Use Of Living Cells Research Articles

The interplay between hyaluronic acid and stem cell secretome boosts pulmonary differentiation in 3D biomimetic microenvironments

Mesenchymal stem cells (MCSs) secretome provide MSC-like therapeutic effects in preclinical models of lung injury, circumventing safety concerns with the use of live cells. Secretome consists of Extracellular Vesicles (EVs), including populations of nano- to micro-sized particles (exosomes and microvesicles) delimited by a phospholipidic bilayer. However, its poor stability and bioavailability severely limit its application. The role of Hyaluronic acid (HA) as potential carrier in biomedical applications has been widely demonstrated. Here, we investigated the interplay between HA and MSCs- secretome blends and their ability to exert a bioactive effect on pulmonary differentiation in a 3D microenvironment mimicking lung niche. To this aim, the physical-chemical properties of HA/Secre blends have been characterized at low, medium and high HA Molecular Weights (MWs), by means of SEM/TEM, DLS, confocal microscopy and FTIR. Collectively physical-chemical properties highlight the interplay between the HA and the EVs. In 3D matrices, HA/Secre blends showed to promote differentiation in pulmonary lineage, improved as the MW of the HA in the blends decreased. Finally, HA/Secre blends' ability to cross an artificial mucus has been demonstrated. Overall, this work provides new insights for the development of future devices for the therapy of respiratory diseases that are still unmet.

Label-Free and Real-Time Optical Detection of Affinity Binding of the Antibody on Adherent Live Cells.

Oblique-incidence reflectivity difference (OIRD) is a novel real-time, label-free, and nondestructive optical detection method and exhibits encouraging application in the detection of antibody/DNA microarrays. In this study, for the first time, an OIRD label-free immunoassay was achieved by using adherent live cells as the probe. The cells were cultured on glass cells, and the affinity binding of antibodies targeted on the HLA class I antigen of the cell surface was detected with an OIRD. The results show that an OIRD is able to detect the binding process of anti-human HLA-A, B, and C antibodies on MDA-MB-231 cells and HUVEC cells. Control experiments and complementary fluorescence analysis confirmed the high detection specificity and good quantitative virtue of the OIRD label-free immunoassay. Label-free OIRD imaging analysis of cell microarrays was further demonstrated successfully, and the underlying optical mechanism was revealed by combining the theoretical modeling. This work explores the use of live cells as probes for an OIRD immunoassay, thus expanding the potential applications of the OIRD in the field of pathological analysis, disease diagnosis, and drug screening, among others.

A Novel, Probe-Based and Instrument-Free Method for Improved Ultrahigh-Throughput Single-Cell Gene Expression Analysis of Peripheral Blood Mononuclear Cells

Single-cell gene expression assays are critical tools for identifying functional subtypes of immune cells, as well as changes within cells resulting from pathology or treatment. However, most current methods are expensive, low in sample throughput, rely on reverse transcription and lack the sensitivity to consistently measure low expressed genes from single cells - preventing measurements of many key immune biomarkers and molecular pathways. Another problem with existing single-cell sequencing methods is that many of the cell-associated reads either fail to map to transcripts or map to uninformative ribosomal and mitochondrial genes. Some methods also require the use of live cells, which means the single-cell assay has to be carried out at the specific time of treatment or when culture is terminated. The aforementioned limitations have contributed to most single-cell sequencing studies being restricted to small numbers of samples and have prevented their wider scale adoption in biopharma, clinical, translational and applied applications. Here we report on the development and performance of a novel combinatorial split-pool-based workflow, TempO-LINC™, that adds cell-identifying molecular barcodes onto high-sensitivity gene expression probes within fixed cells. Cells can be fixed at the end of a treatment, or when obtained from a patient and stably stored for months until they are ready to be processed with other samples through the TempO-LINC assay. All probes within the same fixed cell receive an identical barcode, enabling the reconstruction of single-cell gene expression profiles across as few as several hundred cells and up to 100,000 cells per run. TempO-LINC simultaneously processes up to 96 fixed cell samples per run, has a simple workflow that is amenable to automation and as we will show, has a higher gene detection rate than existing single-cell platforms. We will also show that TempO-LINC has an observed doublet rate of less than 0.5% for 12,500 cells analyzed. Critically, TempO-LINC reduces sample sequencing costs and although we show the assay accurately profiles the whole transcriptome (23,000 transcripts), it can also be targeted to measure only genes/pathways of interest - dramatically reducing sequencing costs yet further. We applied TempO-LINC to assay PBMCs and a human hepatocyte model for toxicology and demonstrated the ability to correctly identify unique cell states and molecular pathways from these populations. We will also present time course and dose response data from compound treatment of cells. All of these features position TempO-LINC as a potential disruptive technology that hematology researchers, clinicians and drug developers can use for new large-scale applications/studies using single-cell sequencing across thousands of samples while obtaining improved data quality.

