Cell Suspension Research Articles

Iodate reduction by marine aerobic bacteria.

Iodate reductase (Idr) gene cluster (idrABP1P2 ) is involved in bacterial iodate (IO3 -) respiration under anaerobic conditions. Putative idr gene clusters are present in both anaerobic and aerobic bacteria; however, the specific physiological roles of idr genes in aerobic bacteria remain unclear. Therefore, in this study, three marine aerobic bacteria with putative idr gene clusters (Roseovarius azorensis, Notoacmeibacter marinus, and Aliiroseovarius sediminilitoris) were grown in the presence of iodate to determine whether they can reduce iodate to iodide (I-). All tested bacteria almost completely reduced 2 mM iodate under static conditions but only reduced 0.1-0.5 mM iodate under shaking conditions. Moreover, the washed cell suspension of R. azorensis reduced iodate only when the cells were pre-grown statically in the presence of iodate. Transcriptional analysis revealed that the expression levels of idrA, idrB, idrP1 , and idrP2 genes were upregulated in R. azorensis when the cells were grown statically in the presence of iodate. Specifically, idrA expression was induced by 0.1 μM iodate and was up to 14-fold higher compared to that of the non-iodate control. These results suggest that marine aerobic bacteria reduce iodate under oxygen-limited conditions, and that this capacity is induced by environmentally relevant levels of iodate in seawater. Our results suggest that marine aerobic bacteria contribute to iodide production in marine surface waters, thereby affecting the global iodine cycling and ozone budget.

Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array.

Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues? We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed. Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation. When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models. Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis. C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest. N/A.

Abstract A030: Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in treatment due to its complex interaction with the tumour microenvironment (TME). Fibroblasts, a crucial component of the TME, influence tumour progression and therapeutic responses. This study explores the diversity of cancer-associated fibroblast (CAF) populations in PDAC tumours versus adjacent normal fibroblasts (ANFs), focussing on myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs). These fibroblast subtypes exhibit distinct characteristics and localisation within the tumour. The research examines the impact of CAFs and ANFs on immune cell behaviour, particularly in relation to adhesion and migration. Methods: Tumour and juxta-tumoral tissue from patients were digested into single cell suspensions. Cells were stained with a 22-plex antibody panel by flow cytometry. Pseudo-emperipolesis assays were assessed by co-culture of lymphocytes with CAF and ANF fibroblasts. Furthermore, these were performed with or without stimulation of fibroblasts with by TGF-b or TNF-a/IFN-g, to promote myCAF or iCAF phenotypes respectively. Gene expression analysis of tumour-derived fibroblast subtypes was assessed by single-cell RNA sequencing using the 10X Genomics platform. The transcriptional profile of spatially resolved fibroblasts which were proximal or distant to tumour epithelium was determined by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Results: Flow cytometry analysis revealed that iCAFs predominated within the tumour stroma compared to myCAFs, although iCAF populations were less prevalent within ANF populations. Upon stimulation with inflammatory cytokines (TNF-α/IFN-γ), CAFs promoted lymphocyte migration in contrast to ANFs. However, TGF-β stimulation of CAF downregulated the migration of lymphocytes. As such, TGF-β or TNF-α/IFN-γ stimulation exhibited antagonistic effects on lymphocyte adhesion and migration in CAFs. Tumour-proximal fibroblasts exhibited myCAFs characteristics expressing α-SMA and PDPN, as well as ECM molecules such as collagen and integrins, suggesting a barrier to lymphocyte migration. Conversely, distal fibroblasts displayed an inflammatory profile, expressing cytokines and chemokines including CCL2, CXCL9, CXCL12, IL6, IL11 and LIF, thereby contributing to the inflammatory milieu within the tumour microenvironment. Additionally, iCAFs demonstrated higher transcription levels of hyaluronan synthase (Has1) and fibulin 2 (FBLN2) which are crucial for ECM formation and remodelling. This study identifies a differential role for iCAFs and myCAFs in the modulate of lymphocyte adhesion and migration within the PDAC microenvironment, highlighting the complex interplay between fibroblast subtypes and immune cell dynamics. Citation Format: Fouzia Zayou, Hayden Pearce, Wayne Croft, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A030.

