Cell Growth Profiles Research Articles

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Cytokine dysregulation in amnestic mild cognitive impairment

The pathophysiology of amnestic Mild Cognitive Impairment (aMCI) is largely unknown, although some papers found signs of immune activation. To assess the cytokine network in aMCI after excluding patients with major depression (MDD) and to examine the immune profiles of quantitative aMCI (qMCI) and distress symptoms of old age (DSOA) scores. A case-control study was conducted on 61 Thai aMCI participants and 60 healthy old adults (both without MDD). The Bio-Plex Pro human cytokine 27-plex test kit was used to assay cytokines/chemokines/growth factors in fasting plasma samples. aMCI is characterized by a significant immunosuppression, and reductions in T helper 1 (Th)1 and T cell growth profiles, the immune-inflammatory responses system, interleukin (IL)1β, IL6, IL7, IL12p70, IL13, GM-CSF, and MCP-1. These 7 cytokines/chemokines exhibit neuroprotective effects at physiologic concentrations. In multivariate analyses, three neurotoxic chemokines, CCL11, CCL5, and CXCL8, emerged as significant predictors of aMCI. Logistic regression showed that aMCI was best predicted by combining IL7, IL1β, MCP-1, years of education (all inversely associated) and CCL5 (positively associated). We found that 38.2% of the variance in the qMCI score was explained by IL7, IL1β, MCP-1, IL13, years of education (inversely associated) and CCL5 (positively associated). The DSOA was not associated with any immune data. An imbalance between lowered levels of neuroprotective cytokines and chemokines, and relative increases in neurotoxic chemokines are key factors in aMCI. Future MCI research should always control for the confounding effects of affective symptoms.

Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media.

Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40-80°C) and pH (7.6-10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.

Production of lignin monomeric alcohols and lipids from oil palm empty fruit bunch by a combination of alkaline nitrobenzene depolymerization and Lipomyces starkeyi bioconversion

The efficient valorization of lignocellulosic biomass into valuable chemicals represents a cornerstone for advancing sustainable bioeconomy practices. This study focuses on the innovative conversion of oil palm empty fruit bunch (OPEFB) into lignin monomeric alcohols and lipids through a novel two-step process combining alkaline nitrobenzene depolymerization and bioconversion by Lipomyces starkeyi. Initially, OPEFB undergoes alkaline nitrobenzene depolymerization to break down complex lignin structures into monomeric aldehydes. The resulting derivates serve as substrates for the bioconversion by L. starkeyi, which is competent at metabolizing lignin-derived compounds into higher-value lignin monomeric alcohols (syringyl, vanillyl, and 4-hydroxybenzyl alcohol) and lipids, simultaneously. The strategy delineates the optimization of depolymerization conditions, sodium hydroxide concentration, nitrobenzene effect, and reaction time to maximize the yield of depolymerized lignin suitable for bioconversion. Subsequently, the bioconversion process toward cell growth, lipid production, and fatty acid profiles in various fermentations by L. starkeyi was explored. The highest syringaldehyde and vanillin obtained reached up to 4.81 mM and 0.64 mM, respectively. Then, it successfully produced 3.57 mM and 0.40 mM of syringyl alcohol and vanillyl alcohol, respectively, with 5.86 g L−1 of the lipid titer. The study demonstrates the potential of this integrated approach to convert OPEFB into valuable lignin-derived biochemicals and lipids, thereby contributing to the development of alternative strategies for biomass utilization efficiently.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Fed-batch performance profiles for mAb production using different intensified N - 1 seed strategies are CHO cell-line dependent.

