Cell Growth Conditions Research Articles

Oxygen uptake rate analysis to evaluate the impact of hydrodynamic stress on the growth of the avian cell line DuckCelt®-T17

Scale-up of bioprocesses involving animal cell culture is hampered by the sensitivity of the cells to hydrodynamic stress, either from agitation or bubble bursting. Here, the hydrodynamic stress experienced by a recent cell line, the DuckCelt®-T17 avian cells, previously used for viral vaccine production, is investigated in shake flasks and in a 3 L bioreactor. Cell stress was assessed by monitoring the dissolved oxygen in the culture medium, which depends on Oxygen Transfer Rate (OTR) and Oxygen Uptake Rate (OUR) during cultivation. Classical parameters such as the maximum growth rate (µmax) and metabolite profiles were also determined. A dynamic model able to predict nutrient consumption, metabolic waste production, viable cell number and OUR was also developed and validated from the data measured in shake flasks. The experiments performed in the stirred tank bioreactor (STBR) show that OUR depended on both the cell growth phase and the stirring conditions. The oxygen consumption of the cells during the exponential growth phase (where there were no nutrient and O2 limitations) was significantly altered at average and maximum shear rates above 70 and 840 s−1, respectively, indicating highly shear-sensitive cells. OUR is a suitable tool to identify the hydrodynamic conditions for robust cell growth. The scale-up criteria to be favored for the DuckCelt®-T17 cell culture in STBRs would be the shear and/or the tip’s speed.

The role of Atoh7/Notch signaling in the differentiation of retinal stem cells into retinal ganglion cells and its potential application in glaucoma treatment

Background: One of the main causes of blindness in persons over 60 is glaucoma, yet there are presently no viable therapies available. The differentiation to retinal ganglion cell (RGC) is a key step in the stem cell-based ocular regeneration therapy that have become a new hotspot in this field. This study aimed to investigate the differentiation pathway of Retinal stem cells (RSCs) and their potential for future application in glaucoma treatment. Methods: Gas pressurization was employed to simulate intraocular pressure (IOP) conditions in vitro, utilizing polyimide membranes as the culture chamber. RSCs were derived from mice and underwent a series of transfection and detection procedures. The expression levels of mRNA and protein were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Cell apoptosis and proportion were assessed through flow cytometry analysis. Immunofluorescence staining was performed to identify cell morphology, growth status, and relative markers. In vivo validation was conducted on glaucoma mouse models, highlighting the regulatory system involving Atoh7/Notch for RSC differentiation. Results: At 40 mmHg, Atoh7 facilitates RGC differentiation through the regulation of Notch expression. Activation of Atoh7/Notch signaling in glaucomatous retina enhances RSC viability and differentiation. Subsequent transplantation of RSCs significantly improves visual acuity in glaucoma animal models, accompanied by increased expression and number of positive cells for RGC markers, as well as activation of Atho7/Notch signaling. Conclusion: The present study elucidates the alterations and impacts of IOP in glaucoma, while also highlighting the potential utility of Atoh7/Notch as a promising avenue for comprehending the underlying mechanisms governing retinal ganglion cell differentiation.

Substrate stiffness modulates extracellular vesicles’ release in a triple-negative breast cancer model

Aim: The microenvironment effect on the tumoral-derived Extracellular Vesicle release, which is of significant interest for biomedical applications, still represents a rather unexplored field. The aim of the present work is to investigate the interrelation between extracellular matrix (ECM) stiffness and the release of small EVs from cancer cells. Here, we focus on the interrelation between the ECM and small extracellular vesicles (sEVs), specifically investigating the unexplored aspect of the influence of ECM stiffness on the release of sEVs. Methods: We used a well-studied metastatic Triple-Negative Breast Cancer (TNBC) cell line, MDA-MB-231, as a model to study the release of sEVs by cells cultured on substrates of different stiffness. We have grown MDA-MB-231 cells on two collagen-coated polydimethylsiloxane (PDMS) substrates at different stiffness (0.2 and 3.6 MPa), comparing them with a hard glass substrate as control, and then we isolated the respective sEVs by differential ultracentrifugation. After checking the cell growth conditions [vitality, morphology by immunofluorescence microscopy, stiffness by atomic force microscopy (AFM)], we took advantage of a multi-parametric approach based on complementary techniques (AFM, Nanoparticle Tracking Analysis, and asymmetric flow field flow fractionation with a multi-angle light scattering detector) to characterize the TNBC-derived sEV obtained in the different substrate conditions. Results: We observe that soft substrates induce TNBC cell softening and rounding. This effect promotes the release of a high number of larger sEVs. Conclusion: Here, we show the role of ECM physical properties in the regulation of sEV release in a TNBC model. While the molecular mechanisms regulating this effect need further investigation, our report represents a step toward an improved understanding of ECM-cell-sEVs crosstalk.