Refrigerated human mesenchymal stromal cells as an alternative to cryostorage for use in clinical investigation.

Mesenchymal stromal cells (MSCs) and other therapeutic cells show efficacy for cardiac damage, neurological disease, chronic lung disease, pediatric graft versus host disease, and several inflammatory conditions. Based on their anti-inflammatory and immune-modulatory activities, responsiveness, and secretion of beneficial factors, cellular therapeutics may provide benefits in acute and chronic traumatic injury. However, the use of live cells presents logistical challenges, especially for military trauma. MSCs are generally shipped and stored frozen but require sterile handling before infusion. This requires skilled personnel and equipment not readily available in a forward medical treatment facility or even a small community hospital. Commercial human bone marrow- and adipose-derived MSCs from multiple donors were cultured under standard conditions, harvested and stored at 4°C in solution for up to 21 days. Cell viability, ATP content, apoptosis, proliferation capability, immunomodulation activity, and responsiveness were assessed after different amounts of time. Human MSCs can be stored at 4°C in MSC culture medium for 14 days while maintaining a reasonable level of viability and function. Both viability and function are reduced when MSCs are stored in crystalloid solutions. This approach makes it feasible to prepare cellular therapeutic agents in a laboratory or commercial facility and ship them under refrigerated conditions. Once they reach their destination, they can be stored at 4°C under conditions similar to blood products. Cells prepared and stored this way could also be used directly with minimal handling, making them more practical for both civilian and military trauma.

Rewiring cell-free metabolic flux in E. coli lysates using a block-push-pull approach.

Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared to their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the 'block-push-pull' concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and toward endogenous ethanol synthesis. The approach is readily adaptable, is relatively rapid and allows for the manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating by-product-forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux toward ethanol. The approach incorporating these block, push and pull strategies maximized the glucose-to-ethanol conversion in an Escherichia coli lysate that initially had low ethanologenic potential. A 10-fold improvement in the percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and completely transforming the consumed input substrate to a desired output product in a lysate-based, cell-free system.

Multiagent Intratumoral Immunotherapy Can Be Effective in A20 Lymphoma Clearance and Generation of Systemic T Cell Immunity

The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.

Extracellular Vesicles: A New Promise for the Prevention of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) despite numerous efforts of neonatologists remains one of the most frequent and long-lasting chronic respiratory diseases consequent to extreme preterm birth. New clinical trials are exploring the possible use of mesenchymal stem cells (MSCs) and especially their products, extracellular vesicles (EVs), that overcome some of the possible issues related to the use of live cells. MSCs already reached clinical implementation; MSC-EVs, on the contrary, showed extremely promising results in the preclinical setting but are still waiting their first in human results that are likely to happen soon. KEY POINTS: · BPD is one of the most frequent complications of preterm birth, and its prevention lacks an effective tool.. · EVs have shown encouraging results in preclinical animal models.. · Technical and biological advancements are needed before routine clinical use..

<i>Eletteria cardamomum</i> and Greenly Synthesized MgO NPs: A Detailed Review of Their Properties and Applications

The use of live cells to synthesize green nanoparticles (NPs) is a promising and novel bio nanotechnology method. Chemical and physical methods are utilized to synthesize NPs; however, biological approaches are preferred because they are ecologically friendly, safe, healthful, cost-effective, accessible, and effective sources of high productivity and purity. Toxic and hazardous substances, as well as the use of external reducing, stabilizing, or capping agents, are not necessary for the green synthesis of NPs. Cardamom seeds, which are little Capsules (fruits) of <i>Elettaria cardamomum</i> (Family (Zingiberaceae)], have been used in herbal medicine to treat a wide range of ailments, including asthma, tooth and gum infections, cataracts, nausea, diarrhea, and cardiac, digestive, and kidney problems. Cardamom capsules have a number of additional health benefits that are important from both a traditional and current pharmacological perspective. MO-NPs (metal oxide nanoparticles) have caused quite a stir in recent years due to their diverse set of properties. <i>Elettaria cardamomum</i> is high in polyphenolic compounds and flavonoids, making it a viable green source for large-scale, low-cost, and environmentally friendly MgO-NP production. UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) with EDS are used to investigate the phytoassisted synthesis of MgO. (Energy dispersive X-ray spectroscopy). Researchers have successfully disseminated suitable MO-NPs for obtaining significant results by leveraging their numerous desirable qualities, such as those found in the pharmaceutical, ceramics, textile, electronic, and fertilizer industries.

