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Despite decades of efforts, state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal. Current dressings adhere to the wound and necessitate debridement. This work describes the first “supramolecular hybrid hydrogel (SHH)” burn dressing that is biocompatible, self-healable, and on-demand dissoluble for easy and trauma-free removal, prepared by a simple, fast, and scalable method. These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution. The SHHs show high mechanical strength (>50 kPa), self-heal rapidly in ∼1 min, and dissolve quickly (4–6 min) using an amantadine hydrochloride (AH) solution that breaks the supramolecular interactions in the SHHs. Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro. The SHHs also do not elicit any significant cytokine response in vitro. Furthermore, in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls. Thus, these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

Objective Chronic skin wounds are one of the most devastating complications in diabetic patients due to the formation of advanced glycation end products (AGEs) resulting from non-enzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger pro-inflammatory signals that impair wound healing in diabetes and contribute to the pathology of chronic skin wounds. Approach We previously developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that acts as a competitive inhibitor of AGEs, and another ELP fusion protein containing stromal cell-derived factor 1 (SDF1) that promotes revascularization. Herein, we report the effects of protein coacervates incorporating both vRAGE-ELP and SDF1-ELP on wound healing in an in vitro diabetes-mimicking cell culture system, and in in vivo in full-thickness wounds on diabetic mice. Results The combination of vRAGE-ELP and SDF1-ELP increased cell metabolic activity in AGE-stimulated endothelial cells, promoted in vitro tube formation and accelerated healing in an in vitro cell migration assay. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure in the combination groups reached almost 100% on post-wounding day 35, compared to 62% and 85% on the same days in animals treated with fibrin gel control and vehicle control consisting of ELP alone. Innovation To our knowledge, this is the first study that attempts to reverse the AGE-RAGE-mediated signaling as well as to promote cell proliferation and vascularization in one single treatment. Conclusion The co-delivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.

We describe the design, generation, and in vitro evaluation of targeted amphipathic fusion peptides and immunoconjugates for the ablation of prostate cancer cells. The overexpression of the prostate-specific membrane antigen (PSMA) was exploited as means to specifically deliver cytotoxic peptides to prostate cancer cells. Cationic amphipathic lytic peptides were chosen as cytotoxic agents due to their ability to depolarize mitochondrial membranes and induce apoptosis. Specific delivery of the lytic peptide was facilitated by PSMA-targeting peptides and antibodies. Our results indicate that although the use of PSMA-targeted peptides only modestly enhanced the cytotoxic activity of the lytic peptide, peptide-antibody conjugates were two orders of magnitude more potent than untargeted peptide. In addition to quantifying the cytotoxic activities of the individual constructs, we also investigated the mechanisms of cell death induced by the fusion peptides and immunoconjugates. Although fusion peptides induced oncotic/necrotic death in cells, treatment with immunoconjugates resulted in apoptotic death. In summary, immunoconjugates based on lytic peptides are a promising class of therapeutics for prostate cancer therapy and warrant further investigation. [Cancer Res 2007;67(13):6368–75]

Supplementary Figures 1-2 from Amphipathic Peptide-Based Fusion Peptides and Immunoconjugates for the Targeted Ablation of Prostate Cancer Cells

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between −4 and −6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (−10 to −15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA dictates human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA targeting of FOXA1 and FOXA2 in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p < 0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < 0.05), all consistent with a partial/transient cell reprogramming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During hPSC-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate complex effects of FOXA1/2 on hepatic GRN effecting de-differentiation and metabolism with applications in studies of cancer, differentiation, and organogenesis.