Development of hydroxyapatite/poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)/gelatin composite nanofibers to improve bone‐forming cell proliferation and mineral deposition

AbstractCalcium phosphate hydroxyapatite (HA) was successfully incorporated into poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)/gelatin (PHBV/GEL) nanofibers through an electrospinning process in a hexafluoroisopropanol solvent (HFIP). The PHBV/GEL was electrospun and collected using a rotating metallic network to develop highly ordered nonwoven mats. The effects of HA addition on the morphologies of the obtained fibers were investigated using SEM. The average diameter of the fibers ranged around 700–900 nm, depending on the HA content. From a mechanical perspective, the addition of HA nanoparticles showed variations in tensile strength and elastic modulus values. The lowest tensile strength measured for neat PHBV/GEL was 0.37 ± 0.08 MPa with an elastic modulus of 9.45 ± 0.65 MPa, whereas the highest tensile strength reported for 5% HA‐loaded PHBV/GEL mats was 1.1 ± 0.4 MPa with an elastic modulus of 16.9 ± 0.39 MPa. In addition, cell viability, and mineral deposition were also studied using MC3T3‐E1 pre‐osteoblasts. The in vitro experiment results showed that the HA loaded‐PHBV/GEL mats provide optimal conditions for cell growth and osteogenic differentiation of MC3T3‐E1 cells. This study reveals the potential of PHBV/GEL/HA matrices in the development of novel guided bone‐regeneration (GBR) membranes for orthopedic and craniomaxillofacial surgery, by providing favorable structural features for attachment, growth, and differentiation of osteoblast cells.Highlights Simple and cheap electrospinning strategy toward network‐like fibrous mats. Microstructure, mechanical, and biological features were evaluated. Electrospun PHBV/GEL/HA promoted calcium phosphate deposition by MC3T3‐E1 cells.

Inhibitory Effect of Combined Exposure to Copper Ions and Polystyrene Microplastics on the Growth of Skeletonema costatum

As two significant pollutants, the increased concentrations of copper ions and microplastics in coastal waters pose a substantial threat to the health of marine ecosystems. This study explored the individual and combined toxic effects of copper ions and microplastics on the marine diatom Skeletonema costatum. The results indicated that copper ions at a concentration of 0.05 mg/L exerted a negative impact on the growth of S. costatum; however, the influence of microplastics on algal cell growth was more complex: there was no significant inhibition of algal cell growth observed within the first 72 h of cultivation, but a pronounced negative effect on cell growth was noted between 72 and 96 h, with high concentrations (10.00 mg/L) of microplastics even causing a premature stagnation phase in algal cell growth. When both pollutants were present simultaneously, their impact on cell growth was similar to that of the microplastic pollution group, yet the growth condition was better than that of the copper ion pollution group. Through investigation of adsorption rates and toxicity mitigation rates, it was found that the adsorption of copper ions by microplastics was a significant reason for the improved growth condition of algal cells in the mixed pollution group compared to the copper ion pollution group. However, in the later stages of cultivation, the inhibitory effect of microspheres on algal cell growth counteracted their mitigating effect on copper ion toxicity. This study provides a reference for evaluating the impact of microplastics and heavy metals on coastal ecosystem health and for revealing the interactions between different pollutants.

