Culture Duration Research Articles

Potential of culturing microalgae Chlorella vulgaris and Nannochloropsis oculata with aquaculture wastewater for simultaneous aquafeed production and wastewater remediation

Aquaculture expansion has resulted in nutrient pollution in aquatic ecosystems, primarily due to nitrogen-rich effluents, leading to eutrophication and degraded water quality. Although conventional wastewater treatment methods are effective, they are often costly and environmentally risky. Microalgae offer a promising alternative, enabling both wastewater remediation and the production of nutrient-rich biomass. However, most research has mainly focused on nutrient removal efficiencies, with relatively little attention given to the quality of the microalgal biomass and its suitability for simultaneous aquafeed production. This study evaluates the growth, nutritional content, and nutrient removal efficiencies of Chlorella vulgaris (C. vulgaris) and Nannochloropsis oculata (N. oculata) in synthetic aquaculture wastewater (AW). The findings reveal that both species showed significant growth in AW and F/2 media, with N. oculata reaching the highest cell density (17.6 × 10⁶ cells/mL) in AW. After seven days, C. vulgaris removed 83.7 ± 0.42% of nutrients in AW and 78.0 ± 4.35% in F/2, while N. oculata achieved 71.3 ± 1.50% and 72.3 ± 10.0%, respectively. Biomass from both species was also rich in protein (35.9–57.4%) and carbohydrates (12.7–40.9%). Particularly, N. oculata produced 46% dw protein and 40.9% dw carbohydrates in aquaculture wastewater, with protein levels higher than most previously reported values in such conditions. Additionally, with over 71% nutrient removal in only seven days, a longer culture duration and higher initial biomass inoculum could further enhance the efficiency. These findings highlight the potential of N. oculata and C. vulgaris for sustainable aquaculture, effectively treating aquaculture wastewater and producing high-quality aquafeed biomass, thereby supporting environmentally friendly and cost-effective practices.

Fracture Behavior and Biocompatibility of Cellulose Nanofiber-Reinforced Poly(vinyl alcohol) Composite Hydrogels Cross-Linked with Borax.

We investigated the fracture behavior of cellulose nanofiber (CNF)-reinforced poly(vinyl alcohol) (PVA) hydrogels cross-linked with borax and the effect of freeze-thaw (FT) cycles on it. The CNF/PVA/Borax hydrogel not subjected to FT achieved a fracture energy of 5.8 kJ m-2 and a dissipative length of 2.3 mm, comparable to those of tough hydrogels. Lacking either CNF or borax remarkably decreased the fracture energy and the dissipative length; CNF contributed to a physical blocking of the crack growth, whereas the complexations between CNF and borate yielded nonlocalization of energy dissipation. Repeated FT cycles markedly improved the mechanical performance of unnotched samples, but they decreased the fracture energy due to the lowering of the dissipative length. Besides, CNF/PVA/Borax hydrogels were suitable for cell scaffold materials. The culture of umbilical cord mesenchymal stem cells (UC-MSCs) revealed a positive correlation between culture duration and the number of UC- MSCs adherent to the material.

Flux balance analysis and peptide mapping elucidate the impact of bioreactor pH on Chinese hamster ovary (CHO) cell metabolism and N-linked glycosylation in the fab and Fc regions of the produced IgG

Culture conditions have a profound impact on therapeutic protein production and glycosylation, a critical therapeutic-quality attribute, especially for monoclonal antibodies (mAbs). While the critical culture parameter of pH has been known since the early 1990s to affect protein glycosylation and production, detailed glycan and metabolic characterization and mechanistic understanding are critically lacking. Here, Chinese Hamster Ovary (CHO) cells were grown in bioreactors at pH 6.75, 7, and 7.25 (± 0.03) to examine how pH affects cell metabolism and site-specific N-linked glycosylation of the produced broadly neutralizing anti-HIV IgG1 mAb. VRC01 has N-linked glycosylation sites in both the Fc region and the Fab region, a situation not previously examined with respect to mAb glycosylation as affected by culture conditions. Using parsimonious Flux Balance Analysis (pFBA) and Flux Variability Analysis (FVA), we dissect and quantitate the impact of pH on cell growth, glucose/lactate metabolism, accumulation of the toxic metabolite ammonia, IgG production rates, and nonessential amino acid metabolism. pFBA revealed that beyond the established mechanism of glutamine conversion to glutamate, ammonia is also produced by the reaction converting serine to pyruvate, especially in the later phases of culture. pFBA also provided insights into the switch from ammonia production to consumption, notably due to depletion of glutamine, and consumption of glutamate and aspartate. We document that culture duration and pH alter the complex bimodal patterns (production/uptake) of several essential and non-essential amino acids. Site-specific N-linked glycan analysis using glycopeptide mapping demonstrated that pH significantly affects the glycosylation profiles of the two IgG1 sites. Fc region glycans were completely fucosylated but did not contain any sialylation. The Fab region glycans were not completely fucosylated but contained sialylated glycans. Bioreactor pH affected both the fucosylation and sialylation indexes in the Fab region and the galactosylation index of the Fc region. However, fucosylation in the Fc region was unaffected thus demonstrating that the effect of pH on site-specific N-linked glycosylation is complex.