Elastin-like peptides (ELPs) are a versatile platform for tissue engineering and drug delivery. In this study, micelle forming ELP chains are genetically fused to three therapeutic molecules, keratinocyte growth factor (KGF), stromal cell-derived growth factor 1 (SDF1), and cathelicidin (LL37), to be used in wound healing. Chronic wounds represent a growing problem worldwide. A combinatorial therapy approach targeting different aspects of wound healing would be beneficial, providing a controlled and sustained release of active molecules, while simultaneously protecting these therapeutics from the surrounding harsh wound environment. The results of this study demonstrate that the conjugation of the growth factors KGF and SDF1, and the antimicrobial peptide LL37 to ELPs does not affect the micelle structure and that all three therapeutic moieties retain their bioactivity in vitro. Importantly, when the combination of these micelle ELP nanoparticles are applied to wounds in diabetic mice, over 90% wound closure is observed, which is significantly higher than when the therapeutics are applied in their naked forms. The application of the nanoparticles designed in this study is the first report of targeting different aspect of wound healing synergistically.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Background: High-powered pulsed electric fields (PEF) may be used for tissue debridement and disinfection, while lower PEF intensities may stimulate beneficial cellular responses for wound healing. We investigated the dual effects of nonuniform PEF on cellular death and stimulation. Methods: Dermal fibroblast or keratinocyte monolayers were exposed to PEF induced by two needle electrodes (2 mm apart). Voltages (100–600 V; 1 Hz; 70 micros pulse width; 90 pulses/cycle) were applied between the two electrodes. Controls consisted of similar monolayers subjected to a scratch mechanical injury. Results: Cell growth and closure of the cell-free gap was faster in PEF-treated cell monolayers versus scratched ones. Media conditioned from cells pre-exposed to PEF, when applied to responder cells, stimulated greater proliferation than media from scratched monolayers. Conclusions: PEF treatment causes the release of soluble factors that promote cell growth, and thus may play a role in the accelerated healing of wounds post PEF.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

Medical robots have demonstrated the ability to manipulate percutaneous instruments into soft tissue anatomy while working beyond the limits of human perception and dexterity. Robotic technologies further offer the promise of autonomy in carrying out critical tasks with minimal supervision when resources are limited. Here, we present a portable robotic device capable of introducing needles and catheters into deformable tissues such as blood vessels to draw blood or deliver fluids autonomously. Robotic cannulation is driven by predictions from a series of deep convolutional neural networks that encode spatiotemporal information from multimodal image sequences to guide real-time servoing. We demonstrate, through imaging and robotic tracking studies in volunteers, the ability of the device to segment, classify, localize and track peripheral vessels in the presence of anatomical variability and motion. We then evaluate robotic performance in phantom and animal models of difficult vascular access and show that the device can improve success rates and procedure times compared to manual cannulations by trained operators, particularly in challenging physiological conditions. These results suggest the potential for autonomous systems to outperform humans on complex visuomotor tasks, and demonstrate a step in the translation of such capabilities into clinical use. Getting safe and fast access to blood vessels is vital to many methods of treatment and diagnosis in medicine. Robot-assisted or even fully autonomous methods can potentially do the task more reliably than humans, especially when veins are hard to detect. In this work, a method is tested that uses deep learning to find blood vessels and track the movement of a patient’s arm.

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.

Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases. Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

