High Cell Concentrations Research Articles

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification.Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL).Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained.Key points• Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield• Use of a TFDF system allowed continuous virus harvesting and clarification• TFDF perfusion system has great potential towards the establishment of an intensified vector production

Measurement and control of foam generation in a mammalian cell culture.

Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody-producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE-15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE-15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE-15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >106 cells/mL. Supplementation of the cultures with SE-15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess.

3D Cell Culture Method in Channel-Free Water-in-Oil Droplets.

A new channel-free water-in-oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long-term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO-based automated 3D cell culture system is developed, it will be a useful tool for organoid research.

Pulmonary passage of canine adipose tissue-derived mesenchymal stem cells through intravenous transplantation in mouse model.

The intravenous administration of adipose tissue-derived mesenchymal stem cells (AdMSCs) in veterinary medicine is an attractive treatment option. On the other hand, it can result in severe complications, including pulmonary thromboembolism (PTE). The present study assessed the occurrence of PTE after the intravenous infusion of canine AdMSCs (cAdMSCs) into experimental animals. Five-week-old male BALB/c hairless mice were categorized into groups labeled A to G. In the control group (A), fluorescently stained 2 × 106 cAdMSCs were diluted in 200 μL of suspension and injected into the tail vein as a single bolus. The remaining groups included the following: group B with 5 × 106 cells, group C with 3 × 106 cells, group D with 1 × 106 cells, group E with 1 × 106 cells injected twice with a one-day interval, group F with 2 × 106 cells in 100 μL of suspension, and group G with 2 × 106 cells in 300 μL of suspension. Group D achieved a 100% survival rate, while none of the subjects in groups B and C survived (p = 0.002). Blood tests revealed a tendency for the D-dimer levels to increase as the cell dose increased (p = 0.006). The platelet count was higher in the low cell concentration groups and lower in the high cell concentration groups (p = 0.028). A histological examination revealed PTE in most deceased subjects (96.30%). PTE was verified, and various variables were identified as potential contributing factors, including the cell dose, injection frequency, and suspension volume.

Collective production of hydrogen sulfide gas enables budding yeast lacking MET17 to overcome their metabolic defect.

Assimilation of sulfur is vital to all organisms. In S. cerevisiae, inorganic sulfate is first reduced to sulfide, which is then affixed to an organic carbon backbone by the Met17 enzyme. The resulting homocysteine can then be converted to all other essential organosulfurs such as methionine, cysteine, and glutathione. This pathway has been known for nearly half a century, and met17 mutants have long been classified as organosulfur auxotrophs, which are unable to grow on sulfate as their sole sulfur source. Surprisingly, we found that met17Δ could grow on sulfate, albeit only at sufficiently high cell densities. We show that the accumulation of hydrogen sulfide gas underpins this density-dependent growth of met17Δ on sulfate and that the locus YLL058W (HSU1) enables met17Δ cells to assimilate hydrogen sulfide. Hsu1 protein is induced during sulfur starvation and under exposure to high sulfide concentrations in wild-type cells, and the gene has a pleiotropic role in sulfur assimilation. In a mathematical model, the low efficiency of sulfide assimilation in met17Δ can explain the observed density-dependent growth of met17Δ on sulfate. Thus, having uncovered and explained the paradoxical growth of a commonly used "auxotroph," our findings may impact the design of future studies in yeast genetics, metabolism, and volatile-mediated microbial interactions.

Failure and out of Specification Manufacturing of Autologous CAR-T Cells Could be Associated with a High Concentration of Total Nucleated Cells, CD3 + Cells and Neutrophils in the Apheresis Product