Mechanically Induced Cavitation in Biological Systems.

Cavitation bubbles form in soft biological systems when subjected to a negative pressure above a critical threshold, and dynamically change their size and shape in a violent manner. The critical threshold and dynamic response of these bubbles are known to be sensitive to the mechanical characteristics of highly compliant biological systems. Several recent studies have demonstrated different biological implications of cavitation events in biological systems, from therapeutic drug delivery and microsurgery to blunt injury mechanisms. Due to the rapidly increasing relevance of cavitation in biological and biomedical communities, it is necessary to review the current state-of-the-art theoretical framework, experimental techniques, and research trends with an emphasis on cavitation behavior in biologically relevant systems (e.g., tissue simulant and organs). In this review, we first introduce several theoretical models that predict bubble response in different types of biological systems and discuss the use of each model with physical interpretations. Then, we review the experimental techniques that allow the characterization of cavitation in biologically relevant systems with in-depth discussions of their unique advantages and disadvantages. Finally, we highlight key biological studies and findings, through the direct use of live cells or organs, for each experimental approach.

Pairing Microwell Arrays with an Affordable, Semiautomated Single-Cell Aspirator for the Interrogation of Circulating Tumor Cell Heterogeneity.

Comprehensive analysis of tumor heterogeneity requires robust methods for the isolation and analysis of single cells from patient samples. An ideal approach would be fully compatible with downstream analytic methods, such as advanced genomic testing. These endpoints necessitate the use of live cells at high purity. A multitude of microfluidic circulating tumor cell (CTC) enrichment technologies exist, but many of those perform bulk sample enrichment and are not, on their own, capable of single-cell interrogation. To address this, we developed an affordable semiautomated single-cell aspirator (SASCA) to further enrich rare-cell populations from a specialized microwell array, per their phenotypic markers. Immobilization of cells within microwells, integrated with a real-time image processing software, facilitates the detection and precise isolation of targeted cells that have been optimally seeded into the microwells. Here, we demonstrate the platform capabilities through the aspiration of target cells from an impure background population, where we obtain purity levels of 90%-100% and demonstrate the enrichment of the target population with high-quality RNA extraction. A range of low cell numbers were aspirated using SASCA before undergoing whole transcriptome and genome analysis, exhibiting the ability to obtain endpoints from low-template inputs. Lastly, CTCs from patients with castration-resistant prostate cancer were isolated with this platform and the utility of this method was confirmed for rare-cell isolation. SASCA satisfies a need for an affordable option to isolate single cells or highly purified subpopulations of cells to probe complex mechanisms driving disease progression and resistance in patients with cancer.

Metabolic Profiling of Escherichia coli-based Cell-Free Expression Systems for Process Optimization.

Biotechnology has transformed the production of various chemicals and pharmaceuticals due to its efficient and selective processes, but it is inherently limited by its use of live cells as "biocatalysts." Cell-free expression (CFE) systems, which use a protein lysate isolated from whole cells, have the potential to overcome these challenges and broaden the scope of biomanufacturing. Implementation of CFE systems at scale will require determining clear markers of lysate activity and developing supplementation approaches that compensate for potential variability across batches and experimental protocols. Towards this goal, we use metabolomics to relate lysate preparation and performance to metabolic activity. We show that lysate processing affects the metabolite makeup of lysates, and that lysate metabolite levels change over the course of a CFE reaction regardless of whether a target compound is produced. Finally, we use this information to develop ways to standardize lysate activity and to design an improved CFE system.