The superiority of innovative spiral-interdigital microelectrode pattern in increasing the sensitivity of tracing synchronization via serum starvation in cellular metabolism

In order to investigate the changes in the properties of the cell culture solution in the effect of cell synchronization via cell starvation (for 12, 24, and 36 h), a new spiral-interdigital pattern of microelectrode as a biosensor has been proposed. Then, to test its superiority, the results of this spiral-interdigital pattern with the results of the commercial pattern have been compared. The cells were selected from breast cancer standard lines (MDA-MB-231). Changes in CV peaks of the secretions were recorded by the spiral-interdigital pattern, in which increasing the interactive surface with homogenous electric paths had been considered by simulation before fabrication. The results of the simulation and experimental procedures showed a meaningful correlation. The occurrence of CV oxidative peaks at about 0.1–0.4 V and reductive peaks at approximately 0 V in the spiral-interdigital biosensor in the starved MDA-MB-231 cell line has been observed. The starvation situation resembles one that does not cause meaningful cell apoptosis or necrosis, and this method is only used to make the cells synchronized. Also, no peak is observed in normal cell growth conditions. In addition, by using the commercial design of the electrodes, no peak is observed in any of the conditions of normal and synchronized growth of the cells. Therefore, it seems that the observed peaks are caused by the agents that are secreted in the cell culture solution in a synchronized situation. Moreover, the design of the new spiral-interdigital electrode can significantly increase the sensitivity of the sensor to receive these peaks due to more space and a uniform electric field.

Transcription factor AbrB regulates ROS generation and clearance in Bacillus licheniformis

Oxidative damage caused by the accumulation of reactive oxygen species (ROS) is one of the main obstacles to the improvement of microbial cell growth and fermentation characteristics under adverse environments. And the antioxidant capacity of cells will increase with the cell growth. Here, we found that a transition state transcription factor AbrB related to changes in cell growth status could regulate the accumulation of ROS and antioxidant capacity in Bacillus licheniformis. The results showed that the accumulation of intracellular ROS was reduced by 23.91 % and the cell survival rates were increased by 1.77-fold under 0.5 mM H2O2 when AbrB was knocked out. We further mapped regulatory target genes of AbrB related to ROS generation or clearance based on our previously analyzed transcriptome sequencing. It proved that AbrB could promote ROS generation via upregulating the synthesis of oxidase and siderophores, and negatively regulating the synthesis of iron chelators (pulcherriminic acid, and H2S). Additionally, AbrB could inhibit ROS clearance by negatively regulating the synthesis of antioxidase (superoxide dismutase, catalase, peroxidase, thioredoxin, thioredoxin reductase) and cysteine. Those results illustrated that the inactivation of AbrB during the stationary phase, along with its control over ROS generation and clearance, might represent a vital self-protection mechanism during cell evolution. Overall, the systematic investigation of the multi-pathway regulation network of ROS generation and clearance highlights the important function of AbrB in maintaining intracellular redox balance.

Stress modulation strategies in Kluyveromyces marxianus: Unravelling the effects of shear force and aeration for enhanced specific ergosterol production

AbstractErgosterol, as a precursor for synthesising useful molecules like vitamin D2, possesses significant physiological functions in both fungal and human systems. In fungi, ergosterol plays a crucial role in stress responses. In contrast to Saccharomyces cerevisiae yeast, the changes in specific ergosterol content of Kluyveromyces marxianus under various stress conditions are less known. This study investigated how ergosterol content changes in response to different stress factors. Carbon to nitrogen (C/N) ratio was examined using experimental design. The effects of aeration and shear force beside constant overall volumetric mass transfer coefficient (KLa) were examined. Cell growth and specific ergosterol content were investigated using ethanol stress during a two-stage fermentation. Based on the results, contradictory settings regarding C/N ratio and shear force were found to be favourable for cell growth and specific ergosterol content. However, increased aeration consistently elevated specific ergosterol content and favoured cell growth as well (2.5-fold and 1.5-fold, respectively). In K. marxianus fermentations, higher ergosterol yield can be achieved through a two-stage fermentation (138.9 mg L−1 compared to 52.9 mg L−1), where the first stage provides favourable conditions for cell growth, and the second stage involves stress (beneficial for ergosterol production) conditions. Conclusions drawn from the two-stage fermentation results suggest that early transitioning of cell growth to the second phase will not result higher adaption and specific ergosterol content compared to the transition at the end of exponential growth phase.

