Hybrid adipocyte-derived exosome nano platform for potent chemo-phototherapy in targeted hepatocellular carcinoma.

The high prevalence and severity of hepatocellular carcinoma (HCC) present a significant menace to human health. Despite the significant advancements in nanotechnology-driven antineoplastic agents, there remains a conspicuous gap in the development of targeted chemotherapeutic agents specifically designed for HCC. Consequently, there is an urgent need to explore potent drug delivery systems for effective HCC treatment. Here we have exploited the interplay between HCC and adipocyte to engineer a hybrid adipocyte-derived exosome platform, serving as a versatile vehicle to specifically target HCC and exsert potent antitumor effect. A lipid-like prodrug of docetaxel (DSTG) with a reactive oxygen species (ROS)-cleavable linker, and a lipid-conjugated photosensitizer (PPLA), spontaneously co-assemble into nanoparticles, functioning as the lipid cores of the hybrid exosomes (HEMPs and NEMPs). These nanoparticles are further encapsuled within adipocyte-derived exosome membranes, enhancing their affinity towards HCC cancer cells. As such, cancer cell uptakes of hybrid exosomes are increased up to 5.73-fold compared to lipid core nanoparticles. Our in vitro and in vivo experiments have demonstrated that HEMPs not only enhance the bioactivity of the prodrug and extend its circulation in the bloodstream but also effectively inhibit tumor growth by selectively targeting hepatocellular carcinoma tumor cells. Self-facilitated synergistic drug release subsequently promoting antitumor efficacy, inducing significant inhibition of tumor growth with minimal side effects. Our findings herald a promising direction for the development of targeted HCC therapeutics.

Ferroptosis inducers: A new frontier in cancer therapy.

Ferroptosis represents a non-apoptotic form of programmed cell death characterized by iron-dependent lipid peroxidation. This cell death modality not only facilitates the direct elimination of cancer cells, but also enhances their susceptibility to other pharmacological anti-cancer agents. The burgeoning interest in ferroptosis has been driven by a growing body of evidence that underscores the efficiency and minimal toxicity of ferroptosis inducers. Traditional inducers, such as erastin and RSL3 have shown substantial promise in clinical applications due to their potent therapeutic effects. Their significant potential of these inducers has spurred the development of a variety of small molecule ferroptosis inducers. These novel inducers boast an enhanced structural variety, improved metabolic stability, the capability to initiate ferroptosis without triggering apoptosis, making them well-suited for in vivo use. Despite these advancements, challenges still remain, particularly concerning the drug delivery, tumor specificity, and circulation duration of these small molecules in vivo. Addressing these challenges, contemporary research has pivoted towards innovative delivery systems tailored for ferroptosis inducers to facilitate precise, targeted, and synegestic therapeutic delivery. This review scrutinizes the latest progress in small molecule ferroptosis inducers and nano drug delivery systems geared towards ferroptosis sensitization. Furthermore, it delineated the prospective therapeutic advantages and the existing hurdles in the development of ferroptosis inducers for malignant tumor treatment.

Modeling dry eye with an air-liquid interface in corneal epithelium-on-a-chip.

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air-liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF), non-steroidal anti-inflammatory drug. Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell-cell junctions. They increased the expression of inflammatory genes (e.g., IL-6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the potential use of AL stimulus within the CEpOC to induce cellular characteristics relevant to DES.

Cyclic Stretching Enhances Angiocrine Signals at Liver Bud Stage from Human Pluripotent Stem Cells in Two-Dimensional Culture.

