Culture Chip Research Articles

3D Interfacial and Spatiotemporal Regulation of Human Neuroepithelial Organoids.

Neuroepithelial (NE) organoids with dorsal–ventral patterning provide a useful three‐dimensional (3D) in vitro model to interrogate neural tube formation during early development of the central nervous system. Understanding the fundamental processes behind the cellular self‐organization in NE organoids holds the key to the engineering of organoids with higher, more in vivo‐like complexity. However, little is known about the cellular regulation driving the NE development, especially in the presence of interfacial cues from the microenvironment. Here a simple 3D culture system that allows generation and manipulation of NE organoids from human‐induced pluripotent stem cells (hiPSCs), displaying developmental phases of hiPSC differentiation and self‐aggregation, first into NE cysts with lumen structure and then toward NE organoids with floor‐plate patterning, is established. Longitudinal inhibition reveals distinct and dynamic roles of actomyosin contractility and yes‐associated protein (YAP) signaling in governing these phases. By growing NE organoids on culture chips containing anisotropic surfaces or confining microniches, it is further demonstrated that interfacial cues can sensitively exert dimension‐dependent influence on luminal cyst and organoid morphology, successful floor‐plate patterning, as well as cytoskeletal regulation and YAP activity. This study therefore sheds new light on how organoid and tissue architecture can be steered through intracellular and extracellular means.

Design and Modeling of a Microfluidic Coral Polyps Culture Chip with Concentration and Temperature Gradients.

Traditional methods of cultivating polyps are costly and time-consuming. Microfluidic chip technology makes it possible to study coral polyps at the single-cell level, but most chips can only be analyzed for a single environmental variable. In this work, we addressed these issues by designing a microfluidic coral polyp culture chip with a multi-physical field for multivariable analyses and verifying the feasibility of the chip through numerical simulation. This chip used multiple serpentine structures to generate the concentration gradient and used a circuit to form the Joule effect for the temperature gradient. It could generate different temperature gradients at different voltages for studying the growth of polyps in different solutes or at different temperatures. The simulation of flow field and temperature showed that the solute and heat could be transferred evenly and efficiently in the chambers, and that the temperature of the chamber remained unchanged after 24 h of continuous heating. The thermal expansion of the microfluidic chip was low at the optimal culture temperature of coral polyps, which proves the feasibility of the use of the multivariable microfluidic model for polyp culture and provides a theoretical basis for the actual chip processing.

An easy-repeat method to build a blood-brain barrier model on a chip with independent TEER detection module

Blood–brain barrier on a chip (BBBoC) has emerged as a promising tool for high-throughput screening of central nervous system drugs. During the drug screening, it is essential to ensure high repeatability and consistency of assays in different culture units or chips with the same structures. For the assembled chip, cells are introduced into the inoculating chamber by perfusion. However, this method leads to differences between BBBoC because of the uncontrollable flow and sedimentation of the cell suspension in initial stages of cell inoculating. In this work, an inoculating-first and assembling-later method was presented to overcome the shortcoming. First, a porous membrane fixed between the PDMS brackets was made to support the cells. Then human brain microvascular endothelial cells and human astrocytes were respectively inoculated in turn onto the two sides of the porous membrane. When cells grew and occupied the porous membrane, the cell support was sandwiched between two glass substrates with microelectrodes for TEER detection. The method ensured high consistency of cell inoculating number on the porous membrane. Subsequently, a blood-brain barrier model in vitro with high trans-endothelial electrical resistance (TEER) value and expression of ZO-1 was achieved.

A Cell Culture Chip with Transparent, Micropillar-Decorated Bottom for Live Cell Imaging and Screening of Breast Cancer Cells.

