Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Novel 3D-Printed Microfluidic Magnetic Platform for Rapid DNA Isolation.

This study presents a novel miniaturized device as a 3D-printed microfluidic magnetic platform specifically designed to manipulate magnetic microparticles in a microfluidic chip for rapid deoxyribonucleic acid (DNA) isolation. The novel design enables the movement of the magnetic particles in the same or opposite directions with the flow or suspends them in continuous flow. A computational model was developed to assess the effectiveness of the magnetic manipulation of the particles. Superparamagnetic monodisperse silica particles synthesized in-house are utilized for the isolation of fish sperm DNA and human placenta DNA. It was demonstrated that the proposed platform can perform DNA isolation within 10 min with an isolation efficiency of 50% at optimum operating conditions.

Evaluation of drug carrier hepatotoxicity using primary cell culture models.

This study aims to establish a primary rat hepatocyte culture model to evaluate dose-dependent hepatotoxic effects of drug carriers (lipopolymer nanoparticles; LPNs) temporal. Primary rat hepatocyte cell cultures were used to determine half-maximal Inhibition Concentrations (IC50) of the drug-carrier library. Drug-carrier library, at concentrations <50 µg/mL, is benign to primary rat hepatocytes as determined using albumin and urea secretions. Albumin, as a hepatic biomarker, exhibited a more sensitive and faster outcome, compared to urea, for the determination of the IC50 value of LPNs. Temporal measurements of hepatic biomarkers including urea and albumin, and rigorous physicochemical (hydrodynamic diameter, surface charge, etc.) characterization, should be combined to evaluate the hepatotoxicity of drug carrier libraries in screens.

Progressive hypoxia-on-a-chip: An in vitro oxygen gradient model for capturing the effects of hypoxia on primary hepatocytes in health and disease.

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3-6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia-inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions.

Deep-supercooling for extended preservation of adipose-derived stem cells.

Cell preservation is an enabling technology for widespread distribution and applications of mammalian cells. Traditional cryopreservation via slow-freezing or vitrification provides long-term storage but requires cytotoxic cryoprotectants (CPA) and tedious CPA loading/unloading, cooling, and recovering procedures. Hypothermic storage around 0-4 °C is an alternative method but only works for a short period due to its high storage temperatures. Here, we report on the deep-supercooling (DSC) preservation of human adipose-derived stem cells at deep subzero temperatures without freezing for extended storage. Enabled by surface sealing with an immiscible oil phase, cell suspension can be preserved in a liquid state at -13 °C and -16 °C for 7 days with high cell viability, retention of stemness, attachment, and multilineage differentiation capacities. These results demonstrate that DSC is an improved short-term preservation approach to provide off-the-shelf cell sources for booming cell-based medicine and bioengineering.

Polyethylene glycol protects primary hepatocytes during supercooling preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Supercooled preservation of cultured primary rat hepatocyte monolayers.

Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.

Extended supercooled storage of red blood cells.

Red blood cell (RBC) transfusions facilitate many life-saving acute and chronic interventions. Transfusions are enabled through the gold-standard hypothermic storage of RBCs. Today, the demand for RBC units is unfulfilled, partially due to the limited storage time, 6 weeks, in hypothermic storage. This time limit stems from high metabolism-driven storage lesions at +1-6 °C. A recent and promising alternative to hypothermic storage is the supercooled storage of RBCs at subzero temperatures, pioneered by our group. Here, we report on long-term supercooled storage of human RBCs at physiological hematocrit levels for up to 23 weeks. Specifically, we assess hypothermic RBC additive solutions for their ability to sustain supercooled storage. We find that a commercially formulated next-generation solution (Erythro-Sol 5) enables the best storage performance and can form the basis for further improvements to supercooled storage. Our analyses indicate that oxidative stress is a prominent time- and temperature-dependent injury during supercooled storage. Thus, we report on improved supercooled storage of RBCs at -5 °C by supplementing Erythro-Sol 5 with the exogenous antioxidants, resveratrol, serotonin, melatonin, and Trolox. Overall, this study shows the long-term preservation potential of supercooled storage of RBCs and establishes a foundation for further improvement toward clinical translation.

