A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening.

Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.

The landscape of drug sensitivity and resistance in sarcoma.

Sarcomas are a family of rare malignancies composed of over 100 distinct histological subtypes. The rarity of sarcoma poses significant challenges in conducting clinical trials to identify effective therapies, to the point that many rarer subtypes of sarcoma do not have standard-of-care treatment. Even for established regimens, there can be substantial heterogeneity in responses. Overall, novel, personalized approaches for identifying effective treatments are needed to improve patient out-comes. Patient-derived tumor organoids (PDTOs) are clinically relevant models representative of the physiological behavior of tumors across an array of malignancies. Here, we use PDTOs as a tool to better understand the biology of individual tumors and characterize the landscape of drug resistance and sensitivity in sarcoma. We collected n=194 specimens from n=126 sarcoma patients, spanning 24 distinct subtypes. We characterized PDTOs established from over 120 biopsy, resection, and metastasectomy samples. We leveraged our organoid high-throughput drug screening pipeline to test the efficacy of chemotherapeutics, targeted agents, and combination therapies, with results available within a week from tissue collection. Sarcoma PDTOs showed patient-specific growth characteristics and subtype-specific histopathology. Organoid sensitivity correlated with diagnostic subtype, patient age at diagnosis, lesion type, prior treatment history, and disease trajectory for a subset of the compounds screened. We found 90 biological pathways that were implicated in response to treatment of bone and soft tissue sarcoma organoids. By comparing functional responses of organoids and genetic features of the tumors, we show how PDTO drug screening can provide an orthogonal set of information to facilitate optimal drug selection, avoid ineffective therapies, and mirror patient outcomes in sarcoma. In aggregate, we were able to identify at least one effective FDA-approved or NCCN-recommended regimen for 59% of the specimens tested, providing an estimate of the proportion of immediately actionable information identified through our pipeline. Highlights: Standardized organoid culture preserve unique sarcoma histopathological featuresDrug screening on patient-derived sarcoma organoids provides sensitivity information that correlates with clinical features and yields actionable information for treatment guidanceHigh-throughput screenings provide orthogonal information to genetic sequencingSarcoma organoid response to treatment correlates with patient response to therapyLarge scale, functional precision medicine programs for rare cancers are feasible within a single institution.

Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

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.

Highly absorptive dressing composed of natural latex loaded with alginate for exudate control and healing of diabetic wounds.

Wounds can take longer to heal in diabetic patients, increasing the risk of infections and other complications. The most common wounds in diabetic patients are diabetic foot ulcers, a severe complication associated with diabetes mellitus. The United States alone spends $18.7 billion annually on care for these wounds including pain and infection management. If improperly managed, infected lesions may require amputation. The enormous cost associated with wound care and the dire consequences if not cared for properly, emphasize the need to develop strategies to accelerate the healing of diabetic foot wounds. Natural rubber latex (NRL), extracted from Hevea brasiliensis (the rubber tree), has been widely applied as a carrier system for several pharmacologically active compounds. Furthermore, it has been shown to encourage angiogenesis, facilitate cell adhesion, and accelerate wound healing. When NRL dressings are applied to wounds of diabetic patients, exudate release is upregulated. The production of exudate is essential to wound healing as it provides the nutrients, proteins, cells, and environment required for regeneration. Despite its benefits, it is necessary to control excess exudate to avoid prolonged healing resulting from dermatitis, maceration of the wound edges, and lesion growth. In order to solve the problem of excessive exudate release induced by NRL membrane application, we aimed to regulate humidity by absorbing excess exudate and increase water vapor transmission. We developed a highly absorptive, permeable, alginate loaded NRL dressing. Adding alginate to NRL membranes, swelling was increased up to 80-fold, absorbing 4.80 g of water per gram of dry membrane. Moreover, water vapor transmission was improved drastically as the material transmitted 480% more water vapor than pure NRL membranes. Furthermore, in vitro tests demonstrated not only that the membranes are biocompatible, but that they also enhance cell proliferation. Through a cell proliferation assay, we observed that fibroblast proliferation was improved by the presence of NRL while the keratinocytes benefit from the presence of alginate. The NRL-alginate dressings have great potential to improve diabetic wound care by accelerating the healing process.