In the recent years, microfabrication technologies have been widely used in cell biology, tissue engineering, and regenerative medicine studies. Today, the implementation of microfabricated devices in cancer research is frequent and advantageous because it enables the study of cancer cells in controlled microenvironments provided by the microchips. Breast cancer is one of the most common cancers in women, and the way breast cancer cells interact with their physical microenvironment is still under investigation. In this study, we developed a transparent cell culture chip (Ch-Pattern) with a micropillar-decorated bottom that makes live imaging and monitoring of the metabolic, proliferative, apoptotic, and morphological behavior of breast cancer cells possible. The reason for the use of micropatterned surfaces is because cancer cells deform and lose their shape and acto-myosin integrity on micropatterned substrates, and this allows the quantification of the changes in morphology and through that identification of the cancerous cells. In the last decade, cancer cells were studied on micropatterned substrates of varying sizes and with a variety of biomaterials. These studies were conducted using conventional cell culture plates carrying patterned films. In the present study, cell culture protocols were conducted in the clear-bottom micropatterned chip. This approach adds significantly to the current knowledge and applications by enabling low-volume and high-throughput processing of the cell behavior, especially the cell–micropattern interactions. In this study, two different breast cancer cell lines, MDA-MB-231 and MCF-7, were used. MDA-MB-231 cells are invasive and metastatic, while MCF-7 cells are not metastatic. The nuclei of these two cell types deformed to distinctly different levels on the micropatterns, had different metabolic and proliferation rates, and their cell cycles were affected. The Ch-Pattern chips developed in this study proved to have significant advantages when used in the biological analysis of live cells and highly beneficial in the study of screening breast cancer cell–substrate interactions in vitro.

An integrated nucleic acid detection method based on a microfluidic chip for collection and culture of rice false smut spores.

Rice false smut spores (RFSS), which are airborne spores caused by Ustilaginoidea virens (U. virens), not only cause severe yield loss and grain quality reduction, but also produce toxins that are harmful to humans and animals. Nucleic acid detection has become the main method for RFSS monitoring due to its high specificity and sensitivity. However, nucleic acid detection requires multiple steps of spore collection, DNA extraction, nucleic acid amplification and detection, which has a high demand for personnel and is hard to link with other intelligent equipment to achieve automation. Microfluidic chip has become an important approach for integrated detection of pathogens owning to miniaturization and integration in recent years. Yet there is a lack of portable methods that integrate the collection of airborne fungal spores and nucleic acid detection. Because RFSS have thick cell walls and require liquid nitrogen grinding to extract DNA, breaking the walls on-chip is difficult. Therefore, the realization of RFSS wall breaking on-chip is a major difficulty and also a very meaningful study. This study uses RFSS as the research object and provides a novel method of culturing RFSS on-chip to solve the problem of hard wall breaking, realizing the integrated detection of RFSS. The mycelium grown by RFSS germination could be easily broken to release DNA for on-chip detection, which eliminates the need for manual DNA extraction and resolves the issue of difficult wall breaking. This chip can collect RFSS based on the aerodynamic theory and achieve gas-liquid coupling through a simple microvalve structure. A micromixer is constructed to mix the liquid, and then accomplish detection quickly by recombinase polymerase amplification and lateral flow dipsticks (RPA-LFD). The detection sensitivity of this method is 1 × 102-1 × 105 CFU ml-1. It can realize the "sample in and answer out" detection of RFSS due to its simple operation, independence from precision instruments, high sensitivity and specificity. The result shows that it can be used for the early detection of RFSS, has great application prospects and is expected to promote the development of on-site instant detection equipment.

An integrated microfluidic chip for alginate microsphere generation and 3D cell culture.

Three-dimensional (3D) hydrogel microspheres have attracted increasing attention as cell culture carriers. The system of hydrogel microspheres provides great advantages for cell growth owing to its high surface-to-volume ratio and biocompatible environment. However, an integrated system that includes microsphere generation, microsphere capture and in situ culture together has not been realized yet. Here we present a multifunctional microfluidic device to accomplish the overall process including cell-laden microsphere generation, online demulsification and dynamic-culture. The microfluidic device can produce massive monodispersed alginate microspheres and allows us to immobilize the alginate microspheres and record bacterial growth. Moreover, the microspheres provide a suitable environment through the mechanical properties of soft tissues, leading to high cell viability, proliferation, activity and biocompatibility. We believe that this versatile and biocompatible platform will provide a more reliable analysis tool for tissue engineering and cell therapy.

An Integrated Nucleic Acid Detection Method Based on Microfluidic Chip for Collection and Culture of Rice False Smut Spores

An Integrated Nucleic Acid Detection Method Based on Microfluidic Chip for Collection and Culture of Rice False Smut Spores

In silico design and fabrication of an SFI chip-based microspheroid culture system.