Manufacturing capacities of commercial autologous chimeric antigen receptor (CAR)-T cells have been significantly increased during the last years, allowing treatment of a larger patient cohort. However, this was associated with the emergence of several failures and out of specifications (OOS), resulting in treatment delay. The aim of our study was to identify early indicators related to apheresis products that can predict CAR-T cells manufacturing failure or OOS. To this end, we analyzed manipulation processes of cellular starting materials (conservation, freezing, cryopreservation) and quality controls (QC) including count and/or concentration of total nucleated cells (TNC), CD3 +, CD4 +, CD8 + cells, platelets, monocytes and neutrophils. We analyzed 802 apheresis products that were collected for the manufacturing of axicabtagene ciloleucel (axi-cel, n=303), tisagenlecleucel (tisa-cel, n=403), brexucatagene autoleucel (brexu-cel, n=44) and idecabtagene vicleucel (ide-cel, n=52). This cohort was classified into 3 groups: failure (CAR-T cells not produced), OOS (CAR-T cells produced but non-compliant) and compliant. We identified 21 failures and 24 OOS, resulting in either no treatment or a significant delay when the OOS were injected (57.6±17.5d versus 45.9±16.3d in compliant group, p=0.0405). Among each CAR-T cells product, 13 (4.3%) axi-cel, 30 (7.4%) tisa-cel, 1 (2.3%) brexu-cel and 1 (1.9%) ide-cel were concerned. The main causes of failures were low viability (33.3%), non-compliant dose (28.6%), microbiological contamination (23.8%) and formulation concerns (14.3%). In OOS, low viability (41.7%), low dose (29.2%), microbiological contamination (4.2%), formulation (12.5%) and safety concerns (8.3%; i.e high vector copy number) were identified. 17 products (13 failures and 4 OOS) were successfully remanufactured without an increased delay comparing to compliant products (47.5±15.9 and 47.3±15.1 respectively versus 45.9±16.3 d). To identify risk factors of manufacturing failure or OOS due to non-compliant viability and/or dose, we analyzed processing and QC of collected cellular starting materials. OOS were associated with a high concentration of TNC (146.7±94.3 vs 61.6±36.1 x10 6/mL, p=0.009) and CD3 + cells (88.6±76.5 vs 26.0±21.9 x10 6/mL, p=0.0112) in cell collections, while failures were observed in apheresis with high neutrophils concentration (14.3±15.03 x10 3/µL vs 6.0±7.6 x10 3/µL, p=0.0195). Interestingly, neutrophils were significantly decreased in apheresis collected for CAR-T cells remanufacturing which succeed (p=0.0156). This preliminary study showed that high TNC, CD3 + cells and neutrophils concentration in the apheresis product might be predictive factors of failures and/or OOS of commercial autologous CAR-T cells manufacturing.

Cultivation of Brackish Water Microalgae for Pig Manure Liquid Digestate Recycling

Sustainability and recycling of agricultural and animal husbandry waste are important. Pig manure contains relatively high concentrations of organic matter, such as nitrate-nitrogen, ammonia nitrogen, and phosphate, and a direct discharge can cause environmental pollution. This study successfully culturing four brackish water microalgae, including Tetraselmis chuii, Isochrysis galbana, Chlorella vulgaris, and Proteomonas sulcata, by using a diluted digestate solution of pig manure (NH3 concentration is lower than 10 mg/L). These microalgae can reach their highest cell concentration within 3–7 days of cultivation. The small microalgae, C. vulgaris and I. galbana, reached a cell density of 2.5 × 107 and 1.5 × 107, respectively, whereas lower cell densities were documented for large microalgae T. chuii (1.4 × 106) and P. sulcata (1.6 × 106). Our findings highlight the feasibility of sustainable treatment of animal manure using brackish water microalgae. These results provide opportunities to reduce freshwater usage and environmental pollutions, and support microalgae production for further aquaculture application.

The Ying and Yang of Ganglioside Function in Cancer.

The plethora of information about the expression of cancer cell-associated gangliosides, their role(s) in signal transduction, and their potential usefulness in the development of cancer treatments makes this an appropriate time to review these enigmatic glycosphingolipids. Evidence, reflecting the work of many, indicates that (1) expression of specific gangliosides, not generally found in high concentrations in most normal human cells, can be linked to certain types of cancer. (2) Gangliosides can affect the ability of cells to interact either directly or indirectly with growth factor receptors, thereby changing such things as a cell's mobility, rate of proliferation, and metastatic ability. (3) Anti-ganglioside antibodies have been tested, with some success, as potential treatments for certain cancers. (4) Cancer-associated gangliosides shed into the circulation can (a) affect immune cell responsiveness either positively or negatively, (b) be considered as diagnostic markers, and (c) be used to look for recurrence. (5) Cancer registries enable investigators to evaluate data from sufficient numbers of patients to obtain information about potential therapies. Despite advances that have been made, a discussion of possible approaches to identifying additional treatment strategies to inhibit metastasis, responsible for the majority of deaths of cancer patients, as well as for treating therapy-resistant tumors, is included.