The elicitation effects of diode and He-Ne laser irradiations on the alleviation of nutrient-deficiency induced damage in anthocyanin-producing red-fleshed apple cell suspension

Purpose We explored the elicitation role of the laser irradiations on the alleviation of nutrient-deficiency induced damage in anthocyanin-producing red-fleshed apple cell suspension in continuous production of anthocyanin. Methods Anthocyanin-producing red-fleshed apple cells were irradiated by 4 intensity levels of red He-Ne (RHNL) and blue diode (BDL) lasers for 20 min. Results Nutrient deficiency indicated negative effect on total soluble proteins (TSP), superoxidase dismutase (SOD) activity, and total phenolics content (TPC) while it displayed a positive effect on malondialdehyde (MDA), total flavonoids content (TFC), O2 -, H2O2 - , and lipoxygenase (LOX) and polyphenol oxidase (PPO) activities in light controls, illustrating oxidative stress. The laser irradiations on suspension cells indicated variable effects on measured parameters and were time of growth-, levels of intensity-, and laser type-dependent. Likewise, the elicitation effects of lasers relied on a critical threshold among ROS generation and antioxidative system which determines the fate of cells against oxidative stress. The same trend was displayed by RHNL at 6.46 mWcm−2 intensity and BDL at 13.73 mWcm−2. These intensities resulted in a significant increase in SOD, APX, POD, and CAT activities and TSP, TPC, TFC, proline, and glycine betaine accumulation, while induced decrease in LOX, and PPO activities and MDA, and ROS generation, alleviating cellular injury from prolonged nutrient deficiency by diminishing lipid peroxidation and oxidative damages of cell membrane. Conclusion Results suggested that lasers application on mitigating nutrient deficiency stress relied on establishing a suitable balance between ROS generation and antioxidative system, which enables the nutrient-starved anthocyanin-producing cells to continuously produce anthocyanin.

Wound Healing and Scar Patterning After Addition of Autologous Skin Cell Suspension to Meshed Grafts

IntroductionA common treatment for large deep-to-full-thickness burns is excision and grafting with a widely meshed split-thickness skin graft (mSTSG). Due to the differential healing of the interstices and adhered split-thickness skin graft, wound patterning and delayed wound healing are common outcomes of this treatment. Delayed healing may increase infection rates and wound care requirements, while wound patterning may be psychologically and aesthetically consequential for patients. Autologous skin cell suspension (ASCS) can be used to “over spray” a meshed autograft. It was hypothesized that the use of ASCS combined with mSTSG would increase the rate of wound healing and decrease patterning in healed burn wounds. MethodsFull-thickness burns or excisional wounds (n = 8 each) were created in red Duroc pigs and received 4:1 mSTSGs after wound bed preparation. Half of the wounds received ASCS and half did not at the time of grafting. Percent re-epithelialization, patterning, rete ridge ratio, cellularity, dermal and epidermal thickness, immunofluorescent S100β staining, and melanin index were assessed for each scar. ResultsWounds that received ASCS exhibited increased rates of re-epithelialization (burn +ACSC versus burn-ASCS; day 3 (53.9 ± 3.1 versus 34.3 ± 3.3, P = 0.009): day 5 (68.1 ± 1.6 versus 40.8 ± 3.2, P < 0.001)). Excision +ASCS versus excision-ASCS; day 7 (98.1 ± 1.2 versus 86.4 ± 2.0, day 7 P = 0.022) compared to wounds not treated with ASCS. There was no difference in rete ridge ratio, cellularity, dermal thickness, epidermal thickness, S100β staining, melanin index, or patterning was measured between wounds that received ASCS and those that did not. ConclusionsThe addition of ASCS to 4:1 mSTSGs leads to increased rate of wound healing but does not impact the degree of patterning in this model, suggesting that ASCS application likely robustly transfers keratinocytes but not functioning melanocytes at acute timepoints.

