-
Microbial Cell Factories Jun 2024With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in... (Review)
Review
With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in the parallelization of microbes cultivations are of great aid, however, for many species down-scaling leaves a metabolic footprint. Yarrowia lipolytica is one such demanding yeast species, for which scaling-down inevitably leads to perturbations in phenotype development. Strictly aerobic metabolism, propensity for filamentation and adhesion to hydrophobic surfaces, spontaneous flocculation, and high acidification of media are just several characteristics that make the transfer of the micro-scale protocols developed for the other microbial species very challenging in this case. It is well recognized that without additional 'personalized' optimization, either MTP-based or single-cell-based protocols are useless for accurate studies of Y. lipolytica phenotypes. This review summarizes the progress in the scaling-down and parallelization of Y. lipolytica cultures, highlighting the challenges that occur most frequently and strategies for their overcoming. The problem of Y. lipolytica cultures down-scaling is illustrated by calculating the costs of micro-cultivations, and determining the unintentionally introduced, thus uncontrolled, variables. The key research into culturing Y. lipolytica in various MTP formats and micro- and pico-bioreactors is discussed. Own recently developed and carefully pre-optimized high-throughput cultivation protocol is presented, alongside the details from the optimization stage. We hope that this work will serve as a practical guide for those working with Y. lipolytica high-throughput screens.
Topics: Yarrowia; High-Throughput Screening Assays
PubMed: 38915032
DOI: 10.1186/s12934-024-02465-3 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Cell culture is a fundamental tool for cell-based assays in biological and preclinical research. The measurements of cell culture, including cell count, viability, and... (Review)
Review
Cell culture is a fundamental tool for cell-based assays in biological and preclinical research. The measurements of cell culture, including cell count, viability, and metabolic activity, can reflect the conditions of cells under culture conditions. The conventional cell culture and detection methods have problems such as high consumption of reagents and samples, inability to monitor cell status in real time, and difficulty in spatiotemporally adjusting the cell microenvironment. A cell impedance sensor measures changes in the electrical impedance of cells through alternating current, enabling real-time monitoring of impedance changes caused by cell activities such as attachment, growth, proliferation, and migration. Microfluidic chips are praised for reducing complex biological processes, integrating multiple analysis modes, and achieving high automation in detection. Integrating microfluidic chips with cell impedance sensors greatly improves the capability and efficiency of cell-related analysis. This review outlines the application of microfluidic chip-based impedance sensors in 2D and 3D cell systems and summarizes the research progress in application of such sensors in research on cell growth, proliferation, viability, metabolic activity, and drug screening. Finally, this review prospects the future development trends and possible challenges, providing ideas for the development of microfluidic chips integrated with electrical impedance sensors in drug screening.
Topics: Electric Impedance; Humans; Biosensing Techniques; Cell Culture Techniques; Microfluidic Analytical Techniques; Cell Proliferation; Cell Survival; Lab-On-A-Chip Devices; Animals
PubMed: 38914492
DOI: 10.13345/j.cjb.230668 -
Microsystems & Nanoengineering 2024Assays mimicking in vitro the concentration gradients triggering biological responses like those involved in fighting infections and blood clotting are essential for...
Assays mimicking in vitro the concentration gradients triggering biological responses like those involved in fighting infections and blood clotting are essential for biomedical research. Microfluidic assays prove especially attractive as they allow precise control of gradient shape allied to a reduction in scale. Conventional microfluidic devices are fabricated using solid plastics that prevent direct access to responding cells. Fluid-walled microfluidics allows the manufacture of circuits on standard Petri dishes in seconds, coupled to simple operating methods; cell-culture medium sitting in a standard dish is confined to circuits by fluid walls made of an immiscible fluorocarbon. We develop and experimentally validate an analytical model of diffusion between two or more aqueous streams flowing at different rates into a fluid-walled conduit with the cross-section of a circular segment. Unlike solid walls, fluid walls morph during flows as pressures fall, with wall shape changing down the conduit. The model is validated experimentally for Fourier numbers < 0.1 using fluorescein diffusing between laminar streams. It enables a priori prediction of concentration gradients throughout a conduit, so allowing rapid circuit design as well as providing bio-scientists with an accurate way of predicting local concentrations of bioactive molecules around responsive and non-responsive cells.
