function may limit the long-term efficacy of this therapeutic approach.

Organoids.

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

3D Tumor Models for Breast Cancer: Whither We Are and What We Need.

Three-dimensional (3D) models have led to a paradigm shift in disease modeling in vitro, particularly for cancer. The past decade has seen a phenomenal increase in the development of 3D models for various types of cancers with a focus on studying stemness, invasive behavior, angiogenesis, and chemoresistance of cancer cells, as well as contributions of its stroma, which has expanded our understanding of these processes. Cancer biology is moving into exploring the emerging hallmarks of cancer, such as inflammation, immune evasion, and reprogramming of energy metabolism. Studies into these emerging concepts have provided novel targets and treatment options such as antitumor immunotherapy. However, 3D models that can investigate the emerging hallmarks are few and underexplored. As commonly used immunocompromised mice and syngenic mice cannot accurately mimic human immunology, stromal interactions, and metabolism and require the use of prohibitively expensive humanized mice, there is tremendous scope to develop authentic 3D tumor models in these areas. Taking the specific case of breast cancer, we discuss the currently available 3D models, their applications to mimic signaling in cancer, tumor-stroma interactions, drug responses, and assessment of drug delivery systems and therapies. We discuss the lacunae in the development of 3D tumor models for the emerging hallmarks of cancer, for lesser-explored forms of breast cancer, and provide insights to develop such models. We discuss how the next generation of 3D models can provide a better mimic of human cancer modeling compared to xenograft models and the scope toward preclinical models and precision medicine.

Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel.

Drug-induced hepatocellular cholestasis leads to altered bile flow. Bile is propelled along the bile canaliculi (BC) by actomyosin contractility, triggered by increased intracellular calcium (Ca2+). However, the source of increased intracellular Ca2+ and its relationship to transporter activity remains elusive. We identify the source of the intracellular Ca2+ involved in triggering BC contractions, and we elucidate how biliary pressure regulates Ca2+ homeostasis and associated BC contractions. Primary rat hepatocytes were cultured in collagen sandwich. Intra-canalicular Ca2+ was measured with fluo-8; and intra-cellular Ca2+ was measured with GCaMP. Pharmacological modulators of canonical Ca2+-channels were used to study the Ca2+-mediated regulation of BC contraction. BC contraction correlates with cyclic transfer of Ca2+ from BC to adjacent hepatocytes, and not with endoplasmic reticulum Ca2+. A mechanosensitive Ca2+ channel (MCC), Piezo-1, is preferentially localized at BC membranes. The Piezo-1 inhibitor GsMTx-4 blocks the Ca2+ transfer, resulting in cholestatic generation of BC-derived vesicles whereas Piezo-1 hyper-activation by Yoda1 increases the frequency of Ca2+ transfer and BC contraction cycles. Yoda1 can recover normal BC contractility in drug-induced hepatocellular cholestasis, supporting that Piezo-1 regulates BC contraction cycles. Finally, we show that hyper-activating Piezo-1 can be exploited to normalize bile flow in drug-induced hepatocellular cholestasis.

Sequential drug delivery for liver diseases.

The liver performs critical physiological functions such as metabolism/detoxification and blood homeostasis/biliary excretion. A high degree of blood access means that a drug's resident time in any cell is relatively short. This short drug exposure to cells requires local sequential delivery of multiple drugs for optimal efficacy, potency, and safety. The high metabolism and excretion of drugs also impose both technical challenges and opportunities to sequential drug delivery. This review provides an overview of the sequential events in liver regeneration and the related liver diseases. Using selected examples of liver cancer, hepatitis B viral infection, fatty liver diseases, and drug-induced liver injury, we highlight efforts made for the sequential delivery of small and macromolecular drugs through different biomaterials, cells, and microdevice-based delivery platforms that allow fast delivery kinetics and rapid drug switching. As this is a nascent area of development, we extrapolate and compare the results with other sequential drug delivery studies to suggest possible application in liver diseases, wherever appropriate.

Effect of Native and Acetylated Dietary Resistant Starches on Intestinal Fermentative Capacity of Normal and Stunted Children in Southern India.

The health benefits of dietary amylase resistant starch (RS) arise from intestinal microbial fermentation and generation of short chain fatty acids (SCFA). We compared the intestinal fermentative capability of stunted and nonstunted ('healthy') children in southern India using two types of RS: high amylose maize starch (HAMS) and acetylated HAMS (HAMSA). Twenty children (10 stunted and 10 healthy) aged 2 to 5 years were fed biscuits containing HAMS (10 g/day) for two weeks followed by a 2-week washout and then HAMSA biscuits (10 g/day) for 2 weeks. Fecal samples were collected at 3-4 day intervals and pH and SCFA analyzed. At entry, stunted children had lower SCFA concentrations compared to healthy children. Both types of RS led to a significant decrease in fecal pH and increase in fecal acetate and propionate in both healthy and stunted children. However, while HAMS increased fecal butyrate in both groups of children, HAMSA increased butyrate in healthy but not stunted children. Furthermore, healthy children showed a significantly greater increase than stunted children in both acetate and butyrate when fed either RS. No adverse effects were reported with either RS. Stunted children have impaired capacity to ferment certain types of RS which has implications for choice of RS in formulations aimed at improving microbial function in stunted children.

A Novel Ex Vivo System Using 3D Polymer Scaffold to Culture Circulating Tumor Cells from Breast Cancer Patients Exhibits Dynamic E-M Phenotypes.