Surface-segregating zwitterionic copolymers to control poly(dimethylsiloxane) surface chemistry.

The use of microfluidic devices in biomedicine is growing rapidly in applications such as organs-on-chip and separations. Polydimethylsiloxane (PDMS) is the most popular material for microfluidics due to its ability to replicate features down to the nanoscale, flexibility, gas permeability, and low cost. However, the inherent hydrophobicity of PDMS leads to the adsorption of macromolecules and small molecules on device surfaces. This curtails its use in "organs-on-chip" and other applications. Current technologies to improve PDMS surface hydrophilicity and fouling resistance involve added processing steps or do not create surfaces that remain hydrophilic for long periods. This work describes a novel, simple, fast, and scalable method for improving surface hydrophilicity and preventing the nonspecific adsorption of proteins and small molecules on PDMS through the use of a surface-segregating zwitterionic copolymer as an additive that is blended in during manufacture. These highly branched copolymers spontaneously segregate to surfaces and rearrange in contact with aqueous solutions to resist nonspecific adsorption. We report that mixing a minute amount (0.025 wt%) of the zwitterionic copolymer in PDMS considerably reduces hydrophobicity and nonspecific adsorption of proteins (albumin and lysozyme) and small molecules (vitamin B12 and reactive red). PDMS blended with these zwitterionic copolymers retains its mechanical and physical properties for at least six months. Moreover, this approach is fully compatible with existing PDMS device manufacture protocols without additional processing steps and thus provides a low-cost and user-friendly approach to fabricating reliable biomicrofluidics.

Alterations in Cytoskeleton and Mitochondria in the Development and Reversal of Steatosis in Human Hepatocytes.

BACKGROUND & AIMS: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. METHODS: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer, in which we fully optimized reagent dosing specifically for human hepatocytes. RESULTS: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network, leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. CONCLUSIONS: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.

Supramolecular hybrid hydrogels as rapidly on-demand dissoluble, self-healing, and biocompatible burn dressings.

Despite decades of efforts, state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal. Current dressings adhere to the wound and necessitate debridement. This work describes the first "supramolecular hybrid hydrogel (SHH)" burn dressing that is biocompatible, self-healable, and on-demand dissoluble for easy and trauma-free removal, prepared by a simple, fast, and scalable method. These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution. The SHHs show high mechanical strength (>50 kPa), self-heal rapidly in ∼1 min, and dissolve quickly (4-6 min) using an amantadine hydrochloride (AH) solution that breaks the supramolecular interactions in the SHHs. Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro. The SHHs also do not elicit any significant cytokine response in vitro. Furthermore, in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls. Thus, these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

Comparing two extracellular additives to facilitate extended storage of red blood cells in a supercooled state.

Background: Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at -4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements. Study Design and Methods: Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0-165 mM of trehalose or 0-165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and -4°C under a layer of paraffin oil to prevent ice formation. Results: PEG400 reduced hemolysis and increased deformability in -4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at -4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control. Discussion: Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.

Propulsion and trapping of microparticles by active cilia arrays.

We model the transport of a microscopic particle via a regular array of beating elastic cilia, whose tips experience an adhesive interaction with the particle's surface. At optimal adhesion strength, the average particle velocity is maximized. Using simulations spanning a range of cilia stiffness and cilia-particle adhesion strength, we explore the parameter space over which the particle can be "released", "propelled", or "trapped" by the cilia. We use a lower-order model to predict parameters for which the cilia are able to "propel" the particle. This is the first study that shows how both stiffness and adhesion strength are crucial for manipulation of particles by active cilia arrays. These results can facilitate the design of synthetic cilia that integrate adhesive and hydrodynamic interactions to selectively repel or trap particulates. Surfaces that are effective at repelling particulates are valuable for antifouling applications, while surfaces that can trap and, thus, remove particulates from the solution are useful for efficient filtration systems.

CYP450 drug inducibility in NAFLD via an in vitro hepatic model: Understanding drug-drug interactions in the fatty liver.

Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model's dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.

ß-Dispersion of blood during sedimentation.

Aggregation of human red blood cells (RBC) is central to various pathological conditions from bacterial infections to cancer. When left at low shear conditions or at hemostasis, RBCs form aggregates, which resemble stacks of coins, known as 'rouleaux'. We experimentally examined the interfacial dielectric dispersion of aggregating RBCs. Hetastarch, an RBC aggregation agent, is used to mimic conditions leading to aggregation. Hetastrach concentration is incrementally increased in blood from healthy donors to measure the sensitivity of the technique. Time lapse electrical impedance measurements were conducted as red blood cells form rouleaux and sediment in a PDMS chamber. Theoretical modeling was used for obtaining complex permittivity of an effective single red blood cell aggregate at various concentrations of hetastarch. Time response of red blood cells' impedance was also studied to parametrize the time evolution of impedance data. Single aggregate permittivity at the onset of aggregation, evolution of interfacial dispersion parameters, and sedimentation kinetics allowed us to distinguish differential aggregation in blood.

Designing oscillating cilia that capture or release microscopic particles.

We use computational modeling to capture the three-dimensional interactions between oscillating, synthetic cilia and a microscopic particle in a fluid-filled microchannel. The synthetic cilia are elastic filaments that are tethered to a substrate and are actuated by a sinusoidal force, which is applied to their free ends. The cilia are arranged in a square pattern, and a neutrally buoyant particle is initially located between these filaments. Our computational studies reveal that, depending on frequency of the beating cilia, the particle can be either driven downward toward the substrate or driven upward and expelled into the fluid above the cilial layer. This behavior mimics the performance of biological cilia used by certain marine animals to extract suspended food particles. The findings uncover a new route for controlling the deposition of microscopic particles in microfluidic devices.

A comparison of hepato-cellular in vitro platforms to study CYP3A4 induction.

In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies.

Shear and extensional deformation of droplets containing polymers and nanoparticles.

We investigate the effects of polymer chains and nanoparticles on the deformation of a droplet in shear and extensional flow using computational modeling that accounts for both the solid and fluid phases explicitly. We show that under shear flow, both the nanoparticles and the encapsulated polymers reduce the shear-induced deformation of the droplet at intermediate capillary numbers. At high capillary numbers, however, long polymer chains can induce the breakup of the droplet. We find that the latter behavior is dependent on the nature of the imposed flow. Specifically, under extensional flow, long polymers inhibit the droplet breakup and reduce deformation. Overall, the findings provide guidelines for tailoring the stability of filled droplets under an imposed flow, and thus, the results can provide useful design rules in a range of technological applications.

Simple Surface Modification of Poly(dimethylsiloxane) via Surface Segregating Smart Polymers for Biomicrofluidics.

Poly(dimethylsiloxane) (PDMS) is likely the most popular material for microfluidic devices in lab-on-a-chip and other biomedical applications. However, the hydrophobicity of PDMS leads to non-specific adsorption of proteins and other molecules such as therapeutic drugs, limiting its broader use. Here, we introduce a simple method for preparing PDMS materials to improve hydrophilicity and decrease non-specific protein adsorption while retaining cellular biocompatibility, transparency, and good mechanical properties without the need for any post-cure surface treatment. This approach utilizes smart copolymers comprised of poly(ethylene glycol) (PEG) and PDMS segments (PDMS-PEG) that, when blended with PDMS during device manufacture, spontaneously segregate to surfaces in contact with aqueous solutions and reduce the hydrophobicity without any added manufacturing steps. PDMS-PEG-modified PDMS samples showed contact angles as low as 23.6° ± 1° and retained this hydrophilicity for at least twenty months. Their improved wettability was confirmed using capillary flow experiments. Modified devices exhibited considerably reduced non-specific adsorption of albumin, lysozyme, and immunoglobulin G. The modified PDMS was biocompatible, displaying no adverse effects when used in a simple liver-on-a-chip model using primary rat hepatocytes. This PDMS modification method can be further applied in analytical separations, biosensing, cell studies, and drug-related studies.