Angiocrine signals during the development and growth of organs, including the liver, intestine, lung, and bone, are essential components of intercellular communication. The signals elicited during the liver bud stage are critical for vascularization and enhanced during the intercellular communication between the cells negative for kinase insert domain receptor (KDR) (KDR- cells) and the cells positive for KDR (KDR+ cells), which constitute the liver bud. However, the use of a human pluripotent stem cell (hPSC)-derived system has not facilitated the generation of a perfusable vascularized liver organoid that allows elucidation of liver development and has great potential for liver transplantation. This is largely owing to the lack of fundamental understanding to induce angiocrine signals in KDR- and KDR+ cells during the liver bud stage. We hypothesized that mechanical stimuli of cyclic stretching/pushing by the fetal heart adjacent to the liver bud could be the main contributor to promoting angiocrine signals in KDR- and KDR+ cells during the liver bud stage. In this study, we show that an organ-on-a-chip platform allows the emulation of an in vivo-like mechanical environment for the liver bud stage in vitro and investigate the role of cyclic mechanical stretching (cMS) to angiocrine signals in KDR- and KDR+ cells derived from hPSCs. RNA sequencing revealed that the expression of genes associated with epithelial-to-mesenchymal transition, including angiocrine signals, such as hepatocyte growth factor (HGF) and matrix metallopeptidase 9 (MMP9), were increased by cMS in cocultured KDR- and KDR+ cells. The expression and secretions of HGF and MMP9 were increased by 1.98- and 1.69-fold and 3.23- and 3.72-fold with cMS in the cocultured KDR- and KDR+ cells but were not increased by cMS in the monocultured KDR- and KDR+ cells, respectively. Finally, cMS during the liver bud stage did not lead to the dedifferentiation of hepatocytes, as the cells with cMS showed hepatic maker expression (CYP3A4, CYP3A7, ALB, and AAT) and 1.71-fold higher CYP3A activity than the cells without cMS, during 12 day-hepatocyte maturation after halting cMS. Our findings provide new insights into the mechanical factors during the liver bud stage and directions for future improvements in the engineered liver tissue.

Brain organoid-on-a-chip to create multiple domains in forebrain organoids.

Brain organoids are three-dimensionally reconstructed brain tissue derived from pluripotent stem cells in vitro. 3D tissue cultures have opened new avenues for exploring development and disease modeling. However, some physiological conditions, including signaling gradients in 3D cultures, have not yet been easily achieved. Here, we introduce Brain Organoid-on-a-Chip platforms that generate signaling gradients that in turn enable the induction of topographic forebrain organoids. This creates a more continuous spectrum of brain regions and provides a more complete mimic of the human brain for evaluating neurodevelopment and disease in unprecedented detail.

Emerging platinum(IV) prodrug nanotherapeutics: A new epoch for platinum-based cancer therapy.

Owing to the unique DNA damaging cytotoxicity, platinum (Pt)-based chemotherapy has long been the first-line choice for clinical oncology. Unfortunately, Pt drugs are restricted by the severe dose-dependent toxicity and drug resistance. Correspondingly, Pt(IV) prodrugs are developed with the aim to improve the antitumor performance of Pt drugs. However, as "free" molecules, Pt(IV) prodrugs are still subject to unsatisfactory in vivo destiny and antitumor efficacy. Recently, Pt(IV) prodrug nanotherapeutics, inheriting both the merits of Pt(IV) prodrugs and nanotherapeutics, have emerged and demonstrated the promise to address the underexploited dilemma of Pt-based cancer therapy. Herein, we summarize the latest fronts of emerging Pt(IV) prodrug nanotherapeutics. First, the basic outlines of Pt(IV) prodrug nanotherapeutics are overviewed. Afterwards, how versatile Pt(IV) prodrug nanotherapeutics overcome the multiple biological barriers of antitumor drug delivery is introduced in detail. Moreover, advanced combination therapies based on multimodal Pt(IV) prodrug nanotherapeutics are discussed with special emphasis on the synergistic mechanisms. Finally, prospects and challenges of Pt(IV) prodrug nanotherapeutics for future clinical translation are spotlighted.

Integrated-gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) afflicts a significant percentage of the population; however, no effective treatments have yet been established because of the unsuitability of in vitro assays and animal experimental models. Here, we present an integrated-gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) by co-culturing human gut and liver cell lines interconnected via microfluidics in a closed circulation loop, for the initiation and progression of NAFLD by treatment with free fatty acids (FFAs) for 1 and 7 days, respectively. Co-cultured Caco-2 gut-mimicking cells and HepG2 hepatocyte-like cells demonstrate the protective effects from apoptosis against FFAs treatment, whereas mono-cultured cells exhibit induced apoptosis. Phenotype and gene expression analyses reveal that the FFAs-treated gut and liver cells accumulated intracellular lipid droplets and show an increase in gene expression associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating the mechanisms of NAFLD.

Critical roles of linker length in determining the chemical and self-assembly stability of SN38 homodimeric nanoprodrugs.