THE RECOMBINANT BACTERIA LACTOCOCCUS LACTIS EXPRESSING SARS-COV-2 SPIKE PROTEIN OR ITS RECEPTOR-BINDING DOMAIN ELICITS AN IMMUNE RESPONSE FOLLOWING ORAL IMMUNISATION OF RATS

Mucosal delivery of antigens represents an effective strategy for the development of vaccines to control diseases with the upper respiratory and/or gastrointestinal tracts as the main routes of transmission. Lactic acid bacteria (LAB) are food-grade bacteria used for the expression of target antigens in the development of mucosal vaccines. Over the last two decades, research on the use of LAB has focused on the construction of genetically modified strains. Current study aimed to develop and examine the recombinant Lactococcus lactis strains, expressing SARS-CoV-2 spike (S) protein or the receptor-binding domain (RBD) of S protein as immunizing antigens.The spike gene from the component II of Sputnik V vaccine and two synthesized coding sequences of the RBD region of SARS-CoV-2 spike protein (319–529 residues named HA-spike and 318–590 residues named mini-spike) were subcloned into expression vector pNZ8121. The resulting plasmids were verified by sequencing and subsequently transformed into competent L. lactis NZ3900 cells. The expression of recombinant proteins in obtained strains was analyzed by western blot using SARS-COV-2 Spike RBD antibodies. The results showed that the spike gene could be efficiently expressed in the cells of recombinant L. lactis. The major 150 kDa band detected in the cell lysate of L. lactis pNZ::spike represented the full-length S protein. Protein bands of 35 kDa and 23 kDa from the lysates of L. lactis pNZ::mini-spike and L. lactis pNZ::HA-spike respectively were detected indicating that the recombinant RBD protein was also successfully expressed. The mini-spike was expressed more abundantly than the HA-spike variant. Based on the growth kinetics and protein expression profiles of recombinant strains, the highest protein yield was obtained under the following conditions: overnight culture was inoculated (1/25 inoculum) and grown until reaching an optical density of 0,9 at 600 nm, then gene expression was induced by adding nisin (1 ng/ml), cells were harvested after 3 h incubation with the inducer. To examine the immunogenicity of recombinant strains, 1 ml of cell suspension (1,5×109 CFU) of L. lactis pNZ::spike, pNZ::mini-spike or L. lactis pNZ::HA-spike was orally administered to Wistar rats. The same volume of peptone water was administered to the control group. Serum was collected from the animals at 28 days and IgG level was determined by ELISA for the quantitative determination of IgG antibodies to SARS-CoV-2 (COVID-19) Spike RBD protein in rat serum. As expected, the serum from control animals lacked antibodies to the RBD antigen. The serum of rats immunised once with cell suspension of the recombinant strains showed the following levels of specific IgG: 124,24±12,56 ng/ml in group pNZ::mini-spike, 165,91±59,13 ng/ml in group pNZ::spike, and 213,60±54,08 ng/ml in group pNZ::HA-spike. The highest IgG level (545,56±230,24 ng/ml) was detected in the serum of rats immunized with ultrasound-treated cell suspension of L. lactis pNZ::spike. No adverse effects were observed in vaccinated animals at regular check-ups throughout the study period.The current results have demonstrated that the RBD and full spike protein were successfully expressed in L. lactis. Oral administration of the recombinant L. lactis expressing S protein or RBD to rats appeared to generate the anti-SARS-CoV-2 antibodies in serum, positioning this bacteria as promising basis for creating oral vaccine.

Glycogen nanoparticles for efficient mRNA transduction to T lymphocytes.

