7-day Culture Research Articles

Prior exposure to alkylating agents negatively impacts testicular organoid formation in cells obtained from childhood cancer patients.

Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)? Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy. Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge. We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls. We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production. Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006). N/A. Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available. Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue. This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.

Phenology and the response of photosynthesis to irradiance and temperature gradient in the herbal drug red alga, Chondria armata (Rhodomelaceae, Ceramiales) from Kagoshima, Japan

Seasonal changes in the size of the herbal drug red alga Chondria armata (Rhodomelaceae, Ceramiales) were investigated in Kagoshima, Japan, which is near the northern distributional limit in the western Pacific. Additionally, its photosynthetic response to irradiance and temperature was examined using dissolved oxygen sensors and a pulse amplitude modulation (PAM)-chlorophyll fluorometer. This alga was observed in tidepools throughout the year; its height and weight were greatest in December and the lowest in April and May. The net photosynthesis of the photosynthesis–irradiance (P–E) curve determined at 28°C quickly saturated at 113 µmol photons m-2 s-1, with minimal inhibition even at 1000 µmol photons m-2 s-1. The gross photosynthesis of the photosynthesis–temperature (P–T) curved over 8 to 40°C, measured at 500 µmol photons m-2 s-1, peaked at 30.1°C and decreased rapidly below 20°C and above 36°C, respectively. Similarly, the effective quantum yield (ΔF/Fm') after a 3-day culture during 4–40°C at 50 µmol photons m-2 s-1 remained stable between 16°C and 32°C but decreased outside of this range. The combined effect of irradiance (200 [low] and 1000 [high] µmol photons m-2 s-1) and temperature (28, 22, and 16°C) revealed that ΔF/Fm' declined during exposure to high irradiance at all temperature treatments. However, it mostly recovered after a subsequent 12-hour period of dim-light acclimation at 28°C and 22°C. In contrast, those at 16°C could not recover, indicating the occurrence of low-temperature light stress. This alga appears to be well-adapted to the irradiance and temperature environment at the study site. However, the winter temperature appears to approach its threshold level, and the occurrence of strong light during the winter might adversely affect the abundance of this alga near its northern distributional limit.

The mechanical, wear, antibacterial properties and biocompatibility of injectable restorative materials under wet challenge

ObjectivesTo evaluate the mechanical, wear, antibacterial properties, and biocompatibility of injectable composite materials. MethodsTwo injectable composite resins (GU and BI), one flowable composite resin (FS), and one flowable compomer (DF), in A2 shade, were tested. Mechanical properties were tested via three-point bending test immediately after preparation and after 1-day, 7-day, 14-day, and 30-day water storage. Under water-PMMA slurry immersion, specimens were subjected to a 3-body wear test (10,000 cycles) against stainless steel balls, while the roughness, wear depth, and volume loss were recorded. After 1-day and 3-day MC3T3-E1 cell culture, cell viability was evaluated with CCK-8 test kits, while the cell morphology was observed under CLSM and SEM. Antibacterial properties on S. mutans were assessed via CFU counting, CLSM, and SEM observation. SPSS 26.0 was used for statistical analysis (α = 0.05). ResultsThe mechanical properties were material-dependent and sensitive to water storage. Flexural strength ranked GU > FS > BI > DF at all testing levels. Three nanocomposites had better wear properties than DF. No significant difference on 1-day cell viability was found, but DF showed significantly lower cell proliferation than nanocomposites on 3-day assessment. GU and FS had more favourable cell adhesion and morphology. CFU counting revealed no significant difference, while FS presented a slightly thicker biofilm and BI showed relatively lower bacteria density. ConclusionsInjectable nanocomposites outperformed the compomer regarding mechanical properties, wear resistance, and biocompatibility. The tested materials presented comparable antibacterial behaviours. Flowable resin-based composites’ performances are affected by multiple factors, and their compositions can be attributed. Clinical SignificanceA profound understanding of the mechanical, wear, and biological properties of the restorative material is imperative for the clinical success of dental restorations. The current study demonstrated superior properties of highly filled injectable composite resins, which imply their wider indications and better long-term clinical performances.