Responses of <i>Unio pictorum</i> to the Presence of Toxic and Non-Toxic Strains of <i>Microcystis aeruginosa</i>

In order to assess the impact of cyanobacteria on mollusks under experimental conditions, the interaction of toxic and non-toxic strains of cyanobacteria Microcystis aeruginosa (Kützing) Kützing and bivalve mollusks Unio pictorum (L., 1758) was studied. Cyanobacteria have a negative effect on bivalve mollusks: 40% death of mollusks and deterioration of their adaptive capacity were recorded when co-cultivated with M. aeruginosa at a high cell concentration. At the same time, there was no difference in the mortality of mollusks incubated with toxic and non-toxic cyanobacteria. A decrease in the content of microcystin LR in the presence of bivalves was revealed. No statistically significant increase in the number of cyanobacteria in the water was noted after transit passage through the digestive system of bivalves.

Comparative physiological study of sea cucumbers from eastern waters of United States.

Sea cucumbers, belonging to the phylum Echinodermata, are known to possess valuable bioactive compounds that have medicinal properties. In several countries, such as Korea, China, and Japan, they are cultured in the aquaculture industries for food and medicinal purposes. Research has shown that different species of sea cucumbers each possesses unique medicinal values. As a result, we strive towards finding species with better health resilience in aquaculture system to be cultured for nutritional and medicinal purposes. In this paper, we compared the physiological and immunological parameters of three species of sea cucumbers, Cucumaria frondosa (C. frondosa), Isostychopus badionotus (I. badionotus), and Pentacta pygmaea (P. Pygmaea) from the waters of the eastern United States as they have not been studied extensively. Four different cells of sea cucumbers, phagocytic, red spherule, white spherule, and vibratile cells, that contribute to their immunity were counted. C. frondosa exhibited the highest concentrations of phagocytic cells, white spherule cells, and vibratile cells, compared to the two other species. Due to its high phagocytic cell concentration, the highest phagocytic capacity was seen in C. frondosa although it was not statistically significant. We also observed that C. frondosa had the highest total cell count and the highest concentration of coelomic protein among the three species. Lastly, C. frondosa possessed the highest lysozyme activity. Taken together, we concluded that C. frondosa is the best of the three species compared to be reared in the aquaculture systems for use in the food and biomedicine industries due to its immunological and physiological properties.

Non-Deterministic Factors Affect Competition Between Thermophilic Autotrophs from Deep-Sea Hydrothermal Vents

