Chinese Hamster Ovary Research Articles

Comparative Study of Biosynthesizing Selenium Nanoparticles by Gum Arabic and Poly Anionic Cellulose to Prevent Radiation-Induced Death in Chinese Hamster Ovary (CHO) Cells

Purpose: In premenopausal women, abdominopelvic radiotherapy may have a direct and profound effect on ovarian function. Stabilized selenium Nanoparticles (NPs) with some natural materials have been demonstrated to have high antioxidant activity and reduce radiation damage as a radioprotector. This study was done to compare the ability for the biosynthesis of selenium NPs by Gum Arabic (Se-GA) and Polyanionic Cellulose (Se-PAC) in the protection of Chinese Hamster Ovary (CHO) cells against radiation damage.
 Materials and Methods: First, Selenium Nanoparticles (SeNPs) were synthesized in the presence of GA and PAC. Then, CHO cells were cultured in-vitro and were randomly divided into six groups in different concentrations of Se-GA and Se-PAC to measure the biocompatibility of NPs. Finally, cells were treated with NPs and radiation (6MV, 2Gy), and the percentage of cell survival was determined by MTT assay. Both NPs with an average size of 20-30 nm and an absorption absorbance peak at about 300 nm using Ultraviolet-Visible (UV–Vis) spectroscopy.
 Results: According to the parametric t-test analysis, Se-GA nanoparticles with a concentration higher than 0.4 ppm significantly increased the radioprotective effect on CHO cells compared to the control group (P<0.05). However, Se-PAC showed no significant increase in radioprotection in contrast to the control group (P>0.05).
 Conclusion: Se-GA nanoparticles have antioxidant properties, and the radiation protection properties of Se-GA nanoparticles are significantly higher than control. Consequently, Se-GA nanoparticles showed promising results and may be able to play the role of a radioprotector.

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

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

A systemic approach to identifying sequence frameworks that decrease mAb production in a transient Chinese hamster ovary cell expression system.

Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their "developability" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these "difficult-to-express" (DTE) mAbs has shown that these phenotypes are driven by post-translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG1κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines.

A Novel Non-Psychoactive Fatty Acid from a Marine Snail, Conus inscriptus, Signals Cannabinoid Receptor 1 (CB1) to Accumulate Apoptotic C16:0 and C18:0 Ceramides in Teratocarcinoma Cell Line PA1

The cannabinoid-type I (CB1) receptor functions as a double-edged sword to decide cell fate: apoptosis/survival. Elevated CB1 receptor expression is shown to cause acute ceramide accumulation to meet the energy requirements of fast-growing cancers. However, the flip side of continual CB1 activation is the initiation of a second ceramide peak that leads to cell death. In this study, we used ovarian cancer cells, PA1, which expressed CB1, which increased threefold when treated with a natural compound, bis(palmitoleic acid) ester of a glycerol (C2). This novel compound is isolated from a marine snail, Conus inscriptus, using hexane and the structural details are available in the public domain PubChem database (ID: 14275348). The compound induced two acute ceramide pools to cause G0/G1 arrest and killed cells by apoptosis. The compound increased intracellular ceramides (C:16 to 7 times and C:18 to 10 times), both of which are apoptotic inducers in response to CB1 signaling and thus the compound is a potent CB1 agonist. The compound is not genotoxic because it did not induce micronuclei formation in non-cancerous Chinese hamster ovarian (CHO) cells. Since the compound induced the cannabinoid pathway, we tested if there was a psychotropic effect in zebrafish models, however, it was evident that there were no observable neurobehavioral changes in the treatment groups. With the available data, we propose that this marine compound is safe to be used in non-cancerous cells as well as zebrafish. Thus, this anticancer compound is non-toxic and triggers the CB1 pathway without causing psychotropic effects.

Online monitoring of the respiration activity in 96-deep-well microtiter plate Chinese hamster ovary cultures streamlines kill curve experiments.

Cell line generation of mammalian cells is a time-consuming and labor-intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource-intensive and time-consuming due to necessary sampling and offline analysis effort. This study, thus, explores the potential of online monitoring the oxygen transfer rate (OTR), as a non-invasive and efficient alternative for kill curve experiments. The OTR is monitored using the Transfer-rate Online Measurement (TOM) system and the micro(μ)-scale Transfer-rate Online Measurement (μTOM) device, which was used for mammalian cells first. It could be shown that the OTR curves for both devices align perfectly, affirming consistent cultivation conditions. The μTOM device proves effective in performing kill curve experiments in 96-deep-well plates without the need for sampling and offline analysis. The streamlined approach reduces medium consumption by 95%, offering a cost-effective and time-efficient solution for kill curve experiments. The study validates the generalizability of the method by applying it to two different CHO cell lines (CHO-K1 and sciCHO) with two antibiotics (puromycin and hygromycin B) each. In conclusion, the broad application of OTR online monitoring for CHO cell cultures in 96-deep-well plates is highlighted. The μTOM device proves as a valuable tool for high-throughput experiments, paving the way for diverse applications, such as media and clone screening, cytotoxicity tests, and scale-up experiments.

