Urine-derived stem cells (USCs) represent a precious tool to be used in a broad field of applications such as research, pre-clinical and clinical studies. Nevertheless, knowledge about these cells, also in view of using them as native USCs or directly reprogrammed into specific cell lines, is poor. We have profiled the transcriptome of native and MyoD induced USCs (from both healthy and DMD subjects) using RNAseq. We found great variability across individuals probably due to the co-presence of distinct cell types, we and other have already described. Specific surface markers to select different USCs subpopulations are still missing, therefore we used the new technology Celector® (StemSel Ltd.). This instrument separates cells based only on their physical properties: dimensions, morphology, density. Three different fractions (F) were obtained and analyzed for canonical mesenchymal markers (CD90, CD73, CD105, CD44) by flow cytometry. CD90 and CD105 were mildly higher in F2 and interestingly, F3 showed an enrichment of CD146 level, a pericyte marker. Further characterization of these two cell populations is ongoing. These preliminary data confirm that USCs are composed by heterogeneous cells and further studies will define the different properties. The fine characterization of USCs populations makes USCs appealing as source of patient-specific cells to be used for different scopes, as mutation detection, or cell reprogramming.

Monoclonal antibodies (mAbs) are promising reagents both for the manufacture of drug substances and for their employment as a drug themselves, but to be approved for utilization, according to FDA recommendations and WHO guidelines, they have to undergo verifications regarding their purity, stability and percentage of aggregates. Moreover, stability tests of lots have to be performed in order to verify molecular size distribution over time and lot-to-lot consistency. Recent works in literature have highlighted the need for suitable, sensitive and reliable complementary analytical techniques for the characterization of mAbs and quantification of aggregates. Size-exclusion chromatography (SEC) is the reference technique in the biopharmaceutical industry for its robustness, high performance and simple use; however it presents some limitations especially toward the separation and detection of aggregates with high molecular weight. On the other hand, flow field-flow fractionation (F4) in its miniaturized version (hollow fiber flow field-flow fractionation, HF5) shows comparable performances with interesting additional advantages: a broad size range, gentle separation mechanism with low dilution factor and higher sensitivity. To propose HF5 as a complementary technique for evaluating aggregates’ content in mAbs samples, a comparative study of both SEC and HF5 performances has been made. In this work, SEC and HF5 were coupled with UV and multi-angle light scattering detection and employed first in separating standard samples of proteins mixture used as a sample model. Then, a screening of mobile phases and an evaluation of separation performances was performed on a therapeutic mAbs formulation, demonstrating the complementarities between SEC and HF5 and their possible use as a separative platform approach for the characterization and quality control of protein drugs.

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model.

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FlFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage, which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FlFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FlFFF of cells, at superior size-based selectivity and reduced analysis time.

HUVECs (human umbilical vein endothelial cells) are a model for prediction of endothelial dysfunction in various diseases. Cultured cells seem to be a good model for studying molecular processes in endothelial tissue. Nevertheless, they cannot reproduce real physiological status because some biological factors necessarily change in culture conditions. As a consequence, it would be useful to improve preparation of the cellular starting materials from real samples, especially when gene expression studies are further required on the cells. In this work we present a new method for the easy isolation of HUVECs from raw cord blood based on the use of the flow-assisted separative technique, gravitational field-flow fractionation (GrFFF) able to give a relatively pure sample for gene analysis without cell culture steps. The method can be applied to cell populations enzymatically removed from the umbilical vein which can be purified from contaminants cells and used directly for gene expression analysis.