T lymphocyte therapies demonstrate significant promise in the treatment of cancer and infectious diseases. An efficient gene delivery system is essential for the safe and reliable introduction of exogenous genes, especially mRNA, into cells to achieve therapeutic purposes. Commercial transfection reagents are suitable for the transduction of plasmids to adherent cells, whereas they are ineffective for suspension cells such as T lymphocytes and for unstable mRNA. Moreover, the cytotoxicity of transfection reagents themselves constitutes an impediment to their application. The challenge of mRNA transduction to T lymphocytes with high efficiency is notably formidable. An innovative transfection strategy is urgently needed. In this study, we synthesized aminated glycogen (AGly) nanoparticles as gene vectors, encapsulating mRNA to facilitate the efficient transfection of T lymphocytes. Compared to commercial transfection reagent PEI, the AGly demonstrated favorable biocompatibility. The positive charge provided AGly with pH buffering ability and mRNA-binding capacity. AGly formed stable nanoparticles with mRNA, which were readily internalized by suspension cells and enhanced the cellular uptake of mRNA. In the T lymphocyte model cell lines (Jurkat cells and HuT 78 cells), AGly demonstrated superior transfection efficiency than that of PEI. Consequently, AGly can emerge as a viable mRNA vector for the efficient transfection of T lymphocytes whilst circumventing the issue of cytotoxicity. The AGly designed in this study provides a novel concept for the exploitation of transfection reagents and proposes a promising methodology for the proficient transfection of T lymphocytes which may significantly contribute to the treatment of cancer and other complex diseases.

Development of Neural Cells and Spontaneous Neural Activities in Engineered Brain-Like Constructs for Transplantation.

Stem cell transplantation has demonstrated efficacy in treating neurological disorders by generating functional cells and secreting beneficial factors. However, challenges remain for current cell suspension injection therapy, including uncontrollable cell distribution, the potential for tumor formation, and limited ability to treat spatial defects. Therefore, implants with programmable cell development, tailored 3D structure, and functionalized biomaterials have the potential to both control cell distribution and reduce or heal spatial defects. Here, a biomimetic material system comprising gelatin, alginate, and fibrinogen has been developed for neural progenitor cell constructs using 3D printing. The resulting constructs exhibit excellent formability, stability, and developmental functions in vitro, as well as biocompatibility and integration into the hippocampus in vivo. The controllability, reproducibility, and material composition of the constructs show potential for use in personalized stem cell-based therapies for defective neurological disorders, neural development research, disease modeling, and organoid-derived intelligent systems.

2PP-Hydrogel Covered Electrodes to Compensate for Media Effects in the Determination of Biomass in a Capillary Wave Micro Bioreactor.

Microbioreactors increase information output in biopharmaceutical screening applications because they can be operated in parallel without consuming large quantities of the pharmaceutical formulations being tested. A capillary wave microbioreactor (cwMBR) has recently been reported, allowing cost-efficient parallelization in an array that can be activated for mixing as a whole. Although impedance spectroscopy can directly distinguish between dead and viable cells, the monitoring of cells in suspension within bioreactors is challenging because the signal is influenced by the potentially varying properties of the culture medium. In order to address this challenge, an impedance sensor consisting of two sets of microelectrodes in a cwMBR is presented. Only one set of electrodes was covered by a two-photon cross-linked hydrogel to become insensitive to the influence of cells while remaining sensitive to the culture medium. With this impedance sensor, the biomass of Saccharomyces cerevisiae could be measured in a range from 1 to 20 g L-1. In addition, the sensor can compensate for a change in the conductivity of the suspension of 5 to 15 mS cm-1. Moreover, the two-photon cross-linking of hydroxyethyl starch methacrylate hydrogel, which has been studied in detail, recommends itself for even much broader sensing applications in miniaturized bioreactors and biosensors.

Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses

Abstract In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (P &amp;gt; 0.05). In contrast, the Coulter counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (P &amp;lt; 0.05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages.

Multiplexed Microfluidic Platform for Parallel Bacterial Chemotaxis Assays.