Recent optimizations of cell culture processes have focused on the final seed scale-up step (N - 1 stage) used to inoculate the production bioreactor (N-stage bioreactor) to enable higher inoculation cell densities (2-20 × 106 cells/mL), which could shorten the production culture duration and/or increase the volumetric productivity. N - 1 seed process intensification can be achieved by either non-perfusion (enriched-batch or fed-batch) or perfusion culture to reach those higher final N - 1 viable cell densities (VCD). In this study, we evaluated how different N - 1 intensification strategies, specifically enriched-batch (EB) N - 1 versus perfusion N - 1, affect cell growth profiles and monoclonal antibody (mAb) productivity in the final N-stage production bioreactor operated in fed-batch mode. Three representative Chinese Hamster Ovary (CHO) cell lines producing different mAbs were cultured using either EB or perfusion N - 1 seeds and found that the N-stage cell growth and mAb productivities were comparable between EB N - 1 and perfusion N - 1 conditions for two of the cell lines but were very different for the third. In addition, within the two similar cell growth cell lines, differences in cell-specific productivity were observed. This suggests that the impact of the N - 1 intensification process on production was cell-line dependent. This study revealed that the N - 1 intensification strategy and the state of seeds from the different N - 1 conditions may affect the outcome of the N production stage, and thus, the choice of N - 1 intensification strategy could be a new target for future upstream optimization of mAb production.

New insights into rare earth element-induced microalgae lipid accumulation: Implication for biodiesel production and adsorption mechanism

A coupling technology for lipid production and adsorption of rare earth elements (REEs) using microalgae was studied in this work. The microalgae cell growth, lipid production, biochemical parameters and lipid profiles were investigated under different REEs (Ce3+, Gd3+and La3+). The results showed that the maximum lipid production was achieved at different concentrations of REEs, with lipid productivities of 300.44, 386.84 and 292.19 mg L−1 d−1 under treatment conditions of 100 μg L−1 Ce3+, 250 μg L−1 Gd3+ and 1 mg L−1 La3+, respectively. Moreover, the adsorption efficiency of Ce3+, Gd3+ and La3+exceeded 96.58 %, 93.06 % and 91.3 % at concentrations of 25–1000 μg L−1, 100–500 μg L−1 and 0.25–1 mg L−1, respectively. In addition, algal cells were able to adsorb 66.2 % of 100 μg L−1 Ce3+, 48.4 % of 250 μg L−1 Gd3+ and 59.9 % of 1 mg L−1 La3+. The combination of extracellular polysaccharide and algal cell wall could adsorb 25.2 % of 100 μg L−1 Ce3+, 44.5 % of 250 μg L−1 Gd3+ and 30.5 % of 1 mg L−1 La3+, respectively. These findings indicated that microalgae predominantly adsorbed REEs through the intracellular pathway. This study elucidates the mechanism of effective lipid accumulation and adsorption of REEs by microalgae under REEs stress conditions. It establishes a theoretical foundation for the efficient microalgae lipid production and REEs recovery from wastewater or waste residues containing REEs.

Plasmonic Functional Assay Platform Determines the Therapeutic Profile of Cancer Cells.

Functional assay platforms could identify the biophysical properties of cells and their therapeutic response to drug treatments. Despite their strong ability to assess cellular pathways, functional assays require large tissue samples, long-term cell culture, and bulk measurements. Even though such a drawback is still valid, these limitations did not hinder the interest in these platforms for their capacity to reveal drug susceptibility. Some of the limitations could be overcome with single-cell functional assays by identifying subpopulations using small sample volumes. Along this direction, in this article, we developed a high-throughput plasmonic functional assay platform to identify the growth profile of cells and their therapeutic profile under therapies using mass and growth rate statistics of individual cells. Our technology could determine populations' growth profiles using the growth rate data of multiple single cells of the same population. Evaluating spectral variations based on the plasmonic diffraction field intensity images in real time, we could simultaneously monitor the mass change for the cells within the field of view of a camera with the capacity of > ∼500 cells/h scanning rate. Our technology could determine the therapeutic profile of cells under cancer drugs within few hours, while the classical techniques require days to show reduction in viability due to antitumor effects. The platform could reveal the heterogeneity within the therapeutic profile of populations and determine subpopulations showing resistance to drug therapies. As a proof-of-principle demonstration, we studied the growth profile of MCF-7 cells and their therapeutic behavior to standard-of-care drugs that have antitumor effects as shown in the literature, including difluoromethylornithine (DFMO), 5-fluorouracil (5-FU), paclitaxel (PTX), and doxorubicin (Dox). We successfully demonstrated the resistant behavior of an MCF-7 variant that could survive in the presence of DFMO. More importantly, we could precisely identify synergic and antagonistic effects of drug combinations based on the order of use in cancer therapy. Rapidly assessing the therapeutic profile of cancer cells, our plasmonic functional assay platform could be used to reveal personalized drug therapies for cancer patients.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum

BackgroundFinding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. MethodsIn this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. ResultsThe findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. ConclusionsThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

In vitro triple culture model of retinoblastoma for pre-clinical investigations.