Device for automated aseptic sampling: Automated sampling solution for future cell and gene manufacturing.

Cell sampling is a key step performed regularly throughout the cell manufacturing process to gather cell samples for cell growth, progress, and characteristics analysis. While the current method of sampling by pipetting in a biosafety cabinet is commonly used, it is labour-intensive and susceptible to contamination risks. We have developed Device for Automated Aseptic Sampling (DAAS), to enable automated, small volume (0.02-1.00mL) aseptic sampling with minimal dead volume primarily for cell and gene therapy manufacturing. The aim of DAAS is to enable an accurate and consistent sampling process, with minimal contamination risks and interruption to the cells in culture. DAAS can potentially interface with other automated solutions to enable automated and streamlined cell manufacturing workflow and reduce overall manufacturing costs. DAAS has been verified as an aseptic sampling solution via repeated microbial ingression tests. It has also been tested for achieving comparable cell density and viability compared to manual pipetting, with negligible cross-sample carryover when used to sample Jurkat cells of different cell concentrations. The application of using DAAS to sample cell periodically and monitor cell growth and viability continuously for prolonged cell culture was successfully demonstrated with Jurkat cell culture in a static culture flask and donor T cell culture in an automated bioreactor system over a culture duration of 10days in a Biosafety Level-2 laboratory. Overall, DAAS presents great potential as an automated and aseptic sampling solution, offering cell and gene therapy manufacturers easier and more frequent access to cell samples with minimal interruptions to the cell culture. This enables close monitoring of cell culture and a more automated, connected and cost-effect cell and gene therapy manufacturing process.

A deep learning model for predicting blastocyst formation from cleavage-stage human embryos using time-lapse images

Efficient prediction of blastocyst formation from early-stage human embryos is imperative for improving the success rates of assisted reproductive technology (ART). Clinics transfer embryos at the blastocyst stage on Day-5 but Day-3 embryo transfer offers the advantage of a shorter culture duration, which reduces exposure to laboratory conditions, potentially enhancing embryonic development within a more conducive uterine environment and improving the likelihood of successful pregnancies. In this paper, we present a novel ResNet-GRU deep-learning model to predict blastocyst formation at 72 HPI. The model considers the time-lapse images from the incubator from Day 0 to Day 3. The model predicts blastocyst formation with a validation accuracy of 93% from the cleavage stage. The sensitivity and specificity are 0.97 and 0.77 respectively. The deep learning model presented in this paper will assist the embryologist in identifying the best embryo to transfer at Day 3, leading to improved patient outcomes and pregnancy rates in ART.

Adolescents’ dietary patterns, their drivers and association with double burden of malnutrition in adolescents: a cross-sectional study in Kenya’s urban slums