Hydrothermal vents provide windows into the rocky subseafloor on Earth and serve as terrestrial analog sites for extraterrestrial environments. By studying patterns of community assembly in hydrothermal vents and using geochemical models, we can better understand how the deep-sea biosphere contributes to local and global biogeochemical cycling and gather valuable information about how similar communities may arise on Earth and beyond Earth. One prevailing thought is that vent microbial community assembly is driven by deterministic factors such as the thermodynamic favorability of redox reactions. We hypothesized that subsurface microbial communities may also be significantly influenced by other factors, such as differential cell yields, varying optimal growth temperatures, and stochasticity. At Axial Seamount in the Pacific Ocean, H2-consuming methanogens of the genera Methanocaldococcus (Topt 82°C) and Methanothermococcus (Topt 65°C) and H2-consuming sulfur reducers of the genus Desulfurobacterium (Topt 72°C) are the most abundant autotrophs that grow optimally at or above 65°C (Fortunato et al. 2017). At one low-temperature hydrothermal vent site, Marker 113, methanogens are the predominant thermophilic autotrophs while at another site, Marker 33, thermophilic autotrophic sulfur reducers predominate. There is no apparent geochemical or thermodynamic explanation for the differences in community composition. In this study, we performed a series of co-culture competition experiments using Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Desulfurobacterium thermolithotrophum HR11 as representative methanogens and sulfur reducers common to hydrothermal vents to explain the variations in community composition between thermophilic autotrophs. M. jannaschii increases its cell yield (cells produced per mole of CH4 produced) when grown on very low H2 concentrations as part of a growth rate-growth yield tradeoff (Topçuoğlu et al. 2019). This increase in cell yield could provide methanogens with a competitive growth advantage over H2-consuming sulfur reducers, who otherwise catalyze a more thermodynamically favorable growth reaction. Competition co-culture experiments were conducted between M. jannaschii and D. thermolithotrophum at 72°C and between M. thermolithotrophicus and D. thermolithotrophum at 65°C, both at 1:1 ratios and initial aqueous H2 concentrations of 1.2 mM (high H2) and 85 μM (low H2) to determine the effects of temperature and H2 availability on autotroph competition. For both methanogens, the growth rate, maximum cell concentration, and total CH4 produced decreased when they were grown in co-culture, at low H2, or both relative to monocultures grown with high H2. The methanogen cell yields generally increased in co-culture and at low H2. At both experimental temperatures, the growth rate of D. thermolithotrophum remained unchanged in co-culture and at low H2 relative to monocultures but the maximum cell concentration decreased in co-culture relative to monocultures at both H2 concentrations. However, at low H2, both in mono- and co-culture, there was no detectable H2S produced by the sulfur reducer suggesting a significant shift in growth yield. At both temperatures and H2 concentrations, the sulfur reducer reached higher cell concentrations than the methanogens. Stochasticity or vent fluid chemistry could lead to early colonization of a vent by methanogens followed by niche exclusion of autotrophic sulfur reducers due to a numerical advantage of the methanogens. Therefore, competitive co-culture experiments were run as before at high H2 with varying initial methanogen:sulfur reducer ratios. At 72°C, D. thermolithotrophum reached the same maximum cell concentration and produced the same amount of H2S in monoculture and co-culture even when the methanogens initially outnumbered the sulfur reducer up to 10,000-fold. M. jannaschii reached a lower maximum cell concentration and produced less CH4 in all co-cultures relative to growth in monoculture. At 65°C, D. thermolithotrophum reached the same maximum cell concentrations and produced the same amount of H2S in monoculture and co-culture when the methanogens initially outnumbered the sulfur reducers up to 100-fold. However, when the methanogens initially outnumbered the sulfur reducers 1,000-fold, M. thermolithotrophicus grew as well as in monoculture and the maximum cell concentration and amount of H2S produced by D. thermolithotrophum was significantly lower than in monoculture and the other co-culture conditions. In conclusion, both methanogens and sulfur reducers shift their redox reactions away from CH4 and H2S production, respectively, and towards biomass production when H2 is limiting. This should be accounted for in thermodynamic predictive models. Furthermore, a combination of growth temperatures lower than the optimum of sulfur reducers and high initial methanogen cell concentrations relative to sulfur reducers can lead to a long-term predominance of methanogens over autotrophic sulfur reducers in vent environments through niche exclusion.

Chemical Composition and Antimicrobial Activity of Lavender (Lavandula angustifolia Mill.), Peppermint (Mentha piperita L.), Raspberry Seed (Rubus idaeus L.), and Ylang-Ylang (Cananga odorata (Lam.) Essential Oils—Towards Hurdle Technologies in the Production of Chocolate Mousse

The growing consumer demand for the development of functional foods with a number of benefits for the consumer has led to a considerable increase in the studies focused on examining different natural agents to be included in the composition of newly developed functional foods. The chemical compositions of the essential oils (EOs) of lavender (Lavandula angustifolia Mill.), peppermint (Mentha piperita L.), raspberry seed (Rubus idaeus L.), and ylang-ylang (Cananga odorata (Lam.)) were determined using gas chromatography with a mass selective detector (GC-MS). The antibacterial and antifungal activities of these EOs were examined using a high-throughput 96-well microplate bioassay procedure and the MICs of each EO against each test microorganism were determined. The results indicated significant antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Salmonella abony NTCC 6017, Pseudomonas aeruginosa NBIMCC 1390, Bacillus subtilis ATCC 19659, Penicillium chrysogenum ATCC 28089, Fusarium moniliforme ATCC 38932, Aspergillus niger ATCC 1015, and Aspergillus flavus ATCC 9643. To explore their potential applications in food preservation, model chocolate mousse food emulsions were prepared that incorporated EOs and/or selected probiotic lactobacilli strains in both free and encapsulated forms. The inclusion of EOs and/or probiotic lactobacilli resulted in enhanced microbial safety and an extended shelf life. Furthermore, the chocolate mousse variants that were biopreserved with the inclusion of probiotic lactobacilli maintained a high viable lactobacillus cell concentration throughout the storage period. As a result, these products would not only be suitable as functional probiotic foods but also as effective delivery vehicles for probiotic lactobacilli.