First report of Bretziella fagacearum infecting chestnut in Michigan.

In the summer of 2021, a 20-year-old 'Colossal' (Castanea sativa × C. crenata hybrid) tree in a commercial chestnut orchard in northwest Michigan suddenly declined. Until 2023, an additional 26 adjacent trees declined, suggesting the occurrence of root-graft transmission of the pathogen. The initial wilting of leaves progressed to complete tree death in about 10 days. Symptoms included wilting, and bronzing, followed by tanning starting at leaf apex and margins, with significant defoliation. Sometimes black-to-brown streaks of discoloration appear in the sapwood, with no signs of mycelial mat production on dead trees. Branches from symptomatic trees in two different areas of the orchard were submitted to Plant and Pest Diagnostics at Michigan State University. Bretziella fagacearum (Bretz) Z.W. de Beer, Marinc., T.A. Duong & M.J. Wingf. was detected in both samples using nested PCR (Wu et al. 2011) and qPCR (Bourgault et al. 2022). The products of the nested PCR were sequenced (GenBank accession nos. OR522695-OR522696) and BLASTn search results showed 100% identity to an ex-type strain of B. fagacearum (MH865866). Surface-sterilized discolored sapwood chips were plated on acidified potato dextrose agar (aPDA). Bretziella fagacearum was consistently recovered; colony and endoconidia morphology aligned with the description of the pathogen (De Beer et al. 2017). A pure culture (BF277) was obtained for inoculation experiments. To confirm pathogenicity, 10 'Colossal' chestnut seedlings (average stem diameter of 9 mm) were inoculated in the greenhouse with a 14-day old culture of BF277. Using a conical drill bit, two 0.4 mm diameter holes were drilled, one was 5 cm above the soil line at a 45° angle and the other was on the opposite side of the stem at least 10 cm above the soil line. A 50-µl conidial suspension (1 × 107 conidia per ml) was applied and the holes were sealed with Parafilm. Five 'Colossal' seedlings were inoculated with sterile water. Leaf epinasty with bent petioles was observed 14 days later. Leaf wilting and necrosis similar to natural infection in the orchard were observed at 24 and 34 days after inoculation, respectively. Water-inoculated control plants showed no symptoms. Bretziella fagacearum was reisolated from symptomatic plants by surface sterilizing leaf petioles with 75% ethanol (30 s), followed by 10% (v/v) bleach (1 min), and two rinses with sterile deionized water (>1 min). Petiole pieces (~1 cm) were plated on aPDA. The pathogen was reisolated from six symptomatic plants and detected using qPCR in the remaining four seedlings. Bretziella fagacearum was not detected in control plants. The identity of the recovered fungus was confirmed following the amplification of the internal transcribed spacer (ITS) from extracted genomic DNA, as described in Chahal et al. 2022. The resulting PCR product was sequenced and assembled into a consensus sequence using Geneious Prime. The consensus sequence (accession no. OR515809) revealed 100% identity to the ex-type of B. fagacearum (KU042044). This is the first record of B. fagacearum infecting chestnut trees in Michigan. Previously, B. fagacearum has been reported infecting Chinese chestnut (C. mollissima) in Missouri (Bretz and Long, 1950). Oak wilt is widely distributed in Michigan and is the predominant disease afflicting red oaks in the Midwestern U.S. Consequently, constant vigilance and monitoring are essential in chestnut orchards to promptly detect and effectively manage potential infections.

Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Single cell analysis of Chinese hamster ovary cells during a bioprocess using a novel dynamic imaging system.

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in-line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6-day bioreactor culture. The system is also capable of sampling at-line, removing the necessity for taking samples off-line and enabling real time monitoring of cells in a bioreactor culture.

First Report ofLasiodiplodia hormozganensisCausing Leaf Spot Disease onAreca catechuin China.