Homodimeric prodrug nanoassemblies (HDPNs) have been widely studied for efficient cancer therapy by virtue of their ultra-high drug loading and distinct nanostructure. However, the development of SN38 HDPNs is still a great challenge due to the rigid planar aromatic ring structure. Improving the structural flexibility of homodimeric prodrugs by increasing the linker length may be a potential strategy for constructing SN38 HDPNs. Herein, three SN38 homodimeric prodrugs with different linker lengths were synthesized. The number of carbon atoms from the disulfide bond to the adjacent ester bond is 1 (denoted as α-SN38-SS-SN38), 2 (ß-SN38-SS-SN38), and 3 (γ-SN38-SS-SN38), respectively. Interestingly, we found that α-SN38-SS-SN38 exhibited extremely low yield and poor chemical stability. Additionally, ß-SN38-SS-SN38 demonstrated suitable chemical stability but poor self-assembly stability. In comparison, γ-SN38-SS-SN38 possessed good chemical and self-assembly stability, thereby improving the tumor accumulation and antitumor efficacy of SN38. We developed the SN38 HDPNs for the first time and illustrated the underlying molecular mechanism of increasing the linker length to enhance the chemical and self-assembly stability of homodimeric prodrugs. These findings would provide new insights for the rational design of HDPNs with superior performance.

Gut-liver-axis microphysiological system for studying cellular fluidic shear stress and inter-tissue interaction.

To clarify the physiological and pathological roles of gut-liver-axis (GLA) in the human body, a GLA microphysiological system (GLA-MPS) holds great potential. However, in current GLA-MPSs, the importance of a physiologically relevant flow for gut and liver cells' cultivation is not fully addressed. In addition, the integration of individual organ perfusion, circulation flow, and organ tissue functions in a single device has not been achieved. Here, we introduce a GLA-MPS by integrating two cell-culture chambers with individually applied perfusion flows and a circulation channel with an on-chip pneumatic micropump under cell-culture chambers via a porous membrane for interconnecting them. We analyzed the fluid shear stress (FSS) with computational fluid dynamics simulations and confirmed that the physiologically relevant FSS could be applied to the gut (Caco-2) (8 × 10-3 dyn cm-2) and liver (HepG2) cells (1.2 × 10-7 dyn cm-2). Under the physiologically relevant flow, the Caco-2 and HepG2 cells in the GLA-MPS maintained a cell survival rate of 95% and 92%, respectively. Furthermore, the expression of functional proteins such as zonula occludens 1 (in Caco-2) and albumin (in HepG2) was enhanced. To demonstrate the GLA interaction, the inflammatory bowel disease was recapitulated by applying lipopolysaccharide for only Caco-2 cells. The inflammatory proteins, such as inducible nitric oxide synthase, were induced in Caco-2 and HepG2 cells. The presented GLA-MPS can be adapted as an advanced in vitro model in various applications for disease modeling associated with inter-tissue interactions, such as inflammatory disease.

Tumor-on-a-chip model for advancement of anti-cancer nano drug delivery system.

Despite explosive growth in the development of nano-drug delivery systems (NDDS) targeting tumors in the last few decades, clinical translation rates are low owing to the lack of efficient models for evaluating and predicting responses. Microfluidics-based tumor-on-a-chip (TOC) systems provide a promising approach to address these challenges. The integrated engineered platforms can recapitulate complex in vivo tumor features at a microscale level, such as the tumor microenvironment, three-dimensional tissue structure, and dynamic culture conditions, thus improving the correlation between results derived from preclinical and clinical trials in evaluating anticancer nanomedicines. The specific focus of this review is to describe recent advances in TOCs for the evaluation of nanomedicine, categorized into six sections based on the drug delivery process: circulation behavior after infusion, endothelial and matrix barriers, tumor uptake, therapeutic efficacy, safety, and resistance. We also discuss current issues and future directions for an end-use perspective of TOCs.

An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells.

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell-culturing time using two defined culturing media containing TGF-ß, and Wnt/ß-catenin pathway inhibitors, and bFGF growth factor over 25 days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-ß and Wnt/ß-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

In vitro culture at 39 °C during hepatic maturation of human ES cells facilitates hepatocyte-like cell functions.

Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. However, these cells do not have hepatic functions comparable to those of hepatocytes in vivo due to insufficient hepatic differentiation. Here we showed that the hepatic functions of hPSC-HLCs were facilitated by applying physiological liver temperatures during hepatic differentiation. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39 °C; the 42 °C treatment caused significantly greater cell death than the 39 °C treatment. We confirmed the improvement of hepatic functions, such as albumin secretion, cytochrome P450 3A activity, and collagen production, without severe cell damage. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs and lead to the realization of drug discovery efforts and drug toxicological tests.

