Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

Ultrastable Iodinated Oil-Based Pickering Emulsion Enables Locoregional Sustained Codelivery of Hypoxia Inducible Factor-1 Inhibitor and Anticancer Drugs for Tumor Combination Chemotherapy.

Tumor hypoxia-associated drug resistance presents a major challenge for cancer chemotherapy. However, sustained delivery systems with a high loading capability of hypoxia-inducible factor-1 (HIF-1) inhibitors are still limited. Here, we developed an ultrastable iodinated oil-based Pickering emulsion (PE) to achieve locally sustained codelivery of a HIF-1 inhibitor of acriflavine and an anticancer drug of doxorubicin for tumor synergistic chemotherapy. The PE exhibited facile injectability for intratumoral administration, great radiopacity for in vivo examination, excellent physical stability (>1 mo), and long-term sustained release capability of both hydrophilic drugs (i.e., acriflavine and doxorubicin). We found that the codelivery of acriflavine and doxorubicin from the PE promoted the local accumulation and retention of both drugs using an acellular liver organ model and demonstrated significant inhibition of tumor growth in a 4T1 tumor-bearing mouse model, improving the chemotherapeutic efficacy through the synergistic effects of direct cytotoxicity with the functional suppression of HIF-1 pathways of tumor cells. Such an iodinated oil-based PE provides a great injectable sustained delivery platform of hydrophilic drugs for locoregional chemotherapy.

Cirrhotic hepatocellular carcinoma-based decellularized liver cancer model for local chemoembolization evaluation.

Transarterial chemoembolization (TACE) is a common treatment for unresectable intermediate stage hepatocellular carcinoma (HCC) and involves the combination of chemotherapy agents and embolic materials to target and block the blood supply to the tumor, leading to localized treatment. However, the selection of clinical chemoembolization agents remains limited, and the effectiveness of various agents is still under investigation. Meanwhile, replicating the complex vasculature and extracellular matrix (ECM) circumstances of HCC in in vitro models for evaluating embolic agents proves to be challenging. Herein, we developed a decellularized cancerous liver model with translucent appearance, a complicated hepatic vascular system and tissue-specific ECM for the evaluation of embolic agents. Inkpad oil and microparticles were used to illustrate different systems of vascular structures between healthy and HCC rats' livers. Quantitative analysis with AngioTool revealed significant differences in vessel density and lacunarity between the two groups. Proteomics showed higher secretion of collagens in the HCC rat liver models than in healthy livers. Utilizing this in vitro model, we investigated the impact of tumor-specific vascular structure and ECM composition on chemoembolization performance, the two key factors inaccessible by currently available drug release testing platforms. Our findings revealed that the presence of an aberrant vascular system and the distorted ECM within the model led to drug retention. This preclinical model holds great promise as a valuable tool for evaluating embolic agents and studying their performance in the tumor microenvironment. STATEMENT OF SIGNIFICANCE: Transarterial chemoembolization (TACE), which employs drug-eluting embolic agents to obstruct the tumor-feeding vessels while locally releasing chemotherapeutic drugs into the tumor, has become the first-line treatment of unresectable liver cancer over past two decades. Nevertheless, the advancement of effective drug-eluting embolic agents has been retarded due to the lack of appropriate in vitro models for assessing the local embolization and chemotherapy performances in TACE. Here we developed a cirrhotic hepatocellular carcinoma-based decellularized liver cancer model, which preserves the aberrant vasculatures and tumor-specific extracellular matrix of liver cancer, for TACE evaluation. This model incorporates a blood flow simulation component to assess the dynamics of drug release behaviors of chemoembolic agents within tumor-mimicking conditions, more accurately replicating the in vivo environment for the locoregional assessments as compared to conventional in vitro models.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.

Chemically Induced Extracellular Ice Nucleation Reduces Intracellular Ice Formation Enabling 2D and 3D Cellular Cryopreservation.