The areca palm (Areca catechu L.) is one of the significant cash crops in Hainan Province (China), and a valuable tropical medicinal plant (Cao et al. 2020). In September 2020, spots were found on about 80% of the area of the leaves in a 1,000-acre plantation of areca palms in Haikou City, Hainan Province, and the average incidence was 25%. Initially, Elliptical or irregular dark brown spots appeared on the leaves, with an average size of about 1.5 cm2. With the further expansion of the disease, the spot turned light brown in the center with dark brown edges and a prominent yellow halo. Later stage of the disease, the spots became grayish-white in the center, with obvious whorls, on which many small black spots (pycnidia) were scattered. Eventually the leaves dried out. Ten leaves with typical symptoms were collected from the field. Lesion marginal tissues (5×5 mm2) were surfaced sterilized in 75% ethanol for 20 s, followed by 4 min in 1% NaClO, rinsed 3 times with sterile water, plated on PDA and incubated at 28 ℃. A fungus was isolated with a 98% isolation rate. This strain was named HNAC-5. Subcultures were 80 mm in diameter, white, villous, and neatly edged, after two days of incubation at 28 ℃ in dark. Pycnidia were solitary or clustered in stromata, with orifices that oozed black liquid. Conidiogenous cells were colorless and short cylindrical. Conidia unicellular, initially hyaline, aseptate, ellipsoid to ovoid with granular content, becoming pigmented, 1-septate with longitudinal striations, and measuring 20-31×10-13 μm (n=100). These morphological characteristics were similar to Lasiodiplodia spp. (Abdollahzadeh et al. 2010). The internal transcribed spacer region of rDNA, β-tubulin gene, and translation elongation gene were amplified using ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R primers, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The resulting sequences were deposited in GenBank under accession numbers OR272043, OR282568, and OR282567. BLAST analysis showed that the three sequences of HNAC-5 were more than 99% similar to strain CBS 124709 of L. hormozganensis. Phylogenetic analysis was performed using the maximum likelihood method based on the three-gene combined dataset with MEGA 7.0 software. The results indicated that HNAC-5 was grouped in the same clade as otherL. hormozganensis Abdollahzadeh, Zare & A.J.L. Phillips. Pathogenicity test was carried out on 15 healthy leaves by in vivo inoculation. Ten leaves were pricked with a sterile needle and divided into group 1 and 2. The remaining five uninjured leaves were group 3. Group 1 and 3 were inoculated with 5-mm-diameter mycelial plugs obtained from 3-day cultures, and group 2 treated with PDA plugs served as controls. Fifteen leaves were cultured at 28°C and 100% relative humidity. After 5 days, leaves of group 1 showed symptoms of the disease and on the tenth day showed the same symptoms as the initial onset of the disease in the field, while leaves of Group 2 and 3 showed no symptoms. Pathogenicity tests were conducted three times with the same results. L. hormozganensiswas re-isolated from the inoculated symptomatic leaves, thus, Koch's postulates were confirmed. In China, L. hormozganensis has been reported to cause Bougainvillea spectabilis Willd. branch blight disease (Li et al. 2015), and Scaevola taccada (Gaertn.) Roxb. leaf spot disease (Zhang et al. 2020). To our knowledge, this is the first report of L. hormozganensiscausing leaf spot disease onA. catechuin China.

Three-Dimensional Microfibrous Scaffold with Aligned Topography Produced via a Combination of Melt-Extrusion Additive Manufacturing and Porogen Leaching for In Vitro Skeletal Muscle Modeling.

