Tunable hybrid hydrogels with multicellular spheroids for modeling desmoplastic pancreatic cancer.

The tumor microenvironment consists of diverse, complex etiological factors. The matrix component of pancreatic ductal adenocarcinoma (PDAC) plays an important role not only in physical properties such as tissue rigidity but also in cancer progression and therapeutic responsiveness. Although significant efforts have been made to model desmoplastic PDAC, existing models could not fully recapitulate the etiology to mimic and understand the progression of PDAC. Here, two major components in desmoplastic pancreatic matrices, hyaluronic acid- and gelatin-based hydrogels, are engineered to provide matrices for tumor spheroids composed of PDAC and cancer-associated fibroblasts (CAF). Shape analysis profiles reveals that incorporating CAF contributes to a more compact tissue formation. Higher expression levels of markers associated with proliferation, epithelial to mesenchymal transition, mechanotransduction, and progression are observed for cancer-CAF spheroids cultured in hyper desmoplastic matrix-mimicking hydrogels, while the trend can be observed when those are cultured in desmoplastic matrix-mimicking hydrogels with the presence of transforming growth factor-ß1 (TGF-ß1). The proposed multicellular pancreatic tumor model, in combination with proper mechanical properties and TGF-ß1 supplement, makes strides in developing advanced pancreatic models for resembling and monitoring the progression of pancreatic tumors, which could be potentially applicable for realizing personalized medicine and drug testing applications.

Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration.

Silk fibroin (SF) is a promising biomaterial for tendon repair, but its relatively rigid mechanical properties and low cell affinity have limited its application in regenerative medicine. Meanwhile, gelatin-based polymers have advantages in cell attachment and tissue remodeling but have insufficient mechanical strength to regenerate tough tissue such as tendons. Taking these aspects into account, in this study, gelatin methacryloyl (GelMA) is combined with SF to create a mechanically strong and bioactive nanofibrous scaffold (SG). The mechanical properties of SG nanofibers can be flexibly modulated by varying the ratio of SF and GelMA. Compared to SF nanofibers, mesenchymal stem cells (MSCs) seeded on SG fibers with optimal composition (SG7) exhibit enhanced growth, proliferation, vascular endothelial growth factor production, and tenogenic gene expression behavior. Conditioned media from MSCs cultured on SG7 scaffolds can greatly promote the migration and proliferation of tenocytes. Histological analysis and tenogenesis-related immunofluorescence staining indicate SG7 scaffolds demonstrate enhanced in vivo tendon tissue regeneration compared to other groups. Therefore, rational combinations of SF and GelMA hybrid nanofibers may help to improve therapeutic outcomes and address the challenges of tissue-engineered scaffolds for tendon regeneration.

pH-Responsive doxorubicin delivery using shear-thinning biomaterials for localized melanoma treatment.

Injectable shear-thinning biomaterials (STBs) have attracted significant attention because of their efficient and localized delivery of cells as well as various molecules ranging from growth factors to drugs. Recently, electrostatic interaction-based STBs, including gelatin/LAPONITE® nanocomposites, have been developed through a simple assembly process and show outstanding shear-thinning properties and injectability. However, the ability of different compositions of gelatin and LAPONITE® to modulate doxorubicin (DOX) delivery at different pH values to enhance the effectiveness of topical skin cancer treatment is still unclear. Here, we fabricated injectable STBs using gelatin and LAPONITE® to investigate the influence of LAPONITE®/gelatin ratio on mechanical characteristics, capacity for DOX release in response to different pH values, and cytotoxicity toward malignant melanoma. The release profile analysis of various compositions of DOX-loaded STBs under different pH conditions revealed that lower amounts of LAPONITE® (6NC25) led to higher pH-responsiveness capable of achieving a localized, controlled, and sustained release of DOX in an acidic tumor microenvironment. Moreover, we showed that 6NC25 had a lower storage modulus and required lower injection forces compared to those with higher LAPONITE® ratios. Furthermore, DOX delivery analysis in vitro and in vivo demonstrated that DOX-loaded 6NC25 could efficiently target subcutaneous malignant tumors via DOX-induced cell death and growth restriction.

Engineering liver microtissues to study the fusion of HepG2 with mesenchymal stem cells and invasive potential of fused cells.