The sensing of and response to ambient chemical gradients by microorganisms via chemotaxis regulates many microbial processes fundamental to ecosystem function, human health, and disease. Microfluidics has emerged as an indispensable tool for the study of microbial chemotaxis, enabling precise, robust, and reproducible control of spatiotemporal chemical conditions. Previous techniques include combining laminar flow patterning and stop-flow diffusion to produce quasi-steady chemical gradients to directly probe single-cell responses or loading micro-wells to entice and ensnare chemotactic bacteria in quasi-steady chemical conditions. Such microfluidic approaches exemplify a trade-off between high spatiotemporal resolution of cell behavior and high-throughput screening of concentration-specific chemotactic responses. However, both aspects are necessary to disentangle how a diverse range of chemical compounds and concentrations mediate microbial processes such as nutrient uptake, reproduction, and chemorepulsion from toxins. Here, we present a protocol for the multiplexed chemotaxis device (MCD), a parallelized microfluidic platform for efficient, high-throughput, and high-resolution chemotaxis screening of swimming microbes across a range of chemical concentrations. The first layer of the two-layer polydimethylsiloxane (PDMS) device comprises a serial dilution network designed to produce five logarithmically diluted chemostimulus concentrations plus a control from a single chemical solution input. Laminar flow in the second device layer brings a cell suspension and buffer solution into contact with the chemostimuli solutions in each of six separate chemotaxis assays, in which microbial responses are imaged simultaneously over time. The MCD is produced via standard photography and soft lithography techniques and provides robust,repeatable chemostimulus concentrations across each assay in the device. This microfluidic platform provides a chemotaxis assay that blends high-throughput screening approaches with single-cell resolution to achieve a more comprehensive understanding of chemotaxis-mediated microbial processes. Key features • Microchannel master molds are fabricated using photolithography techniques in a clean room with a mask aligner to fabricate multilevel feature heights. • The microfluidic device is fabricated from PDMS using standard soft lithography replica molding from the master molds. • The resulting microchannel requires a one-time calibration of the driving inlet pressures, after which devices from the same master molds have robust performance. • The microfluidic platform is optimized and tested for measuring chemotaxis of swimming prokaryotes.

Endophyte-based fungal elicitors for enhanced production of valepotriates and sesquiterpenoids in leaf cell suspension cultures of Valeriana jatamansi Jones.

The immense therapeutic value of Valeriana jatamansi is attributed to the presence of bioactive secondary metabolites (valepotriates and sesquiterpenoids). Its over-exploitation in wild habitats resulted in extensive depletion, necessitating alternative approaches to produce its therapeutic metabolites. This study sought to assess the ability of endophytes of V. jatamansi to boost the biosynthesis of secondary metabolites in the leaf-cell suspension (LCS) culture of V. jatamansi. A total of 11 fungal endophytes were isolated from the rhizomes of V. jatamansi. Isolated endophytes were found to belong to phylum Ascomycota, Basidiomycota, and Mucoromycota. Supplementation of extracts of endophyte Phaeosphaeriaceae sp. VRzFB, Mucor griseocyanus VRzFD, Penicillium raistrickii VRzFK, and Penicillium sajarovii VRzFL in the LCS culture of V. jatamansi increased the fresh cell biomass by 19.6%-39.1% and dry cell biomass by 23.4%-37.8%. Most of the endophytes' extract could increase the content of valepotriates (26.5%-76.5% valtrate and 40.5%-77.9% acevaltrate) and sesquiterpenoids (19.9%-61.1% hydroxyl valerenic acid) in LCS culture. However, only two endophytes, Irpex lacteus VRzFI and Fusarium oxysporum VRzFF, could increase the sesquiterpenoids acetoxy valerenic acid (36.9%-55.3%). In contrast, some endophytes' extracts caused negative or no significant effect on the cell biomass and targeted metabolites. Increased secondary metabolites were corroborated with increased expression of iridoid biosynthesis genes in LCS culture. Production of H2O2 and lipid peroxidation was also varied with different endophytes indicating the modulation of cellular oxidative stress due to endophyte elicitors. The results suggest the distinct effect of different fungal endophytes-extract on LCS culture, and endophytes can serve as biotic elicitors for increasing the secondary metabolite production in plant in vitro systems.