Recent advances in 3D bioprinting of musculoskeletal tissues.

The musculoskeletal system is essential for maintaining posture, protecting organs, facilitating locomotion, and regulating various cellular and metabolic functions. Injury to this system due to trauma or wear is common, and severe damage may require surgery to restore function and prevent further harm. Autografts are the current gold standard for the replacement of lost or damaged tissues. However, these grafts are constrained by limited supply and donor site morbidity. Allografts, xenografts, and alloplastic materials represent viable alternatives, but each of these methods also has its own problems and limitations. Technological advances in three-dimensional (3D) printing and its biomedical adaptation, 3D bioprinting, have the potential to provide viable, autologous tissue-like constructs that can be used to repair musculoskeletal defects. Though bioprinting is currently unable to develop mature, implantable tissues, it can pattern cells in 3D constructs with features facilitating maturation and vascularization. Further advances in the field may enable the manufacture of constructs that can mimic native tissues in complexity, spatial heterogeneity, and ultimately, clinical utility. This review studies the use of 3D bioprinting for engineering bone, cartilage, muscle, tendon, ligament, and their interface tissues. Additionally, the current limitations and challenges in the field are discussed and the prospects for future progress are highlighted.

Screening Cancer Immunotherapy: When Engineering Approaches Meet Artificial Intelligence.

Immunotherapy is a class of promising anticancer treatments that has recently gained attention due to surging numbers of FDA approvals and extensive preclinical studies demonstrating efficacy. Nevertheless, further clinical implementation has been limited by high variability in patient response to different immunotherapeutic agents. These treatments currently do not have reliable predictors of efficacy and may lead to side effects. The future development of additional immunotherapy options and the prediction of patient-specific response to treatment require advanced screening platforms associated with accurate and rapid data interpretation. Advanced engineering approaches ranging from sequencing and gene editing, to tumor organoids engineering, bioprinted tissues, and organs-on-a-chip systems facilitate the screening of cancer immunotherapies by recreating the intrinsic and extrinsic features of a tumor and its microenvironment. High-throughput platform development and progress in artificial intelligence can also improve the efficiency and accuracy of screening methods. Here, these engineering approaches in screening cancer immunotherapies are highlighted, and a discussion of the future perspectives and challenges associated with these emerging fields to further advance the clinical use of state-of-the-art cancer immunotherapies are provided.

Microengineered poly(HEMA) hydrogels for wearable contact lens biosensing.

Microchannels in hydrogels play an essential role in enabling a smart contact lens. However, microchannels have rarely been created in commercial hydrogel contact lenses due to their sensitivity to conventional microfabrication techniques. Here, we report the fabrication of microchannels in poly(2-hydroxyethyl methacrylate) (poly(HEMA)) hydrogels that are used in commercial contact lenses with a three-dimensional (3D) printed mold. We investigated the corresponding capillary flow behaviors in these microchannels. We observed different capillary flow regimes in these microchannels, depending on their hydration level. In particular, we found that a peristaltic pressure could reinstate flow in a dehydrated channel, indicating that the motion of eye-blinking may help tears flow in a microchannel-containing contact lens. Colorimetric pH and electrochemical Na+ sensing capabilities were demonstrated in these microchannels. This work paves the way for the development of microengineered poly(HEMA) hydrogels for various biomedical applications such as eye-care and wearable biosensing.

A Patch of Detachable Hybrid Microneedle Depot for Localized Delivery of Mesenchymal Stem Cells in Regeneration Therapy.