Enzyme adds antibody to cell surface

The use of live cells as therapies could become more widespread if there were easy ways to modify cell surfaces. A team led by Peng Wu of Scripps Research in California now reports a single-step chemoenzymatic reaction for attaching antibodies to cell surfaces using a bacterial fucosyltransferase enzyme from Helicobacter pylori (ACS Cent. Sci. 2018, DOI: 10.1021/acscentsci.8b00552). The researchers discovered that the enzyme can accept a broad range of substrates, so they attached an antibody to the enzyme’s normal substrate, guanosine diphosphate fucose. The enzyme successfully transfers the combination, even though it’s significantly larger than the usual substrate on its own. The reaction takes less than 10 minutes. The researchers demonstrated the method by attaching trastuzumab, an antibody used for cancer treatment, to the surface of natural killer (NK) immune cells. Trastuzumab–NK conjugates are more effective than unmodified NK cells in killing cancer cells both in cell culture and in

Influence of Dyes on Metal Removal: a Study Using Live and Dead Cells of Penicillium simplicissimum in Single-Metal and Dye-Metal Mixtures

The metal removal efficiency of live and dead fungal cells of Penicillium simplicissimum (isolate 10, KP713758) was tested against 100 ppm toxic metals Cd, Cu, Pb, Zn and trivalent Cr in single-metal solutions and in mixtures with 50 ppm triphenylmethane (TPM) dyes (Cotton Blue, Crystal Violet, Methyl Violet, and Malachite Green). Results revealed that live cells were more effective at removing metals from single (10.8–70.0%) and dye-metal mixtures (5.3–62.2%) than dead cells (5.1–27.4%). The dyes in dye-metal mixtures influenced metal uptake capacity. All four dyes inhibited the uptake of Cd (0.9–7.1%), Zn (5.3–7.7%), Cu (5.0–25.8%) and Cr (47.3–60.7%) by live cells. The exception was the enhance removal of Pb by live cells (48.6 to 56.3–62.2%) in the presence of dye (MG or CB). Pb uptake was not affected by MV (51.8%), but was inhibited by CV (31.9%). For dead cells, uptake of metals (Cd, Cr and Zn) in single (3.2–27.4%) and dye-metal solutions (0.7–27.4%) was similar, whilst removal of Cu (6.4–13.5%) and Pb (33.6–44.0%) was inhibited by all four dyes. P. simplicissimum is concluded to have potential to remove toxic metals even in the presence of TPM dyes, with preferred use of live cells than dead cells.

Thermoresponsive Microgel Coatings as Versatile Functional Compounds for Novel Cell Manipulation Tools.

For the effective use of live cells in biomedicine as in vitro test systems or in biotechnology, non-invasive cell processing and characterisation are key elements. Thermoresponsive polymer coatings have been demonstrated to be highly beneficial for controlling the interaction of adherent cells through their cultivation support. However, the widespread application of these coatings is hampered by limitations in their adaptability to different cell types and because the full range of applications has not yet been fully explored. In the work presented here, we address these issues by focusing on three different aspects. With regard to the first aspect, by using well-defined laminar flow in a microchannel, a highly controllable and reproducible shear force can be applied to adherent cells. Employing this tool, we demonstrate that cells can be non-invasively detached from a support using a defined shear flow. The second aspect relates to the recent development of simple methods for patterning thermoresponsive coatings. Here, we show how such patterned coatings can be used for improving the handling and reliability of a wound-healing assay. Two pattern geometries are tested using mouse fibroblasts and CHO cells. In terms of the third aspect, the adhesiveness of cells depends on the cell type. Standard thermoresponsive coatings are not functional for all types of cells. By coadsorbing charged nanoparticles and thermoresponsive microgels, it is demonstrated that the adhesion and detachment behaviour of cells on such coatings can be modulated.

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen

We have developed a new high-content cytotoxicity assay using live cells, called “ImageTOX.” We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

Preservation of phytase enzyme produced by anaerobic rumen bacteria, Mitsuokella jalaludinii

Poultry feed consists of feed ingredients like soybean meal and corn, which contain high levels of phytate that is poorly utilised especially by the monogastric animals that lack of phytase. Hence, phytase has been extensively applied as a feed supplement in poultry production due to the efficiency of this enzyme in improving phosphorous (P) availability, thus reducing P excretion to the environment as well as reducing the feed cost by reducing inorganic P supplementation. Mitsuokella jalaludinii, an obligate anaerobe, Gram-negative rumen bacterium, produces high phytase activity. Birds supplemented with bacterial preparation of M. jalaludinii showed comparable performance to that of commercial phytase. However, the anaerobic nature of this bacterium renders difficulty in the use of live cells as feed supplement in commercial poultry production. Therefore, this study was conducted to determine a suitable method to preserve phytase activity of M. jalaludinii regardless of cells viability. Mitsuokella jalaludinii was grown in MF medium under anaerobic condition and the cells were subjected to various treatments to preserve the enzyme, including bead beating, compressed air, moist heat, dry heat and freeze-drying under aerobic condition. The results showed that the total number of viable cells were significantly (p&lt;0.05) reduced when the cells were subjected to bead beating, whereas no viable cells were detected for compressed air, moist heat, dry heat and freeze-drying. Bead beating, compressed air, moist heat and dry heat treatments resulted in the reduction of phytase activity. However, only freeze-drying method was able to preserve high level of phytase activity significantly (p&lt;0.05).