Skeletal muscle tissue (SMT) has a highly hierarchical and anisotropic morphology, featuring aligned and parallel structures at multiple levels. Various factors, including trauma and disease conditions, can compromise the functionality of skeletal muscle. The in vitro modeling of SMT represents a useful tool for testing novel drugs and therapies. The successful replication of SMT native morphology demands scaffolds with an aligned anisotropic 3D architecture. In this work, a 3D PCL fibrous scaffold with aligned morphology was developed through the synergistic combination of Melt-Extrusion Additive Manufacturing (MEAM) and porogen leaching, utilizing PCL as the bulk material and PEG as the porogen. PCL/PEG blends with different polymer ratios (60/40, 50/50, 40/60) were produced and characterized through a DSC analysis. The MEAM process parameters and porogen leaching in bi-distilled water allowed for the development of a micrometric anisotropic fibrous structure with fiber diameters ranging from 10 to 100 µm, depending on PCL/PEG blend ratios. The fibrous scaffolds were coated with Gelatin type A to achieve a biomimetic coating for an in vitro cell culture and mechanically characterized via AFM. The 40/60 PCL/PEG scaffolds yielded the most homogeneous and smallest fibers and the greatest physiological stiffness. In vitro cell culture studies were performed by seeding C2C12 cells onto a selected scaffold, enabling their attachment, alignment, and myotube formation along the PCL fibers during a 14-day culture period. The resultant anisotropic scaffold morphology promoted SMT-like cell conformation, establishing a versatile platform for developing in vitro models of tissues with anisotropic morphology.

Optimized Decellularization of a Porcine Fasciocutaneaous Flap.

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.

Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively. Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity. Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p<0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p<0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions. Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues. Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 237.

CAR-T cell expansion platforms yield distinct T cell differentiation states

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis

ObjectivesTo assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. MethodsThe study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. ResultsOnly one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8–89.8%) and the negative percentage agreement was 100% (95% CI, 97.2–100%). The positive predictive value was extremely high (100%; 95% CI, 97.6–100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7–88.2%), partially explained by the missing target species in the panel. DiscussionThe BF-JIP showed high performances to detect pathogens involved in acute arthritis.

First report of Calonectria ilicicola causing red crown rot of soybean in Indiana.

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.

First Report of Leaf Blight of Camellia japonica Caused by Diaporthe fukushii in Tennessee and the United States.

Japanese camellia (Camellia japonica), is an important ornamental species that has an increasing economic value in China, Japan, Australia and the USA (Vela et al. 2013). Leaf blight symptoms were observed on 20-year-old C. japonica 'April Tryst' leaves collected from a research plot in McMinnville, TN in March 2022. Leaf blight first appeared in the leaf tips and was irregular in shape (2 to 3 cm in diameter). Affected areas displayed gray color discoloration with a deep black margin and gradually expanded in size along the leaf margin, eventually causing leaf death and defoliation. Dark brown globose to subglobose conidiomata (pycnidia) were observed abundantly on the infected leaves (Fig. 1a). Disease severity was 25 to 50% of leaf area and incidence was 10% out of 60 plants. Three leaves were collected from each symptomatic plant and the surface disinfected with 10% NaOCl for 60 s, washed thrice with distilled water, and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG4744 and FBG6184 on PDA, 15 days after incubation at 25°C (light/dark: 12/12h) were white to pale grey with dense and felted mycelium with concentric zonation. Spherical black pycnidia were observed on the concentric rings 2-3 weeks after incubation. Alpha conidia were on average 7.15 × 4.82 µm (4.89 to 9.37 µm × 2.91 to 6.74 µm) in size and were aseptate, hyaline, smooth, and ellipsoidal (n=50). Beta conidia were not observed. Pathogen identity was confirmed by extracting total DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old cultures. Primer pairs ITS1/ITS4 (White et al. 1990), T1/T222 and EF1/EF2 (Stefańczyk et al. 2016) were used to amplify and sequence the ribosomal internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factors 1-α (EF1-α) genetic markers, respectively. The sequences (GenBank accession nos. OR607729, ITS; OR608485, BT; OR608487, EF1-α) were 100% similar to Diaporthe fukushii (=Phomopsis fukushii) in the NCBI nr/nt database (JQ807450: ITS; MG812590: BT, and MG281573: EF1-α). A phylogenetic analysis was performed using concatenated sequences of ITS, BT, and EF1-α of D. fukushii and other closely related taxa retrieved from GenBank (Fig. 2). Pathogenicity tests were performed on 1-year-old 10 healthy potted plants of C. japonica 'April Tryst' per isolate (Mathew et al. 2015; Yang et al. 2019). One leaf per plant was wounded with a sterilized 0.2-mm needle. PDA plugs (5 mm) taken from 7-day old cultures of FBG4744 and FBG6184 isolates were deposited on the wounded leaves and covered with moist cotton (Yang et al. 2019; Zhao et al. 2020). Ten additional plants were used as control and sterile PDA plugs were placed on the wounded leaves. Plants were covered with clear plastic bags and kept inside a greenhouse at 21 to 23°C, 70% RH, 16 h photoperiod. All inoculated leaves exhibited blight symptoms 14 days after inoculation (Fig. 1b) while control plants remained asymptomatic (Fig. 1c). The pathogen was reisolated from all the inoculated leaves and was confirmed as D. fukushii using morphological and molecular tools. Diaporthe species (D. tulliensis, D. passiflorae and D. perseae) have been previously reported to cause leaf spot on Camellia sinensis in Taiwan (Ariyawansa et al. 2021), but to our knowledge, this is the first report of leaf blight of C. japonica caused by Diaporthe fukushii in Tennessee and the United States. Identification of this novel disease is important in developing necessary management approaches.