The majority of the cancer-associated deaths is due to metastasis-the spread of tumors to other organs. Circulating tumor cells (CTCs), which are shed from the primary tumor into the circulation, serve as precursors of metastasis. CTCs have now gained much attention as a new prognostic and diagnostic marker, as well as a screening tool for patients with metastatic disease. However, very little is known about the biology of CTCs in cancer metastasis. An increased understanding of CTC biology, their heterogeneity, and interaction with other cells can help towards a better understanding of the metastatic process, as well as identify novel drug targets. Here we present a novel ex vivo 3D system for culturing CTCs from breast cancer patient blood samples using porous poly(ε-caprolactone) (PCL) scaffolds. As a proof of principle study, we show that ex vivo culture of 12/16 (75%) advanced stage breast cancer patient blood samples were enriched for CTCs identified as CK+ (cytokeratin positive) and CD45- (CD45 negative) cells. The deposition of extracellular matrix proteins on the PCL scaffolds permitted cellular attachment to these scaffolds. Detection of Ki-67 and bromodeoxyuridine (BrdU) positive cells revealed proliferating cell population in the 3D scaffolds. The CTCs cultured without prior enrichment exhibited dynamic differences in epithelial (E) and mesenchymal (M) composition. Thus, our 3D PCL scaffold system offers a physiologically relevant model to be used for studying CTC biology as well as for individualized testing of drug susceptibility. Further studies are warranted for longitudinal monitoring of epithelial-mesenchymal transition (EMT) in CTCs for clinical association.

Tissue mimetic 3D scaffold for breast tumor-derived organoid culture toward personalized chemotherapy.

Breast cancer cell lines lose the inherent gene expression profiles of their source tumor and when cultured as monolayers (two-dimensional) are unable to represent patient tumors. Thus, we engineered a biochemico- and mechano-mimetic three-dimensional (3D) culture platform for primary breast cancer cells by decellularizing cancer-associated fibroblasts (CAFs) cultured on 3D macroporous polymer scaffolds to recapitulate tumor behavior and drug response more realistically. The presence of the CAF-derived extracellular matrix deposited on the polycaprolactone scaffold promoted cell attachment and viability, which is ascribed to higher levels of phosphorylated Focal Adhesion Kinase that mediates cell attachment via integrins. Single cells from primary breast cancers self-organized into tumoroids on prolonged culture. Response of the tumoroids to two chemotherapeutic drugs, doxorubicin and mitoxanthrone, varied significantly across patient samples. This model could be used as an ex vivo platform to culture primary cells toward developing effective and personalized chemotherapy regimens.

Inflammatory Role of Cancer-Associated Fibroblasts in Invasive Breast Tumors Revealed Using a Fibrous Polymer Scaffold.

Inflammation in cancer fuels metastasis and worsens prognosis. Cancer-associated fibroblasts (CAFs) present in the tumor stroma play a vital role in mediating the cascade of cancer inflammation that drives metastasis by enhancing angiogenesis, tissue remodeling, and invasion. In vitro models that faithfully recapitulate CAF-mediated inflammation independent of coculturing with cancer cells are nonexistent. We have engineered fibrous matrices of poly(ε-caprolactone) (PCL) that can maintain the manifold tumor-promoting properties of patient-derived CAFs, which would otherwise require repetitive isolation and complex coculturing with cancer cells. On these fibrous matrices, CAFs proliferated and remodeled the extracellular matrix (ECM) in a parallel-patterned manner mimicking the ECM of high-grade breast tumors and induced stemness in breast cancer cells. The response of the fibroblasts was observed to be sensitive to the scaffold architecture and not the polymer composition. The CAFs cultured on fibrous matrices exhibited increased activation of the NF-κB pathway and downstream proinflammatory gene expression compared to CAFs cultured on conventional two-dimensional (2D) dishes and secreted higher levels of proinflammatory cytokines such as IL-6, GM-CSF, and MIP-3α. Consistent with this, we observed increased infiltration of inflammatory cells to the tumor site and enhanced invasiveness of the tumor in vivo when tumor cells were injected admixed with CAFs grown on fibrous matrices. These data suggest that CAFs better retain their tumor-promoting proinflammatory properties on fibrous polymeric matrices, which could serve as a unique model to investigate the mechanisms of stroma-induced inflammation in cancer progression.

MiRNomics Reveals Breast Cancer Cells Cultured on 3D Scaffolds Better Mimic Tumors in Vivo than Conventional 2D Culture.

Tissue-engineering-based three-dimensional (3D) models offer several advantages over conventional two-dimensional (2D) cultures and can mimic tissues in vivo. Although studies have analyzed the changes in the expression of genes and proteins that might mediate in vivo-like signaling, the changes in the post-transcriptional control of gene expression that are critical in fine-tuning of signaling events has never been studied. In this study, we used next-generation sequencing (NGS) to analyze the changes in the post-transcriptional regulation in MDA-MB-231 breast cancer cells cultured on 3D scaffolds. The changes in the expression of several known microRNAs were similar to the changes reported in highly invasive cancers and their profiles highly correlated with xenotumors and human breast tumors. To elucidate the role of miRNAs in modulating metastatic potential, we integrated the miRNA and the mRNA microarray data and developed networks for major pathways implicated in metastasis. From these networks, we identified several key miRNA-mRNA interactions that might contribute to the invasive behavior and aid in developing a miRNA signature for highly invasive breast cancers. This report on the differential regulation of miRNAs in breast cancer cells cultured on scaffolds demonstrates that 3D culture better mimics the tissue in vivo with novel insights into the roles of miRNAs in modulating metastatic progression.

Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive in Vitro Model for Breast Cancer Metastasis.

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly(ε-caprolactone) (PCL) scaffolds of modulus 7.0 ± 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency in vitro and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell-cell and cell-matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIF1 signaling pathways-all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis.