Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts.

Static cold storage (SCS), the current clinical gold standard for organ preservation, provides surgeons with a limited window of time between procurement and transplantation. In vascularized composite allotransplantation (VCA), this time limitation prevents many viable allografts from being designated to the best-matched recipients. Machine perfusion (MP) systems hold significant promise for extending and improving organ preservation. Most of the prior MP systems for VCA have been built and tested for large animal models. However, small animal models are beneficial for high-throughput biomolecular investigations. This study describes the design and development of a multiparametric bioreactor with a circuit customized to perfuse rat abdominal wall VCAs. To demonstrate its concept and functionality, this bioreactor system was employed in a small-scale demonstrative study in which biomolecular metrics pertaining to graft viability were evaluated non-invasively and in real time. We additionally report a low incidence of cell death from ischemic necrosis as well as minimal interstitial edema in machine perfused grafts. After up to 12 h of continuous perfusion, grafts were shown to survive transplantation and reperfusion, successfully integrating with recipient tissues and vasculature. Our multiparametric bioreactor system for rat abdominal wall VCA provides an advanced framework to test novel techniques to enhance normothermic and sub-normothermic VCA preservations in small animal models.

Predicting mini-tablet dissolution performance utilizing X-ray computed tomography.

Mini-tablets (MTs) have been utilized as an alternative to monolithic tablets due to their ease of use for pediatric populations, dose flexibility and tailoring of drug release profiles. Similar to monolithic tablets, MTs can develop film coat and internal core defects during manufacturing processes that may adversely affect their dissolution performance. The use of x-ray computed microtomography (XRCT) is well documented for monolithic tablets as a means of identifying internal defects, but applications to MTs have not been well studied. In this study, we have developed a workflow that analyzes reconstructed XRCT images of enteric-coated mini-tablets using deep learning convolutional neural networks. This algorithm was utilized to extract key physical features of individual MTs, such as micro-crack volume and enteric coat thickness. By performing dissolution studies on individual MTs, correlations were established based on the physical parameters obtained by XRCT and the dissolution performance, enabling prediction of dissolution performance utilizing non-destructive imaging data. This workflow provides insight into the physical variability of MT populations that are generated during manufacturing, enabling optimization of critical tableting and coating parameters to achieve the target dissolution criteria. Through this mechanistic understanding, quality is built into the final drug product through rational development of formulation and process parameters.

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Investigation of inter- and intra-rater reliability using digital dental software for prosthodontics crown preparations.

INTRODUCTION: Since the introduction of the digital dental assessment systems, there has been a shift towards this trend as it appears to provide a more accurate, reliable, time efficient, and reproducible assessment. The Planmeca Emerald scanner coupled with Romexis Compare software allows students and staff to objectively assess individual crown preparations, receive numerical values of key dimensions, and subsequently undergo comparison with ideal crown preparation dimensions. OBJECTIVES: To measure the inter- and intra-rater reliability using the intra-oral scanner and Romexis Compare for prosthodontic crown preparations, and to evaluate the possible implementation of this software as a grading and self-assessment tool in a preclinical setting. METHODS: The Planmeca scanner and Romexis Compare were used to compare the difference between 30 experimental preparations (n = 15 anterior teeth, n = 15 posterior teeth) with their respective unprepared typodont teeth. Three student examiners (W., K., V.) independently scanned the typodont teeth in pre-formed standardized and non-standardized putty jigs. Each preparation was measured from facial, lingual, incisal/occlusal, and margin surfaces. A second trial was completed after 2 weeks to assess intra-rater reliability. The data were tabulated, graphed, and analyzed using SPSS and GraphPad Prism. RESULTS: The results of the study show greater consistencies in inter-operator measurements for anterior teeth. Some variations, however, were found in posterior teeth measurements between the operators. The results of the intra-rater measurements appear to be relatively consistent. CONCLUSION: With some limitations, Romexis Compare can be used as a reliable and repeatable method for objective and consistent evaluation of student prosthodontic preparations in a preclinical setting.

Myoblast maturity on aligned microfiber bundles at the onset of strain application impacts myogenic outcomes.

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5-7 rather than Days 3-7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. STATEMENT OF SIGNIFICANCE: This is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.

Advances in Perfusion Systems for Solid Organ Preservation.

In the past, a diagnosis of organ failure would essentially be a death sentence for patients. With improved techniques for organ procurement and surgical procedures, transplantations to treat organ failure have become standard medical practice. However, while the demand for organs has skyrocketed, the donor pool has not kept pace leading to long recipient waiting lists. Organ preservation provides a means to increase the number of available transplantable organs. However, there are significant drawbacks associated with cold storage, the current gold standard. To address the short-comings due to diffusional limitations, engineers have developed cold perfusion systems. More recently, there has been a significant trend towards the development of near-normothermic systems to enhance the functional preservation of solid organs including livers, lungs, hearts, kidneys, and vascularized composite allotransplants. Here we review recent advances in the development of perfusion systems for the preservation of solid organs. We provide a brief history of organ transplantation, the limitations of existing systems, and describe research being done to develop commercially available perfusion systems to enhance organ preservation.

Single-sperm sequencing reveals the accelerated mitochondrial mutation rate in male Daphnia pulex (Crustacea, Cladocera).

Mutation rate in the nuclear genome differs between sexes, with males contributing more mutations than females to their offspring. The male-biased mutation rates in the nuclear genome is most likely to be driven by a higher number of cell divisions in spermatogenesis than in oogenesis, generating more opportunities for DNA replication errors. However, it remains unknown whether male-biased mutation rates are present in mitochondrial DNA (mtDNA). Although mtDNA is maternally inherited and male mtDNA mutation typically does not contribute to genetic variation in offspring, male mtDNA mutations are critical for male reproductive health. In this study, we measured male mtDNA mutation rate using publicly available whole-genome sequences of single sperm of the freshwater microcrustacean Daphnia pulex Using a stringent mutation detection pipeline, we found that the male mtDNA mutation rate is 3.32 × 10-6 per site per generation. All the detected mutations are heteroplasmic base substitutions, with 57% of mutations converting G/C to A/T nucleotides. Consistent with the male-biased mutation in the nuclear genome, the male mtDNA mutation rate in D. pulex is approximately 20 times higher than the female rate per generation. We propose that the elevated mutation rate per generation in male mtDNA is consistent with an increased number of cell divisions during male gametogenesis.

Imaging and Clinical Characteristics Predict Near-Term Disablement From Bone Metastases: Implications for Rehabilitation.

OBJECTIVE: To distinguish which patients with bone metastases are at risk for near-term disablement in order to assist clinicians in assessing the appropriateness of referrals for rehabilitation services. DESIGN: Prospective cohort study. SETTING: National Cancer Institute-designated comprehensive cancer center imbedded in a tertiary medical center. PARTICIPANTS: Data were collected from members (n=78) of a patient cohort (N=311) with stage IIIB or IV non-small-cell lung cancer or extensive-stage small-cell lung cancer who developed new or progressive imaging-confirmed bone metastases during the 2-year course of the study. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Functional capabilities were assessed at 3- to 4-week intervals over the study's 2-year duration with the Activity Measure for Post-Acute Care Computer Adaptive Testing. RESULTS: Seventy-eight participants developed new or progressive bone metastases during the study. Most were men, and 83% had non-small-cell lung cancer. Metastases were most frequently located in the ribs (n=62), pelvis (n=49), or the thoracic (n=60) and lumbar spine (n=44). While neither the number of bone metastases nor their specific location was associated with near-term changes in patient mobility, their association with pain or a focal neurologic deficit was strongly associated with large declines in mobility. Similarly, patients whose imaging studies revealed new metastases and the expansion of established metastases were more likely to lose mobility. CONCLUSIONS: The total burden, specific locations, and overall distribution of bone metastases did not predict disablement. Patients with lung cancer-associated bone metastases are at markedly increased risk for declining mobility when their metastases are expanding in size and increasing in number, or are associated with pain or with new neurologic deficits.

Controlled Endolysosomal Release of Agents by pH-responsive Polymer Blend Particles.

PURPOSE: A key step of delivering extracellular agents to its intracellular target is to escape from endosomal/lysosomal compartments, while minimizing the release of digestive enzymes that may compromise cellular functions. In this study, we examined the intracellular distribution of both fluorecent cargoes and enzymes by a particle delivery platform made from the controlled blending of poly(lactic-co-glycolic acid) (PLGA) and a random pH-sensitive copolymer. METHODS: We utilized both microscopic and biochemical methods to semi-quantitatively assess how the composition of blend particles affects the level of endosomal escape of cargos of various sizes and enzymes into the cytosolic space. RESULTS: We demonstrated that these polymeric particles enabled the controlled delivery of cargos into the cytosolic space that was more dependent on the cargo size and less on the composition of blend particles. Blend particles did not induce the rupture of endosomal/lysosomal compartments and released less than 20% of endosomal/lysosomal enzymes. CONCLUSIONS: This study provides insight into understanding the efficacy and safety of a delivery system for intracellular delivery of biologics and drugs. Blend particles offer a potential platform to target intracellular compartments while potentially minimizing cellular toxicity.

Symptom Burden and Functional Gains in a Cancer Rehabilitation Unit.

BACKGROUND/AIMS: To determine if there is a relationship between patient symptoms and functional improvement on inpatient rehabilitation. METHODS: Retrospective review of medical records at an American tertiary referral-based cancer center of all patients admitted to an inpatient rehabilitation unit between 3/1/2013-5/20/2013. Main outcome measures included the Edmonton Symptom and Assessment Scale (ESAS) and Functional Independence Measure (FIM). FINDINGS: The medical records for 71 unique cancer rehabilitation inpatients were analyzed. Statistical analysis of total admission ESAS on total FIM change found no significant relationships. The symptom burden of the patients was mild. Patients demonstrated statistically significant improvements in function and symptoms during inpatient rehabilitation. The mean change in total FIM and total ESAS were an increase of 19.20 and decrease of 7.41 respectively. Statistically significant changes occurred in fatigue, sleep, pain, and anxiety. CONCLUSION: Both symptom and functional scores improved significantly during inpatient rehabilitation. However, no significant relationships were found between symptoms at admission and improvement in FIM.

Polymer blend particles with defined compositions for targeting antigen to both class I and II antigen presentation pathways.

Defense against many persistent and difficult-to-treat diseases requires a combination of humoral, CD4(+) , and CD8(+) T-cell responses, which necessitates targeting antigens to both class I and II antigen presentation pathways. In this study, polymer blend particles are developed by mixing two functionally unique polymers, poly(lactide-co-glycolide) (PLGA) and a pH-responsive polymer, poly(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate) (DMAEMA-co-PAA-co-BMA). Polymer blend particles are shown to enable the delivery of antigens into both class I and II antigen presentation pathways in vitro. Increasing the ratio of the pH-responsive polymer in blend particles increases the degree of class I antigen presentation, while maintaining high levels of class II antigen presentation. In a mouse model, it is demonstrated that a significantly higher and sustained level of CD4(+) and CD8(+) T-cell responses, and comparable antibody responses, are elicited with polymer blend particles than PLGA particles and a conventional vaccine, Alum. The polymer blend particles offer a potential vaccine delivery platform to generate a combination of humoral and cell-mediated immune responses that insure robust and long-lasting immunity against many infectious diseases and cancers.

Comparison of efficacy of pulverization and sterile paper point techniques for sampling root canals.

INTRODUCTION: The purpose of this study was to compare the efficacy of the pulverization and sterile paper point techniques for sampling root canals using 5.25% NaOCl/17% EDTA and 1.3% NaOCl/MTAD (Dentsply, Tulsa, OK) as irrigation regimens. METHODS: Single-canal extracted human teeth were decoronated and infected with Enterococcus faecalis. Roots were randomly assigned to 2 irrigation regimens: group A with 5.25% NaOCl/17% EDTA (n = 30) and group B with 1.3% NaOCl/MTAD (n = 30). After chemomechanical debridement, bacterial samplings were taken using sterile paper points and pulverized powder of the apical 5 mm root ends. RESULTS: The sterile paper point technique did not show growth in any samples. The pulverization technique showed growth in 24 of the 60 samples. The Fisher exact test showed significant differences between sampling techniques (P < .001). The sterile paper point technique showed no difference between irrigation regimens. However, 17 of the 30 roots in group A and 7 of the 30 roots in group B resulted in growth as detected by pulverization technique. Data showed a significant difference between irrigation regimens (P = .03) in pulverization technique. CONCLUSIONS: The pulverization technique was more efficacious in detecting viable bacteria. Furthermore, this technique showed that 1.3% NaOCl/MTAD regimen was more effective in disinfecting root canals.

Selectively targeting the toll-like receptor 9 (TLR9)--IRF 7 signaling pathway by polymer blend particles.

Signaling through toll-like receptor 9 (TLR9) has been exploited for cancer therapy. The stimulation of TLR9 leads to two bifurcating signaling pathways - NF-κB-dependent pro-inflammatory cytokines pathway and IRF-7-dependent type I interferons (IFNs) pathway. In this study, we employ polymer blend particles to present the synthetic ligand, CpG oligonucleotides (CpG ODNs), to TLR9. The polymer blend particles are made from the blend of pH-insensitive and pH-sensitive copolymer. By tailoring the composition of the pH-sensitive polymer, CpG ODNs are presented to TLR9 in a way that only activates the IRF-7 signaling pathway. CpG ODNs have been used for cancer therapy in both preclinical and clinical studies. The selective activation of IRF-7 could potentially enhance the apoptosis of tumor cells and immunological control of tumor progression without inadvertently activating NF-κB-dependent oncogenesis.

Effect of the poly(ethylene glycol) (PEG) density on the access and uptake of particles by antigen-presenting cells (APCs) after subcutaneous administration.

Lymphatic trafficking of particles to the secondary lymphoid organs, such as lymph nodes, and the cell types that particles access are critical factors that control the quality and quantity of immune responses. In this study, we evaluated the effect of PEGylation on the lymphatic trafficking and accumulation of particles in draining lymph nodes (dLNs) as well as the cell types that internalized particles. As a model system, 200 nm polystyrene (PS) particles were modified with different densities of poly(ethylene glycol) (PEG) and administered subcutaneously to mice. PEGylation enhanced the efficiency of particle drainage away from the injection site as well as the access of particles to dendritic cells (DCs). The accumulation of particles in dLNs was dependent on the PEG density. PEGylation also enhanced uptake by DCs while reducing internalization by B cells at the single cell level. Our results indicate that PEGylation facilitated the trafficking of particles to dLNs either through enhanced trafficking in lymphatic vessels or by enhanced internalization by migratory DCs. This study provides insight into utilizing PEGylated particles for the development of synthetic vaccines.

Selective local delivery of RANK siRNA to bone phagocytes using bone augmentation biomaterials.

Fracture healing and fracture fixation in the context of osteoporosis is extremely difficult. To inhibit osteoclast-induced bone resorption and associated implant loosening in this pathology, we describe a local delivery strategy to delivery RNA interfering technology to bone sites to target and down-regulate osteoclast formation and function. Resorbable polymer, poly(lactic-co-glycolic acid) (PLGA) microparticles were exploited as a passive phagocyte-targeting carrier to deliver RANK siRNA to both osteoclast precursors and osteoclasts - the professional phagocytes in bone. These natural phagocytes internalize micron-sized particles while most other non-targeted cells in bone cannot. PLGA-siRNA microparticles were dispersed within biomedical grade calcium-based injectable bone cement clinically used in osteoporosis as a bone augmentation biomaterial for fragility fracture prevention and fixation. siRNA released from this formulation in vitro retains bioactivity against the cell target, RANK, in cultured osteoclast precursor cells, inhibiting their progression toward the osteoclastic phenotype. These data support the proof-of-concept to utilize a clinically relevant approach to locally deliver siRNA to phagocytes in bone and improve fragility fracture healing in the context of osteoporosis. This local delivery system delivers siRNA therapeutics directly to osteoporosis sites from clinically familiar injected bone augmentation materials but could be extended to other injectable biomaterials for local siRNA delivery.

Activation of inflammasomes by tumor cell death mediated by gold nanoshells.

Gold nanoshell-enabled photothermal therapy (NEPTT) utilizes the efficient thermal conversion of near infrared (NIR) light for the ablation of cancer cells. Cancer therapies that combine cell killing with the induction of a strong immune response against the dying tumor cells have been shown to increase therapeutic efficacy in the clearance and regression of cancers. In this study, we assessed the ability of dying cells generated by in vitro NEPTT to activate inflammasome complexes. We quantified levels of major danger-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uric acid, released from tumor cells treated by NEPTT. The amount of DAMPs released was dependent on the dose of nanoshells internalized by cells. However, under all the employed conditions, the levels of generated DAMPs were insufficient to activate inflammasome complexes and to induce the production of pro-inflammatory cytokines (i.e. IL-1ß). The results from this study provide insights into the development of nanoplasmonics for combining both photothermal therapy and immunotherapy to eradicate cancers.

Effects of particle size on toll-like receptor 9-mediated cytokine profiles.

Biomaterials interface with toll-like receptor (TLR) 9-mediated innate immunity in a wide range of medical applications, such as tissue implants and drug delivery systems. The stimulation of TLR9 can lead to two different signaling pathways, resulting in the generation of proinflammatory cytokines (i.e. IL-6) and/or type I interferons (IFNs, i.e. IFN-α). These two categories of cytokines differentially influence both innate and adaptive immunity. Although particle size is known to be a critical parameter of biomaterials, its role in TLR9-mediated cytokine profiles is not clear. Here, we examined how the size of biomaterials impacted cytokine profiles by using polystyrene particles of defined sizes as model carriers for TLR9 agonists (CpG oligonucleotides (CpG ODNs)). CpG ODNs bound to nano- to submicro- particles stimulated the production of both IL-6 and IFN-α, while those bound to micro particles resulted in IL-6 secretions only. The differential TLR9-mediated cytokine profiles were attributed to the pH of endosomes that particles trafficked to. The magnitude of IFN-α production was highly sensitive to the change in endosomal pH in comparison to that of IL-6. Our results define two critical design variables, size and the ability to modulate endosomal pH, for the engineering of biomaterials that potentially interface with TLR9-mediated innate immunity. The fine control of these two variables will allow us to fully exploit the beneficial facets of TLR9-mediated innate immunity while minimizing undesirable side effects.

Enabling customization of non-viral gene delivery systems for individual cell types by surface-induced mineralization.

Delivering genes to mediate functions of cells is a crucial technology for both basic science and clinical applications. Though numerous non-viral gene delivery systems have been developed, the diversity of mammalian cells poses a great challenge to the material design. Here, we demonstrate that surface-induced mineralization represents a promising approach to systematically customize DNA delivery with respect to the characteristics of cells. We initially examined gene transfer in nine cell types derived from different tissues and organisms by surface-induced DNA-doped calcium carbonate nanocomposites derived from a library of mineral solutions. Subsequently, we correlated gene transfer efficiency with cellular uptake, pH responsiveness of nanocomposites, and phagosomal pH of individual cell types. Based on the correlation, we were able to optimize the DNA delivery to the cell types of interest. Surface-induced mineralization possesses great potential for customizing gene transfer in realizing gene- and cell-based therapy and probing functions of genes.

A microfluidic manipulator for enrichment and alignment of moving cells and particles.

Grooved structures have been widely studied in particle separation and fluid mixing in microfluidic channel systems. In this brief report, we demonstrate the use of patterning flows produced by two different sorts of grooved surfaces: single slanted groove series (for enrichment patterns) and V-shaped groove series (for focusing patterns), into a microfluidic device to continuously manipulate the flowing particles, including microbeads with 6 microm, 10 microm, and 20 microm in diameter and mouse dendritic cells of comparable sizes to the depth of the channel. The device with grooved channels was developed and fabricated by soft-lithographic techniques. The particle distributions after passing through the single slanted grooves illustrate the size-dependent enrichment profiles. On the other hand, particles passing through the V-shaped grooves show focusing patterns downstream, for the combination effect from both sides of single slanted grooves setup side-by-side. Compared with devices utilizing sheath flows, the focusing patterns generated in this report are unique without introducing additional flow control. The alignment of the concentrated particles is expected to facilitate the visualization of sizing and counting in cell-based devices. On the other hand, the size-dependent patterns of particle distributions have the potential for the application of size-based separation.

Detecting and differentiating microbes by dendritic cells for the development of cell-based biosensors.

Dendritic cells (DCs) are a specialized family of antigen presenting cells. They play critical roles in sensing and processing microbial information through a series of pattern recognition receptors (PPRs), including the well-characterized toll-like receptors (TLRs). In this study, we demonstrated the utilization of a DC cell line, DC2.4, as a cell source for the detection and differentiation of microbes towards the development of cell-based biosensors. As a proof of principle, the gram-negative bacteria Escherichia coli K12 strain D21 and its lipopolysaccharide (LPS) mutants were used as model targets. The stimulation of DCs by bacterial strains was monitored by the production of nitric oxide (NO), and the colorimetric Greiss assay was used to quantify the level of NO produced. Our results demonstrated that DCs could detect and differentiate microbes with subtle differences in the composition of specific cell surface components, i.e. LPS, within minutes. Though the current colorimetric-based NO assay limited the detection sensitivity, we showed that DCs were able to detect as low as 2-3 bacteria per cell. Furthermore, compared to macrophages, DCs were superior in discriminating LPS mutants. Our study demonstrates that DCs possess great potential as cell sources for the development of novel cell-based biosensors for detecting microbes with high selectivity and sensitivity and rapid responsiveness. In addition, when DCs are coupled with other biosensor platforms, higher sensitivity can be expected.