SIRT3 protects endometrial receptivity in patients with polycystic ovary syndrome.

The sirtuin family is well recognized for its crucial involvement in various cellular processes. Nevertheless, studies on its role in the human endometrium are limited. This study aimed to explore the expression and localization of the sirtuin family in the human endometrium, focusing on sirtuin 3 (SIRT3) and its potential role in the oxidative imbalance of the endometrium in polycystic ovary syndrome (PCOS). Endometrial specimens were collected from both patients with PCOS and controls undergoing hysteroscopy at the Center for Reproductive Medicine, Peking University Third Hospital, from July to August 2015 and used for cell culture. The protective effects of SIRT3 were investigated, and the mechanism of SIRT3 in improving endometrial receptivity of patients with PCOS was determined using various techniques, including cellular bioenergetic analysis, small interfering ribonucleic acid (siRNA) silencing, real-time quantitative polymerase chain reaction, Western blot, immunofluorescence, immunohistochemistry, and flow cytometry analysis. The sirtuin family was widely expressed in the human endometrium, with SIRT3 showing a significant increase in expression in patients with PCOS compared with controls (P <0.05), as confirmed by protein and gene assays. Concurrently, endometrial antioxidant levels were elevated, while mitochondrial respiratory capacity was reduced, in patients with PCOS (P <0.05). An endometrial oxidative stress (OS) model revealed that the downregulation of SIRT3 impaired the growth and proliferation status of endometrial cells and reduced their receptivity to day 4 mouse embryos. The results suggested that SIRT3 might be crucial in maintaining normal cellular state by regulating antioxidants, cell proliferation, and apoptosis, thereby contributing to enhanced endometrial receptivity. Our findings proposed a significant role of SIRT3 in improving endometrial receptivity in patients with PCOS by alleviating OS and regulating the balance between cell proliferation and apoptosis. Therefore, SIRT3 could be a promising target for predicting and improving endometrial receptivity in this patient population.

A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

Mechanism and clinical progression of solid tumors bone marrow metastasis.

The rich blood supply of the bone marrow provides favorable conditions for tumor cell proliferation and growth. In the disease's early stages, circulating tumor cells can escape to the bone marrow and form imperceptible micro metastases. These tumor cells may be reactivated to regain the ability to grow aggressively and eventually develop into visible metastases. Symptomatic bone marrow metastases with abnormal hematopoiesis solid tumor metastases are rare and have poor prognoses. Treatment options are carefully chosen because of the suppression of bone marrow function. In this review, we summarized the mechanisms involved in developing bone marrow metastases from tumor cells and the clinical features, treatment options, and prognosis of patients with symptomatic bone marrow metastases from different solid tumors reported in the literature.

Simulation of microbial growth based on Euler’s method

Microorganisms such as bacteria, fungi and yeast produce valuable metabolites when they are grown in suitable culture conditions. The cultivation condition affects the cell growth, metabolism, and product production in a sophisticated and nonlinear way. Therefore, in this research, the growth of Lactococcus lactis NZ9000 in response to the growth conditions was simulated using different growth models. The objective was to simulate the effect of temperature, agitation speed, carbon and nitrogen sources, on the cell growth using the exponential model, logistic and Monod equations. All equations were solved according to the Euler’s method using MATLAB R2021a for simulation. The experimental data used for the simulation were from literature. The accuracy of the model was expressed as percentage relative error between the maximum value of experimental and simulated data. Simulation results shows that the optimum conditions for cell growth was achieved at temperature 27°C, agitation speed of 100 rpm with glucose and peptone as the carbon and nitrogen sources respectively equation. The maximum cell concentration by logistic equation gives the lowest percentage error of 6.40% and 0.33% for the effect of temperature and agitation respectively. While Monod equation give the closest accuracy of 1.84% and 7.11% for carbon and nitrogen sources respectively. Thus, it was shown that the complexity of the microorganism growth was able to be simulated using suitable model such as logistic equation with the lowest relative error.