Increasing evidence from cancer cell fusion with different cell types in the tumor microenvironment has suggested a probable mechanism for how metastasis-initiating cells could be generated in tumors. Although human mesenchymal stem cells (hMSCs) have been known as promising candidates to create hybrid cells with cancer cells, the role of hMSCs in fusion with cancer cells is still controversial. Here, we fabricated a liver-on-a-chip platform to monitor the fusion of liver hepatocellular cells (HepG2) with hMSCs and study their invasive potential. We demonstrated that hMSCs might play dual roles in HepG2 spheroids. The analysis of tumor growth with different fractions of hMSCs in HepG2 spheroids revealed hMSCs' role in preventing HepG2 growth and proliferation, while the hMSCs presented in the HepG2 spheroids led to the generation of HepG2-hMSC hybrid cells with much higher invasiveness compared to HepG2. These invasive HepG2-hMSC hybrid cells expressed high levels of markers associated with stemness, proliferation, epithelial to mesenchymal transition, and matrix deposition, which corresponded to the expression of these markers for hMSCs escaping from hMSC spheroids. In addition, these fused cells were responsible for collective invasion following HepG2 by depositing Collagen I and Fibronectin in their surrounding microenvironment. Furthermore, we showed that hepatic stellate cells (HSCs) could also be fused with HepG2, and the HepG2-HSC hybrid cells possessed similar features to those from HepG2-hMSC fusion. This fusion of HepG2 with liver-resident HSCs may propose a new potential mechanism of hepatic cancer metastasis.

Cancer-on-a-Chip for Modeling Immune Checkpoint Inhibitor and Tumor Interactions.

Cancer immunotherapies, including immune checkpoint inhibitor (ICI)-based therapies, have revolutionized cancer treatment. However, patient response to ICIs is highly variable, necessitating the development of methods to quickly assess efficacy. In this study, an array of miniaturized bioreactors has been developed to model tumor-immune interactions. This immunotherapeutic high-throughput observation chamber (iHOC) is designed to test the effect of anti-PD-1 antibodies on cancer spheroid (MDA-MB-231, PD-L1+) and T cell (Jurkat) interactions. This system facilitates facile monitoring of T cell inhibition and reactivation using metrics such as tumor infiltration and interleukin-2 (IL-2) secretion. Status of the tumor-immune interactions can be easily captured within the iHOC by measuring IL-2 concentration using a micropillar array where sensitive, quantitative detection is allowed after antibody coating on the surface of array. The iHOC is a platform that can be used to model and monitor cancer-immune interactions in response to immunotherapy in a high-throughput manner.

Biodegradable microneedle patch for transdermal gene delivery.

The skin houses a developed vascular and lymphatic network with a significant population of immune cells. Because of the properties of the skin, nucleic acid delivery through the tissue has the potential to treat a range of pathologies, including genetic skin conditions, hyperproliferative diseases, cutaneous cancers, wounds, and infections. This work presents a gelatin methacryloyl (GelMA) microneedle (MN)-based platform for local and controlled transdermal delivery of plasmid DNA (pDNA) with high transfection efficiency both in vitro and in vivo. Intracellular delivery of the nucleic acid cargo is enabled by poly(ß-amino ester) (PBAE) nanoparticles (NPs). After being embedded in the GelMA MNs, sustained release of DNA-encapsulated PBAE NPs is achieved and the release profiles can be controlled by adjusting the degree of crosslinking of the GelMA hydrogel. These results highlight the advantages and potential of using PBAE/DNA NP-embedded GelMA MN patches (MN/PBAE/DNA) for successful transdermal delivery of pDNA for tissue regeneration and cancer therapy.

Microfluidic-Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications.

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high-efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label-free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor-intensive steps of labeling molecular signatures of cells. In general, microfluidic-based cell sorting approaches can separate cells using "intrinsic" (e.g., fluid dynamic forces) versus "extrinsic" external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label-free microfluidic-based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic-based cell separation methods are discussed.

In Vitro Human Liver Model of Nonalcoholic Steatohepatitis by Coculturing Hepatocytes, Endothelial Cells, and Kupffer Cells.