Retinoblastoma (Rb) is a rare cancer of the retina that occurs during early childhood. The disease is relatively rare but aggressive, accounting for ∼3% of childhood cancers. Treatment modalities encompass the administration of large doses of chemotherapeutic drugs, which result in multiple side-effects. Therefore, it is essential to have safe and effective newer therapies and suitable physiologically relevant, alternative-to-animal, in vitro cell culture-based models to enable rapid and efficient evaluation of potential therapies. This investigation was focused on the development of a triple co-culture model comprising Rb, retinal epithelium, and choroid endothelial cells, using a protein coating cocktail, to recapitulate this ocular cancer under in vitro conditions. This resulting model was used for screening drug toxicity, based on the growth profile of Rb cells, using carboplatin as the model drug. Further, a combination of bevacizumab and carboplatin was evaluated using the developed model, to lower the concentration of carboplatin and thereby reduce its physiological side-effects. The effect of drug treatment on the triple co-culture was assessed by increase in the apoptotic profile of Rb cells. Further, the barrier properties were found to be lower with a decrease in the angiogenetic signals that included expression of vimentin. Measurement of cytokine levels signified reduced inflammatory signals due to the combinatorial drug treatment. These findings validated that the triple co-culture Rb model was suitable for evaluating anti-Rb therapeutics and could thereby decrease the immense load on animal trials, which are the primary screens employed for evaluating retinal therapies.

High cell density culture of Neochloris oleoabundans in novel horizontal thin-layer algal reactor: Effects of localized aeration, nitrate concentration and mixing frequency

A horizontal thin-layer algal reactor was designed, constructed and tested for high cell density culture of an oil-rich green alga, Neochloris oleoabundans, at a thickness of 6 mm. A hollow fiber membrane sparger was used for localized aeration with CO2-enriched air, which allowed control of culture pH at a narrow range of pH 7.0–7.5. The optimal nitrate concentration under the investigated conditions was 345 mg-N/L with a cell density of ∼6.6 × 108 cells/mL and biomass concentration of ∼8 g/L. The volumetric and areal biomass productivities were 1.81 g/L/day and 10.85 g m− 2/day, respectively, and the maximum volumetric and areal productivities 3.57 g/L/day and 21.42 g m− 2/day, respectively. Intermittent mixing at a frequency of once every two hours (five min mixing each time) produced a cell growth profile similar to that of continuous mixing. Such intermittent mixing resulted in 98.38 % saving on mixing energy and 33.9 % reduction in water evaporation. It was observed that metabolic stress such as high pH and high nitrate concentration could inhibit cell division while causing cell size to enlarge abnormally. The results indicate that it is technically feasible to have high cell density culture of photoautotrophic microalgae with intermittent mixing and no culture circulation, opening the door towards the development of open algal farming systems of low capital, operational, and maintenance costs.

Magnetoelastic Monitoring System for Tracking Growth of Human Mesenchymal Stromal Cells.

Magnetoelastic sensors, which undergo mechanical resonance when interrogated with magnetic fields, can be functionalized to measure various physical quantities and chemical/biological analytes by tracking their resonance behaviors. The unique wireless and functionalizable nature of these sensors makes them good candidates for biological sensing applications, from the detection of specific bacteria to tracking force loading inside the human body. In this study, we evaluate the viability of magnetoelastic sensors based on a commercially available magnetoelastic material (Metglas 2826 MB) for wirelessly monitoring the attachment and growth of human mesenchymal stromal cells (hMSCs) in 2D in vitro cell culture. The results indicate that the changes in sensor resonance are linearly correlated with cell quantity. Experiments using a custom-built monitoring system also demonstrated the ability of this technology to collect temporal profiles of cell growth, which could elucidate key stages of cell proliferation based on acute features in the profile. Additionally, there was no observed change in the morphology of cells after they were subjected to magnetic and mechanical stimuli from the monitoring system, indicating that this method for tracking cell growth may have minimal impact on cell quality and potency.