PubMed: 38911344
DOI: 10.1038/s41378-024-00698-1 -
Frontiers in Immunology 2024The immune memory is one of the defensive strategies developed by both unicellular and multicellular organisms for ensuring their integrity and functionality. While the... (Review)
Review
The immune memory is one of the defensive strategies developed by both unicellular and multicellular organisms for ensuring their integrity and functionality. While the immune memory of the vertebrate adaptive immune system (based on somatic recombination) is antigen-specific, encompassing the generation of memory T and B cells that only recognize/react to a specific antigen epitope, the capacity of vertebrate innate cells to remember past events is a mostly non-specific mechanism of adaptation. This "innate memory" can be considered as germline-encoded because its effector tools (such as innate receptors) do not need somatic recombination for being active. Also, in several organisms the memory-related information is integrated in the genome of germline cells and can be transmitted to the progeny for several generations, but it can also be erased depending on the environmental conditions. Overall, depending on the organism, its environment and its living habits, innate immune memory appears to be a mechanism for achieving better protection and survival against repeated exposure to microbes/stressful agents present in the same environment or occurring in the same anatomical district, able to adapt to changes in the environmental cues. The anatomical and functional complexity of the organism and its lifespan drive the generation of different immune memory mechanisms, for optimal adaptation to changes in the living/environmental conditions. The concept of innate immunity being non-specific needs to be revisited, as a wealth of evidence suggests a significant degree of specificity both in the primary immune reaction and in the ensuing memory-like responses. This is clearly evident in invertebrate metazoans, in which distinct scenarios can be observed, with both non-specific (immune enhancement) or specific (immune priming) memory-like responses. In the case of mammals, there is evidence that some degree of specificity can be attained in different situations, for instance as organ-specific protection rather than microorganism-specific reaction. Thus, depending on the challenges and conditions, innate memory can be non-specific or specific, can be integrated in the germline and transmitted to the progeny or be short-lived, thereby representing an exceptionally plastic mechanism of defensive adaptation for ensuring individual and species survival.
Topics: Animals; Immunologic Memory; Immunity, Innate; Humans; Germ Cells; Adaptation, Physiological
PubMed: 38903500
DOI: 10.3389/fimmu.2024.1386578 -
Journal of Pharmaceutical and... Jun 2024Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies...
Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development.
PubMed: 38901155
DOI: 10.1016/j.jpba.2024.116301 -
Journal of Extracellular Vesicles Jun 2024Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated...
Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID-biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on-chip antifouling-immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID-biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1-cell adhesion molecule (L1CAM) as a target biomarker. The EVID-biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1 pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC = 0.973. EVID-biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300 µL) and an assay time of 1.5 h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.
Topics: Parkinson Disease; Humans; Extracellular Vesicles; Neural Cell Adhesion Molecule L1; Biomarkers; Male; Female; Liquid Biopsy; Aged; Middle Aged
PubMed: 38898558
DOI: 10.1002/jev2.12467 -
Applied Microbiology and Biotechnology Jun 2024The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device...
The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.
Topics: CHO Cells; Cricetulus; Escherichia coli; Animals; Saccharomyces cerevisiae; Acoustics; Suspensions; Lab-On-A-Chip Devices
PubMed: 38896136
DOI: 10.1007/s00253-024-13215-1 -
Frontiers in Bioengineering and... 2024
PubMed: 38895556
DOI: 10.3389/fbioe.2024.1432352 -
APL Bioengineering Jun 2024Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to...
Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and no automation. Here, we introduce the sliding insert micropipette aspiration method, which permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.
PubMed: 38894959
DOI: 10.1063/5.0193333 -
Sensors (Basel, Switzerland) Jun 2024Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry...
Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.
Topics: alpha-Galactosidase; Dried Blood Spot Testing; Humans; Fabry Disease; Transistors, Electronic; Biosensing Techniques; Infant, Newborn; Neonatal Screening
PubMed: 38894470
DOI: 10.3390/s24113681