3D cell assemblies such as spheroids reproduce the in vivo state more accurately than traditional 2D cell monolayers and are emerging as tools to reduce or replace animal testing. Current cryopreservation methods are not optimized for complex cell models, hence they are not easily banked and not as widely used as 2D models. Here we use soluble ice nucleating polysaccharides to nucleate extracellular ice and dramatically improve spheroid cryopreservation outcomes. This protects the cells beyond using DMSO alone, and with the major advantage that the nucleators function extracellularly and hence do not need to permeate the 3D cell models. Critical comparison of suspension, 2D and 3D cryopreservation outcomes demonstrated that warm-temperature ice nucleation reduces the formation of (fatal) intracellular ice, and in the case of 2/3D models this reduces propagation of ice between adjacent cells. This demonstrates that extracellular chemical nucleators could revolutionize the banking and deployment of advanced cell models.

Near Infrared-Absorbing Nanoparticle-Mediated Endovascular Photothermal Precision Embolization of Tumor Feeding Vessels for Starvation Treatment.

Photothermal therapy has attracted enormous attention as an efficient treatment modality in cancer ablation but still encounters a major bottleneck due to the limited penetration depth of light inside tissues. To overcome the challenge of deep tissue penetration, we present a strategy of endovascular photothermal precision embolization (EPPE), which employs an endovascular optical fiber to induce local embolization only in the entrance of feeding vessels through photothermal heating for the purpose of fully blocking the blood supply of the whole tumor. In EPPE, we apply a highly efficient and biocompatible photothermal agent, i.e., near-infrared (NIR)-light-absorbing diketopyrrolopyrrole-dithiophene-based nanoparticle, which exhibits a high cell-killing efficacy at a concentration of 200 µg/mL using 808 nm laser irradiation of 0.5 W/cm2 within 5 min in both 2D cell culture and a 3D tumor spheroid model. We verify the feasibility of EPPE in an ex vivo organ-structured recellularized liver model and further confirm the in vivo efficacy of the photothermal treatment in a rat liver model. The photothermal treatment combined with the embolization effect holds promise to serve as an effective starvation therapy to treat tumors of varying sizes and locations.

A 3D Tumor-Mimicking In Vitro Drug Release Model of Locoregional Chemoembolization Using Deep Learning-Based Quantitative Analyses.

Primary liver cancer, with the predominant form as hepatocellular carcinoma (HCC), remains a worldwide health problem due to its aggressive and lethal nature. Transarterial chemoembolization, the first-line treatment option of unresectable HCC that employs drug-loaded embolic agents to occlude tumor-feeding arteries and concomitantly delivers chemotherapeutic drugs into the tumor, is still under fierce debate in terms of the treatment parameters. The models that can produce in-depth knowledge of the overall intratumoral drug release behavior are lacking. This study engineers a 3D tumor-mimicking drug release model, which successfully overcomes the substantial limitations of conventional in vitro models through utilizing decellularized liver organ as a drug-testing platform that uniquely incorporates three key features, i.e., complex vasculature systems, drug-diffusible electronegative extracellular matrix, and controlled drug depletion. This drug release model combining with deep learning-based computational analyses for the first time permits quantitative evaluation of all important parameters associated with locoregional drug release, including endovascular embolization distribution, intravascular drug retention, and extravascular drug diffusion, and establishes long-term in vitro-in vivo correlations with in-human results up to 80 d. This model offers a versatile platform incorporating both tumor-specific drug diffusion and elimination settings for quantitative evaluation of spatiotemporal drug release kinetics within solid tumors.

Engineering orthotopic tumor spheroids with organ-specific vasculatures for local chemoembolization evaluation.

Developing a three-dimensional (3D) in vitro tumor model with vasculature systems suitable for testing endovascular interventional therapies remains a challenge. Here we develop an orthotopic liver tumor spheroid model that captures the organ-level complexity of vasculature systems and the extracellular matrix to evaluate transcatheter arterial chemoembolization (TACE) treatment. The orthotopic tumor spheroids are derived by seeding HepG2 cell colonies with controlled size and location surrounding the portal triads in a decellularized rat liver matrix and are treated by clinically relevant drug-eluting beads embolized in a portal vein vasculature while maintaining dynamic physiological conditions with nutrient and oxygen supplies through the hepatic vein vasculature. The orthotopic tumor model exhibits strong drug retention inside the spheroids and embolization location-dependent cellular apoptosis responses in an analogous manner to in vivo conditions. Such a tumor spheroid model built in a decellularized scaffold containing organ-specific vasculatures, which closely resembles the unique tumor microenvironment, holds the promise to efficiently assess various diagnostic and therapeutic strategies for endovascular therapies.

Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants.

Spheroids are a powerful tool for basic research and to reduce or replace in vivo (animal) studies but are not routinely banked nor shared. Here, we report the successful cryopreservation of hepatocyte spheroids using macromolecular (polyampholyte) cryoprotectants supplemented into dimethyl sulfoxide (DMSO) solutions. We demonstrate that a polyampholyte significantly increases post-thaw recovery, minimizes membrane damage related to cryo-injury, and remains in the extracellular space making it simple to remove post-thaw. In a model toxicology challenge, the thawed spheroids matched the performance of fresh spheroids. F-actin staining showed that DMSO-only cryopreserved samples had reduced actin polymerization, which the polyampholyte rescued, potentially linked to intracellular ice formation. This work may facilitate access to off-the-shelf and ready-to-use frozen spheroids, without the need for in-house culturing. Readily accessible 3-D cell models may also reduce the number of in vivo experiments.

Two Rac1 pools integrate the direction and coordination of collective cell migration.

Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

Compartmentalized PGRP expression along the dipteran Bactrocera dorsalis gut forms a zone of protection for symbiotic bacteria.

All metazoan guts are subject to opposing pressures wherein the immune system must eliminate pathogens while tolerating the presence of symbiotic microbiota. The Imd pathway is an essential defense against invading pathogens in insect guts, but tolerance mechanisms are less understood. Here, we find PGRP-LB and PGRP-SB express mainly in the anterior and middle midgut in a similar pattern to symbiotic Enterobacteriaceae bacteria along the Bactrocera dorsalis gut. Knockdown of PGRP-LB and PGRP-SB enhances the expression of antimicrobial peptide genes and reduces Enterobacteriaceae numbers while increasing abundance of opportunistic pathogens. Microbiota numbers recover to normal levels after the RNAi effect subsided. In contrast, high expression of PGRP-LC in the foregut allows increased antibacterial peptide production to efficiently filter the entry of pathogens, protecting the symbiotic bacteria. Our study describes a mechanism by which regional expression of PGRPs construct a protective zone for symbiotic microbiota while maintaining the ability to fight pathogens.

Shape memory micro-anchors with magnetic guidance for precision micro-vascular deployment.

Transcatheter medical micro-devices through circulatory system show great potential for therapy but lack strategies to stably anchor them at the desired site in vascularized tissues to take actions. Here a shape memory functionalized biodegradable magnetic micro-anchor (SM2A) is developed to achieve magnetic guided endovascular localization through precisely controlled shape transformation. The SM2A comprises anisotropic polylactide-based microparticle embedded with superparamagnetic Fe3O4 nanoparticles, exhibiting thermally activated tunable shape recovery modes at a body-friendly temperature range to accomplished an efficient endovascular anchoring effect in both decellularized liver organ and rabbit ear embolization models. The SM2A can be anchored at the target micro-vessel, exhibiting a controlled radial expansion of the vessel wall yielding with estimated stresses of 7-26 kPa in contact stress and 38-218 kPa in von Mises stress. The SM2A is a promising platform to incorporate diagnostic or therapeutic agents for precision deployment and in-situ action.

Shape-Anisotropic Microembolics Generated by Microfluidic Synthesis for Transarterial Embolization Treatment.

Particulate embolic agents with calibrated sizes, which employ interventional procedures to achieve endovascular embolization, have recently attracted tremendous interest in therapeutic embolotherapies for a wide plethora of diseases. However, the particulate shape effect, which may play a critical role in embolization performances, has been rarely investigated. Here, polyvinyl alcohol (PVA)-based shape-anisotropic microembolics are developed using a facile droplet-based microfluidic fabrication method via heat-accelerated PVA-glutaraldehyde crosslinking reaction at a mild temperature of 38 ° C. Precise geometrical controls of the microembolics are achieved with a nearly capsule shape through regulating surfactant concentration and flow rate ratio between dispersed phase and continuous phase in the microfluidics. Two specific models are employed, i.e., in vitro decellularized rabbit liver embolization model and in vivo rabbit ear embolization model, to systematically evaluate the embolization behaviors of the nonspherical microembolics. Compared to microspheres of the same volume, the elongated microembolics demonstrated advantageous endovascular navigation capability, penetration depth and embolization stability due to their comparatively smaller radial diameter and their central cylindrical part providing larger contact area with distal vessels. Such nonspherical microembolics present a promising platform to apply shape anisotropy to achieve distinctive therapeutic effects for endovascular treatments.