A feed enrichment strategy targeting the tricarboxylic acid cycle for increasing monoclonal antibody production and alleviating ammonia accumulation in Chinese hamster ovary cell culture

In this study, we demonstrated a simple strategy for increasing mAb production by enriching an existing feed with TCA cycle intermediates and precursors. TCA cycle intermediates/precursors were tested individually and as mixtures, termed as ‘Enriched Feeds’. The enriched feed mixtures were tested in two compositions comprising: (i) only TCA intermediates/precursors that individually increased mAb titre (EF_Partial) and, (ii) all TCA intermediates/precursors (EF_Complete). These mixtures were also tested for initiation at two phases: (i) from Day 3 (start of exponential phase) and, (ii) from Day 6 (start of stationary phase), and at two concentration levels (high and low). Our results showed that the EF_Partial mixtures were more efficient in increasing mAb titre as compared to the EF_Complete mixtures. The EF_Partial_D6_Low mixture gave the highest titre increase of 55 %, as compared to the control culture. Additionally, a decrease in ammonia production was observed immediately upon the supply of the enriched feeds. In the individual assessment, succinate supplementation resulted in a superior cell growth profile and also exhibited the most significant reduction in ammonia. The individually tested TCA compounds and mixtures also did not impact mAb quality. Therefore, in this study, we demonstrate that a simple approach of enriching feeds with TCA cycle intermediates/precursors effectively increases mAb production by > 50 % without affecting quality attributes.

From fertilised oocyte to cultivated meat – harnessing bovine embryonic stem cells in the cultivated meat industry

Global demand for animal protein is on the rise, but many practices common in conventional production are no longer scalable due to environmental impact, public health concerns, and fragility of food systems. For these reasons and more, a pressing need has arisen for sustainable, nutritious, and animal welfare–conscious sources of protein, spurring research dedicated to the production of cultivated meat. Meat mainly consists of muscle, fat, and connective tissue, all of which can be sourced and differentiated from pluripotent stem cells to resemble their nutritional values in muscle tissue. In this paper, we outline the approach that we took to derive bovine embryonic stem cell lines (bESCs) and to characterise them using FACS (fluorescence-activated cell sorting), real-time PCR and immunofluorescence staining. We show their cell growth profile and genetic stability and demonstrate their induced differentiation to mesoderm committed cells. In addition, we discuss our strategy for preparation of master and working cell banks, by which we can expand and grow cells in suspension in quantities suitable for mass production. Consequently, we demonstrate the potential benefits of harnessing bESCs in the production of cultivated meat.

Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μm Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

Sustain release of loaded insulin within biomimetic hydrogel microsphere for sciatic tissue engineering in vivo

We developed insulin loaded biomimetic microsphere by laccase-mediated crosslinking using a microfluidic device in the water-in-oil emulsion system as an injectable vehicle for the repair of sciatic tissue. Aqueous polymeric solution of phenol-substituted hyaluronic acid (HAPh) and collagen (ColPh) containing insulin and laccase flowed from the inner channel into oil flow within an outer channel which leads formation of hydrogel microsphere. The physical properties of prepared specimens including swelling rate, mechanical resistance and the prolonged release rate of microspheres proved applicability of fabricated vehicles for tissue engineering and drug delivery systems. The growth profile and behavior of cells in microspheres indicated cytocompatibility of the method and prepared vehicles for microtissue development. Histopathological examination revealed a significant increase in axonal regeneration, and remyelination process in injured sciatic nerve following treatment with HAPh/ColPh microspheres containing insulin compared to control groups. Also, the functional characteristic of sciatic tissue showed that the presence of biomimetic microsphere and insulin simultaneously had improved sciatic tissue functions including functional sciatic index (SFI) values, reaction to hot plate and muscle weight of rats. In summary, the results proved that composite biomimetic microspheres containing insulin effectively improved nerve regeneration in the rat model.

