CHO-K1 Cells Research Articles

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO–K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO–K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

PurposesThe objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. MethodsTwo lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. ResultsOur findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. ConclusionThese results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.

Matrix attachment regions enhance transgene expression by manipulating position-dependent effects in stably transfected CHO-K1 cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluorescent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recombinant protein production in CHO-K1 cells. This insight underscores the importance of strategic MAR incorporation in vector design for optimized recombinant protein expression.

Efficient generation of a stable CHO-K1 cell line overexpressing the human water channel aquaporin-5 as tool to generate therapeutic antibodies

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren’s syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid–liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

In vitro activation of the CB1 receptor by the semi-synthetic cannabinoids hexahydrocannabinol (HHC), hexahydrocannabinol acetate (HHC-O) and hexahydrocannabiphorol (HHC-P).

Semi-synthetic cannabinoids (SSCs) including hexahydrocannabinol (HHC) are emerging on the drug market and sold openly as purportedly legal replacements for cannabis and Δ9-THC. By the beginning of 2024, 24 European countries had identified HHC, often sold openly in edibles (foods/candy), vapes and low-THC cannabis flowers and resins. The SSC market is developing rapidly, with HHC acetate (HHC-O), hexahydrocannabiphorol (HHC-P) and others recently identified. These developments may mark the first major change in the market for 'legal' replacements to cannabis since 'Spice' containing synthetic cannabinoids, such as JWH-018, emerged in 2008. Currently, there are some data available on the pharmacology of SSCs, which is crucial for understanding their effects, evaluating health risks and informing public health responses. This study focused on characterizing the in vitro activation of the human CB1 receptor by the (R)- and (S)-epimers of HHC, HHC-P and HHC-O. Using recombinant CHO-K1 cells expressing the human CB1 receptor, the potency (EC50) and efficacy were determined. It was established that (9R)-HHC and (9R)-HHC-P activated the CB1 receptor as partial agonists and with five and two times lower potency compared to JWH-018, respectively, while the (S)-epimers exhibited even lower potency. The (R)-epimer of HHC-O activate the CB1 receptor to even lesser extent and the (S)-epimer showed no activation. For HHC and HHC-P, all epimers exhibited similar level of efficacy. This available evidence suggests cannabimimetic effects of the tested SSC with the exception for the acetates that likely function as pro-drugs in vivo.

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

The physiological levels of epigallocatechin gallate (EGCG) enhance the Cd-induced oxidative stress and apoptosis in CHO-K1 cells

Currently, the increasing pollution of the environment by heavy metals is observed, caused both by natural factors and those related to human activity. They pose a significant threat to human health and life. It is therefore important to find an effective way of protecting organisms from their adverse effects. One potential product showing a protective effect is green tea. It has been shown that EGCG, which is found in large amounts in green tea, has strong antioxidant properties and can therefore protect cells from the adverse effects of heavy metals. Therefore, the aim of the study was to investigate the effect of EGCG on cells exposed to Cd. In the study, CHO-K1 cells (Chinese hamster ovary cell line) were treated for 24 h with Cd (5 and 10 µM) and EGCG (0.5 and 1 µM) together or separately. Cell viability, ATP content, total ROS activity, mitochondrial membrane potential and apoptosis potential were determined. The results showed that, in tested concentrations, EGCG enhanced the negative effect of Cd. Further analyses are needed to determine the exact mechanism of action of EGCG due to the small number of publications on the subject and the differences in the results obtained in the research.

Mutational analysis of pig tissue factor pathway inhibitor α to increase anti-coagulation activity in pig-to-human xenotransplantation.

Blood coagulation mediated by pig tissue factor (TF), which is expressed in pig tissues, causes an instant blood-mediated inflammatory reaction during pig-to-human xenotransplantation. Previously, we generated a soluble pig tissue factor pathway inhibitor α fusion immunoglobulin (TFPI-Ig) which inhibits pig TF activity more efficiently than human TFPI-Ig in human plasma. In this study, we generated several pig TFPI-Ig mutants and tested the efficacy of these mutants in preventing pig-to-human xenogeneic blood coagulation. Structurally important amino acid residues of pig TFPI-Ig were changed into different residues by site-directed mutagenesis. Subsequently, a retroviral vector encoding each cDNA of several pig TFPI-Ig mutants was cloned and transduced into CHO-K1 cells. After establishing stable cell lines expressing each of the pig TFPI-Ig mutants, soluble proteins were produced and purified for evaluating their inhibitory effects on pig TF-mediated blood coagulation in human plasma. The replacement of K36 and K257 with R36 and H257, respectively, in pig TFPI-Ig more efficiently blocked pig TF activity in human plasma when compared with the wild-type pig TFPI-Ig. These results may provide additional information to understand the structure of pig TFPIα, and an improved pig TFPI-Ig variant that more efficiently blocks pig TF-mediated blood coagulation during pig-to-human xenotransplantation.

The Protective Role of Magnoliae Flos in Preventing Ovotoxicity and Managing Ovarian Function: An In Vitro and In Vivo Study.

Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.