The emergence of microfluidic devices and computational fluid dynamics (CFD) has propelled the need for next-generation biomimetic cell culture platforms that are flexible for monitoring and regulation. Therefore, this study evaluated a CFD application in an in silico-designed and spheroid-based flow integration 3D cell culture chip (SFI chip) to illustrate cell culture, drug screening, cytokine delivery, and differentiation of cells in a platform that partially recapitulates the natural environment. Our results show that a flow rate of 0.05 mL h-1 or less induced no physical stress in the SFI chip (15 mm), and uniform cell spheroids (approximately 200 μm) were formed across the platform. The cultured cells were tested in several experimental contexts (co-culture, drug screening, cytokine delivery, and differentiation), demonstrating the usefulness of computational simulation in expediting discovery and simple and effective means to scale the production of standardized cell spheroids cultured under dynamic and natural conditions. Advanced cell culture technologies can be used to accelerate research and discovery and the preclinical and clinical development of cell and cell-free therapies for urgent medical needs.

Ceiling culture chip reveals dynamic lipid droplet transport during adipocyte dedifferentiation via actin remodeling.

Adipocyte dedifferentiation has recently gained attention as a process underpinning adipocyte plasticity; however, a lack of suitable experimental platforms has hampered studies into the underlying mechanisms. Here, we developed a microscope-mountable ceiling culture chip that provides a stable yet tunable culture environment for long-term live-imaging of dedifferentiating adipocytes. A detailed spatiotemporal analysis of mature adipocyte dedifferentiation utilizing the culture platform and Cre-recombinase tracers revealed the involvement of dynamic actin remodeling for lipid droplet (LD) secretion during adipocyte dedifferentiation. Additionally, Hippo, Hedgehog, and PPARγ signaling pathways were identified as potent regulators of adipocyte dedifferentiation. Contrary to the belief that adult adipocytes are relatively static, we show that adipocytes are very dynamic, relying on actin-driven mechanical forces to execute LD extrusion and intercellular LD transfer processes.

Tumor-intrinsic FABP5 is a novel driver for colon cancer cell growth via the HIF-1 signaling pathway

Dysfunctional lipid metabolism is a known cause of cancer development and progression, yet little is known about the underlying molecular mechanisms that contribute to cancer progression. In this study, we demonstrate that fatty acid binding protein 5 (FABP5) is elevated in colon cancer tissue and this increased expression is linked to upregulation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway. Under physiologically in vivo mimicked conditions via a polydimethylsiloxane (PDMS)-based three-dimensional (3D) culture chip, FABP5-knockdown colon cancer cells exhibited attenuated cell growth throughout the culture period. FABP5 was found to regulate HIF-1α protein levels and gene expression levels within the HIF-1α signaling pathway under hypoxic conditions. Our results provide evidence that supports the use of FABP5 as a prognostic factor in colon cancer. The FABP5/HIF-1α axis is a promising target for ameliorating fatty acid-triggered cancer progression.

Adhesive-Based Fabrication Technique for Culture of Lung Airway Epithelial Cells with Applications in Cell Patterning and Microfluidics.

This work describes a versatile and cost-effective cell culture method for micropatterning and growing adherent cells on porous membranes using pressure-sensitive double-sided adhesives. This technique also allows cell culture using conventional methods and their easy integration into microfluidic chip devices. Adhesives can be used to form different patterns of cultured cells, which can be used for cell proliferation and wound-healing models. To demonstrate the viability of our system, we evaluate the toxicity effect of five different adhesives on two distinct airway epithelial cell lines and show functional applications for cell patterning and microfluidic cell culture chip fabrication. We developed a sandwiched microfluidic device that enabled us to culture cells in a submerged condition and transformed it into a dynamic platform when required. The viability of cells and their inflammatory responses to IL-1β stimulation were investigated. Our technique is applicable for conventional culturing of cells, widely available in biomedical research labs, while enabling the introduction of perfusion for an advanced dynamic cell culture model when needed.

Mechanical Stress Induces a Transient Suppression of Cytokine Secretion in Astrocytes Assessed at the Single-Cell Level with a High-Throughput Microfluidic Chip.

