A thermoplastic microfluidic microphysiological system to recapitulate hepatic function and multicellular interactions.

Hepatic in vitro platforms ranging from multi-well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next-generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high-throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue-culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media-to-cell ratio in comparison with multi-well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte-Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic-based high-throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi-cellular interactions and therapeutic responses.

Microfluidic Cell Culture Platforms to Capture Hepatic Physiology and Complex Cellular Interactions.

Animal models such as rats and primates provide body-wide information for drug and metabolite responses, including organ-specific toxicity and any unforeseen side effects on other organs. Although effective in the drug-screening process, their translatability to humans is limited because of the lack of high concordance and correlation among enzymatic mechanisms, cellular mechanisms, and resulting toxicities. A significant mode of failure for safety prediction in drug screening is hepatotoxicity, resulting in ∼30% of all safety-related drug failures and withdrawals from the market. The liver is a multifunctional organ with diverse metabolic, secretory, and inflammatory response roles and is essential for maintaining key body functions. Conventional cell culture platforms (such as multiwell plate cultures) and metabolic enzyme systems (microsomes, cytochrome P450 enzymes) have been routinely used to assess drug pharmacokinetics and metabolism. However, current in vitro models often fail to recapitulate the complexity and dynamic nature of human tissues, imposing a heavy reliance on in vivo testing using preclinical species that have metabolic processes, disease mechanisms, and modes of toxicity distinct from humans. Recently, microphysiological systems (MPS) have gained attention as powerful tools with the potential to generate human-relevant information that can supplant and fill the gap of knowledge between preclinical animal models and simpler, conventional in vitro cell culture systems. Developments in microfabrication technologies for generating complex microfluidic systems, along with the ability to establish and maintain multicellular models to capture dynamic, human-relevant behavior, have provided new avenues to generate such physiologically relevant systems. These MPS platforms, when designed and developed with in vivo-derived design parameters, have the potential to capture key aspects and better mimic organ functionality. In this review, we discuss developments in microtechnologies for fabricating, establishing, and maintaining hepatic cell culture systems, with a specific focus on models that aim to capture in vivo physiology in vitro. By designing microscale systems to impart specific in vivo physiologic parameters, it is possible to create a dynamic system that can capture multiple aspects of the hepatic microenvironment, bringing us closer to a comprehensive in vitro testing platform for hepatic responses and toxicities.

A microfluidic in-line ELISA for measuring secreted protein under perfusion.

Recent progress in the development of microfluidic microphysiological systems such as 'organs-on-chips' and microfabricated cell culture is geared to simulate organ-level physiology. These tissue models leverage microengineering technologies that provide capabilities of presenting cultured cells with input signals in a more physiologically relevant context such as perfused flow. Proteins that are secreted from cells have important information about the health of the cells. Techniques to quantify cellular proteins include mass spectrometry to ELISA (enzyme-linked immunosorbent assay). Although our capability to perturb the cells in the microphysiological systems with varying inputs is well established, we lack the tools to monitor in-line the cellular responses. User intervention for sample collection and off-site is cumbersome, causes delays in obtaining results, and is especially expensive because of collection, storage, and offline processing of the samples, and in many case, technically impractical to carry out because of limitated sample volumes. To address these shortcomings, we report the development of an ELISA that is carried out in-line under perfusion within a microfluidic device. Using this assay, we measured the albumin secreted from perfused hepatocytes without and under stimulation by IL-6. Since the method is based on a sandwich ELISA, we envision broad application of this technology to not just organs-on-chips but also to characterizing the temporal release and measurement of soluble factors and response to drugs.

Long-term maintenance of a microfluidic 3D human liver sinusoid.

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPA's initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies.

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers.

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Abeta1-42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Abeta monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their beta-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Abeta-mediated toxicity.

Polyphenolic glycosides and aglycones utilize opposing pathways to selectively remodel and inactivate toxic oligomers of amyloid ß.

Substantial evidence suggests that soluble prefibrillar oligomers of the Aß42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aß isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aß oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aß oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aß soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aß oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aß oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aß oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aß fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aß oligomers (which are indistinguishable in size and morphology to Aß soluble oligomers) or promote aggregation of freshly disaggregated Aß peptide; thus revealing that they are specific for remodeling toxic Aß conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aß-mediated toxicity.

A GFP complementation system for monitoring and directing nanomaterial mediated protein delivery to human cellular organelles.

Protein-based therapeutics are gaining importance for their biocompatibility and activity toward specific targets. When these targets are intracellular, it is critical to deliver biomolecules to sites in the cell cytoplasm while retaining biomolecule activity in the complex cellular milieu. However, intracellular protein delivery is not viable unless accompanied by an active uptake mechanism or carrier mediated delivery. Moreover, once entry into the cell is achieved, detection of the biomolecule requires laborious techniques that lack real-time measurement. We have developed a fluorescence-based complementary protein delivery sensing system using split green fluorescence protein (GFP(1-10) and GFP(11)) fragments, which can be used as an indicator for protein delivery and retention of activity, and as a means to pinpoint subcellular localization. We demonstrate in vitro localized delivery by expressing the GFP(11) fragment onto the mitochondrial outer membrane of human cells, and using a model carrier (15 nm silica nanoparticles) to deliver GFP(1-10) and image trafficking and mitochondrial localization of protein delivery. Our results indicate that nanoscale materials can be used as protein carriers for targeting cell constituents including functional molecules, signaling pathways, and organelles. We envision that this GFP complementation system is ideally suited for directing nanoparticle-based delivery of drugs and other bioactive molecules into subcellular locations within cells, which can impact protein-protein interactions, signal transduction pathways, and organelle function in vitro within the context of high-throughput screening protocols.