Gut fungal community and its probiotic effect on Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) is a destructive horticultural pest which causes considerable economic losses every year. A collection of microorganisms live within the B. dorsalis gut, and they are involved in its development, physiology, and behavior. However, knowledge regarding the composition and function of the gut mycobiota in B. dorsalis are still limited. Here, we comprehensively characterized the gut mycobiota in B. dorsalis across different developmental stages. High-throughput sequencing results showed a significant difference in fungal species abundance and diversity among different developmental stages of B. dorsalis. Quantitative polymerase chain reaction and culture-dependent methods showed that yeast species was the dominant group in the larval stage. We isolated 13 strains of yeast from the larval gut, and found that GF (germ-free) larvae mono-associated with strain Hanseniaspora uvarum developed faster than those mono-associated with other tested fungal strains. Supplementing the larval diet with H. uvarum fully rescued B. dorsalis development, shortened the larval developmental time, and increased adult wing lengths, as well as the body sizes and weights of both pupae and adults. Thus, our study highlights the close interactions between gut fungi, especially H. uvarum, and B. dorsalis. These findings can be applied to the sterile insect technique program to promote host development during mass insect rearing.

Generative Adversarial Training with Dual-Attention for Vascular Segmentation and Topological Analysis.

The vascular topology is of vital importance in building a chemotherapy model for the liver cancer in rats. And segmentation of vessels in the liver is an indispensable part of vessels' topological analysis. In this paper, we proposed and validated a novel pipeline for segmenting liver vessels and extracting their skeletons for topological analysis. We employed a dual-attention based U-Net trained in a generative adversarial network (GAN) fashion to obtain precise segmentations of vessels. For subsequent topological analysis, the vessels' skeletons are extracted and classified according to their lengths and bifurcation orders. Based on 40 samples with carefully-annotated ground truth labels, our experiments revealed consistent superiority in terms of both segmentation accuracy and topology correctness, demonstrating the robustness of the proposed pipeline.

Visualization and Evaluation of Chemoembolization on a 3D Decellularized Organ Scaffold.

Transarterial chemoembolization (TACE) has emerged as the mainstay treatment for patients suffering from unresectable intermediate hepatocellular carcinoma and also holds the potential to treat other types of hypervascular cancers such as renal cell carcinoma. However, an in vitro model for evaluating both embolic performance and drug-release kinetics of the TACE embolic agents is still lacking since the current models greatly simplified the in vivo vascular systems as well as the extracellular matrices (ECM) in the organs. Here, we developed a decellularized organ model with preserved ECM and vasculatures as well as a translucent appearance to investigate chemoembolization performances of a clinically widely used embolic agent, i.e., a doxorubicin-loaded ethiodised oil (EO)-based emulsion. We, for the first time, utilized an ex vivo model to evaluate the liquid-based embolic agent in two organs, i.e., liver and kidneys. We found that the EO-based emulsion with enhanced stability by incorporating an emulsifier, i.e., hydrogenated castor oil-40 (HCO), showed an enhanced occlusion level and presented sustained drug release in the ex vivo liver model, suggesting an advantageous therapeutic effect for TACE treatment of hepatocellular carcinoma. In contrast, we observed that drug-release burst happened when applying the same therapy in the kidney model even with the HCO emulsifier, which may be explained by the presence of the specific renal vasculature and calyceal systems, indicating an unfavorable effect in the renal tumor treatment. Such an ex vivo model presents a promising template for chemoembolization evaluation before in vivo experiments for the development of novel embolic agents.

Proposal for a novel elliptical ultrasonic aspirator and its fundamental performance in cartilage removal.