Triple Negative Breast Cancer (TNBC): Cannabis sativa-Role of Phytocannabinoids

This review paper highlights the role of phytocannabinoids in controlling triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), which has clinical features that include high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. TNBC is one of the most aggressive subtypes of breast cancer that is often associated with poor patient outcomes because of the development of metastases in secondary organisms like in the brain, bone, and lungs. Triple-negative breast cancer (TNBC) is a kind of breast cancer that does not have any of the receptors that are commonly found in breast cancer. The highest number of TNBC cases has been recorded in India. Currently, TNBC is highly prevalent among Indian women and develops approximately 20% to 43% of all patients with breast cancer. Triple-negative breast cancer (TNBC) is an aggressive malignancy that requires effective targeted drug therapy. Patients with TNBC develop metastasis and recurrence over time and have reduced survival compared to patients with other subtypes of breast cancer. Pre-clinical studies have demonstrated that phytocannabinoids exert important antitumor properties in the main breast cancer subtypes, particularly in TNBC, where different phytocannabinoids and synthetic cannabinoids have shown interesting therapeutic actions. Neither CBD nor THC are universally efficacious in reducing cancer cell viability. Focusing on in vitro studies, the effect of CBD on cancer cell viability ranges from no effect, to a modest reduction, and to significant cytotoxicity depending on concentrations, cancer cell lines, cell growth conditions, the performed assays, and the time of CBD exposure. However, it should also be noted that research into the efficacy, dosage and drug safety of cannabinoids in tumor therapy still has a long way to go, especially with regard to human clinical trials to be conducted, through which alone the benefits and advantages for cancer patients but also possible risks can be defined.

Simvastatin-loaded 3D aerogel scaffolds promote bone regeneration.

It is imperative to design a suitable material for bone regeneration that emulates the microstructure and compositional framework of natural bone while mitigating the shortcomings of current repair materials. The aim of the study is to synthesize a 3D aerogel scaffold composed of PLCL/gelatin electro-spun nanofiber loaded with Simvastatin and investigate its biocompatibility as well as its performance in cell proliferation and ossification differentiation. PLCL/gelatin nanofibers were fabricated in coaxial electrospinning with simvastatin added. Fibers were fragmented, pipetted into molds, frozen, and dried. The morphology of fibers and contact angles in 4 groups of PLCL, PLCL@S, 3D-PLCL, and 3D-PLCL@S was observed and compared. MC3T3-E1 cells were planted at the four materials to observe cell growth status, and ALP and ARS tests were conducted to compare the ossification of cells. TEM scanning showed the coaxial fiber of the inner PLCL and outer gelatin. The mean diameter of the PLCL/gelatin fibers is 561 ± 95nm and 631 ± 103nm after the drug loading. SEM showed the fibers in the 3D-PLCL@S group were more curled and loose with more space interlaced. The contact angle in this group was 27.1°, the smallest one. Drug release test demonstrated that simvastatin concentration in the 3D-PLCL@S could remain at a relatively high level compared to the control group. The cell proliferation test showed that MC3T3-EI cells could embed into the scaffold deeply and exhibit higher viability in the 3D-PLCL@S group than other groups. The ossification tests of ALP and ARS also inferred that the 3D-PLCL@S scaffold could offer a better osteogenic differentiation matrix. The PLCL/gelatin aerogel scaffold, when loaded with Simvastatin, demonstrates a more pronounced potential in enhancing osteoblast proliferation and osteogenic differentiation. We hypothesize that this scaffold could serve as a promising material for addressing bone defects.

Metformin and Glucose Concentration as Limiting Factors in Retinal Pigment Epithelial Cell Viability and Proliferation.

Metformin is a well-established drug for the treatment of type 2 diabetes; however, the mechanism of action has not been well described and many aspects of how it truly acts are still unknown. Moreover, regarding in vitro experiments, the glycaemic status when metformin is used is generally not considered, which, added to the suprapharmacological drug concentrations that are commonly employed in research, has resulted in gaps of its mechanism of action. The aim of this study was to determine how glucose and metformin concentrations influence cell culture. Considering that diabetic retinopathy is one of the most common complications of diabetes, a retinal pigment epithelial cell line was selected, and cell viability and proliferation rates were measured at different glucose and metformin concentrations. As expected, glucose concentration by itself positively influenced cell proliferation rates. When the metformin was considered, results were conditioned, as well, by metformin concentration. This conditioning resulted in cell death when high concentrations of metformin were used under physiological concentrations of glucose, while this did not happen when clinically relevant concentrations of metformin were used independently of glucose status. Our study shows the importance of in vitro cell growth conditions when drug effects such as metformin's are being analysed.