Scaling‐up of an Insect Cell‐based Virus Production Process in a Novel Single‐use Bioreactor with Flexible Agitation

AbstractA novel single‐use bioreactor was recently introduced to the market that is agitated by impellers suspended on flexible ropes rather than a rigid shaft. Computational fluid dynamics (CFD) models were created and validated by particle image velocimetry (PIV) to predict the bioreactor's fluid flow and mixing. The data were then used to scale‐up a Spodoptera frugiperda, subclone 9 (Sf9) insect cell‐based production of recombinant adeno‐associated virus (AAV) from a benchtop glass bioreactor to the single‐use system with 30 L working volume. This viral vector is one of the most commonly used in gene therapies. The volumetric power input was kept constant while maintaining reasonable mixing times and shear stresses between the scales. Peak cell densities of up to 7.2 × 106 cells mL−1 and maximum virus titers of 1.7 × 1011 vg mL−1 were achieved. Similar cell growth and metabolite profiles further proved the successful process transfer between the two geometrically non‐similar bioreactor systems. The pilot bioreactor yielded between 3.3 and 4.8 × 1015 vg that, depending on the therapy, can be sufficient for the treatment of a single patient.

Derivation of an anti-cancer drug nanocarrier using a malonic acid-based deep eutectic solvent as a functionalization agent

Deep eutectic solvents (DESs) are novel ‘green’ solvents that have recently gained considerable interest from diverse sectors of the scientific community. They are considered biocompatible, chemically stable, biodegradable, low-volatility, and non-flammable. In addition, the physicochemical properties of DESs are highly tunable and can be customized to meet the needs of a particular mission, including for its novel application in drug delivery systems. Here, graphene functionalized with the DES choline chloride:malonic acid was investigated for anti-cancer activity after loading with the drug doxorubicin. The DES-functionalized graphene demonstrated high drug loading efficiency and exhibited destructive effects against cancerous cells. Real-time cell growth analysis confirmed its cytotoxicity against cancerous cells over time. All told, the combination of choline chloride and malonic acid shows great promise as a green functionalizing agent for a nano-drug carrier, owing to its lower cytotoxicity, higher doxorubicin loading capacity, and inhibition of cancer cell growth profile.

Transcriptomic and Functional Evidence for Differential Effects of MCF-7 Breast Cancer Cell-Secretome on Vascular and Lymphatic Endothelial Cell Growth.

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-β signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC.

Delirium due to hip fracture is associated with activated immune-inflammatory pathways and a reduction in negative immunoregulatory mechanisms

BackgroundThe objectives of this study were to delineate whether delirium in older adults is associated with activation of the immune-inflammatory response system (IRS) as indicated by activation of M1, T helper (Th)1, and Th17 profiles, and/or by reduced activities of the compensatory immunoregulatory system (CIRS), including Th2 and T regulatory profiles.MethodsWe recruited 65 older adult patients with a low energy impact hip fracture who underwent hip fracture operation. The CAM-ICU and the Delirium Rating Scale, Revised-98-Thai version (DRS-R-98) were assessed pre-operatively and 1, 2 and 3 days after surgery. Blood samples (day 1 and 2) post-surgery were assayed for cytokines/chemokines using a MultiPlex assay and the neutrophil/lymphocyte ratio.ResultsWe found that delirium and/or the DRS-R-98 score were associated with IRS activation as indicated by activated M1, Th1, Th17 and T cell growth profiles and by attenuated CIRS functions. The most important IRS biomarkers were CXCL8, interleukin (IL)-6, and tumor necrosis factor-α, and the most important CIRS biomarkers were IL-4 and soluble IL-1 receptor antagonist. We found that 42.5% of the variance in the actual changes in the DRS-R-98 score (averaged from day 1 to day 3) was explained by T cell growth factors, baseline DRS-R-98 scores and age. An increase in the NLR reflects overall IRS, M1, Th1, Th17, and Th2 activation.ConclusionsPost-hip surgery delirium is associated with activated IRS pathways and appears especially in patients with lowered CIRS functions.