Mesenchymal stem cells (MSCs) have been widely used for regenerative therapy. In most current clinical applications, MSCs are delivered by injection but face significant issues with cell viability and penetration into the target tissue due to a limited migration capacity. Some therapies have attempted to improve MSC stability by their encapsulation within biomaterials; however, these treatments still require an enormous number of cells to achieve therapeutic efficacy due to low efficiency. Additionally, while local injection allows for targeted delivery, injections with conventional syringes are highly invasive. Due to the challenges associated with stem cell delivery, a local and minimally invasive approach with high efficiency and improved cell viability is highly desired. In this study, we present a detachable hybrid microneedle depot (d-HMND) for cell delivery. Our system consists of an array of microneedles with an outer poly(lactic-co-glycolic) acid (PLGA) shell and an internal gelatin methacryloyl (GelMA)-MSC mixture (GMM). The GMM was characterized and optimized for cell viability and mechanical strength of the d-HMND required to penetrate mouse skin tissue was also determined. MSC viability and function within the d-HMND was characterized in vitro and the regenerative efficacy of the d-HMND was demonstrated in vivo using a mouse skin wound model.

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.

Thrombolytic Agents: Nanocarriers in Controlled Release.

Thrombosis is a life-threatening pathological condition in which blood clots form in blood vessels, obstructing or interfering with blood flow. Thrombolytic agents (TAs) are enzymes that can catalyze the conversion of plasminogen to plasmin to dissolve blood clots. The plasmin formed by TAs breaks down fibrin clots into soluble fibrin that finally dissolves thrombi. Several TAs have been developed to treat various thromboembolic diseases, such as pulmonary embolisms, acute myocardial infarction, deep vein thrombosis, and extensive coronary emboli. However, systemic TA administration can trigger non-specific activation that can increase the incidence of bleeding. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses. To overcome these limitations, various types of nanocarriers have been introduced that enhance the pharmacokinetic effects by protecting the TA from the biological environment and targeting the release into coagulation. The nanocarriers show increasing half-life, reducing side effects, and improving overall TA efficacy. In this work, the recent advances in various types of TAs and nanocarriers are thoroughly reviewed. Various types of nanocarriers, including lipid-based, polymer-based, and metal-based nanoparticles are described, for the targeted delivery of TAs. This work also provides insights into issues related to the future of TA development and successful clinical translation.

Design and Simulation of an Integrated Centrifugal Microfluidic Device for CTCs Separation and Cell Lysis.

Separation of circulating tumor cells (CTCs) from blood samples and subsequent DNA extraction from these cells play a crucial role in cancer research and drug discovery. Microfluidics is a versatile technology that has been applied to create niche solutions to biomedical applications, such as cell separation and mixing, droplet generation, bioprinting, and organs on a chip. Centrifugal microfluidic biochips created on compact disks show great potential in processing biological samples for point of care diagnostics. This study investigates the design and numerical simulation of an integrated microfluidic device, including a cell separation unit for isolating CTCs from a blood sample and a micromixer unit for cell lysis on a rotating disk platform. For this purpose, an inertial microfluidic device was designed for the separation of target cells by using contraction-expansion microchannel arrays. Additionally, a micromixer was incorporated to mix separated target cells with the cell lysis chemical reagent to dissolve their membranes to facilitate further assays. Our numerical simulation approach was validated for both cell separation and micromixer units and corroborates existing experimental results. In the first compartment of the proposed device (cell separation unit), several simulations were performed at different angular velocities from 500 rpm to 3000 rpm to find the optimum angular velocity for maximum separation efficiency. By using the proposed inertial separation approach, CTCs, were successfully separated from white blood cells (WBCs) with high efficiency (~90%) at an angular velocity of 2000 rpm. Furthermore, a serpentine channel with rectangular obstacles was designed to achieve a highly efficient micromixer unit with high mixing quality (~98%) for isolated CTCs lysis at 2000 rpm.

Gut-on-a-chip: Current progress and future opportunities.

