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A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000 L, and greater, to meet commercial demand. However, the concern of the ‘shear sensitivity’ of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10 g/L are now reported with cell densities greater than 2 × 10 7  cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of ‘shear sensitivity’ has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, “upper limit” to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.

Anemia and iron deficiency continue to be the most prevalent nutritional disorders in the world, affecting billions of people in both developed and developing countries. The initial diagnosis of anemia is typically based on several markers, including red blood cell (RBC) count, hematocrit and total hemoglobin. Using modern hematology analyzers, erythrocyte parameters such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), etc. are also being used. However, most of these commercially available analyzers pose several disadvantages: they are expensive instruments that require significant bench space and are heavy enough to limit their use to a specific lab and lead to a delay in results, making them less practical as a point-of-care instrument that can be used for swift clinical evaluation. Thus, there is a need for a portable and economical hematology analyzer that can be used at the point of need. In this work, we evaluated the performance of a system referred to as the cell tracking velocimetry (CTV) to measure several hematological parameters from fresh human blood obtained from healthy donors and from sickle cell disease subjects. Our system, based on the paramagnetic behavior that deoxyhemoglobin or methemoglobin containing RBCs experience when suspended in water after applying a magnetic field, uses a combination of magnets and microfluidics and has the ability to track the movement of thousands of red cells in a short period of time. This allows us to measure not only traditional RBC indices but also novel parameters that are only available for analyzers that assess erythrocytes on a cell by cell basis. As such, we report, for the first time, the use of our CTV as a hematology analyzer that is able to measure MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), the percentage of hypochromic cells (which is an indicator of insufficient marrow iron supply that reflects recent iron reduction), and the correlation coefficients between these metrics. Our initial results indicate that most of the parameters measured with CTV are within the normal range for healthy adults. Only the parameters related to the red cell volume (primarily MCV and RDW) were outside the normal range. We observed significant discrepancies between the MCV measured by our technology (and also by an automated cell counter) and the manual method that calculates MCV through the hematocrit obtained by packed cell volume, which are attributed to the artifacts of plasma trapping and cell shrinkage. While there may be limitations for measuring MCV, this device offers a novel point of care instrument to provide rapid RBC parameters such as iron stores that are otherwise not rapidly available to the clinician. Thus, our CTV is a promising technology with the potential to be employed as an accurate, economical, portable and fast hematology analyzer after applying instrument-specific reference ranges or correction factors.

The use of functionalized magnetic particles for the detection or separation of multiple chemicals and biomolecules from biofluids continues to attract significant attention. After their incubation with the targeted substances, the beads can be magnetically recovered to perform analysis or diagnostic tests. Particle recovery with permanent magnets in continuous-flow microdevices has gathered great attention in the last decade due to the multiple advantages of microfluidics. As such, great efforts have been made to determine the magnetic and fluidic conditions for achieving complete particle capture; however, less attention has been paid to the effect of the channel geometry on the system performance, although it is key for designing systems that simultaneously provide high particle recovery and flow rates. Herein, we address the optimization of Y-Y-shaped microchannels, where magnetic beads are separated from blood and collected into a buffer stream by applying an external magnetic field. The influence of several geometrical features (namely cross section shape, thickness, length, and volume) on both bead recovery and system throughput is studied. For that purpose, we employ an experimentally validated Computational Fluid Dynamics (CFD) numerical model that considers the dominant forces acting on the beads during separation. Our results indicate that rectangular, long devices display the best performance as they deliver high particle recovery and high throughput. Thus, this methodology could be applied to the rational design of lab-on-a-chip devices for any magnetically driven purification, enrichment or isolation.

Hematological analysis is crucial for diagnosing and monitoring blood-related disorders. Nevertheless, conventional hematology analyzers remain confined to laboratory settings due to their high cost, substantial space requirements, and maintenance needs. Herein, we present a portable cell tracking velocimetry (CTV) device for the precise measurement of the magnetic susceptibility of biological entities at the single-cell level, focusing on red blood cells (RBCs) in this work. The system integrates a microfluidic channel positioned between permanent magnets that generate a well-defined magnetic field gradient (191.82 TA/mm2). When the cells are injected into the chamber, their particular response to the magnetic field is recorded and used to estimate their properties and quantify their intracellular hemoglobin (Hb) concentration. We successfully track over 400 RBCs per condition using imaging and trajectory analysis, enabling detailed characterizations of their physical and magnetic properties. A comparison of the mean corpuscular hemoglobin measurements revealed a strong correlation between our CTV system and standard ultraviolet–visible (UV-Vis) spectrophotometry (23.1 ± 5.8 pg vs. 22.4 ± 3.9 pg, p > 0.05), validating the accuracy of our measurements. The system’s single-cell resolution reveals population distributions unobtainable through conventional bulk analysis methods. Thus, this portable CTV technology provides a rapid, label-free approach for magnetic cell characterization, offering new possibilities for point-of-care hematological analysis and field-based research applications.

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

This research was funded by Texas Tech University via the HEF New Faculty Startup, NRUF Startup, and Core Research Support Fund. Cristina González-Fernández thanks the Spanish Ministry of Universities for the Margarita Salas postdoctoral fellowship (grants for the requalification of the Spanish university system for 2021–2023, University of Cantabria), funded by the European Union-NextGenerationEU.

Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV-1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre-miR-199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre-miR-199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre-miR-199a. The TAT-TAR interaction enhanced the loading of the miR-199a into EVs by 65-fold. Endogenously loaded EVs were ineffective at delivering active miR-199a-3p therapeutic to recipient SK-Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.