Generation and Gene Expression Profiles of Grevy's Zebra Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) can serve as a biological resource for functional and conservation research for various species. This realization has led to the generation of iPSCs from many species, including those identified as endangered. However, the understanding of species variation in mammalian iPSCs remains largely unknown. To gain insight into species variation in iPSCs, we generated iPSCs from a new species Grevy's zebra (Equus grevyi; gz-iPSCs), which has been listed as endangered in the IUCN (International Union for Conservation of Nature) Red List. We isolated primary fibroblast cells from an individual and successfully reprogrammed them into iPSCs. The generated gz-iPSCs continued to grow under primed-type culture condition and showed pluripotency and differentiation potential. To describe the molecular characteristics of gz-iPSCs, we performed RNA sequencing analysis. The gz-iPSC transcriptome showed robust expression of pluripotency-associated genes reported in human and mouse, suggesting evolutionary conservation among the species. This study provides insight into the iPSCs from a rare species and helps the understanding of the gene expression basis underlying mammalian pluripotent stem cells.

Nanocasting of fibrous morphology on a substrate for long-term propagation of human induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) can be self-renewed for many generations on nanofibrous substrates. Herein, a casting method is developed to replicate the nanofibrous morphology into a thin layer of polymethylsiloxane (PDMS). The template is obtained by electrospinning and chemical crosslinking of gelatin nanofibers on a glass slide. The replicas of the template are surface-functionalized by gelatin and used for propagation of hiPSCs over tenth generations. The performance of the propagated hiPSCs is checked by immunofluorescence imaging, flowcytometry, and RT-PCR, confirming the practicability of this method. The results are also compared to those obtained using electrospun nanofiber substrates. Inherently, the PDMS replica is of low stiffness and can be reproduced easily. Compared to other patterning techniques, casting is more flexible and cost-effective, suggesting that this method might find applications in cell-based assays that rely on stringent consideration of both substrate stiffness and surface morphology.

Triglyceride-Mimetic Structure-Gated Prodrug Nanoparticles for Smart Cancer Therapy.

Off-target drug release and insufficient drug delivery are the main obstacles for effective anticancer chemotherapy. Prodrug-based self-assembled nanoparticles bioactivated under tumor-specific conditions are one of the effective strategies to achieve on-demand drug release and effective tumor accumulation. Herein, stimuli-activable prodrugs are designed yielding smart tumor delivery by combination of the triglyceride-mimic (TG-mimetic) prodrug structure and disulfide bond. Surprisingly, these prodrugs can self-assemble into uniform nanoparticles (NPs) with a high drug loading (over 40%) and accumulate in tumor sites specifically. The super hydrophobic TG structure can act as a gate that senses lipase to selectively control over NP dissociation and affect the glutathione-triggered prodrug activation. In addition, the impacts of the double bonds in the prodrug NPs on parent drug release and the following cytotoxicity, pharmacokinetics, and antitumor efficiency are further demonstrated. Our findings highlight the promising potential of TG-mimetic structure-gated prodrug nanoparticles for tumor-specific drug delivery.

Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration.

Skeletal muscle regeneration requires extracellular matrix (ECM) remodeling, including an acute and transient breakdown of collagen that produces gelatin. Although the physiological function of this process is unclear, it has inspired the application of gelatin to injured skeletal muscle for a potential pro-regenerative effect. Here, we investigated a bi-phasic effect of gelatin in skeletal muscle regeneration, mediated by the hormetic effects of reactive oxygen species (ROS). Low-dose gelatin stimulated ROS production from NADPH oxidase 2 (NOX2) and simultaneously upregulated the antioxidant system for cellular defense, reminiscent of the adaptive compensatory process during mild stress. This response triggered the release of the myokine IL-6, which stimulates myogenesis and facilitates muscle regeneration. By contrast, high-dose gelatin stimulated ROS overproduction from NOX2 and the mitochondrial chain complex, and ROS accumulation by suppressing the antioxidant system, triggering the release of TNFα, which inhibits myogenesis and regeneration. Our results have revealed a bi-phasic role of gelatin in regulating skeletal muscle repair mediated by intracellular ROS, the antioxidant system and cytokine (IL-6 and TNFα) signaling.