Objective: The incidence of severe infectious complications after burn injury increases mortality by 40%. However, traditional approaches for managing burn infections are not always effective. High-voltage, pulsed electric field (PEF) treatment shortly after a burn injury has demonstrated an antimicrobial effect in vivo; however, the working parameters and long-term effects of PEF treatment have not yet been investigated. Approach: Nine sets of PEF parameters were investigated to optimize the applied voltage, pulse duration, and frequency or pulse repetition for disinfection of Pseudomonas aeruginosa infection in a stable mouse burn wound model. The bacterial load after PEF administration was monitored for 3 days through bioluminescence imaging. Histological assessments and inflammation response analyses were performed at 1 and 24 h after the therapy. Results: Among all tested PEF parameters, the best disinfection efficacy of P. aeruginosa infection was achieved with a combination of 500 V, 100 μs, and 200 pulses delivered at 3 Hz through two plate electrodes positioned 1 mm apart for up to 3 days after the injury. Histological examinations revealed fewer inflammatory signs in PEF-treated wounds compared with untreated infected burns. Moreover, the expression levels of multiple inflammatory-related cytokines (interleukin [IL]-1α/β, IL-6, IL-10, leukemia inhibitory factor [LIF], and tumor necrosis factor-alpha [TNF-α]), chemokines (macrophage inflammatory protein [MIP]-1α/β and monocyte chemoattractant protein-1 [MCP-1]), and inflammation-related factors (vascular endothelial growth factor [VEGF], macrophage colony-stimulating factor [M-CSF], and granulocyte–macrophage colony-stimulating factor [G-CSF]) were significantly decreased in the infected burn wound after PEF treatment. Innovation: We showed that PEF treatment on infected wounds reduces the P. aeruginosa load and modulates inflammatory responses. Conclusion: The data presented in this study suggest that PEF treatment is a potent candidate for antimicrobial therapy for P. aeruginosa burn infections.

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, i.e. hypoxia, can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous micro-tissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3-6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability, in this platform, decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in reactive oxygen species (ROS) accumulation and hypoxiainducible factor-1α (HIF1α) expression was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions.

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at –6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at –4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood. Preservation of human livers at subzero temperatures with ice-free supercooling extends ex vivo organ life.

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin (Hb)-based oxygen carriers have shown potential in increasing oxygen delivery to aid wound healing. Macrophages also take up Hb, thus altering their phenotype and the regulation of inflammation. Herein, we compared the effect of Hb and polymerized Hbs (PolyHbs) on the phenotype of human macrophages. Macrophages were incubated with Hb or different forms of PolyHbs, and the inflammatory secretion profile was analyzed. PolyHbs were produced by polymerizing Hb in the relaxed (R) or tense (T) quaternary state and by varying the molar ratio of the glutaraldehyde crosslinking agent to Hb. Hb decreased the secretion of most measured factors. PolyHb treatment led to generally similar secretion profiles; however, Hb had more similar trends to R-state PolyHb. Ingenuity pathway analysis predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb prepared with a 30:1 (glutaraldehyde:Hb) polymerization ratio. When tested in diabetic mouse wounds, T-state PolyHb resulted in the greatest epidermal thickness and vascular endothelial CD31 staining. Thus, the effects of PolyHb on macrophages are affected by the polymerization ratio and the quaternary state, and T-state PolyHb yields secretion profiles that are most beneficial in wound healing.

Here we present a model to estimate the interaction free energy contribution of each amino acid residue of a given protein. Protein interaction energy is described in terms of per-residue interaction factors, [Formula: see text]. Multibody interactions are implicitly captured in [Formula: see text] through the combination of amino acid terms ([Formula: see text]) guided by local conformation indices ([Formula: see text]). The model enables construction of an interaction factor heat map for a protein in a given fold, allows prima facie assessment of the degree of residue–residue interaction, and facilitates a qualitative and quantitative evaluation of protein association properties. The model was used to compute thermal stability of T4 bacteriophage lysozyme mutants across seven sites. Qualitative assessment of mutational effects provides a straightforward rationale regarding whether a particular site primarily perturbs native or non-native states, or both. The presented model was found to be in good agreement with experimental mutational data ([Formula: see text]) and suggests an approach by which to convert structure space into energy space.

Irreversible electroporation of cell membrane with pulsed electric fields is an emerging physical method for disinfection that aims to reduce the doses and volumes of used antibiotics for wound healing. Here we report on the design of the IGBT-based pulsed electric field generator that enabled eradication of multidrug resistant Pseudomonas aeruginosa PAO1 on the gel. Using a concentric electric configuration we determined that the lower threshold of the electric field required to kill P. aeruginosa PAO1 was 89.28 ± 12.89 V mm−1, when 200 square pulses of 300 µs duration are delivered at 3 Hz. These parameters disinfected 38.14 ± 0.79 mm2 area around the single needle electrode. This study provides a step towards the design of equipment required for multidrug-resistant bacteria disinfection in patients with pulsed electric fields.

Donation after circulatory death (DCD) liver grafts are known to be predisposed to primary nonfunction and ischemic cholangiopathy. Many DCD grafts are discarded because of older donor age or long warm ischemia times. Thus, it is critical to improve the quality of DCD liver grafts. Here, we have tested whether an enriched oxygen carrier added to the preservation solution can prolong graft survival and reduce biliary damage.We assessed the adenosine triphosphate (ATP) content decay of mouse liver grafts after cold ischemia, warm ischemia, and combined warm+cold ischemia. In addition, we used a rat model of liver transplantation to compare survival of DCD grafts preserved in high-oxygen solution (preoxygenated perfluorocarbon [PFC] + University of Wisconsin [UW] solution) versus lower oxygen solution (preoxygenated UW solution).Adenosine triphosphate levels under UW preservation fall to less than 10% after 30 minutes of warm ischemia. Preoxygenated UW solution with PFC reached a significantly higher PaO2. After 45 minutes of warm ischemia in oxygenated UW + PFC solution, grafts showed 63% higher levels of ATP (P = 0.011). In addition, this was associated with better preservation of morphology when compared to grafts stored in standard UW solution. Animals that received DCD grafts preserved in higher oxygenation solution showed improved survival: 4 out of 6 animals survived long-term whereas all control group animals died within 24 hours.The additional oxygen provided by PFC during static cold preservation of DCD livers can better sustain ATP levels, and thereby reduce the severity of ischemic tissue damage. PFC-based preservation solution extends the tolerance to warm ischemia, and may reduce the rate of ischemic cholangiopathy.

Cancer is a devastating disease that takes the lives of hundreds of thousands of people every year. Due to disease heterogeneity, standard treatments, such as chemotherapy or radiation, are effective in only a subset of the patient population. Tumors can have different underlying genetic causes and may express different proteins in one patient versus another. This inherent variability of cancer lends itself to the growing field of precision and personalized medicine (PPM). There are many ongoing efforts to acquire PPM data in order to characterize molecular differences between tumors. Some PPM products are already available to link these differences to an effective drug. It is clear that PPM cancer treatments can result in immense patient benefits, and companies and regulatory agencies have begun to recognize this. However, broader changes to the healthcare and insurance systems must be addressed if PPM is to become part of standard cancer care.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) affect 25% of the world population. NAFLD is predicted to soon become the main cause of liver morbidity and transplantation. The disease is characterized by a progressive increase of lipid accumulation in hepatocytes, which eventually induce fibrosis and inflammation, and can ultimately cause cirrhosis and hepatic carcinoma. Here, we created a patterned model of NAFLD on a chip using free fatty acid gradients to recapitulate a spectrum of disease conditions in a single continuous liver tissue. We established the NAFLD progression via quantification of intracellular lipid accumulation and transcriptional levels of fatty acid transporters and NAFLD pathogenesis markers. We then used this platform to create oxygen driven steatosis zonation mimicking the sinusoidal lipid distribution on a single continuous tissue and showed that this fat zonation disappears under progressed steatosis, in agreement with in vivo observations and recent computational studies. While we focus on free fatty acids and oxygen as the drivers of NAFLD, the microfluidic platform here is extensible to simultaneous use of other drivers.

End-stage liver diseases lead to mortality of millions of patients, as the only treatment available is liver transplantation and donor scarcity means that patients have to wait long periods before receiving a new liver. In order to minimize donor organ scarcity, a promising bioengineering approach is to decellularize livers that do not qualify for transplantation. Through decellularization, these organs can be used as scaffolds for developing new functional organs. In this process, the original cells of the organ are removed and ideally should be replaced by patient-specific cells to eliminate the risk of immune rejection. Induced pluripotent stem cells (iPSCs) are ideal candidates for developing patient-specific organs, yet the maturity and functionality of iPSC-derived cells do not match those of primary cells. In this study, we introduced iPSCs into decellularized rat liver scaffolds prior to the start of differentiation into hepatic lineages to maximize the exposure of iPSCs to native liver matrices. Through exposure to the unique composition and native 3D organization of the liver microenvironment, as well as the more efficient perfusion culture throughout the differentiation process, iPSC differentiation into hepatocyte-like cells was enhanced. The resulting cells showed significantly higher expression of mature hepatocyte markers, including important CYP450 enzymes, along with lower expression of fetal markers, such as AFP. Importantly, the gene expression profile throughout the different stages of differentiation was more similar to native development. Our study shows that the native 3D liver microenvironment has a pivotal role to play in the development of human-origin hepatocyte-like cells with more mature characteristics.

Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. We developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates at around 30–31 °C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells, including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastase in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds.

Burn injury mediated hypermetabolic syndrome leads to increased mortality among severe burn victims, due to liver failure and muscle wasting. Metabolic changes may persist up to 2 years following the injury. Thus, understanding the underlying mechanisms of the pathology is crucially important to develop appropriate therapeutic approaches. We present detailed metabolomic and lipidomic analyses of the liver and muscle tissues in a rat model with a 30% body surface area burn injury located at the dorsal skin. Three hundred and thirty-eight of 1587 detected metabolites and lipids in the liver and 119 of 1504 in the muscle tissue exhibited statistically significant alterations. We observed excessive accumulation of triacylglycerols, decreased levels of S-adenosylmethionine, increased levels of glutamine and xenobiotics in the liver tissue. Additionally, the levels of gluconeogenesis, glycolysis, and tricarboxylic acid cycle metabolites are generally decreased in the liver. On the other hand, burn injury muscle tissue exhibits increased levels of acyl-carnitines, alpha-hydroxyisovalerate, ophthalmate, alpha-hydroxybutyrate, and decreased levels of reduced glutathione. The results of this preliminary study provide compelling observations that liver and muscle tissues undergo distinctly different changes during hypermetabolism, possibly reflecting liver–muscle crosstalk. The liver and muscle tissues might be exacerbating each other’s metabolic pathologies, via excessive utilization of certain metabolites produced by each other.

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a "defatting" drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation.

In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies.

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation.

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, is known to degrade the glycosaminoglycan (GAG) side chains of CSPGs and promote tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37°C under dilute conditions. To overcome this limitation, we report the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature. The copolymer designs, which are based on chain length and composition of the copolymers, were identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian Process Regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24 hrs at 37°C. We demonstrated significant improvements in REA in three iterations of active learning while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promoted residual ChABC activity near 30%, even after one week and notably outperformed other common stabilization methods for ChABC. Together, these results highlight a promising pathway towards sustained tissue regeneration.

The demand for skin rejuvenation procedures has progressively increased in the past decade. Additionally, clinical trials have shown that current therapies might cause downtime and side effects in patients including prolonged erythema, scarring, and dyspigmentation. The goal of this study was to explore the effect of partial irreversible electroporation (pIRE) with pulsed electric fields in aged skin rejuvenation as a novel, non-invasive skin resurfacing technique. In this study, we used an experimental model of aged rats. We showed that treatment with pIRE promoted keratinocyte proliferation and blood flow in aged rat skin. We also found significant evidence indicating that pIRE reformed the dermal extracellular matrix (ECM). Both the collagen protein and fibre density in aged skin increased after pIRE administration. Furthermore, using an image-processing algorithm, we found that the collagen fibre orientation in the histological sections did not change, indicating a lack of scar formation in the treated areas. The results showed that pIRE approach could effectively stimulate keratinocyte proliferation, ECM synthesis, and angiogenesis in an aged rat model.