Establishment of an Animal Model of Oral Squamous Cell Carcinoma Invading the Mandible.

To establish an animal model of oral squamous cell carcinoma invading the mandible through multi-sample experiments that verified the stability, repeatability, tumorigenicity and mandible destruction rate of the model. Oral squamous cell carcinoma cell suspension was injected into the outer side of the mandible through the anterior edge of the masseter muscle of naked mice to observe the tumourforming process. Then, the anatomical, histological and imaging examinations were carried out to determine whether the tumour had invaded the mandible. By comparing the tumour growth of multiple groups of various squamous cell carcinoma cells (CAL27, HN6 and HN30 cells), the changes in body weight and characteristics of tumour formation were compared, and the experience was summarised to further verify the stability, repeatability, tumour formation rate and arch damage rate of the model. The subsequent specimens of tumour-bearing nude mice were validated once the model had been established. In vitro, tumour tissue wrapped around the mandible's tumour-bearing side, and the local texture was tough with no resistance to acupuncture. Haematoxylin and eosin staining revealed that squamous cells were infiltrating the mandible in both the horizontal and sagittal planes. Microcomputed tomography results showed that the mandible on the tumour-bearing side displayed obvious erosion damage. Cell lines with various passage rates clearly had diverse tumour-bearing life cycles. This study successfully established an animal model of oral squamous cell carcinoma invasion of the mandible. The model has excellent biological stability, repeatability, tumorigenesis rate and mandible destruction rate.

Recent Updates on the Management of Split-thickness Skin Graft Donor Sites.

This article is a narrative review of split-thickness skin graft donor site (STSG-DS) management since the international guidelines were created in 2018. Although many new interventions have been developed, there is a lack of quality, multicentered clinical trials to produce updated evidence-based recommendations. Electronic databases, including Google Scholar, Web of Science, Medline, and PubMed, were searched by two independent researchers for literature regarding STSG-DS management published from 2018 through 2022, using specific terms in the text, title, and abstract. The primary endpoint assessed was STSG-DS healing, as measured by wound epithelialization. Thirty-one articles were selected, including three systemic review/meta-analyses, five case series, 14 randomized controlled clinical trials, six observational studies, and three nonrandomized trials. Novel interventions for STSG-DS management included in this article describe applications of human amniotic membranes, acellular dermal matrices, cell suspensions and growth factors, biomaterials, electromagnetic radiation, and natural products. Various interventions have shown promise for STSG-DS management since the creation of the 2018 international guidelines; however, studies with more standardized protocols and endpoints are needed to produce up-to-date, evidence-based recommendations and improve outcomes for patients undergoing split-thickness skin grafting.

Changes in bioenergetic characteristics of the murine lymphoma cells under the action of a thiazole derivative in complex with polymeric nanoparticles

Background. Mitochondria can influence cancer cells both indirectly via reactive oxygen species mediation and directly through mitochondrial biogenesis. Energy production in mitochondria is crucial as it facilitates the synthesis of essential molecules needed for cellular biosynthesis, growth, and proliferation. The development of new anticancer drugs that target the energy metabolism of tumor cells shows promise in cancer treatment. Our study aimed to investigate how the thiazole derivative N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1), the polymeric nanoparticles based on the polyethylene glycol (PEG-PN, Th5), and their complex with BF1 (Th6) affect respiration and mitochondrial membrane potential in murine NK/Ly tumor cells. Materials and Methods. The study was performed on white wild-type male mice with grafted NK/Ly lymphoma. The test substances were added to the cell suspension at a final concentration of 10 μM and incubated for 15 min at 37 °C. Oxygen uptake rates in NK/Ly cells were measured using a polarographic method with Clark electrode. Changes in mitochondrial membrane potential were assessed using the tetramethylrhodamine methyl ester fluorescence dye. The fluorescence intensity was evaluated using the ImageJ computer program. Results. After incubating NK/Ly cells with BF1 (10 µM), Th5, or the BF1 + PEG-PN complex (Th6) for 15 min, no changes were observed in glucose-fueled basal respiration. However, the Th6 complex significantly activated FCCP-stimulated respiratory processes in NK/Ly lymphoma cells. Fluorescent microscopy data indicated that BF1 or Th5 alone did not affect mitochondrial membrane potential values. However, the Th6 complex significantly decreased mitochondrial membrane potential, suggesting a reduction in NK/Ly cell viability. Conclusions The investigated complex of thiazole derivative BF1 with PEG-based polymeric nanoparticles may realize its cytotoxic effect by depolarization of mitochondrial membrane in NK/Ly lymphoma cells.