The liver has a complex and unique microenvironment with multiple cell-cell interactions and internal vascular networks. Although nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with multiple phases, no proper model could fully recapitulate the in vivo microenvironment to understand NAFLD progression. Here, an in vitro human liver model of NAFLD by coculturing human hepatocytes, umbilical vein endothelial cells (HUVECs), and Kupffer cells (KCs) into spheroids is presented. Analysis of indirect cross-talk using conditioned media between steatotic spheroids-composed of hepatocellular carcinoma-derived cells (HepG2) and HUVECs-and mouse KCs reveals that the latter can be activated showing increased cell area, elevated production of reactive oxygen species (ROS), and proinflammatory cytokines. Spheroids incorporating human KCs (HKCs) can also be induced into steatotic stage by supplementing fat. Steatotic spheroids with/without HKCs show different levels of steatotic stages through lipid accumulation and ROS production. Steatotic spheroids made from an immortalized hepatic progenitor cell line (HepaRG) compared to those made from HepG2 cells display similar trends of functionality, but elevated levels of proinflammatory cytokines, and improved reversibility of steatosis. The in vitro human liver system proposed makes strides in developing a model to mimic and monitor the progression of NAFLD.

Engineering Precision Medicine.

Advances in genomic sequencing and bioinformatics have led to the prospect of precision medicine where therapeutics can be advised by the genetic background of individuals. For example, mapping cancer genomics has revealed numerous genes that affect the therapeutic outcome of a drug. Through materials and cell engineering, many opportunities exist for engineers to contribute to precision medicine, such as engineering biosensors for diagnosis and health status monitoring, developing smart formulations for the controlled release of drugs, programming immune cells for targeted cancer therapy, differentiating pluripotent stem cells into desired lineages, fabricating bioscaffolds that support cell growth, or constructing "organs-on-chips" that can screen the effects of drugs. Collective engineering efforts will help transform precision medicine into a more personalized and effective healthcare approach. As continuous progress is made in engineering techniques, more tools will be available to fully realize precision medicine's potential.

Organ-on-a-Chip for Cancer and Immune Organs Modeling.

Bridging the gap between findings in preclinical 2D cell culture models and in vivo tissue cultures has been challenging; the simple microenvironment of 2D monolayer culture systems may not capture the cellular response to drugs accurately. Three-dimensional organotypic models have gained increasing interest due to their ability to recreate precise cellular organizations. These models facilitate investigation of the interactions between different sub-tissue level components through providing physiologically relevant microenvironments for cells in vitro. The incorporation of human-sourced tissues into these models further enables personalized prediction of drug responses. Integration of microfluidic units into the 3D models can be used to control their local environment, dynamic simulation of cell behaviors, and real-time readout of drug testing data. Cancer and immune system related diseases are severe burdens to our health care system and have created an urgent need for high-throughput, and effective drug development plans. This review focuses on recent progress in the development of "cancer-on-a-chip" and "immune organs-on-a-chip" systems designed to study disease progression and predict drug-induced responses. Future challenges and opportunities are also discussed.

A Human Liver-on-a-Chip Platform for Modeling Nonalcoholic Fatty Liver Disease.

The liver possesses a unique microenvironment with a complex internal vascular system and cell-cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2-HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2-HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat-based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.

Minimally Invasive and Regenerative Therapeutics.

Advances in biomaterial synthesis and fabrication, stem cell biology, bioimaging, microsurgery procedures, and microscale technologies have made minimally invasive therapeutics a viable tool in regenerative medicine. Therapeutics, herein defined as cells, biomaterials, biomolecules, and their combinations, can be delivered in a minimally invasive way to regenerate different tissues in the body, such as bone, cartilage, pancreas, cardiac, skeletal muscle, liver, skin, and neural tissues. Sophisticated methods of tracking, sensing, and stimulation of therapeutics in vivo using nano-biomaterials and soft bioelectronic devices provide great opportunities to further develop minimally invasive and regenerative therapeutics (MIRET). In general, minimally invasive delivery methods offer high yield with low risk of complications and reduced costs compared to conventional delivery methods. Here, minimally invasive approaches for delivering regenerative therapeutics into the body are reviewed. The use of MIRET to treat different tissues and organs is described. Although some clinical trials have been performed using MIRET, it is hoped that such therapeutics find wider applications to treat patients. Finally, some future perspective and challenges for this emerging field are highlighted.

Biodegradable Gelatin Methacryloyl Microneedles for Transdermal Drug Delivery.