BackgroundThe double burden of malnutrition (DBM) during adolescence is associated with growth and developmental impairment and risk of non-communicable diseases. There is limited evidence on adolescent’s dietary patterns (DPs), and how they contribute to DBM in urban low income contexts in sub Saharan Africa. This study assessed DPs of adolescents, their drivers and association with DBM in Kenya’s urban slums.MethodsAnthropometric, socio-demographic and dietary-intake data were collected through a cross-sectional survey of 621 adolescents from three major urban slums in Nairobi, Kenya. DPs were derived using principal component analysis. Multinomial-logistic-regression was used to assess the association between the DPs, individual and environmental factors and DBM.ResultsTwo DPs were identified: traditional DP (whole grains/cereals, rice, fruits, legumes/nuts, and water) and transitioning DP (refined cereals (maize/wheat), vegetables, meat, tea/coffee, sweet ultra-processed/deep fried snacks). Adolescents from Mathare (the largest slum) were more likely to adhere to the traditional DP (RRR = 3.43; 95% CI 1.85–6.37). Cultural background (Luo) had a positive association (RRR = 4.28; 95% CI 1.97–9.32), while longer residency in the slum (> 10 years) had a negative association (RRR = 0.47; 95% CI 0.25–0.90) with transitioning DP. The transitioning DP had a positive (non-linear) association with overweight/obesity in girls (RRR = 2.79; 95% CI 1.16–6.71). The DPs were not associated with thinness or stunting.ConclusionThe DPs indicate various stages of nutrition transition of adolescent diets, which are influenced by cultural background, neighbourhood and duration of stay in the slum. Transitioning DP may expose adolescents to the risks of overweight/obesity in the long-term.

Optimal incubation duration of liquid cultures for assessing culture negative conversion in patients with Mycobacterium avium complex and Mycobacterium abscessus pulmonary diseases.

Mycobacterial liquid culturing typically requires six weeks or longer, primarily because of the slow growth rate of Mycobacterium tuberculosis. This study aimed to evaluate the potential of shortening the duration of mycobacterial liquid culturing in healthcare settings with high prevalence rates of non-tuberculous mycobacteria. We retrospectively analyzed the relationship between mycobacterial species and time to positive testing of liquid cultures from sputum samples using the Mycobacteria Growth Indicator Tube system over a 3.5-year period beginning in July 2020 at a university hospital in Japan. We analyzed 15,147 sputum culture samples and found a 1.1% positivity rate for Mycobacterium tuberculosis complex, while the rates for Mycobacterium avium complex and Mycobacterium abscessus were 17.6% and 2.1%, respectively. The median time to positivity was 17 days for Mycobacterium tuberculosis complex, 9 days for Mycobacterium avium complex, and 4 days for Mycobacterium abscessus. Comparing a 4-week culture period with an eight-week period, the positivity rates for Mycobacterium avium complex and Mycobacterium abscessus were 97.0% and 99.4%, respectively. In settings with a high incidence of non-tuberculous mycobacteria, the basic liquid culturing period can be safely shortened to 4 weeks without significantly compromising detection sensitivity, except for the samples that are highly suspected to contain tuberculosis, extremely slow-growing mycobacteria, smear-positive, or nucleic acid amplification testing positive.

Autophagy and Akt-Stimulated Cellular Proliferation Synergistically Improve Antibody Production in CHO Cells.

Over the past decade, engineered producer cell lines have led 10-fold increases in antibody yield, based on an improved understanding of the cellular machinery influencing cell health and protein production. With prospects for further production improvements, increased antibody production would enable a significant cost reduction for life-saving therapies. In this study, we strategized methods to increase cell viability and the resulting cell culture duration to improve production lifetimes. By overexpressing the cell surface adenosine A2A receptor (A2AR), the Akt pathway was activated, resulting in improved cellular proliferation. Alternatively, by inducing autophagy through temperature downshift, we were able to significantly enhance cellular-specific productivity, with up to a three-fold increase in total antibody production as well as three-fold higher cell-specific productivity. Interestingly, the expression levels of the autophagy pathway protein Beclin-1 appeared to correlate best with the total antibody production, of autophagy-related proteins examined. Thus, during cell clonal development Beclin-1 levels may serve as a marker to screen for conditions that optimize antibody titer.

Polysaccharide with anticancer activity from Grifola frondosa cultured in industrial wastewater of Agaricus bisporus.