Investigation of fouling in perfusion cell culture processes using alternating tangential flow filtration

Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short-term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9–0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88 %).

In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes.

Extracellular vesicles (EVs) have garnered much interest due to their fundamental role in intracellular communication and their potential utility in clinical diagnostics and as biotherapeutic vectors. Of particular relevance is the subset of EVs referred to as exosomes, ranging in size from 30 to 150 nm, which contain incredible amounts of information about their cell of origin, which can be used to track the progress of disease. As a complementary action, exosomes can be engineered with therapeutic cargo to selectively target diseases. At present, the lack of highly efficient methods of isolation/purification of exosomes from diverse biofluids, plants, and cell cultures is a major bottleneck in the fundamental biochemistry, clinical analysis, and therapeutic applications. Equally impactful, the lack of effective in-line means of detection/characterization of isolate populations, including concentration and sizing, is limiting in the applications. The method presented here couples hydrophobic interaction chromatography (HIC) performed on polyester capillary-channeled polymer (C-CP) fiber columns followed by in-line optical absorbance and multi-angle light scattering (MALS) detection for the isolation and characterization of EVs, in this case present in the supernatant of Chinese hamster ovary (CHO) cell cultures. Excellent correlation was observed between the determined particle concentrations for the two detection methods. C-CP fiber columns provide a low-cost platform (< $5 per column) for the isolation of exosomes in a 15-min workflow, with complementary absorbance and MALS detection providing very high-quality particle concentration and sizing information.

Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1’s involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets.

Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein)

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP’s influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands.Key points• YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity.• YAP5SA regulates genes involved in proliferation, survival, and mTOR activation.• YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells

AbstractCell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration

High-yield production of therapeutic proteins using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

DR10627, a Novel Dual Glucagon‑like Peptide‑1 and Gastric Inhibitory Polypeptide Receptor Agonist for the Treatment of Obesity and Type 2 Diabetes Mellitus.

Diabetes and obesity are momentous risk factors threatening people's lives and health. Currently available incretin analogue glucagon-like peptide 1 (GLP-1) possesses huge hypoglycemic effect with the unsatisfactory effect of weight loss. Co-agonists targeting GLP-1R plus glucagon receptor (GCGR) or gastric inhibitory polypeptide receptor (GIPR) show synergistic benefits in glycaemic control and weight loss. Here, we describe a novel dual GIP and GLP-1 receptor agonist, DR10627, and performed a preclinical assessment of it. The agonistic ability of DR10627 was indirectly assessed by inducing cAMP accumulation in Chinese hamster ovary (CHO) cells transfected with GLP-1R or GIPR in vitro. The plasma pharmacokinetics of DR10627 were analysed in cynomolgus monkeys. The OGTTs were performed in Sprague‑Dawley (SD) rats. The glucose lowering effects were evaluated by repeated administration of DR10627 in diabetic (db/db) mice for 4 weeks. The effects of anti-obesity and improving metabolism of DR10627 were evaluated by repeated administration of DR10627 in diet-induced obesity (DIO) mice for 57 days. DR10627 had the capacity to activate both GLP-1R and GIPR in vitro. The terminal half-life of DR10627 was found to be approximately 4.19-5.8 h in cynomolgus monkeys. DR10627 had a great improvement in oral glucose tolerance in SD rats. Moreover, DR10627 had a potent glucose-lowering effect in db/db mice, and the hypoglycemic effect of 18 nmol/kg DR10627 was better than that of 50 nmol/kg liraglutide. In addition, 10 and 30 nmol/kg DR10627 possessed the ability of potentiating the weight-loss, lipid-lowing efficacy and improving metabolism to a greater extent than 80 nmol/kg liraglutide. Preclinical assessment demonstrated that administration of DR10627 resulted in glucose lowering in SD rats and db/db mice, and substantial body weight reduction and metabolism improvement in DIO mice. DR10627 is a promising agent deserving further investigation for the treatment of type 2 diabetes and obesity.