Navacaprant, a novel and selective kappa opioid receptor antagonist, has no agonist properties implicated in opioid-related abuse

Kappa opioid receptors (KORs) are implicated in the pathophysiology of various psychiatric and neurological disorders creating interest in targeting the KOR system for therapeutic purposes. Accordingly, navacaprant (NMRA-140) is a potent, selective KOR antagonist being evaluated as a treatment for major depressive disorder. In the present report, we have extended the pharmacological characterization of navacaprant by further demonstrating its selective KOR antagonist properties and confirming its lack of agonist activity at KORs and related targets involved in opioid-related abuse. Using CHO–K1 cells expressing human KOR, mu (MOR), or delta (DOR) opioid receptors, navacaprant demonstrated selective antagonist properties at KOR (IC50 = 0.029 μM) versus MOR (IC50 = 3.3 μM) and DOR (IC50 > 10 μM) in vitro. In vivo, navacaprant (10–30 mg/kg, i.p.) dose-dependently abolished KOR-agonist induced analgesia in the mouse tail-flick assay. Additionally, navacaprant (10, 30 mg/kg, p.o.) significantly reduced KOR agonist–stimulated prolactin release in mice and rats, confirming KOR antagonism in vivo. Navacaprant showed no agonist activity at any opioid receptor subtype (EC50 > 10 μM) in vitro and exhibited no analgesic effect in the tail-flick assays at doses ≤100 mg/kg, p.o. thereby confirming a lack of opioid receptor agonist activity in vivo. Importantly, navacaprant did not alter extracellular dopamine concentrations in the nucleus accumbens shell of freely-moving rats following doses ≤100 mg/kg, p.o., whereas morphine (10, 20 mg/kg, i.p.) significantly increased dopamine levels. These results demonstrate that navacaprant is a KOR-selective antagonist with no pharmacological properties implicated in opioid-related abuse.

A fluorescently-tagged tick kinin neuropeptide triggers peristalsis and labels tick midgut muscles

Ticks are blood-feeding arthropods that require heme for their successful reproduction. During feeding they also acquire pathogens that are subsequently transmitted to humans, wildlife and/or livestock. Understanding the regulation of tick midgut is important for blood meal digestion, heme and nutrient absorption processes and for aspects of pathogen biology in the host. We previously demonstrated the activity of tick kinins on the cognate G protein-coupled receptor. Herein we uncovered the physiological role of the kinin receptor in the tick midgut. A fluorescently-labeled kinin peptide with the endogenous kinin 8 sequence (TMR-RK8), identical in the ticks Rhipicephalus microplus and R. sanguineus, activated and labeled the recombinant R. microplus receptor expressed in CHO-K1 cells. When applied to the live midgut the TMR-RK8 labeled the kinin receptor in muscles while the labeled peptide with the scrambled-sequence of kinin 8 (TMR-Scrambled) did not. The unlabeled kinin 8 peptide competed TMR-RK8, decreasing confocal microscopy signal intensity, indicating TMR-RK8 specificity to muscles. TMR-RK8 was active, inducing significant midgut peristalsis that was video-recorded and evaluated with video tracking software. The TMR-Scrambled peptide used as a negative control did not elicit peristalsis. The myotropic function of kinins in eliciting tick midgut peristalsis was established.

A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

Single-chain human follicle-stimulating hormone with a di-N-glycosylated linker

The classic way to produce single-chain (sc) glycoprotein hormones is to fuse their two subunits through the carboxy-terminal peptide (CTP) from human Choriogonadotropin (hCG). The CTP confers a longer half-life to single-chain hormones thanks to its four O-glycosyl side chains. However, unlike syncytiotrophoblastic cells, most cells used for recombinant protein production do not transfer O-glycosyl chains efficiently. We thus choose to fuse the hFSH subunits with a linker comprising two N-glycosyl side chains (sc-hFSH LNN) or none (sc-hFSH L0N), that were generated using two expression systems, HEK293 and CHO K1 cells. Their production levels and biological activities were tested and compared. Both expression systems successfully produced biologically active sc-hFSH, but, in our hands, CHO K1 cells yielded about 30-fold higher amounts of recombinant protein than HEK293 cells. Moreover, sc-hFSH L0N was considerably less expressed than sc-hFSH LNN in both cell types. Our data show that sc-hFSH L0N and sc-hFSH LNN produced from both cell lines stimulate cAMP and progesterone production in mLTC cells expressing hFSH receptors and exhibit similar B/I (in vitro Bioactivity/Immuno activity) ratios. Finally, the ratio of in vivo/in vitro bioactivities for sc-hFSH LNN relative to natural pituitary heterodimeric hFSH increased 8-fold, most likely because of a longer half-life in the blood.

Novel Ferrocene-Containing Triacyl Derivative of Resveratrol Protects Ovarian Cells from Toxicity Caused by Ortho-Substituted Polychlorinated Biphenyls

Polychlorinated biphenyls (PCBs) can induce neurotoxicity, immunotoxicity, reproductive toxicity, genotoxicity, and carcinogenicity (IARC group 1 Carcinogens). Scientific data suggest that resveratrol possesses the ability to attenuate ortho-PCB-induced toxicity. Recently, a novel ferrocene-containing triacyl derivative of resveratrol (RF) was synthesized and in this study, its potential to protect CHO-K1 cells from selected PCB congeners (75 µM) was evaluated. Cell viability/proliferation was observed by Trypan Blue (TB), Neutral Red (NR), Kenacid Blue (KB), and MTT bioassays, ROS formation by fluorescent probes, and the extent of apoptosis by flow cytometry. All applied bioassays confirmed that RF (2.5–100 μM) remarkably improves viability in PCB 153-treated cells with an increase in cell survival almost up to control levels. This effect was not determined after PCB 77 exposure, although ROS formation was decreased at RF ≥ 50 µM. Apoptosis was significant (p &lt; 0.05) for both congeners. In PCB 77-treated cells, RF did not suppress the induction of cell death. The intended protective effect of RF was evident when cells were treated with PCB 153, and this correlates with results obtained for cell viability. Compared to resveratrol, the novel RF showed promising results in terms of improved biological activity and cell protection against PCB 153 toxicity at all concentrations tested.

Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection.

For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO-K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).