Culture conditions for Grifola frondosa using Agaricus bisporus industrial wastewater as a medium were optimized using Plackett-Burman and Box-Behnken methodologies. Plackett-Burman screening identified culture temperature, shaking speed, and wastewater solubility as the key factors influencing G. frondosa biomass. The Box-Behnken design then established optimal fermentation conditions: 23.7 °C, 202 rpm shaking speed, 10.4 % wastewater solubility, pH 6, 150 mL culture volume in a 250 mL flask, 9-day culture duration, and 10 % inoculation volume. Polysaccharides were isolated and purified from both the mycelia and the culture medium. UV and molecular weight analysis determined that the polysaccharides AIPGF01, NEPGF01, AEPGF01, and NIPGF01 were homogeneous, with molecular weights of 234.7 kDa, 125.2 kDa, 80.0 kDa, and 48.6 kDa, respectively. GC revealed that NIPGF01 and AIPGF01 contained only glucose, while NEPGF01 and AEPGF01 comprised both mannose and glucose in ratios of 12.37:83.69 and 10.06:85.78, respectively. FT-IR confirmed the presence of β-glycosidic linkages in all polysaccharides. NIPGF01 exhibited strong anticancer activity, significantly inhibiting human gastric cancer (MGC80-3) and liver cancer (Hep G2) cells. Apoptosis rates at 50 μg/mL, 200 μg/mL, and 800 μg/mL of NIPGF01 were 38.5 ± 3.5 %, 61.4 ± 4.3 %, and 79.2 ± 5.5 %, respectively. Flow cytometry indicated that NIPGF01 induced apoptosis in MGC80-3 cells by arresting them in the S and G2 phases, leading to a marked reduction in G1 phase cells.

Hypergravity as a Possible Way of Bone Tissue Engineering in Osteoblastic Differentiation from Mesenchymal Stem Cells: A Systematic Review

Background: Tissue engineering development has become a highlight in recent decades. One of the key areas of focus is producing mature bone tissue to overcome orthopedic problems, such as bone defects. Various cultures have been implemented on stem cells; some induce osteoblastic differentiation markers, while others have the opposite effect. Microgravity has been proven in several studies to inhibit the expression of osteogenic differentiation markers. Conversely, hypergravity is expected to have the opposite impact, supporting stem cells in the osteogenesis pathway. Methods: A literature search was conducted using online databases including Sciencedirect, PubMed, and Proquest, covering the period from 2008 to 2022. This search considered only experimental studies published in English. The keywords used in this research were "hypergravity" and "mesenchymal stem cell." All acquired data were processed and analyzed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (2020). Results: Initially, 190 studies were collected from online databases based on relevant keywords. After screening, 5 studies were included in the final analysis, focusing on hypergravity treatment and its effects on mesenchymal stem cells (MSCs). Conclusion: Hypergravity shows a significant and strong impact on osteoblastic differentiation. This study revealed that a gravity force of 30G and a culture duration of 7 to 14 days are the most optimal combination for inducing osteoblastic differentiation in MSCs.

Stocking Hatchery-Produced Mola (<i>Amblypharyngodon mola</i>) Seed in Carp Polyculture Ponds: A Performance Assessment in Odisha, India

Small indigenous fish species (SIS) such as mola (Amblypharyngodon mola) regarded as natural ‘superfoods,’ due to their high nutrient content, offer a promising avenue for addressing the nutritional deficiencies in India. Over the past decade, integrating mola into conventional carp polyculture systems has emerged as a promising nutrition-sensitive innovation, significantly enhancing micronutrient intake, particularly for women and children. However, the lack of hatchery-reared mola seed has hindered the sustainability of this approach. Recently, this bottleneck was addressed by developing a hatchery-based mass seed production protocol for mola, which was introduced into carp farming systems on a pilot basis. This study evaluates the performance of hatchery-produced mola seed inhomestead carp polyculture ponds in Odisha, India by using a field survey. Of the surveyed farmers, 24% purchased mola spawn, while 76% opted for mola fry. The average pond size of surveyed farmers was 0.32 ha with an average culture duration of 9months. Farmers stocked mola spawn at an average density of 283,583/ha and fry at 22,190/ha. Results showed that 93% of farmers achieved successful mola production, with an average mola yield of 194 kg/ha and total production of 2831 kg/ha. Molacontributed an average of 9% to the total fish production reaching a maximum contribution of up to 26%. The mola fish contributed significantly to the household in terms of additional revenue. Household consumption averaged 14 kg of mola per year, with a per capita consumption of 3 kg. Increasing pond size and optimizing stocking density emerge as critical factors positively impacting mola production and income. Overall, this study demonstrates the potential of hatchery-produced mola seed in promoting nutrition-sensitive aquacul-ture in Odisha, offering valuable insights for its scaling up.