Biocompatible and bioresponsive microneedles (MNs) are emerging technology platforms for sustained drug release with a potential to be a key player in transdermal delivery of therapeutics. In this paper, an innovative biodegradable MNs patch for the sustained delivery of drugs using a polymer patch, which can adjust delivery rates based on its crosslinking degree, is reported. Gelatin methacryloyl (GelMA) is used as the base for engineering biodegradable MNs. The anticancer drug doxorubicin (DOX) is loaded into GelMA MNs using the one molding step. The GelMA MNs can efficiently penetrate the stratum corneum layer of a mouse cadaver skin. Mechanical properties and drug release behavior of the GelMA MNs can be adjusted by tuning the degree of crosslinking. The efficacy of the DOX released from the GelMA MNs is tested and the anticancer efficacy of the released drugs against melanoma cell line A375 is demonstrated. Since GelMA is a versatile material in engineering tissue scaffolds, it is expected that the GelMA MNs can be used as a platform for the delivery of various therapeutics.

Integrin-Mediated Interactions Control Macrophage Polarization in 3D Hydrogels.

Adverse immune reactions prevent clinical translation of numerous implantable devices and materials. Although inflammation is an essential part of tissue regeneration, chronic inflammation ultimately leads to implant failure. In particular, macrophage polarity steers the microenvironment toward inflammation or wound healing via the induction of M1 and M2 macrophages, respectively. Here, this paper demonstrates that macrophage polarity within biomaterials can be controlled through integrin-mediated interactions between human monocytic THP-1 cells and collagen-derived matrix. Surface marker, gene expression, biochemical, and cytokine profiling consistently indicate that THP-1 cells within a biomaterial lacking cell attachment motifs yield proinflammatory M1 macrophages, whereas biomaterials with attachment sites in the presence of interleukin-4 (IL-4) induce an anti-inflammatory M2-like phenotype and propagate the effect of IL-4 in induction of M2-like macrophages. Importantly, integrin α2ß1 plays a pivotal role as its inhibition blocks the induction of M2 macrophages. The influence of the microenvironment of the biomaterial over macrophage polarity is further confirmed by its ability to modulate the effect of IL-4 and lipopolysaccharide, which are potent inducers of M2 or M1 phenotypes, respectively. Thus, this study represents a novel, versatile, and effective strategy to steer macrophage polarity through integrin-mediated 3D microenvironment for biomaterial-based programming.

A liver-on-a-chip platform with bioprinted hepatic spheroids.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair.

The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Organs-on-a-chip: a new tool for drug discovery.

INTRODUCTION: The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as 'organs-on-a-chip', have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue. AREAS COVERED: The aim of this article is to provide an overview of the organs-on-a-chip systems that have been recently developed. First, the authors introduce single-organ platforms, focusing on the most studied organs such as liver, heart, blood vessels and lung. Later, the authors briefly describe tumor-on-a-chip platforms and highlight their application for testing anti-cancer drugs. Finally, the article reports a few examples of other organs integrated in microfluidic chips along with preliminary multiple-organs-on-a-chip examples. The article also highlights key fabrication points as well as the main application areas of these devices. EXPERT OPINION: This field is still at an early stage and major challenges need to be addressed prior to the embracement of these technologies by the pharmaceutical industry. To produce predictive drug screening platforms, several organs have to be integrated into a single microfluidic system representative of a humanoid. The routine production of metabolic biomarkers of the organ constructs, as well as their physical environment, have to be monitored prior to and during the delivery of compounds of interest to be able to translate the findings into useful discoveries.

Elastomeric Recombinant Protein-based Biomaterials.

Elastomeric protein-based biomaterials, produced from elastin derivatives, are widely investigated as promising tissue engineering scaffolds due to their remarkable properties including substantial extensibility, long-term stability, self-assembly, high resilience upon stretching, low energy loss, and excellent biological activity. These elastomers are processed from different sources of soluble elastin such as animal-derived soluble elastin, recombinant human tropoelastin, and elastin-like polypeptides into various forms including three dimensional (3D) porous hydrogels, elastomeric films, and fibrous electrospun scaffolds. Elastin-based biomaterials have shown great potential for the engineering of elastic tissues such as skin, lung and vasculature. In this review, the synthesis and properties of various elastin-based elastomers with their applications in tissue engineering are described.