Organ-on-a-chip technology tries to mimic the complexity of native tissues in vitro. Important progress has recently been made in using this technology to study the gut with and without microbiota. These in vitro models can serve as an alternative to animal models for studying physiology, pathology, and pharmacology. While these models have greater physiological relevance than two-dimensional (2D) cell systems in vitro, endocrine and immunological functions in gut-on-a-chip models are still poorly represented. Furthermore, the construction of complex models, in which different cell types and structures interact, remains a challenge. Generally, gut-on-a-chip models have the potential to advance our understanding of the basic interactions found within the gut and lay the foundation for future applications in understanding pathophysiology, developing drugs, and personalizing medical treatments.

A Novel Negative Pressure Isolation Device for Aerosol Transmissible COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic creates a need to protect health care workers (HCWs) from patients undergoing aerosol-generating procedures which may transmit the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Existing personal containment devices (PCDs) may protect HCWs from respiratory droplets but not from potentially dangerous respiratory-generated aerosols. We describe a new PCD and its aerosol containment capabilities. The device ships flat and folds into a chamber. With its torso drape and protective arm sleeves mounted, it provides contact, droplet, and aerosol isolation during intubation and cardiopulmonary resuscitation (CPR). Significantly improved ergonomics, single-use workflow, and ease of removal distinguish this device from previously published designs.

Biodegradable ß-Cyclodextrin Conjugated Gelatin Methacryloyl Microneedle for Delivery of Water-Insoluble Drug.

Transdermal delivery of water-insoluble drugs via hydrogel-based microneedle (MN) arrays is crucial for improving their therapeutic efficacies. However, direct loading of water-insoluble drug into hydrophilic matrices remains challenging. Here, a biodegradable MN array patch that is fabricated from naturally derived polymer conjugates of gelatin methacryloyl and ß-cyclodextrin (GelMA-ß-CD) is reported. When curcumin, an unstable and water-insoluble anticancer drug, is loaded as a model drug, its stability and solubility are improved due to the formation of an inclusion complex. The polymer-drug complex GelMA-ß-CD/CUR can be formulated into MN arrays with sufficient mechanical strength for skin penetration and tunable drug release profile. Anticancer efficacy of released curcumin is observed in three-dimensional B16F10 melanoma models. The GelMA-ß-CD/CUR MN exhibits relatively higher therapeutic efficacy through more localized and deeper penetrated manner compared with a control nontransdermal patch. In vivo studies also verify biocompatibility and degradability of the GelMA-ß-CD MN arrays patch.

Stimuli-Responsive Delivery of Growth Factors for Tissue Engineering.

Growth factors (GFs) play a crucial role in directing stem cell behavior and transmitting information between different cell populations for tissue regeneration. However, their utility as therapeutics is limited by their short half-life within the physiological microenvironment and significant side effects caused by off-target effects or improper dosage. "Smart" materials that can not only sustain therapeutic delivery over a treatment period but also facilitate on-demand release upon activation are attracting significant interest in the field of GF delivery for tissue engineering. Three properties are essential in engineering these "smart" materials: 1) the cargo vehicle protects the encapsulated therapeutic; 2) release is targeted to the site of injury; 3) cargo release can be modulated by disease-specific stimuli. The aim of this review is to summarize the current research on stimuli-responsive materials as intelligent vehicles for controlled GF delivery; Five main subfields of tissue engineering are discussed: skin, bone and cartilage, muscle, blood vessel, and nerve. Challenges in achieving such "smart" materials and perspectives on future applications of stimuli-responsive GF delivery for tissue regeneration are also discussed.

Gelatin Methacryloyl Microneedle Patches for Minimally Invasive Extraction of Skin Interstitial Fluid.