Brain cells are constantly subjected to mechanical signals. Astrocytes are the most abundant glial cells of the central nervous system (CNS), which display immunoreactivity and have been suggested as an emerging disease focus in the recent years. However, how mechanical signals regulate astrocyte immunoreactivity, and the cytokine release in particular, remains to be fully characterized. Here, human neural stem cells are used to induce astrocytes, from which the release of 15 types of cytokines are screened, and nine of them are detected using a protein microfluidic chip. When a gentle compressive force is applied, altered cell morphology and reinforced cytoskeleton are observed. The force induces a transient suppression of cytokine secretions including IL-6, MCP-1, and IL-8 in the early astrocytes. Further, using a multiplexed single-cell culture and protein detection microfluidic chip, the mechanical effects at a single-cell level are analyzed, which validates a concerted downregulation by force on IL-6 and MCP-1 secretions in the cells releasing both factors. This work demonstrates an original attempt of employing the protein detection microfluidic chips in the assessment of mechanical regulation on the brain cells at a single-cell resolution, offering novel approach and unique insights for the understanding of the CNS immune regulation.

Organ-on-a-chip: Determine feasibility of a human liver microphysiological model to assess long-term steroid metabolites in sports drug testing.

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the "organ-on-a-chip" principle. In a multichannel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time points of up to 14 days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3'OH-stanozolol-glucuronide and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in postadministration urine specimens. In conclusion, this pilot study indicates that the "organ-on-a-chip" technology provides a high degree of conformity with traditional human oral administration studies, providing a promising approach for metabolic profiling in sports drug testing.

A Novel SimpleDrop Chip for 3D Spheroid Formation and Anti-Cancer Drug Assay.

Cell culture is important for the rapid screening of anti-cancer drug candidates, attracting intense interest. Traditional 2D cell culture has been widely utilized in cancer biological research. However, 3D cellular spheroids are able to recapitulate the in vivo microenvironment of tissues or tumors. Thus far, several 3D cell culture methods have been developed, for instance, the hanging drop method, spinner flasks and micropatterned plates. Nevertheless, these methods have been reported to have some disadvantages, for example, medium replacement is inconvenient or causes cellular damage. Here, we report on an easy-to-operate and useful micro-hole culture chip (SimpleDrop) for 3D cellular spheroid formation and culture and drug analysis, which has advantages over the traditional method in terms of its ease of operation, lack of shear force and environmentally friendliness. On this chip, we observed the formation of a 3D spheroid clearly. Three drugs (paclitaxel, cisplatin and methotrexate) were tested by both cell viability assay and drug-induced apoptotic assay. The results show that the three drugs present a similar conclusion: cell viability decreased over time and concentration. Moreover, the apoptotic experiment showed a similar trend to the live/dead cell assay, in that the fraction of the apoptotic and necrotic cells correlated with the concentration and time. All these results prove that our SimpleDrop method is a useful and easy method for the formation of 3D cellular spheroids, which shows its potential for both cell–cell interaction research, tissue engineering and anticancer drug screening.

Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments.

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 μm–1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 μm–1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus

Abstract Human respiratory syncytial virus (HRSV) is one of the main drivers of lower respiratory tract illness in infants and young children worldwide. There are no licensed vaccines for HRSV available. Exosomes are cell-derived extracellular nanovesicles containing various biomolecules for intracellular communication. Exosomes secreted by antigen presenting cells such as dendritic cells can elicit immune responses by carrying MHC-I with antigenic peptide complex and other co-stimulating factors. Therefore, exosomes have emerged as potential vaccines to prevent viral infections or to treat cancers. Our prior work has demonstrated that a PDMS microfluidic culture chip device can be used to generate dendritic cell-derived tumor antigenic exosomes, with the capacity to activate tumor-specific CD8+ T cells. Herein, we further extended the work to surface engineer dendritic cell-derived exosomes with antigenic peptides from RSV (M187–195 and NS161–75). A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses in vitro and in vivo. In vitro assays demonstrated that surface engineered exosomes successfully carried the engineered peptides of interest and had the capacity to elicit IFNγ production by activated, virus-specific CD8+ T cells. However, vaccination with engineered exosomes did not prime an in vivo activation of antigen-specific CD8+ T cell response, although surface engineered exosomes exhibit the safe administration. Additional experiments are necessary to optimize the potency of surface engineered exosomes as a vaccine platform for the prevention of viral infections such as RSV.