A broadly applicable method for quantifying cardiomyocyte cell division identifies proliferative events following myocardial infarction

Cardiomyocyte proliferation is a challenging metric to assess. Current methodologies have limitations in detecting the generation of new cardiomyocytes and technical challenges that reduce widespread applicability. Here, we describe an improved cell suspension and imaging-based methodology that can be broadly employed to assess cardiomyocyte cell division in standard laboratories across a multitude of model organisms and experimental conditions. We highlight additional metrics that can be gathered from the same cell preparations to enable additional relevant analyses to be performed. We incorporate additional antibody stains to address potential technical concerns of miscounting. Finally, we employ this methodology with a dual-thymidine analog-labeling approach to a post-infarction murine model, which allowed us to robustly identify unique cycling events, such as cardiomyocytes undergoing multiple rounds of cell division.

Effective and durable repigmentation for stable vitiligo: A randomized within-subject controlled trial assessing treatment with autologous skin cell suspension transplantation

BackgroundVitiligo lesions are often challenging to repigment with conventional medical therapies. Surgical autologous melanocyte transfer methods can be utilized for stable vitiligo but demand specialized skills and equipment. A point-of-care autologous cell harvesting device was designed enabling simple preparation of autologous skin cell suspension (ASCS) containing melanocytes, keratinocytes, and fibroblasts providing a straightforward approach for cellular transplantation. ObjectiveTo evaluate the safety and effectiveness of ASCS for repigmentation of stable vitiligo lesions among adults. MethodsA US multicenter, randomized, within-subject controlled trial compared ASCS to NB-UVB only (Control) in similar vitiligo lesions. ASCS was applied after laser skin resurfacing and followed by NB-UVB treatment. The primary effectiveness endpoint was the proportion of lesions achieving ≥80% repigmentation at week-24. Repigmentation durability was assessed at week-52. ResultsAmong 25 subjects, 36% of ASCS-treated lesions achieved ≥80% repigmentation at week-24 compared to 0% for Control (p<0.025), with durability through week-52. The safety profile of ASCS was acceptable, with favorable patient- and investigator-reported outcomes. LimitationsStudy sample size limited robust subgroup analyses. ConclusionApplication of ASCS is a safe and effective treatment for repigmentation of stable vitiligo lesions with the potential to improve health-related quality of life and reduce burden of disease.

Benefits of Aerosolized, Point-of-care, Autologous Skin Cell Suspension (ASCS) for the Closure of Full-thickness Wounds From Thermal and Nonthermal Causes: Learning Curves From the First 50 Consecutive Cases at an Urban, Level 1 Trauma Center.