The extraction of interstitial fluid (ISF) from skin using microneedles (MNs) has attracted growing interest in recent years due to its potential for minimally invasive diagnostics and biosensors. ISF collection by absorption into a hydrogel MN patch is a promising way that requires the materials to have outstanding swelling ability. Here, a gelatin methacryloyl (GelMA) patch is developed with an 11 × 11 array of MNs for minimally invasive sampling of ISF. The properties of the patch can be tuned by altering the concentration of the GelMA prepolymer and the crosslinking time; patches are created with swelling ratios between 293% and 423% and compressive moduli between 3.34 MPa and 7.23 MPa. The optimized GelMA MN patch demonstrates efficient extraction of ISF. Furthermore, it efficiently and quantitatively detects glucose and vancomycin in ISF in an in vivo study. This minimally invasive approach of extracting ISF with a GelMA MN patch has the potential to complement blood sampling for the monitoring of target molecules from patients.

Combined Effects of Electric Stimulation and Microgrooves in Cardiac Tissue-on-a-Chip for Drug Screening.

Animal models and traditional cell cultures are essential tools for drug development. However, these platforms can show striking discrepancies in efficacy and side effects when compared to human trials. These differences can lengthen the drug development process and even lead to drug withdrawal from the market. The establishment of preclinical drug screening platforms that have higher relevancy to physiological conditions is desirable to facilitate drug development. Here, a heart-on-a-chip platform, incorporating microgrooves and electrical pulse stimulations to recapitulate the well-aligned structure and synchronous beating of cardiomyocytes (CMs) for drug screening, is reported. Each chip is made with facile lithographic and laser-cutting processes that can be easily scaled up to high-throughput format. The maturation and phenotypic changes of CMs cultured on the heart-on-a-chip is validated and it can be treated with various drugs to evaluate cardiotoxicity and cardioprotective efficacy. The heart-on-a-chip can provide a high-throughput drug screening platform in preclinical drug development.

Rhodamine Conjugated Gelatin Methacryloyl Nanoparticles for Stable Cell Imaging.

Fluorescent nanomaterials have been widely used in biological imaging due to their selectivity, sensitivity, and noninvasive nature. These characteristics make the materials suitable for real-time and in situ imaging. However, further development of highly biocompatible nanosystems with long-lasting fluorescent intensity and photostability is needed for advanced bioimaging. We have used electrospraying to generate gelatin methacryloyl (GelMA)-based fluorescent nanoparticles (NPs) with chemically conjugated rhodamine B (RB). The extent of conjugation can be controlled by varying the mass ratio of RB and GelMA precursors to obtain RB-conjugated GelMA (RB-GelMA) NPs with optimal fluorescent properties and particle size. These NPs exhibited superior biocompatibility when compared with pure RB in in vitro cell viability and proliferation assays using multiple cell types. Moreover, RB-GelMA NPs showed enhanced cell internalization and improved brightness compared with unconjugated RB. Our experiments demonstrate that engineered RB-GelMA NPs can be used as a biocompatible fluorescent label for bioimaging.

Non-transdermal microneedles for advanced drug delivery.

Microneedles (MNs) have been used to deliver drugs for over two decades. These platforms have been proven to increase transdermal drug delivery efficiency dramatically by penetrating restrictive tissue barriers in a minimally invasive manner. While much of the early development of MNs focused on transdermal drug delivery, this technology can be applied to a variety of other non-transdermal biomedical applications. Several variations, such as multi-layer or hollow MNs, have been developed to cater to the needs of specific applications. The heterogeneity in the design of MNs has demanded similar variety in their fabrication methods; the most common methods include micromolding and drawing lithography. Numerous materials have been explored for MN fabrication which range from biocompatible ceramics and metals to natural and synthetic biodegradable polymers. Recent advances in MN engineering have diversified MNs to include unique shapes, materials, and mechanical properties that can be tailored for organ-specific applications. In this review, we discuss the design and creation of modern MNs that aim to surpass the biological barriers of non-transdermal drug delivery in ocular, vascular, oral, and mucosal tissue.