Understanding the Role of Biofilms in Acute Recurrent Tonsillitis through 3D Bioprinting of a Novel Gelatin-PEGDA Hydrogel.

Acute recurrent tonsillitis is a chronic, biofilm-related infection that is a significant burden to patients and healthcare systems. It is often treated with repeated courses of antibiotics, which contributes to antimicrobial resistance. Studying biofilms is key to understanding this disease. In vitro modelling using 3D bioprinted hydrogels is a promising approach to achieve this. A novel gelatin-PEGDA pseudomonas fluorescens-laden bioink was developed and bioprinted in a 3D hydrogel construct fabricated using computer-aided design to mimic the tonsillar biofilm environment. The bioprinted constructs were cultured at 37 °C in lysogeny broth for 12 days. Bacterial growth was assessed by spectrophotometry. Cellular viability analysis was conducted using optical fluorescence microscopy (FDA/PI staining). A biocompatible 3D-printed bacteria-laden hydrogel construct was successfully fabricated. Bacterial growth was observed using optical fluorescence microscopy. A live/dead cellular-staining protocol demonstrated bacterial viability. Results obtained after the 12-day culture period showed higher bacterial growth in the 1% gelatin concentration construct compared to the 0% control. This study demonstrates the first use of a bacteria-laden gelatin-PEGDA hydrogel for biofabrication of a 3D-printed construct designed to model acute recurrent tonsillitis. Initiating a study with clinically relevant ex vivo tonsil bacteria will be an important next step in improving treatment of this impactful but understudied disease.

Phytotoxicity effect of a highly toxic isolate of Alternaria alternata metabolites from Iran

Alternaria species produce several mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin (ATX), tentoxin (TTX) and tenuazonic acid (TeA). This research aimed to isolate and identify mycotoxins from highly toxic Alternaria alternata (w19) and A. tennuisima isolates and their phytotoxicity effects. Fungal metabolites were extracted from 21-day cultures of Alternaria in a Czapek broth medium with the organic solvent chloroform/acetone and identified using the HPLC method. Alternaria metabolites were infiltrated in vivo into several plant leaves for phytotoxicity detection. The study investigated the impact of temperature, time, and metabolite concentration on phytotoxicity using the detached leaf infiltration technique. Five mycotoxins (TTX, TeA, ALT, AOH, and AME) were detected in A. alternata W19 isolate with 959.24, 102.03, 24.01, 9.04, and 2.44 ppm, respectively. A. tennuisima produce these toxins in a lower concentration. Infiltration of fungal metabolites induced leaf chlorosis and necrosis, which differs based on temperature, concentration and plant species. Based on our knowledge, this is the first report of Alternaria mycotoxins in Iran and a highly toxic isolate of A. alternata with rapid phytotoxicity on a wide range of susceptible hosts.