Cardiopulmonary protective properties of decorated Ag NPs on lignin modified magnetic nanoparticles mediated by Cydonia in CCD-19Lu, WI-38, BEAS-2B, HCASMC, HCAEC, and HPAEC cell lines

Advances in silver nanoparticles and nanotechnology, which can realize sensitive diagnostic modalities, efficient medical treatment, and better prognosis as well as less adverse effects on non-target tissues, provide an amazing window in the field of cardiopulmonary diseases. The present investigation describes a green procedure for the in situ immobilizing silver nanoparticles over calcium lignosulfonate modified Fe3O4 magnetic nanoparticles mediated by Cydonia root extract (Fe3O4@lignosulfonate-Ag NPs) and its catalytic and subsequent biological performances. The characterization of synthesized Fe3O4@lignosulfonate-Ag NPs were analyzed using XRD, VSM, ICP, SEM, EdaX, elemental mapping and TEM. In the medicinal part of the present research, the lung (CCD-19Lu, WI-38, and BEAS-2B) and heart (HCASMC, HCAEC, and HPAEC) cell viability was determined by trypan blue assay. The caspase activity colorimetric assay kit and Rhodamine123 fluorescence dye were used to determine the caspase-3 activity and mitochondrial membrane potential, respectively. Apoptosis and DNA fragmentation were determined by the TUNEL test. In addition, the inflammatory cytokines concentrations were evaluated by the Rat inflammatory cytokine assay kit. Fe3O4@lignosulfonate-Ag NPs-treated cell cutlers decreased significantly (p ≤ 0.01) the caspase-3 activity, inflammatory cytokines concentrations, and DNA fragmentation, and enhanced the mitochondrial membrane potential and cell viability in the high concentration of Methotrexate-treated CCD-19Lu, WI-38, BEAS-2B, HCASMC, HCAEC, and HPAEC cells. In the antioxidant test, the IC50 of Fe3O4@lignosulfonate-Ag NPs and BHT against DPPH free radicals were 168 and 54 µg/mL, respectively. Finally, Fe3O4@lignosulfonate-Ag NPs may be used as a cardiopulmonary protective supplement to treat cardiopulmonary diseases.

Methanethiol: A Scent Mark of Dysregulated Sulfur Metabolism in Cancer.

In order to cope with increased demands for energy and metabolites as well as to enhance stress resilience, tumor cells develop various metabolic adaptations, representing a hallmark of cancer. In this regard, the dysregulation of sulfur metabolism that may result in elevated levels of volatile sulfur compounds (VSCs) in body fluids, breath, and/or excretions of cancer patients has recently gained attention. Besides hydrogen sulfide (H2S), methanethiol is the predominant cancer-associated VSC and has been proposed as a promising biomarker for non-invasive cancer diagnosis. Gut bacteria are the major exogenous source of exposure to this foul-smelling toxic gas, with methanethiol-producing strains such as Fusobacterium nucleatum highly abundant in the gut microbiome of colorectal carcinoma (CRC) patients. Physiologically, methanethiol becomes rapidly degraded through the methanethiol oxidase (MTO) activity of selenium-binding protein 1 (SELENBP1). However, SELENBP1, which is considered a tumor suppressor, is often downregulated in tumor tissues, and this has been epidemiologically linked to poor clinical outcomes. In addition to impaired removal, an increase in methanethiol levels may derive from non-enzymatic reactions, such as a Maillard reaction between glucose and methionine, two metabolites enriched in cancer cells. High methionine concentrations in cancer cells may also result in enzymatic methanethiol production in mitochondria. Moreover, enzymatic endogenous methanethiol production may occur through methyltransferase-like protein 7B (METTL7B), which is present at elevated levels in some cancers, including CRC and hepatocellular carcinoma (HCC). In conclusion, methanethiol contributes to the scent of cancer as part of the cancer-associated signature combination of volatile organic compounds (VOCs) that are increasingly being exploited for non-invasive early cancer diagnosis.