Additive manufacturing of a 3D vascular chip based on cytocompatible hydrogel

Development of a three-dimensional (3D) vascular microfluidic chip is one of the important challenges for the study vascular-related diseases and new drugs. To date, however, realization of in vivo-like blood vessel chips based on soft- or photo-lithographic fabrication methods have limitations such as complex fabrication processes, tedious manual labor, and frequent assembly failure. In this study, we describe the development of a 3D cell culture chip by utilizing a stereolithographic printer. The microfluidic chip was fabricated using a resin made of 30% (v/v in water) poly(ethylene glycol) diacrylate (MW = 700) (30% PEG-DA-700), a UV initiator (Irgacure-819 (IRG)) and a photosensitizer (2-isopropylthioxanthone (ITX)) for constructing a cytocompatible hydrogel structure. The 3D-printed cell culture chip is transparent, enabling cell culture and observation of cell proliferation. Endothelial cells were cultured to mimic in vivo-like blood vessel architecture within the 3D-printed chip. After EAhy926 cell cultivation, cell viability was confirmed by a live/dead cell assay and cultured cells were stained with Calcein-AM (Green) and Ethidium homodim-1 (Red). In addition, endothelial cells were successfully cultured in a 3D-printed vascular tube-like architecture in PEG-DA-700. Creating a vascular microfluidic chip with 30% PEG-DA-700 hydrogel using automated 3D-printing processes reduces fabrication steps and time, avoids any assembly and bonding, and economizes manufacturing cost. We believe our stereolithographic 3D-printing method for fabricating a 3D-printed vascular chip provides a cost-effective, convenient, simple, and reproducible route to vascular-related disease modelling and drug screening analyses.

A Three-Dimensional Microfluidic Device for Monitoring Cancer and Chemotherapy-Associated Platelet Activation.

Platelet activation and the risk of thrombosis are increased in cancer patients, especially after chemotherapy. Our previous studies indicated that chemotherapy-induced platelet activation is largely due to endothelial cell damage. Thus, simple in vitro tests, such as aggregometry, are not desirable tests to predict platelet responsiveness to different chemotherapeutic agents because other contributory factors, such as tumor cells, endothelial cells, and the flow rate of platelets, also contribute to the formation of cancer-associated thrombosis. Therefore, developing a platelet detection system, which includes all possible risk parameters, is necessary. In the present study, we described a microengineered microfluidic system that contained a drug concentration generator, cancer cell culture chip, and three-dimensional (3D) circular microvascular model covered with a confluent endothelial layer and perfused with human platelets at a stable flow rate. Doxorubicin was injected through two injection sites. Endothelial cell injury was evaluated by counting, cell cytoskeleton observation, and the level of IACM1 and ET-1 in endothelial cells or a culture medium. Prestained platelets were perfused into the artificial blood vessel, and platelet-endothelial cell adhesion was measured. We found that (i) MCF7 cell-released factors had a cytotoxicity effect on both endothelial cells and platelets. (ii) We confirmed that doxorubicin-induced platelet activation was endothelial cell-dependent. (iii) A lower dosage of doxorubicin (0–2.0 μM) induced platelet activation, while a higher dosage of doxorubicin (2.0–4.0 μM) led to platelet death. Our findings indicated that platelet-endothelial cell adhesion could be used as a diagnostic marker of platelet activation, providing a simple and rapid detective way to predict platelet responsiveness before or during chemotherapy.

Three-Dimensional Motor Nerve Organoid Generation.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

Fabrication of a hydroxyapatite-PDMS microfluidic chip for bone-related cell culture and drug screening

Bone is an important part of the human body structure and plays a vital role in human health. A microfluidic chip that can simulate the structure and function of bone will provide a platform for bone-related biomedical research. Hydroxyapatite (HA), a bioactive ceramic material, has a similar structure and composition to bone mineralization products. In this study, we used HA as a microfluidic chip component to provide a highly bionic bone environment. HA substrates with different microchannel structures were printed by using ceramic stereolithography (SLA) technology, and the minimum trench width was 50 μm. The HA substrate with microchannels was sealed by a thin polydimethylsiloxane (PDMS) layer to make a HA-PDMS microfluidic chip. Cell culture experiments demonstrated that compared with PDMS, HA was more conducive to the proliferation and osteogenic differentiation of the human foetal osteoblast cell line (hFOB). In addition, the concentration gradient of the model drug doxorubicin hydrochloride (DOX) was successfully generated on a Christmas tree structure HA-PDMS chip, and the half maximal inhibitory concentration (IC50) of DOX was determined. The findings of this study indicate that the HA-PDMS microfluidic chip has great potential in the field of high-throughput bone-related drug screening and bone-related research.