An ultrasonic aspirator with elliptical vibration on the end face is presented in this paper. The ultrasonic vibration relied on a specially designed Langevin transducer. The end face of the transducer was screwed into a surgical tool capable of changing amplitude, which requires resonance to provide sufficient vibration amplitude for cutting cartilage or other biological tissues. Based on the coupling of two vertical bending vibrations, the new design produced the elliptical ultrasonic vibration on the end face of the surgical tool, which differed from the axial vibration of a traditional ultrasonic aspirator. The design greatly increased the mechanical effect of the aspirator. A specially designed blade was employed to remove the cartilage tissue. A prototype device was designed to operate at 20 kHz with a displacement amplitude of 150 µm at 200 V. The measurements of the vibrational and impedance responses showed the mechanical and electrical characteristics of the aspirator. Cutting tests on mice costal cartilage were subsequently conducted. Histological analysis displayed a higher proportion of live cells in the region around the cut site, which exhibited a higher removal efficiency.

Comparative genomics of Klebsiella michiganensis BD177 and related members of Klebsiella sp. reveal the symbiotic relationship with Bactrocera dorsalis.

BACKGROUND: Bactrocera dorsalis is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary, and human health importance. Irradiation of pupae in SIT can reduce the ecological fitness of the sterile insects. Our previous study has shown that a gut bacterial strain BD177 that could restore ecological fitness by promoting host food intake and metabolic activities. RESULTS: Using long-read sequence technologies, we assembled the complete genome of K. michiganensis BD177 strain. The complete genome of K. michiganensis BD177 comprises one circular chromosome and four plasmids with a GC content of 55.03%. The pan-genome analysis was performed on 119 genomes (strain BD177 genome and 118 out of 128 published Klebsiella sp. genomes since ten were discarded). The pan-genome includes a total of 49305 gene clusters, a small number of 858 core genes, and a high number of accessory (10566) genes. Pan-genome and average nucleotide identity (ANI) analysis showed that BD177 is more similar to the type strain K. michiganensis DSM2544, while away from the type strain K. oxytoca ATCC13182. Comparative genome analysis with 21 K. oxytoca and 12 K. michiganensis strains, identified 213 unique genes, several of them related to amino acid metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism in BD177 genome. CONCLUSIONS: Phylogenomics analysis reclassified strain BD177 as a member of the species K. michiganensis. Comparative genome analysis suggested that K. michiganensis BD177 has the strain-specific ability to provide three essential amino acids (phenylalanine, tryptophan and methionine) and two vitamins B (folate and riboflavin) to B. dorsalis. The clear classification status of BD177 strain and identification of unique genetic characteristics may contribute to expanding our understanding of the symbiotic relationship of gut microbiota and B. dorsalis.

Decellularized liver as a translucent ex vivo model for vascular embolization evaluation.

Transarterial chemoembolization (TACE) is the preferred treatment for patients with unresectable intermediate stage hepatocellular carcinoma, however currently the development of embolic agents for TACE lacks in vitro models that closely represent the sophisticated features of the organ and the vascular systems therein. In this study, we presented a new strategy using an ex vivo liver model to provide a translucent template for evaluating embolic agents of TACE. The ex vivo liver model was developed through decellularizion of rat liver organs with preserved liver-specific vasculatures and improved transmittance of the whole liver up to 23% at 550 nm. Using this model, we investigated the embolization performances of both liquid and particle-based embolic agents, including penetration depth, embolization end-points, injection pressure and spatial distribution dynamics. We found that the embolization endpoint of liquid embolic agent such as ethiodised oil was strongly dependent on the injection pressure, and the pressure quickly built up when reaching the capillary endings, which could cause embolic agent leaking and potential tissue damages. In contrast, for particle-based embolic agents such as poly-dl-lactide microparticles and CalliSpheres® beads, their embolization endpoints were mainly determined by the particle size, whereas the particle densities close to the endpoints dramatically dropped down, which with the penetration depth represented two critical factors determining the embolic distribution. Such a decellularized organ model may open a new route to visually and quantitatively characterize embolization effects of various embolotherapies.

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.