Engineering of functional vascularized liver tissues holds great promise in addressing donor organ shortage for transplantation. Whole organ decellularization is a cell removal method that retains the native vascular structures of the organ such that it can be anastomosed with the recipient circulation after recellularization with healthy cells. However, a main hurdle to successful implantation of bioengineered organ is the inability to efficiently re-endothelialize the vasculature with a functional endothelium, resulting in blood clotting which is the primary cause of failure in early transplant studies. Here, we present an efficient approach for enhancing re-endothelialization of decellularized rat liver scaffolds by conjugating the REDV cell-binding domain to improve attachment of endothelial cells (EC) on vascular wall surfaces. In order to facilitate expression and purification of the peptide, REDV was fused with elastin-like peptide (ELP) that confers thermally triggered aggregation behavior to the fusion protein. After validating the adhesive properties of the REDV-ELP peptide, we covalently coupled REDV-ELP to the blood vasculature of decellularized rat livers and seeded EC using perfusion of the portal vein. We showed that REDV-ELP increased cell attachment, spreading and proliferation of EC within the construct resulting in uniform endothelial lining of the scaffold vasculature. We further observed that REDV-ELP conjugation dramatically reduced platelet adhesion and activation. Altogether, our results demonstrate that this method allowed functional re-endothelialization of liver scaffold and show great potential toward the generation of functional bioengineered liver for long-term transplantation. Statement of Significance There is a critical need for novel organ replacement therapies as the grafts for transplantation fall short of demand. Recent advances in tissue engineering, through the use of decellularized scaffolds, have opened the possibility that engineered grafts could be used as substitutes for donor livers. However, successful implantation has been challenged by the inability to create a functional vasculature. Our research study reports a new strategy to increase efficiency of endothelialization by increasing the affinity of the vascular matrix for endothelial cells. We functionalized decellularized liver scaffold using elastin-like peptides grafted with REDV cell binding domain. We showed that REDV-ELP conjugation improve endothelial cell attachment and proliferation within the scaffold, demonstrating the feasibility of re-endothelializing a whole liver vasculature using our technique.

Emerging data highlight the critical role for the innate immune system in the progression of nonalcoholic fatty liver disease (NAFLD). Connexin 32 (Cx32), the primary liver gap junction protein, is capable of modulating hepatic innate immune responses and has been studied in dietary animal models of steatohepatitis. In this work, we sought to determine the association of hepatic Cx32 with the stages of human NAFLD in a histologically characterized cohort of 362 patients with NAFLD. We also studied the hepatic expression of the genes and proteins known to interact with Cx32 (known as the connexome) in patients with NAFLD. Last, we used three independent dietary mouse models of nonalcoholic steatohepatitis to investigate the role of Cx32 in the development of steatohepatitis and fibrosis. In a univariate analysis, we found that Cx32 hepatic expression associates with each component of the NAFLD activity score and fibrosis severity. Multivariate analysis revealed that Cx32 expression most closely associated with the NAFLD activity score and fibrosis compared to known risk factors for the disease. Furthermore, by analyzing the connexome, we identified novel genes related to Cx32 that associate with NAFLD progression. Finally, we demonstrated that Cx32 deficiency protects against liver injury, inflammation, and fibrosis in three murine models of nonalcoholic steatohepatitis by limiting initial diet-induced hepatoxicity and subsequent increases in intestinal permeability. Conclusion: Hepatic expression of Cx32 strongly associates with steatohepatitis and fibrosis in patients with NAFLD. We also identify novel genes associated with NAFLD and suggest that Cx32 plays a role in promoting NAFLD development. (Hepatology Communications 2018;2:786-797).