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.

Correction: A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.

Correction for 'A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions' by Kelly Tan et al., Lab Chip, 2019, 19, 1556-1566, DOI: .

A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.

Microphysiological systems (MPSs) are dynamic cell culture systems that provide micro-environmental and external cues to support physiologically relevant, organ-specific functions. Recent progresses in MPS fabrication technologies have enabled the development of advanced models to capture microenvironments with physiological relevance, while increasing throughput and reducing material-based artefacts. In addition to conventional cell culture systems, advanced MPSs are emerging as ideal contenders for disease modeling and incorporation into drug screening. Since liver is a central organ for drug metabolism, liver-on-chip models have been developed to recapitulate hepatic microenvironment with varying complexities, while allowing long-term culture. Recently, we have developed a novel thermoplastic, oxygen-permeable MPS for primary human hepatocyte (PHH) culture. Herein, we have adapted and extended the MPS to a) a 96 microfluidic array (PREDICT-96 array) and b) integrated a novel, ultra-low volume, re-circulating pumping system (PREDICT-96 pump) - collectively known as the PREDICT-96 platform. The PREDICT-96 platform conforms to the industrial standard 96-well footprint and enables media re-circulation. First, we demonstrate the introduction of PHHs into the PREDICT-96 array using standard handling procedures for multi-well plates and allow cells to stabilize in static conditions. Next, we introduce recirculating flow into the bottom channel (using PREDICT-96 pump) to mimic mass transport in vivo. Our results indicate an increase in metabolic and secretory functions of PHHs in the PREDICT-96 platform, and their maintenance over 10 days of flow. Furthermore, long-term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and capture cellular responses to chronic stimuli. The low-volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi-cellular co-cultures and adaptation, integration with high-throughput analytical tools.

The protein-nanomaterial interface.

Developments in the past few years have illustrated the potentially revolutionizing impact of nanomaterials, especially in biomedical imaging, drug delivery, biosensing and the design of functional nanocomposites. Methods to effectively interface proteins with nanomaterials for realizing these applications continue to evolve. Proteins are being used to control both the synthesis and assembly of nanomaterials. There has also been an increasing interest in understanding the influence of nanomaterials on the structure and function of proteins. Understanding and controlling the protein-nanomaterial interface will be crucial for designing functional protein-nanomaterial conjugates and assemblies.

Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics.

Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses.

Multi-omic network-based interrogation of rat liver metabolism following gastric bypass surgery featuring SWATH proteomics.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

Emerging In Vitro Liver Technologies for Drug Metabolism and Inter-Organ Interactions.

In vitro liver models provide essential information for evaluating drug metabolism, metabolite formation, and hepatotoxicity. Interfacing liver models with other organ models could provide insights into the desirable as well as unintended systemic side effects of therapeutic agents and their metabolites. Such information is invaluable for drug screening processes particularly in the context of secondary organ toxicity. While interfacing of liver models with other organ models has been achieved, platforms that effectively provide human-relevant precise information are needed. In this concise review, we discuss the current state-of-the-art of liver-based multiorgan cell culture platforms primarily from a drug and metabolite perspective, and highlight the importance of media-to-cell ratio in interfacing liver models with other organ models. In addition, we briefly discuss issues related to development of optimal liver models that include recent advances in hepatic cell lines, stem cells, and challenges associated with primary hepatocyte-based liver models. Liver-based multiorgan models that achieve physiologically relevant coupling of different organ models can have a broad impact in evaluating drug efficacy and toxicity, as well as mechanistic investigation of human-relevant disease conditions.

Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response.

The liver is a central organ in the human body, and first line of defense between host and external environment. Liver response to any external perturbation is a collective reaction of resident liver cells. Most of the current in vitro liver models focus on hepatocytes, the primary metabolic component, omitting interactions and cues from surrounding environment and non-parenchymal cells (NPCs). Recent studies suggest that contributions of NPCs are vital, particularly in disease conditions, and outcomes of drugs and their metabolites. Along with hepatocytes, NPCs-Kupffer (KC), sinusoidal endothelial (LSEC) and stellate cells (SC) are major cellular components of the liver. Incorporation of primary cells in in vitro liver platforms is essential to emulate the functions of the liver, and its overall response. Herein, we isolate individual NPC cell fractions from rat livers and co-culture them in a transwell format incorporating primary rat hepatocytes with LSECs, SCs, and KCs. Our results indicate that the presence and contributions of multiple cells within the co-culture capture the interactions between hepatocytes and NPC, and modulates the responses to inflammatory stimulus such as LPS. The isolation and co-culture methods could provide a stable platform for creating in vitro liver models that provide defined functionality beyond hepatocytes alone.

Engineered Trehalose Permeable to Mammalian Cells.

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

A novel low-volume two-chamber microfabricated platform for evaluating drug metabolism and toxicity.

To evaluate drug and metabolite efficacy on a target organ, it is essential to include metabolic function of hepatocytes, and to evaluate metabolite influence on both hepatocytes and the target of interest. Herein, we have developed a two-chamber microfabricated device separated by a membrane enabling communication between hepatocytes and cancer cells. The microscale environment created enables cell co-culture in a low media-to-cell ratio leading to higher metabolite formation and rapid accumulation, which is lost in traditional plate cultures or other interconnected models due to higher culture volumes. We demonstrate the efficacy of this system by metabolism of tegafur by hepatocytes resulting in cancer cell toxicity.

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells.

Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of non-parenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for ∼ 80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.

Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells.

Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. Key message: Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131. ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor. Oxidative stress and inflammation promote cell surface expression of CD131. ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes. Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.