Si/SiO2 silicon-on-insulator based scaffold for interfacing with cerebral organoids

Cerebral organoids are hSPC derived tissue complexes that self-organize over an extended period and begin to recapitulate certain features of in vivo neurogenesis, and there is a need to develop tools that allow for the physical and electrical interrogation of these cell complexes as they mature. Presented here is a method of manufacturing thin, porous, scalable silicon scaffolds as a structure to support multi-electrode arrays using industry standard semiconductor fabrication techniques. Mechanical tests by nanoindentation demonstrate the scaffold as linearly elastic with an approximated effective stiffness of 51.6 MPa. Biological cell studies indicate that mature Cerebral Organoids are capable of cellular expansion through, and partially absorbing, the scaffold, with 0.125 mm3 ± 0.027 of new tissue generation over a 5-day culture period. The structure is designed for compatibility with front end of line and back end of line semiconductor processes for future integration with metal electrodes, which enables deep tissue access in multi-cellular structures.

Evaluation of the genotoxicity, cytotoxicity, and bioactivity of calcium silicate-based cements

BackgroundAs calcium silicate-based cements (CSCs) have found success in various vital pulp therapy applications, several new CSC products have emerged. This study aimed to assess the genotoxicity, cytotoxicity, and bioactivity of four CSCs by comparing the newly introduced materials Bio MTA+ and MTA Cem with previously studied materials, Biodentine and NeoMTA.MethodsGenotoxicity was evaluated using the micronucleus (MN) assay in human peripheral blood lymphocyte cells, measuring MN frequency and nuclear division index (NDI). Cytotoxicity was assessed in human dental pulp stem cells through the Water-Soluble Tetrazolium Salt-1 (WST-1) colorimetric assay. Bioactivity was determined by ELISA, measuring the levels of angiogenic and odontogenic markers (BMP-2, FGF-2, VEGF, and ALP). Statistical analyses included ANOVA, Dunnet and Sidak tests, and Wald chi-square test. (p < .05).ResultsThe MN frequency in the groups was significantly lower than that in the positive control group (tetraconazole) (p < .05). NDI values decreased with increasing concentration (p < .05). Bio MTA+ and NeoMTA showed decreased cell viability at all concentrations in 7-day cultures (p < .01). All materials increased BMP-2, FGF-2, and VEGF levels, with Biodentine and NeoMTA showing the highest levels of BMP-2 and FGF-2 on day 7. Biodentine displayed the highest VEGF levels on day 7. Biodentine and NeoMTA groups exhibited significantly higher ALP activity than the Bio MTA+ and MTA Cem groups by day 7.ConclusionBio MTA+ and MTA Cem demonstrated no genotoxic or cytotoxic effects. Moreover, this study revealed bioactive potentials of Bio MTA+ and MTA Cem by enhancing the expression of angiogenic and osteogenic growth factors.

Development of a Nanoparticle System for Controlled Release in Bioprinted Respiratory Scaffolds.

The use of nanoparticle systems for the controlled release of growth factors is a promising approach to mimicking of the biochemical environment of native tissues in tissue engineering. However, sustaining growth factor release inside an appropriate therapeutic window is a challenge, particularly in bioprinted scaffolds. In this study, a chitosan-coated alginate-based nanoparticle system loaded with hepatocyte growth factor was developed and then incorporated into bioprinted scaffolds. The release kinetics were investigated with a focus on identifying the impact of the chitosan coating and culture conditions. Our results demonstrated that the chitosan coating decreased the release rate and lessened the initial burst release, while culturing in dynamic conditions had no significant impact compared to static conditions. The nanoparticles were then incorporated into bioinks at various concentrations, and scaffolds with a three-dimensional (3D) structure were bioprinted from the bioinks containing human pulmonary fibroblasts and bronchial epithelial cells to investigate the potential use of a controlled release system in respiratory tissue engineering. It was found that the bioink loaded with a concentration of 4 µg/mL of nanoparticles had better printability compared to other concentrations, while the mechanical stability of the scaffolds was maintained over a 14-day culture period. The examination of the incorporated cells demonstrated a high degree of viability and proliferation with visualization of the beginning of an epithelial barrier layer. Taken together, this study demonstrates that a chitosan-coated alginate-based nanoparticle system allows the sustained release of growth factors in bioprinted respiratory tissue scaffolds.

Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4°C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.