Small volume bone marrow aspirates with high progenitor cell concentrations maximize cell therapy dose manufacture and substantially reduce donor hemoglobin loss

BackgroundBone marrow (BM) transplantation is a life-saving therapy for hematological diseases, and the BM harbors also highly useful (progenitor) cell types for novel cell therapies manufacture. Yet, the BM collection technique is not standardized.MethodsBenchmarking our collection efficiency to BM collections worldwide (N = 1248), we noted a great variability of total nucleated cell (TNC) yields in BM products (HPC-M) with superior performance of our center, where we have implemented a small volume aspirate policy. Thus, we next prospectively aimed to assess the impact of BM collection technique on HPC-M quality. For each BM collection (N = 20 donors), small volume (3 mL) and large volume (10 mL) BM aspirates were sampled at 3 time points and analyzed for cell composition.ResultsCompared to large volume aspirates, small volume aspirates concentrated more TNCs, immune cells, platelets, hematopoietic stem/progenitor cells, mesenchymal stromal cells (MSCs), and endothelial progenitors. Inversely, the hemoglobin concentration was higher in large volume aspirates indicating more hemoglobin loss. Manufacturing and dosing scenarios showed that small volume aspirates save up to 42% BM volume and 44% hemoglobin for HPC-M donors. Moreover, MSC production efficiency can be increased by more than 150%.ConclusionsWe propose to consider small volume BM aspiration as standard technique for BM collection.

Boronic Acids and Beyond: ROS-Responsive Prodrugs as Tools for a Safer and More Effective Cancer Chemotherapy

AbstractDespite significant scientific advances and the wide variety of available treatments, cancer remains a major cause of death worldwide. Chemotherapy, which is frequently one of the first-line treatments, frequently suffers from low selectivity to cancer cells, leading to the appearance of important side effects. Thus, it becomes imperative to develop a new generation of targeted alternatives that spare the healthy tissues by delivering the cytotoxic payloads safely and selectively to cancer cells. In this respect, prodrugs that are activated by tumor-specific stimuli have attracted significant attention. Despite being a hallmark of cancer and present in high concentrations in cancer cells, reactive oxygen species (ROS) have been rather underexplored as a stimulus for the preparation of targeted prodrugs, particularly when compared with an acidic pH or glutathione. Despite their lower expression, ROS have recently been gaining substantial consideration, with various ROS-responsive prodrugs already reported with meaningful performances both in vitro and in vivo. This review aims to provide critical insights into this strategy by discussing the various available functional groups (with an important focus on boronic acids and their esters), their mechanisms of action, examples of their applications, advantages, limitations, and future challenges.1 Introduction2 Boronic Acids and Boronate Esters2.1 Histone Deacetylase Inhibitors2.2 DNA Alkylating Agents2.3 Selective Estrogen Receptor Modulators and Selective Estrogen Receptor Degraders2.4 ROS Inducers2.5 Prodrugs Based on Other Types of Anticancer Drugs3 Other ROS-Responsive Moieties3.1 Thiazolidinones3.2 1,3-Oxathiolanes3.3 Selenium Ethers3.4 Sulfur-Containing ROS-Responsive Moieties4 Summary and Future Perspectives

Automated Raman feed-back control of multiple supplemental feeds to enable an intensified high inoculation density fed-batch platform process.

Biologics manufacturing is increasingly moving toward intensified processes that require novel control strategies in order to achieve higher titers in shorter periods of time compared to traditional fed-batch cultures. In order to implement these strategies for intensified processes, continuous process monitoring is often required. To this end, inline Raman spectroscopy was used to develop partial least squares models to monitor changes in residual concentrations of glucose, phenylalanine and methionine during the culture of five different glutamine synthetase piggyBac® Chinese hamster ovary clones cultured using an intensified high inoculation density fed-batch platform process. Continuous monitoring of residual metabolite concentrations facilitated automated feed-rate adjustment of three supplemental feeds to maintain glucose, phenylalanine, and methionine at desired setpoints, while maintaining other nutrient concentrations at acceptable levels across all clones cultured on the high inoculation density platform process. Furthermore, all clones cultured on this process achieved high viable cell concentrations over the course of culture, indicating no detrimental impacts from the proposed feeding strategy. Finally, the automated control strategy sustained cultures inoculated at high cell densities to achieve product concentrations between 5 and 8.3g/L over the course of 12days of culture.