Gravfeltet i Hålandsmarka, Time kommune, SV-Norge

In 2008, a pre-development excavation examined a grave field on the ridge of Hålandsmarka. The burial ground was in use from the Early Bronze Age to the end of the Iron Age, and the excavation provided an opportunity to gain new insights into the long-term use of a grave field. The Hålandsmarka excavation allowed archaeologists to explore not only reuse of the grave field throughout this period, but also variations in burial form, treatment of the corpse, and grave goods over shorter timespans. The aim of the project has been to challenge the normative approach to graves as containers of a single burial event, and call attention to construction processes and other practices demonstrating the repeated use and transformation of the graves and their surroundings. Graves can be viewed as places of recurring engagement between the living and the dead, and as opportunities to explore the importance, duration and entanglement of material culture. Hålandsmarka should not be considered as a unique site: the results presented here are heavily dependent on our choice of archaeological methods, and we have the responsibility to be aware of the potential for complexity in both past and present practices when planning future excavations. The findings from Hålandsmarka call attention to the multi-temporal and gathered nature of grave fields in particular, as well as the archaeological record in general.

Piezoelectric silk fibroin nanofibers: Structural optimization to enhance piezoelectricity and biostability for neural tissue engineering

Silk fibroin (SF) has garnered significant interest in tissue engineering due to its excellent biocompatibility and bioactivity. Notably, SF exhibits piezoelectric properties that can be harnessed to electrically stimulate cells, enhancing tissue morphogenesis. However, a systematic approach to control SF's piezoelectricity and biodegradation, and its application in neural morphogenesis, has not been fully explored. In this study, SF was electrospun into nanofibers of various sizes and subjected to a post-electrospinning chemical treatment to examine the effects of phase composition, especially the β-sheet formation, on the piezoelectricity and biostability of SF. The optimized SF scaffolds having a nanofiber diameter of 750 nm and a scaffold thickness of 250 µm allowed for the maintenance of nanofibrous structure for at least 6 weeks in aqueous environments while maintaining piezoelectric potential outputs of at least 200 mVp-p. This enabled cell membrane depolarization over an extended cell culture duration, facilitating the functional maturation of human neural stem cells (hNSCs). Specifically, acoustic piezo-activation of the SF scaffolds, resulting in mechano-electrical stimulation (MES) of the cells, accelerated their differentiation into neurons, astrocytes, and especially oligodendrocytes, leading to robust axon myelination within two weeks. Furthermore, MES enhanced neural connectivity and functional maturity, as evidenced by significant increases in excitatory and inhibitory synapse formation (46 % and 58 %, respectively), action potential amplitude (252 %), and velocity (210 %) compared to static control conditions. These findings demonstrate the potential of biodegradable electrospun SF nanofibers with balanced piezoelectricity and biostability for advancing neural tissue engineering.

Blood-rsCDM: a new rapid and simplified carbapenemase detection method for detecting carbapenemases in Enterobacterales directly from positive blood cultures

ObjectiveWe aim to validate and evaluate a new rapid and simplified method, called Blood-rsCDM, for the detection and characterization of carbapenemase using 3-aminophenylboronic acid (APBA) and ethylenediaminetetraacetic acid (EDTA) β-lactamase inhibitors from positive blood cultures.MethodWe utilized a panel of 172 Enterobacterales strains, including blaKPC (77), blaNDM (48), blaIMP (9), blaVIM (2), blaOXA-181 (2), blaKPC and blaNDM (6), as well as 28 carbapenem-susceptible Enterobacterales isolates, to assess the performance of Blood-rsCDM and the EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined using specific inhibitors at 4 h and 6 h by Blood-rsCDM.ResultsBlood-rsCDM exhibited a sensitivity of 97.9% at both time points, with a specificity of 100%, regardless of the culture duration. The sensitivity of eCIM was 94.4%, with a specificity of 100%. Blood-rsCDM accurately characterized KPC-producing isolates as 77/77, metallo-β-lactamases (MBLs) as 58/59, and KPC and NDM carbapenemases as 6/6 at 4 h. There was no difference in results between the 4 h and 6 h time points. However, Blood-rsCDM could not differentiate OXA-181-producing strains. For eCIM, the characterization numbers for KPC-, OXA-181-, and MBLs-producing strains were 77/77, 2/2, and 57/59, respectively, but it failed to detect the coproduction of KPC and NDM isolates.ConclusionBlood-rsCDM accurately discriminates carbapenemase within 4 h and is capable of directly differentiating multi-enzyme (KPC and NDM) presence from positive blood culture broths. Therefore, Blood-rsCDM represents a rapid, simple, easy-to-read, and accurate tool that can be utilized in resource-limited settings.