Optimal condition-based harvesting policies for biomanufacturing operations with failure risks

ABSTRACTThe manufacture of biological products from live systems such as bacteria, mammalian, or insect cells is called biomanufacturing. The use of live cells introduces several operational challenges including batch-to-batch variability, parallel growth of both desired antibodies and unwanted toxic byproducts in the same batch, and random shocks leading to multiple competing failure processes. In this article, we develop a stochastic model that integrates the cell-level dynamics of biological processes with operational dynamics to identify optimal harvesting policies that balance the risks of batch failures and yield/quality tradeoffs in fermentation operations. We develop an infinite horizon, discrete-time Markov decision model to derive the structural properties of the optimal harvesting policies. We use IgG1 antibody production as an example to demonstrate the optimal harvesting policy and compare its performance against harvesting policies used in practice. We leverage insights from the optimal policy to propose smart stationary policies that are easier to implement in practice.

Asymmetric biodegradable microdevices for cell-borne drug delivery.

Use of live cells as carriers for drug-laden particulate structures possesses unique advantages for drug delivery. In this work, we report on the development of a novel type of particulate structures called microdevices for cell-borne drug delivery. The microdevices were fabricated by soft lithography with a disklike shape. Each microdevice was composed of a layer of biodegradable thermoplastic such as poly(lactic-co-glycolic acid). One face of the thermoplastic layer was covalently grafted with a cell-adhesive polyelectrolyte such as poly-l-lysine. This asymmetric structure allowed the microdevices to bind to live cells through bulk mixing without causing cell aggregation. Moreover, the cell-microdevice complexes were largely stable, and the viability and proliferation ability of the cells were not affected by the microdevices over a week. In addition, sustained release of a mock drug from the microdevices was demonstrated. This type of microdevice promises to be clinically useful for sustained intravascular drug delivery.

Biosorption and Bioaccumulation of Cu2+ from Aqueous Solution Using Living M. amorphae Isolated from Mine Tailings

Mining generates large amounts of wastewater, which may contain elevated levels of potentially toxic metals. We evaluated the use of live cells of Mesorhizobium amorphae CCNWGS0123 as a new, relatively inexpensive and environment-friendly adsorbent material for removal of toxic metals from aqueous solutions, such as mine tailings leachate. Maximum copper uptake and removal efficiency was attained at a dosage of 0.5 g L−1, a pH of 5.0, an agitation speed of 150 rpm, and at a temperature of 28 °C for an initial Cu2+ concentration of 100 mg L−1; equilibrium was achieved within 30 min. The adsorption isothermal data matched Langmuir and Freundlich isotherms well, with correlation coefficients of 0.931 and 0.932, respectively. FT-IR analysis indicated that many functional groups with a negative charge on the cell surface were involved in the interaction between live CCNWGS0123 and copper ions. Scanning electron microscope results confirmed that there was some copper sediment on the cell surface, causing deformation, aggregation, and cell surface damage. Cu2+ accumulation was related to cell growth, and copper uptake was not only due to cell-surface binding, but also to intracellular accumulation. Live cells of CCNWGS0123 are a new biosorbent that have the potential to be economical when small batches of biomass are used to remove Cu2+ from an aqueous solution.

Ligand mediated transcriptional regulation using DNA aptamers in cell free systems

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. Critical to these efforts is the availability of a large library of ligand sensitive gene regulatory systems and the ability to rapidly test and evaluate these networks. When compared to the use of live cells, designing such systems in a cell free context offers a potentially simpler and more flexible context. Here we describe a strategy to regulate T7 RNA polymerase using DNA aptamers in a cell free context. We test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. We utilized a DNA aptamer that binds thrombin as a model system for demonstrating the feasibility of the approach. We show that for the hybrid T7‐aptamer promoter, thrombin addition results in up to a 5‐fold reduction in gene expression. We further demonstrate that gene expression is dependent on the thrombin concentration and can be tuned by altering the position of the aptamer relative to the transcription start site. These results pave the way for expanding the library of ligands that can be used for regulating gene expression and can enable facile engineering of gene networks in cell free systems. This research was supported by NIH Grant EB000657