Maturation and refinement of the maculae and foveae in the Anolis sagrei lizard

The fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of photoreceptor cells, which accounts for a large degree of visual acuity in primates. The maturation of this primate visual acuity area is characterized by the shallowing and widening of the foveal pit, a decrease in the diameter of the rod-free zone, and an increase in photoreceptor cells packing after birth. Maturation occurs concurrently with progressing age, increasing eye size, and retinal length/area. These observations have led to the hypothesis that the maturation of the fovea might be a function of mechanical variables that remodel the retina. However, this has never been explored outside of primates. Here, we take advantage of the Anolis sagrei lizard, which has a bifoveated retina, to study maturation of the fovea and macula. Eyes were collected from male and female lizards–hatchling, 2-month, 4-month, 6-month, and adult. We found that Anolis maculae undergo a maturation process somewhat different than what has been observed in primates. Anole macular diameters actually increase in size and undergo minimal photoreceptor cell packing, possessing a near complete complement of these cells at the time of hatching. As the anole eye expands, foveal centers experience little change in overall retina cell density with most cell redistribution occurring at macular borders and peripheral retina areas. Gene editing technology has recently been developed in lizards; this study provides a baseline of normal retina maturation for future genetic manipulation studies in anoles.

Application-Oriented Bulk Cryopreservation of Human iPSCs in Cryo Bags Followed by Direct Inoculation in Scalable Suspension Bioreactors for Expansion and Neural Differentiation.

Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells' fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.

Applicable pharmacokinetic study: Development and validation of bioanalytical LC-MS/MS method for the simultaneous quantification of cytarabine and glasdegib used for the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is characteristic by high concentrations of immature bone marrow myeloid cells. Glasdegib (DaurismoTM, GSB) in combination with low dose cytarabin (DepocytTM, CTB) have recently been approved by the US Food and Drug Administration for the treatment of newly diagnosed AML in patients over 75 years of age or who have comorbidities that are prohibitive to intensive induction chemotherapy. However, the pharmacokinetic characteristics of the CTB and GSB is yet unknown. This study developed a selective, sensitive and fast analytical method using liquid chromatography-electrospray ionization (ESI) with tandem mass spectrometry (LC-MS/MS) to simultaneously quantify of CTB and GSB in rat plasma using duvelisib as internal standard (IS). The LC-MS/MS instrument was performed in the ESI interface operating at positive ionization and multiple reaction monitoring (MRM) mode. The CTB and GSB with the IS were extracted from rat plasma by using cation exchange solid-phase extraction cartridge before the analysis. The cartridge gave high recovery rates for both drugs without interference from plasma endogenous. Chromatographic separation was performed on reversed-phase polar-100 column (100 mm × 2.1 mm, 3 µm), with an isocratic mobile phase consisted of a mixture of water containing 0.1% formic acid and acetonitrile (85:15, v/v, pH 3.2) at a flow rate of 0.4 mL/min and the process of analysis was run for 3.5 min. The developed method was validated as per the FDA guidelines over a linear concentration range of 5–3000, for both CTB and GSB with high correlation coefficient (r2 ≥ 0.99). The lower limits of detection (LLOD) were 2.0 ng/mL for both drugs. The overall recoveries of CTB and GSB from rat plasma were in the range of 93.93–101.43%, and the mean relative standard deviation (RSD) of the results was 2.99%. The validated-method was successfully applied for the first time, to a pharmacokinetic study on concurrent oral administration of CTB and GSB in rats (12.0 mg/kg of CTB and 8.5 mg/kg of GSB). The maximum plasma concentration (Cmax) for CTB and GSB was 2312.23 ± 448.26 ng/mL and 1710.61 ± 166.04 ng/mL achieved at 2.83 ± 0.14 h and 2.39 ± 0.10 h, respectively. The AUC0-∞ for CTB and GSB was found to be 8592.49 ± 1714.12 and 4527.67 ± 390.01 ng/mL.h; respectively. The elimination half-life (t1/2kel) of CTB and GSB in rat plasma, were determined to be 2.00 ± 0.19 h and 1.58 ± 0.13 h, respectively. The newly developed approach stands out for its high extraction recovery and absence of matrix interference. Findings further demonstrated the high sensitivity of the developed method, which allowed for effective routine tests in pharmacokinetic investigations with LLOD of 2.0 ng/mL and a run time of 3.5 min.