To determine the utility of Autologous Skin Cell Suspension (ASCS) in closing full-thickness (FT) defects from injury and infection. Although ASCS has documented success in closing partial-thickness burns, far less is known about the efficacy of ASCS in FT defects. Fifty consecutive patients with FT defects (burn 17, necrotizing infection 13, crush 7, degloving 5, and other 8) underwent closure with the bilayer technique of 3:1 widely meshed, thin, split-thickness skin graft and 80:1 expanded ASCS. End points were limb salvage rate, donor site reduction, operative and hospital throughput, incidence of complications, and re-epithelialization by 4, 8, and 12 weeks. Definitive wound closure was achieved in 76%, 94%, and 98% of patients, at 4, 8, and 12 weeks, respectively. Limb salvage occurred in 42/43 patients (10 upper and 33 lower extremities). The mean area grafted was 435cm 2 ; donor site size was 212cm 2 , representing a potential reduction of 50%. The mean surgical time was 71 minutes; the total operating room time was 124 minutes. The mean length of stay was 26.4 days; the time from grafting to discharge was 11.2 days. Four out of 50 patients (8%) required 6 reoperations for bleeding (1), breakdown (4), and amputation (1). Four out of 50 patients (8%) developed hypertrophic scarring, which responded to silicone sheeting (2) and laser resurfacing (2). The mean follow-up was 92.7 days. When used for the closure of FT wounds, point-of-care ASCS is effective and safe. Benefits include rapid re-epithelialization, high rate of limb salvage, reduction of donor site size and morbidity, and low incidence of hypertrophic scarring.

Scorpiones, Scolopendra and Gekko Inhibit Lung Cancer Growth and Metastasis by Ameliorating Hypoxic Tumor Microenvironment via PI3K/AKT/mTOR/HIF-1α Signaling Pathway.

To investigate whether Buthus martensii karsch (Scorpiones), Scolopendra subspinipes mutilans L. Koch (Scolopendra) and Gekko gecko Linnaeus (Gekko) could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α (PI3K/AKT/mTOR/HIF-1α) signaling pathway. Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models, with rapamycin and cyclophosphamide as positive controls. Carboxy methyl cellulose solutions of Scorpiones, Scolopendra and Gekko were administered intragastrically as 0.33, 0.33, and 0.83 g/kg, respectively once daily for 21 days. Fluorescent expression were detected every 7 days after inoculation, and tumor growth curves were plotted. Immunohistochemistry was performed to determine CD31 and HIF-1α expressions in tumor tissue and microvessel density (MVD) was analyzed. Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1α signaling pathway-related proteins. Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in mice. Scorpiones, Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α (all P<0.01). Moreover, Scorpiones, Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase (p70S6K) (P<0.05 or P<0.01). In addition, they also decreased the expression of CD31, MVD, bFGF, TGF-β1 and VEGF compared with the model group (P<0.05 or P<0.01). Scorpiones, Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1α signaling pathway.

Analysis of magnetic resonance contrast agent entrapment following reversible electroporation in vitro.

Administering gadolinium-based contrast agent before electroporation allows the contrast agent to enter the cells and enables MRI assessment of reversibly electroporated regions. The aim of this study was evaluation of contrast agent entrapment in Chinese hamster ovary (CHO) cells and comparison of these results with those determined by standard in vitro methods for assessing cell membrane permeability, cell membrane integrity and cell survival following electroporation. Cell membrane permeabilization and cell membrane integrity experiments were performed using YO-PRO-1 dye and propidium iodide, respectively. Cell survival experiments were performed by assessing metabolic activity of cells using MTS assay. The entrapment of gadolinium-based contrast agent gadobutrol inside the cells was evaluated using T1 relaxometry of cell suspensions 25 min and 24 h after electroporation and confirmed by inductively coupled plasma mass spectrometry. Contrast agent was detected 25 min and 24 h after the delivery of electric pulses in cells that were reversibly electroporated. In addition, contrast agent was present in irreversibly electroporated cells 25 min after the delivery of electric pulses but was no longer detected in irreversibly electroporated cells after 24 h. Inductively coupled plasma mass spectrometry showed a proportional decrease in gadolinium content per cell with shortening of T1 relaxation time (R 2 = 0.88 and p = 0.0191). Our results demonstrate that the contrast agent is entrapped in cells exposed to reversible electroporation but exits from cells exposed to irreversible electroporation within 24 h, thus confirming the hypothesis on which detection experiments in vivo were based.