Automated ELISA On-Chip for the Detection of Anti-SARS-CoV-2 Antibodies.

The COVID-19 pandemic has been the most critical public health issue in modern history due to its highly infectious and deathly potential, and the limited access to massive, low-cost, and reliable testing has significantly worsened the crisis. The recovery and the vaccination of millions of people against COVID-19 have made serological tests highly relevant to identify the presence and levels of SARS-CoV-2 antibodies. Due to its advantages, microfluidic-based technologies represent an attractive alternative to the conventional testing methodologies used for these purposes. In this work, we described the development of an automated ELISA on-chip capable of detecting anti-SARS-CoV-2 antibodies in serum samples from COVID-19 patients and vaccinated individuals. The colorimetric reactions were analyzed with a microplate reader. No statistically significant differences were observed when comparing the results of our automated ELISA on-chip against the ones obtained from a traditional ELISA on a microplate. Moreover, we demonstrated that it is possible to carry out the analysis of the colorimetric reaction by performing basic image analysis of photos taken with a smartphone, which constitutes a useful alternative when lacking specialized equipment or a laboratory setting. Our automated ELISA on-chip has the potential to be used in a clinical setting and mitigates some of the burden caused by testing deficiencies.

Steroid 5 alpha-reductase 2 enzyme variants, biomass exposure and tobacco use in Mexican patients with prostate cancer.

The presence of the genetic variants of the steroid 5-alpha reductase 2 enzyme, which is encoded by the SRD5A2 gene, has been associated with an increased risk of developing prostate cancer among certain ethnic groups. However, these molecular studies have not been conducted on the Mexican population. The analysis of the genetic variants, rs9282858 and rs523349, was performed in 101 males with prostate cancer and 100 healthy controls classified as males without prostate abnormalities (n=60) and males with benign prostatic hyperplasia (n=40), to identify a probable association with this cancer type in the Northeast Mexican population. An association was identified between prostate cancer and biomass exposure [P=0.012; odds ratio (OR), 2.89; confidence interval (CI)=1.21-6.88] and tobacco use (P=0.028; OR=1.88; CI=1.07-3.31), while no association was observed between cancer development and the rs9282858 variant, or between a protective effect and the rs523349 variant. Notably, an association was identified between rs523349 and biomass exposure (P=0.013, OR=3.17; CI=1.23-8.17 for the G risk allele, and OR=0.32, CI=0.12-0.81 for the C protective allele) using the dominant genetic model. To the best of our knowledge, the present study was the first of its type to investigate the Mexican population with prostate cancer.

Characterization of a novel automated microfiltration device for the efficient isolation and analysis of circulating tumor cells from clinical blood samples.

The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 out of 8 samples from patients diagnosed with metastatic prostate cancer (mean ± SEM = 21 ± 2.957 CTCs/mL, median = 21 CTCs/mL), demonstrating the potential clinical utility of this device.

High-Throughput Automated Microscopy of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have the potential of becoming the gold standard marker for cancer diagnosis, prognosis and monitoring. However, current methods for its isolation and characterization suffer from equipment variability and human operator error that hinder its widespread use. Here we report the design and construction of a fully automated high-throughput fluorescence microscope that enables the imaging and classification of cancer cells that were labeled by immunostaining procedures. An excellent agreement between our machine vision-based approach and a state-of-the-art microscopy equipment was achieved. Our integral approach provides a path for operator-free and robust analysis of cancer cells as a standard clinical practice.

"Antimicrobial and antiproliferative activity of essential oil, aqueous and ethanolic extracts of Ocimum micranthum Willd leaves".

BACKGROUND: Ocimum micranthum Willd is a plant used in traditional medicine practiced in the region of the Yucatan peninsula. In particular, it is used for the treatment of cutaneous infections and wound healing, however there are currently no existing scientific studies that support these applications. The aim of the present study was to evaluate the antimicrobial and the in vitro proliferative activity (on healthy mammalian cell lines) of the essential oil and extracts (aqueous and ethanolic) of this plant. METHODS: The minimal inhibitory concentration (MIC) of essential oil and aqueous and ethanolic extracts of Ocimum micranthum leaves against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans was determined using the microdilution technique. The in vitro proliferative activity of human fibroblast (hFB) and Chinese hamster ovary (CHO-K1) cells treated with these extracts was evaluated using the MTT test. The hFB cell line was also evaluated using Trypan Blue assay. RESULTS: Candida albicans was more susceptible to the ethanolic extract and the aqueous extract (MIC value of 5 µL/mL and 80 µL/mL respectively). In the case of Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, the MIC of the aqueous and ethanolic extract was 125 µL/mL. The aqueous extract showed a significant (p < 0.05) antiproliferative effect on hFB cells at a concentration of 4%, with cell proliferation percentage values of 73.56% and 20.59% by MTT method and Trypan Blue assay, respectively; the same effect was observed for the ethanolic extract at concentration from 0.06% to 0.25% using MTT method and at a concentration from 0.125% to 0.25% using Trypan Blue assay. In CHO-K1 cells an antiproliferative effect was observed at a concentration of 8% of aqueous extract and from 0.06% to 0.25% of ethanolic extract using the MTT method. CONCLUSION: These assays showed that low concentrations of essential oil and extracts of Ocimum micranthum leaves are sufficient to cause an antiproliferative effect on the hFB cell line but do not produce an antimicrobial effect against the microorganisms evaluated. More studies are necessary to improve understanding of the mechanism of action of the compounds implicated in the bioactivities shown by the crude extracts.

Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells-the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2-4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for µmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27-1.08 × 10(7) cell/mL and 0.72-2.79 × 10(6) cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking-Piret model (d[mAb]/dt = αd[X]/dt + ß[X]) with values of α = 7.65 × 10(-7) µg/cell and ß = 7.68 × 10(-8) µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode.

Supercritical CO2 foaming of thermoplastic materials derived from maize: proof-of-concept use in mammalian cell culture applications.

BACKGROUND: Foams are high porosity and low density materials. In nature, they are a common architecture. Some of their relevant technological applications include heat and sound insulation, lightweight materials, and tissue engineering scaffolds. Foams derived from natural polymers are particularly attractive for tissue culture due to their biodegradability and bio-compatibility. Here, the foaming potential of an extensive list of materials was assayed, including slabs elaborated from whole flour, the starch component only, or the protein fraction only of maize seeds. METHODOLOGY/PRINCIPAL FINDINGS: We used supercritical CO2 to produce foams from thermoplasticized maize derived materials. Polyethylene-glycol, sorbitol/glycerol, or urea/formamide were used as plasticizers. We report expansion ratios, porosities, average pore sizes, pore morphologies, and pore size distributions for these materials. High porosity foams were obtained from zein thermoplasticized with polyethylene glycol, and from starch thermoplasticized with urea/formamide. Zein foams had a higher porosity than starch foams (88% and 85%, respectively) and a narrower and more evenly distributed pore size. Starch foams exhibited a wider span of pore sizes and a larger average pore size than zein (208.84 vs. 55.43 µm2, respectively). Proof-of-concept cell culture experiments confirmed that mouse fibroblasts (NIH 3T3) and two different prostate cancer cell lines (22RV1, DU145) attached to and proliferated on zein foams. CONCLUSIONS/SIGNIFICANCE: We conducted screening and proof-of-concept experiments on the fabrication of foams from cereal-based bioplastics. We propose that a key indicator of foamability is the strain at break of the materials to be foamed (as calculated from stress vs. strain rate curves). Zein foams exhibit attractive properties (average pore size, pore size distribution, and porosity) for cell culture applications; we were able to establish and sustain mammalian cell cultures on zein foams for extended time periods.

Induction of apoptosis in colon cancer cells treated with isorhamnetin glycosides from Opuntia ficus-indica pads.

(OFI) contains health-promoting compounds like flavonoids, being the isorhamnetin glycosides the most abundant. We evaluated the effect of OFI extracts with different isorhamnetin glycosides against two different human colon cancer cells (HT-29 and Caco2). The extracts were obtained by alkaline hydrolysis with NaOH at 40 °C during 15, 30 or 60 min. Tri and diglycosides were the most abundant isorhamnetin glycosides, therefore these compounds were isolated to compare their cytotoxic effect with the obtained from the extracts. The OFI extracts and purified isorhamnetin glycosides were more cytotoxic against HT-29 cells than Caco2 cells. OFI-30 exhibited the lowest IC50 value against HT-29 (4.9 ± 0.5 µg/mL) and against Caco2 (8.2 ± 0.3 µg/mL). Isorhamnetin diglycosides IG5 and IG6 were more cytotoxic than pure isorhamnetin aglycone or triglycosides when they were tested in HT-29 cells. Bioluminescent analysis revealed increased activity of caspase 3/7 in OFI extracts-treated cells, particularly for the extract with the highest concentration of isorhamnetin triglycosides. Flow cytometry analysis confirmed that OFI extract and isorhamnetin glycosides induced a higher percentage of apoptosis in HT-29 than in Caco2, while isorhamnetin was more apoptotic in Caco2. This research demonstrated that glycosilation affected antiproliferative effect of pure isorhamnetin glycosides or when they are mixed with other phytochemicals in an extract obtained from OFI.

Continuous flow micro-bioreactors for the production of biopharmaceuticals: the effect of geometry, surface texture, and flow rate.

We used continuous flow micro-devices as bioreactors for the production of a glycosylated pharmaceutical product (a monoclonal antibody). We cultured CHO cells on the surface of PMMA/PDMS micro-channels that had been textured by micromachining and coated with fibronectin. Three different micro-channel geometries (a wavy channel, a zigzag channel, and a series of donut-shape reservoirs) were tested in a continuous flow regime in the range of 3 to 6 µL min(-1). Both the geometry of the micro-device and the flow rate had a significant effect on cell adhesion, cell proliferation, and monoclonal antibody production. The most efficient configuration was a series of donut-shaped reservoirs, which yielded mAb concentrations of 7.2 mg L(-1) at residence times lower than one minute and steady-state productivities above 9 mg mL(-1) min(-1). These rates are at about 3 orders of magnitude higher than those observed in suspended-cell stirred tank fed-batch bioreactors.

A biopharmaceutical plant on a chip: continuous micro-devices for the production of monoclonal antibodies.

We report a proof-of-principle for the use of micro-devices as continuous bioreactors for the production of monoclonal antibodies. We culture CHO cells on the surface of PMMA "zigzag" channels textured with semi-spherical cavities coated with fibronectin, observing steady-state productivities 100 times higher than those observed in full scale systems.

A simple eccentric stirred tank mini-bioreactor: mixing characterization and mammalian cell culture experiments.

In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5-100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple-easy to assemble, easy to use, easy to clean-cell culture mini-bioreactors for lab-scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini-bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini-bioreactor were comparable to those observed for 6-well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini-bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini-bioreactor.