Biofabrication of biomimetic undulating microtopography at the dermal-epidermal junction and its effects on the growth and differentiation of epidermal cells.

The undulating microtopography located at the junction of the dermis and epidermis of the native skin is called rete ridges (RRs), which plays an important role in enhancing keratinocyte function, improving skin structure and stability, and providing three-dimensional (3D) microenvironment for skin cells. Despite some progress in recent years, most currently designed and manufactured tissue-engineered skin models still cannot replicate the RRs, resulting in a lack of biological signals in the manufactured skin models. In this study, a composite manufacturing method including electrospinning, 3D printing, and functional coating was developed to produce the epidermal models with RRs. Polycaprolactone (PCL) nanofibers were firstly electrospun to mimic the extracellular matrix environment and be responsible for cell attachment. PCL microfibers were then printed onto top of the PCL nanofibers layer by 3D printing to quickly prepare undulating microtopography and finally the entire structures were dip-coated with gelatin hydrogel to form a functional coating layer. The morphology, chemical composition, and structural properties of the fabricated models were studied. The results proved that the multi-process composite fabricated models were suitable for skin tissue engineering. Live and dead staining, cell counting kit-8 (CCK-8) as well as histology (haematoxylin and eosin (HE) methodology) and immunofluorescence (primary and secondary antibodies combination assay) were used to investigate the viability, metabolic activity, and differentiation of skin cells forin vitroculturing.In vitroresults showed that each model had high cell viability, good proliferation, and the expression of differentiation marker. It was worth noting that the sizes of the RRs affected the cell growth status of the epidermal models. In addition, the unique undulation characteristics of the epidermal-dermal junction can be reproduced in the developed epidermal models. Overall, thesein vitrohuman epidermal models can provide valuable reference for skin transplantation, screening and safety evaluation of drugs and cosmetics.

Multi-DNA-Modified Double-Network Hydrogel with Customized Microstructure: A Novel System for Living Circulating Tumor Cells Capture and Real-Time Detection.

The precise and effective isolation of living circulating tumor cells (CTCs) from peripheral blood, followed by their real-time monitoring, is crucial for diagnosing cancer patients. In this study, a cell-imprinted double-network (DN) hydrogel modified with circular multi-DNA (CMD), coined the CMD-imprinted hydrogel with fixed cells as templates (CMD-CIDH), was developed. The hydrogel featured a customized surface for proficient capture of viable CTCs and in situ real-time fluorescent detection without subsequent release. The customized surface, constructed using polyacrylamide/chitosan DN hydrogel as the matrix on the cell template, had a dense network structure, thereby ensuring excellent stability and a low degradation rate. Optimal capture efficiencies, recorded at 93 ± 3% for MCF-7 cells and 90 ± 2% for Hela cells, were achieved by grafting the CMD and adjusting the nodule size on the customized surface. The capture efficiency remained significantly high at 67 ± 11% in simulated breast cancer patient experiments even at a minimal concentration of 5 cells mL-1. Furthermore, CMD grafted onto the surface produced a potent fluorescence signature, enabling in situ real-time fluorescent detection of the target cell's growth state even in complex environments. The customized surface is highly efficient for screening CTCs in peripheral blood and has promising potential for setting up the CTCs culture.

Ovarian cancer cells regulate their mitochondrial content and high mitochondrial content is associated with a poor prognosis

Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient’s prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice.In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.

Generation of Bidimensional and Three-Dimensional Muscle Culture Systems.

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.

A non-canonical Puf3p-binding sequence regulates CAT5/COQ7 mRNA under both fermentable and respiratory conditions in budding yeast.

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3'-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p's direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ10 deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.