Diagnostic blood testing is the most commonly performed clinical procedure in the world, and influences the majority of medical decisions made in hospital and laboratory settings. However, manual blood draw success rates are dependent on clinician skill and patient physiology, and results are generated almost exclusively in centralized labs from large-volume samples using labor-intensive analytical techniques. This paper presents a medical device that enables end-to-end blood testing by performing blood draws and providing diagnostic results in a fully automated fashion at the point-of-care. The system couples an image-guided venipuncture robot, developed to address the challenges of routine venous access, with a centrifuge-based blood analyzer to obtain quantitative measurements of hematology. We first demonstrate a white blood cell assay on the analyzer, using a blood mimicking fluid spiked with fluorescent microbeads, where the area of the packed bead layer is correlated with the bead concentration. Next we perform experiments to evaluate the pumping efficiency of the sample handling module. Finally, studies are conducted on the integrated device — from blood draw to analysis — using blood vessel phantoms to assess the accuracy and repeatability of the resulting white blood cell assay.

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes.NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD+ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

A novel engineering strategy to improve autoantibody detection with peptide fragments derived from the parent antigen is presented. The model system studied was the binding of the putative p53 TAD peptide antigen (residues 46–55) to its cognate anti-p53 antibody, ab28. Each engineered peptide contained the full decapeptide epitope and differed only in the flanking regions. Since minimal structural information was available to guide the design, a simple epitope:paratope binding model was applied. The Hidden Symmetry Model, which we recently reported, was used to guide peptide design and estimate per-residue contributions to interaction free energy as a function of added C- and N-terminal flanking peptides. Twenty-four peptide constructs were designed, synthesized, and assessed for binding affinity to ab28 by surface plasmon resonance, and a subset of these peptides were evaluated in a simulated immunoassay for limit of detection. Many peptides exhibited over 200-fold enhancements in binding affinity and improved limits of detection. The epitope was reevaluated and is proposed to be the undecapeptide corresponding to residues 45–55. HSymM calculated binding free energy and experimental data were found to be in good agreement (R2 > 0.75).

Growth factor therapy is a promising approach for chronic diabetic wounds, but strategies to efficiently and cost-effectively deliver active molecules to the highly proteolytic wound environment remain as major obstacles. Here, we re-engineered keratinocyte growth factor (KGF) and the cellular protective peptide ARA290 into a protein polymer suspension with the purpose of increasing their proteolytic resistance, thus their activity in vivo. KGF and ARA290 were fused with elastin-like peptide (ELP), a protein polymer derived from tropoelastin, that confers the ability to separate into a colloidal suspension of liquid-like coacervates. ELP fusion did not diminish peptides activities as demonstrated by ability of KGF-ELP to accelerate keratinocyte proliferation and migration, and ARA290-ELP to protect cells from apoptosis. We examined the healing effect of ARA290-ELP and KGF-ELP alone or in combination, in a full-thickness diabetic wound model. In this model, ARA290-ELP was found to accelerate healing, notably by increasing angiogenesis in the wound bed. We further showed that co-delivery of ARA290 and KGF, with the 1:4 KGF-ELP to ARA290-ELP ratio, was the most effective wound treatment with the fastest healing rate, the thicker granulation tissue and regenerated epidermis after 28 days. Overall, this study shows that ARA290-ELP and KGF-ELP constitute promising new therapeutics for treatment of chronic wounds.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (108–109 cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

The obesity epidemic continues to escalate each year in the United States more than anywhere else in the world. The existing pharmaceutical and other nonsurgical treatments for morbid obesity produce suboptimal physiologic outcomes compared with those of Roux-en-Y gastric bypass (RYGB) surgery. RYGB has been the gold standard of bariatric surgery because the beneficial long-term outcomes, which include sustainable weight loss and type 2 diabetes mellitus (T2DM) resolution, are far superior to those obtained with other bariatric surgeries. However, the current understanding of RYGB's mechanisms of actions remains limited and incomplete. There is an urgent need to understand these mechanisms as gaining this knowledge may lead to the development of innovative and less invasive procedures and/or medical devices, which can mirror the favorable outcomes of RYGB surgery. In this review, we highlight current observations of the metabolic and physiologic events following RYGB, with a particular focus on the role of the anatomical reconfiguration of the gastrointestinal tract after RYGB.