Insights into the transformation of natural organic matter during UV/peroxydisulfate treatment by FT-ICR MS and machine learning: Non-negligible formation of organosulfates

Natural organic matter (NOM) is a major sink of radicals in advanced oxidation processes (AOPs) and understanding the transformation of NOM is important in water treatment. By using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in conjunction with machine learning, we comprehensively investigated the reactivity and transformation of NOM, and the formation of organosulfates during the UV/peroxydisulfate (PDS) process. After 60 min UV/PDS treatment, the CHO formula number and dissolved organic carbon concentration significantly decreased by 83.4% and 74.8%, respectively. Concurrently, the CHOS formula number increased substantially from 0.7% to 20.5%. Machine learning identifies DBE and AImod as the critical characteristics determining the reactivity of NOM during UV/PDS treatment. Furthermore, linkage analysis suggests that decarboxylation and dealkylation reactions are dominant transformation pathways, while the additions of SO3 and SO4 are also non-negligible. According to SHAP analysis, the m/z, number of oxygens, DBE and O/C of NOM were positively correlated with the formation of organosulfates in UV/PDS process. 92 organosulfates were screened out by precursor ion scan of HPLC-MS/MS and verified by UPLC-Q-TOF-MS, among which, 7 organosufates were quantified by authentic standards with the highest concentrations ranging from 2.1 to 203.0 ng L‒1. In addition, the cytotoxicity of NOM to Chinese Hamster Ovary (CHO) cells increased by 13.8% after 30 min UV/PDS treatment, likely responsible for the formation of organosulfates. This is the first study to employ FT-ICR MS combined with machine learning to identify the dominant NOM properties affecting its reactivity and confirmed the formation of organosulfates from sulfate radical oxidation of NOM.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 μM and Vmax, 0.95 μmol/min/mg) and AICAR (Km, 34.81 μM and Vmax, 0.56 μmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 μM and Vmax, 2.87 μmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 μM and 34.2 μM, respectively) compared to AICAR (Kd, 83.4 μM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. Wildtype and MR-deficient mice were exposed to lipopolysachharide (LPS), after which the protein content in their lung epithelial lining fluid (ELF) was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the ELF using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the thrombospondin family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as uPARAP. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the thrombospondin family.

Experimental analysis of lifelines in a 15,000 L bioreactor by means of Lagrangian sensor particles

This study employs Lagrangian Sensor Particles (LSPs) with a diameter of 40 mm equipped with a pressure sensor to investigate cell lifelines in a 15,000L stirred tank reactor (STR) with three Elephant Ear impellers. The Stokes number of the LSPs is approx. 0.004 on a macro-scale. The vertical probability of presence, axial velocity profiles, circulation time distributions, and residence time distributions are quantified to analyze single-phase mixing heterogeneities, detect hydrodynamic compartments and conduct a Lagrangian regime analysis. Results reveal a similarly distributed probability of presence in the vertical reactor center but emphasize the LSP’s sensitivity to fluctuating densities. Axial velocity distributions illustrate characteristic impeller-induced flow patterns, and circulation time distributions identify three compartments with comparatively shorter times in the axial center. Residence time distributions exhibit a similar compartmentalized profile. Moreover, the study estimates a potential oxygen deprivation zone for CHO cells in the upper compartment and demonstrates the LSP’s efficacy in characterizing impeller systems. Contrary to literature, the ratio of examined global mixing times to circulation times is 1.0, highlighting macro-scale mixing. The research underscores that LSPs offer crucial insights into industrial-scale STRs, specifically for determining hydrodynamic compartments without having optical access.

Exploring the effects of resin particle sizes on enhancing antibody binding capacity of a hybrid biomimetic ligand

Exploring the effects of resin particle sizes on enhancing antibody binding capacity of a hybrid biomimetic ligand

Hybridized quantum dot, silica, and gold nanoparticles for targeted chemo-radiotherapy in colorectal cancer theranostics

Multimodal nanoparticles, utilizing quantum dots (QDs), mesoporous silica nanoparticles (MSNs), and gold nanoparticles (Au NPs), offer substantial potential as a smart and targeted drug delivery system for simultaneous cancer therapy and imaging. This method entails coating magnetic GZCIS/ZnS QDs with mesoporous silica, loading epirubicin into the pores, capping with Au NPs, PEGylation, and conjugating with epithelial cell adhesion molecule (EpCAM) aptamers to actively target colorectal cancer (CRC) cells. This study showcases the hybrid QD@MSN-EPI-Au-PEG-Apt nanocarriers (size ~65 nm) with comprehensive characterizations post-synthesis. In vitro studies demonstrate the selective cytotoxicity of these targeted nanocarriers towards HT-29 cells compared to CHO cells, leading to a significant reduction in HT-29 cell survival when combined with irradiation. Targeted delivery of nanocarriers in vivo is validated by enhanced anti-tumor effects with reduced side effects following chemo-radiotherapy, along with imaging in a CRC mouse model. This approach holds promise for improved CRC theranostics.

Deciphering Metabolic Pathways in High-Seeding-Density Fed-Batch Processes for Monoclonal Antibody Production: A Computational Modeling Perspective

Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.