Serum

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

Blood derivatives are the biofluids of choice for metabolomic clinical studies since blood can be collected with low invasiveness and is rich in biological information. However, the choice of the blood collection tubes has an undeniable impact on the plasma and serum metabolic content. Here, we compared the metabolomic and lipoprotein profiles of blood samples collected at the same time and place from six healthy volunteers but using different collection tubes (each enrolled volunteer provided multiple blood samples at a distance of a few weeks/months): citrate plasma, EDTA plasma, and serum tubes. All samples were analyzed via nuclear magnetic resonance spectroscopy. Several metabolites showed statistically significant alterations among the three blood matrices, and also metabolites' correlations were shown to be affected. The effects of blood collection tubes on the lipoproteins' profiles are relevant too, but less marked. Overcoming the issue associated with different blood collection tubes is pivotal to scale metabolomics and lipoprotein analysis at the level of epidemiological studies based on samples from multicenter cohorts. We propose a statistical solution, based on regression, that is shown to be efficient in reducing the alterations induced by the different collection tubes for both the metabolomic and lipoprotein profiles.

There is a strong body of evidence by several translational studies which demonstrate the potential of circulating miRNAs as a potential biomarker in oncology. However, recent reports documented varying stability of these small RNA molecules in serum samples. The aim of our pilot study was to evaluate the stability of miRNAs in serum in relation to food intake and sample storage. Serum miRNA expression levels of 16 different miRNAs from 8 healthy volunteers were quantified by real-time PCR. 4 samples from each donor were analysed-2 samples (fasting, in the morning and after food intake, at noon) were analysed within 24h and 2 samples (fasting and after food intake, at noon) were stored at -80°C for 14 days and subsequently analysed. Student´s t-test was used to determine significant differences. The detectability of the distinct miRNA as a surrogate for the stability of these small RNA molecules was slightly altered by the storage conditions, but only a miRNA 22-3p, out of the analysed 16 miRNAs, shows significant lower dCq expression (3.821 vs. 4.530; p<0,01) by qPCR dependent on storage conditions (-80°C vs. 4°C). However, miRNA levels were not affected by food intake. The difference between samples taken in the morning (fasting) and at noon (after a normal meal) did not show any significant differences. MiRNAs can be considered to be a relatively stable tool in laboratory diagnostics, but clearly every new assay needs thorough evaluation. The stability of miRNAs documented here in healthy volunteers shows their potential in the search for innovative biomarkers in oncology.

Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise in vitro into blastocyst-like structures called blastoids. Naive hPSC maintenance routinely relies on co-culture with mouse embryonic fibroblast (MEFs) as feeder cells, a method prone to variability and analytical confounders. Here, we describe a feeder-free culture system based on serum coating that supports long-term maintenance of naive hPSCs. Across five laboratories, 30 serum batches were evaluated for the expansion of eight naive hPSCs lines for up to 25 passages. Mass spectrometry analysis identified fibronectin and collagens as extracellular matrix proteins consistently present in serum coating. Cells cultured on serum coating displayed growth kinetics, clonogenic capacity, mutation rates, and global gene expression profiles comparable to MEF-based cultures. Importantly, serum-cultured naive hPSCs efficiently underwent germ layer specification, retained trophectoderm competence, and generated blastoids with efficiency similar to MEF-based cultures. Collectively, serum coating provides a scalable, cost-effective, and robust alternative to feeder-based systems, preserving genomic stability and developmental potential while eliminating MEF-associated disadvantages and variability. This platform facilitates large-scale applications of naive hPSCs and enables more reproducible mechanistic studies.