Evaluation of the Effects of Solvents Used in the Fabrication of Microfluidic Devices on Cell Cultures.

Microfluidic microphysiological systems (MPSs) or "organs-on-a-chip" are a promising alternative to animal models for drug screening and toxicology tests. However, most microfluidic devices employ polydimethylsiloxane (PDMS) as the structural material; and this has several drawbacks. Cyclo-olefin polymers (COPs) are more advantageous than PDMS and other thermoplastic materials because of their low drug absorption and autofluorescence. However, most COP-based microfluidic devices are fabricated by solvent bonding of the constituent parts. Notably, the remnant solvent can affect the cultured cells. This study employed a photobonding process with vacuum ultraviolet (VUV) light to fabricate microfluidic devices without using any solvent and compared their performance with that of solvent-bonded systems (using cyclohexane, dichloromethane, or toluene as the solvent) to investigate the effects of residual solvent on cell cultures. Quantitative immunofluorescence assays indicated that the coating efficiencies of extracellular matrix proteins (e.g., Matrigel and collagen I) were lower in solvent-bonded COP devices than those in VUV-bonded devices. Furthermore, the cytotoxicity of the systems was evaluated using SH-SY5Y neuroblastoma cells, and increased apoptosis was observed in the solvent-processed devices. These results provide insights into the effects of solvents used during the fabrication of microfluidic devices and can help prevent undesirable reactions and establish good manufacturing practices.

Spatiotemporal determination of metabolite activities in the corneal epithelium on a chip.

The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of metabolism and determine the transportation of endogenous and exogenous molecules across the corneal epithelium barrier. This study introduces multiple corneal epitheliums on a chip namely, Corneal Epithelium on a Chip (CEpOC), which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. We observed the spatiotemporal secretion of biologically relevant metabolites (i.e., antioxidant, glutathione and uric acid) as well as the depletion of essential nutrients such as amino acids and vitamins mimicking the in vivo molecules trafficking across the human corneal epithelium. Through the shifts of extracellular metabolites and quantitative analysis of mRNA associated with transporters, we were able to investigate the secretion and transportation activities across the polarized barrier in a correlation with the expression of corneal transporters. Thus, CEpOC can provide a non-invasive, simple, yet effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as advanced experimental model of the human corneal epithelium.

Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems.

A microphysiological system (MPS) holds great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform faces several challenges in terms of the materials used (e.g. polydimethylsiloxane; PDMS). For instance, absorption of drug candidates and fluorescent dyes into PDMS, as well as the effect elicited by materials on cultured cells, can cause inaccurate or misleading results in cell assays. The use of PDMS also poses challenges for mass production and long-term storage of fabricated MPSs. Hence, to circumvent these issues, herein we describe the development of a cyclo olefin polymer (COP)-based MPS using photobonding processes and vacuum ultraviolet (VUV), designated as COP-VUV-MPS. COP is an amorphous polymer with chemical/physical stability, high purity and optical clarity. Due to the thermostability and high modulus of COP, the metal molding processes was applied for mass production of MPSs without deformation of microstructures and with quick fabrication cycle time (approx. 10 min/cycle). Moreover, VUV photobonding process with an excimer light at a 172nm wavelength allowed assembling COP materials without the use of additional solvents and tapes, which might cause cell damages. In comparison with the conventional MPS made of PDMS (PDMS-MPS), COP-VUV-MPS showed improved chemical resistance without causing molecule absorption. Moreover, COP-VUV-MPS maintained the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes or gene expression. These results suggest that COP-VUV-MPS may be broadly applicable for the advancement of MPS and applications in drug development, as well asin vitrotoxicological testing.

Untargeted LC-MS Metabolomics for the Analysis of Micro-scaled Extracellular Metabolites from Hepatocytes.

Metabolome analysis in micro physiological models is a challenge due to the low volume of the cell culture medium (CCM). Here, we report a LC-MS-based untargeted metabolomics protocol for the detection of hepatocyte extracellular metabolites from micro-scale samples of CCM. Using a single LC-MS method we have detected 57 metabolites of which 27 showed >2-fold shifts after 72-hour incubation. We demonstrate that micro-scale CCM samples can be used for modelling micro-physiological temporal dynamics in metabolite intensities.