Microbial Community Structure in the Taklimakan Desert: The Importance of Nutrient Levels in Medium and Culture Methods.

Although the Taklimakan Desert lacks the necessary nutrients and conditions to support an extensive ecosystem, it is a treasure trove of extremophile resources with special structures and functions. We analyzed the bacterial communities using oligotrophic medium and velvet cloth replicate combined with an extended culture duration. We isolated numerous uncultured microorganisms and rare microorganisms belonging to genera not often isolated or recently described, such as Aliihoeflea, Halodurantibacterium, and Indioceanicola. A total of 669 strains were isolated from the soil of the Taklimakan Desert, which were classified into 5 phyla, 7 classes, 25 orders, 42 families, 83 genera, and 379 species. Among them, 148 strains were potential new species. Our data show that even when working with samples from extreme environments, simple approaches are still useful for cultivating stubborn microbes. Through comparing the isolation effects of different nutrient levels on microbial diversity and abundance, the results show that reducing the nutrient level of the medium was more conducive to improving the culturability of microorganisms in low-nutrient environments, while the high-nutrient medium was more suitable for the isolation of dominant fast-growing strains. This study helps to better reflect the diversity of microbial resources and lays a foundation for the further research and utilization of soil microbial resources in the Taklimakan Desert.

12331 Overcoming Limitations Of Identifying Proliferating β-cells In Dispersed Primary Islets Culture For Drug Discovery

Abstract Disclosure: H. Xu: None. S. Rebecca: None. S. Lee: None. J.P. Annes: None. Tremendous efforts have been made with screening platforms to identify islet β-cell mitogens that may be used to treat diabetes. However, promising compounds induce profound proliferation of islet non-β-cells in available screening platforms, calling for both β-cell targeted drug discovery and rigorous identification of β-cell identity to reduce false positivity. First, we used an established drug discovery dispersed islet cell-based platform(2D) to test the replication-promoting activity of GNF2133, a selective DYRK1A inhibitor that promotes β-cell proliferation, in isolated islets from human, rat and mouse. After 72 h in vitro culture, replication of non-β cells (PDX/Insulin negative) contributed to over 60% of the total replicating cells (Ki67+) in all species (human: 64%, rat: 71%, mouse: 67%). Mouse islets, which exhibit the greatest non-β-cell replication response upon GNF2133 treatment (4-fold above baseline, [50 nM]), were selected for subsequent studies. Next, we evaluated identification efficiency of β cell markers in this image-based platform. β cells, selected by the quantification value of insulin intensity, comprised 60-70% of total cell population at 48 h, 50-60% at 72 h and 20-30% at 96 h (p< 0.0001 for both comparisons: 48 h vs 72 h, 72 h vs 96 h). Basal replication of β-cells (Insulin+Ki67+) was 2.80%, 2.32%, 0.85% at 48 h, 72 h and 96 h, respectively. By comparison, the proportion of non-β cells showed an increasing trend as 2.48%, 6.49%, 8.68%, correspondingly. Identification with PDX1, another β cell marker, exhibited the same findings. Prolonged culture of dispersed islet cells, as these results indicate, favors growth of non-β-cells over β-cells. In addition, proliferating β-cells express lower levels of differentiation markers makes their discernment from replicating non-β cells more challenging. 窗体顶端 To minimize the duration of monolayer culture, replication induction was measured using intact islets with addition of BrdU during the last 24 h of culture. Then, islets were either directly fixed (3D) or dispersed and cultured for an additional 36 hour before being fixed (3D+2D). Immunostaining showed 70-80% PDX1 positive cells from both methods, which resembles in vivo β cell distribution within primary mouse islets. Basal β-cell replication in 3D+2D method was 0.30%, similar to in vivo BrdU labelling level. Upon mitogen treatment, PDX1+BrdU+ cells increased to 6.57% in 3D+2D method, slightly lower than 10.02% in 3D assay (p<0.05) yet significantly higher than 1.15% in 2D assay (<0.01). In summary, we developed a 3D+2D assay that enhances β-cell survival and the β-cell replication-induction response. This platform, while offering reduced throughput, provides a robust platform for discovery of β-cell replication-promoting compounds. Further validation with human islets is needed. Presentation: 6/2/2024

Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

A dynamically loaded ex vivo model to study neocartilage and integration in human cartilage repair.