Bioluminescent (BL) reporter proteins are widely employed in different types of multicolor assays thanks to their peculiar features such as high quantum yield and absence of toxicity when expressed in cells or whole organisms. In particular luciferases are the most common reporter proteins enabling ultrasensitive detection in diverse biotechnological applications. Various bioluminescent proteins have been recently isolated from luminescent organisms and the corresponding genes have been cloned. Wild-type luciferases from fireflies, sea pansies, worms, bacteria and their mutants with improved emission and thermostability are in fact suitable for many applications such as cell-based assays, in vivo imaging, energy transfer-based assays and as new bioluminescent probes in association with quantum dots. Concerning cell-based assays, very few works employing multicolor reporter proteins have been reported in literature. The main bottleneck consists in the spectral unmixing that, when using more than two luciferases emitting at a different wavelength and requiring the same substrate, does not permit to completely separate the signals. luciferases with red emission (e.g., the red mutants of P. pyralis and L. italica luciferases) may represent the best choice for bioluminescent in vivo imaging since light of wavelengths from 600 to 1100 nm is readily transmitted through tissues.

Field-flow fractionation is a separation technique characterized by a retention mechanism which makes it suitable for sorting cells over a short analysis time, with low sample carry-over and preserving cell viability. Thanks to its high sensitivity, chemiluminescence detection is suitable for the quantification of just a few cells expressing chemiluminescence or bioluminescence. In this work, different formats for coupling gravitational field-flow fractionation and chemiluminescence detection are explored to achieve ultra-sensitive cell detection in the framework of cell sorting. The study is carried out using human red blood cells as model sample. The best performance is obtained with the on-line coupling format, performed in post-column flow-injection mode. Red cells are isolated from diluted whole human blood in just a few minutes and detected using the liquid phase chemiluminescent reaction of luminol catalysed by the red blood cell heme. The limit of detection is a few hundred injected cells. This is lower than the limit of detection usually achieved by means of conventional colorimetric/turbidimetric methods, and it corresponds to a red blood cell concentration in the injected sample of five orders of magnitude lower than in whole blood.

On-line, continuous chemiluminescence detection has been recently applied to gravitational field-flow fractionation as a new method suitable for flow-assisted assays based on the separation between the analyte in solution and the analyte bound to micrometer-sized particles. In this work, f low field-flow fractionation coupled with off line chemiluminescence detection is exploited for the development of flow-assisted, multianalyte assays. Micrometer-sized, polystyrene beads coated with enzymes suitable for chemiluminescence detection, such as horseradish peroxidase and alkaline phosphatase, are used as model samples. The high size-based selectivity of flow field-flow fractionation and specificity of chemiluminescence detection make possible, in a single run with very short analysis time, the simultaneous, high sensitivity detection of the different enzymes linked to beads of different size.

Chemiluminescence detection has already been combined with different separation techniques such as HPLC and capillary electrophoresis. In this work, it was applied to gravitational field-flow fractionation, a low-cost, flow-assisted separation technique for micronsized particles suited to further on-line detection of the separated analytes. Horseradish peroxidase was used as model sample, either free in solution or immobilized onto micronsized, polystyrene beads. The chemiluminescent substrates were added directly into the mobile phase, and the continuous, steady-state chemiluminescence generated during elution was detected on-line by either a flow-through luminometer or a CCD camera. Ultra-low detection limits, two orders of magnitude lower than those achievable with spectrophotometric detection, were found. The possibility to fully separate and quantitate free and bead-immobilized enzymes is reported, as a step towards the development of multianalyte, ultra-sensitive, micronsized beads-based flow-assisted immunoassays.

Interest in low-cost, analytical-scale, highly efficient, and sensitive separation methods for cells and bacteria has recently been increasing. Field-flow fractionation is well suited to the separation of different types of cells, including bacteria. High performance hollow fiber flow field-flow fractionation of such samples is demonstrated here for the first time with potentially disposable channels and high-sensitivity UV/Vis detectors. In this first application, hollow fiber flow field-flow fractionation is used to fractionate bacteria of biotechnological interest such as deactivated Vibrio cholerae, which are employed for whole-bacteria vaccine production. Quite short analysis times, high reproducibility, and low limits of detection are found. Retention of Vibrio cholerae is shown to depend on the mobile phase composition. Two serologically different Vibrio cholerae strains are partly distinguished by their fractogram profiles.

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFlFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFlFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, AsFlFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.