Skin scar formation is a complex process that results in the formation of dense extracellular matrix (ECM) without normal skin appendages such as hair and glands. The absence of a scarless healing model in adult mammals prevents the development of successful therapies. We show that irreversible electroporation of skin drives its regeneration with all accessory organs in normal adult rats. Pulsed electric fields at 500 V, with 70 μs pulse duration and 1000 pulses delivered at 3 Hz, applied through two electrodes separated by 2 mm lead to massive cell death. However, the ECM architecture of the skin was preserved. Six months after the ablation, the epidermis, sebaceous glands, panniculus carnosus, hair follicles, microvasculature and arrector pili muscle were altogether re-formed in the entire ablated area. These results suggest a key role of the ECM architecture in the differentiation, migration and signalling of cells during scarless wound healing. Copyright © 2016 John Wiley & Sons, Ltd.

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that "defatting" may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4-6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of L-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of L-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.

Hydrogel microcapsules provide miniaturized and biocompatible niches for three-dimensional (3D) in vitro cell culture. They can be easily generated by droplet-based microfluidics with tunable size, morphology, and biochemical properties. Therefore, microfluidic generation and manipulation of cell-laden microcapsules can be used for 3D cell culture to mimic the in vivo environment towards applications in tissue engineering and high throughput drug screening. In this review of recent advances mainly since 2010, we will first introduce general characteristics of droplet-based microfluidic devices for cell encapsulation with an emphasis on the fluid dynamics of droplet breakup and internal mixing as they directly influence microcapsule’s size and structure. We will then discuss two on-chip manipulation strategies: sorting and extraction from oil into aqueous phase, which can be integrated into droplet-based microfluidics and significantly improve the qualities of cell-laden hydrogel microcapsules. Finally, we will review various applications of hydrogel microencapsulation for 3D in vitro culture on cell growth and proliferation, stem cell differentiation, tissue development, and co-culture of different types of cells.

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents.

The three classical core technologies for the preservation of live mammalian biospecimens—slow freezing, vitrification and hypothermic storage—limit the biomedical applications of biospecimens. In this Review, we summarize the principles and procedures of these three technologies, highlight how their limitations are being addressed via the combination of microfabrication and nanofabrication, materials science and thermal-fluid engineering and discuss the remaining challenges. This Review examines classical methods for the preservation of mammalian biospecimens and the associated mechanisms of cryoinjury and discusses how the methods’ applicability limitations are being addressed.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

Cell preservation is an enabling technology for widespread distribution and applications of mammalian cells. Traditional cryopreservation via slow-freezing or vitrification provides long-term storage but requires cytotoxic cryoprotectants (CPA) and tedious CPA loading/unloading, cooling, and recovering procedures. Hypothermic storage around 0–4 °C is an alternative method but only works for a short period due to its high storage temperatures. Here, we report on the deep-supercooling (DSC) preservation of human adipose-derived stem cells at deep subzero temperatures without freezing for extended storage. Enabled by surface sealing with an immiscible oil phase, cell suspension can be preserved in a liquid state at −13 °C and −16 °C for 7 days with high cell viability, retention of stemness, attachment, and multilineage differentiation capacities. These results demonstrate that DSC is an improved short-term preservation approach to provide off-the-shelf cell sources for booming cell-based medicine and bioengineering.

Improving organ preservation and extending the preservation time would have game-changing effects on the current practice of organ transplantation. Machine perfusion has emerged as an improved preservation technology to expand the donor pool, assess graft viability and ensure adequate graft function. However, its efficacy in extending the preservation time is limited. Subzero organ preservation does hold the promise to significantly extend the preservation time and recent advances in cryobiology bring it closer to clinical translation. In this review, we aim to broaden the perspective in the field from a focus on these individual technologies to that of a systems engineering. This would enable the creation of a preservation process that integrates the benefits of machine perfusion with those of subzero preservation, with the ultimate goal to provide on demand availability of donor organs through organ banking.