Articular cartilage injuries in the knee can lead to post-traumatic osteoarthritis if untreated, causing debilitating problems later in life. Standard surgical treatments fail to ensure long lasting repair of damaged cartilage, often resulting in fibrotic tissue. While there is a vast amount of research into cartilage regeneration, integrating engineered implants with cartilage remains a challenge. As cartilage is a load bearing tissue, it is imperative to evaluate tissue repair strategies and their ability to integrate under mechanical loading. This work established a dynamically loaded ex vivo model of cartilage repair using human cartilage explants. The model was used to assess the efficacy of a stem cell therapy delivered in a bioadhesive hydrogel comprised of photocrosslinkable gelatin methacryloyl (GelMA) and microbial transglutaminase to repair the model defect. Extensive neocartilage production and integration were observed via histology and immunohistochemistry after 28days chondrogenic culture. Analysis of culture media allowed monitoring of glycosaminoglycan and type II collagen production over time. A mechanical assessment of integration via a push out test showed a 15-fold increase in push out strength over the culture duration. The model was successful in exhibiting robust chondrogenesis with transglutaminase or without, and under both culture conditions. The work also highlights several limitations of ex vivo models and challenges of working with bioreactors that must be overcome to increase their utility. This ex vivo model has the potential to delay the need for costly pre-clinical studies and provide a more nuanced assessment of cartilage repair strategies than is possible in vivo.

Ultrasound Induces Similar Temporal Endothelial Expression Patterns of eNOS and KLF2 as Normal Flow

ObjectiveTo determine the sensitivity of vascular endothelial cells to long durations of low-intensity pulsed ultrasound (LIPUS) compared to normal flow and identify the duration that maximizes expression of two mechanosensitive genes related to healthy endothelial function, endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 2 (KLF2). MethodsCustom ultrasound exposure tanks were developed and the acoustic field was characterized. Human umbilical vein endothelial cells were seeded into culture plates and exposed to LIPUS at a frequency of 1 MHz and acoustic pressure of 217 kPa for 20 min, 1 h, 6 h, 9 h, or 24 h. As a comparator, other cells were exposed to normal flow. RT-qPCR was used to assess mRNA expression of eNOS and KLF2. ResultsMaximum eNOS and KLF2 expression occurred at 6 h and was localized to the beam path. Both genes exhibited qualitatively similar patterns of expression under LIPUS compared to normal flow. LIPUS induced a more rapid beneficial response compared to normal flow, but flow induced higher expression of both genes. eNOS expression after 6 h of LIPUS was dependent on RNA yield and culture duration prior to experiments. ConclusionEndothelial cells exposed to longer durations of LIPUS than typically employed exhibited greater expression of beneficial genes. The temporal gene expression patterns resulting from LIPUS and normal flow suggest activation of similar signaling pathways. However, LIPUS also caused increased RNA yield that may be linked to proliferation, which would suggest more of a wound healing than atheroprotective phenotype.

Establishment of an in-vitro inflammatory bowel disease model using immunological differentiation of Caco-2 cells

Studies on intestinal cell differentiation, particularly in dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD), have predominantly focused on the disruption of intestinal crypts and suppressive effects on the intestinal microbiota; however, repeated administration of DSS is required to induce inflammatory pathology, and there is a lack of observation of early responses and consideration of differentiation stages. Although colonic adenocarcinoma (Caco-2) cells can be used as intestinal cell models, research on these cells in an immature state is limited. We, therefore, investigated the relationship between Caco-2 cell culture duration and immunological differentiation using α-defensin5 (DEFA5) as an indicator of intestinal immunity and differentiation. Changes in protein and gene expression levels in response to DSS were examined at each differentiation stage. Expression of immune- and differentiation-related proteins, including DEFA5 and lysozyme, was evident from Day 8 of culture. Immune responses to DSS varied with the differentiation stage, affecting cell viability and cytokine expression.•Caco-2 cell culture duration correlates with the differentiation stage of Paneth cells.•DSS exposure elicits different effects depending on the differentiation stage.•Our in-vitro model of IBD facilitates the characterization of the cell differentiation process and provides a methodology to help elucidate the causal mechanisms of IBD.