Organs-on-a-chip最新文献

The discipline of microphysiometry emerged at the end of the 1980s and has been progressing towards today's organs on chips and microphysiological systems approaches. The presented work reviews the development of cellular model from cellular monolayers toward 3D multi-cellular tissue constructs, along with the maturation of sensor principles and technologies. A modular classification into cellular models, biochip, climate control and fluidic system, and control & data acquisition is introduced. The experimental conditions and aspects of data processing are discussed and reproducibility issues such as the use of chemically defined cell culture medium are addressed. A brief review of applications and an outlook on current challenges in the field conclude the review paper.

Antisense oligonucleotides (ASOs) are highly biologically stable and specific therapeutic molecules that interfere with mRNA transcription and as a result effectively reduce the expression of a protein of interest. ASOs have low drug clearance and are retained in tissues. This is particularly evident in the kidneys where they accumulate in the renal proximal tubules. Receptor-mediated endocytosis (RME) plays a role in ASO uptake, however the membrane receptors facilitating the process and the full mechanism behind these transport and subsequent renal retention is still poorly understood. In the present study we employ a proximal tubule-on-a-chip (PTOC) that recapitulates the highly polarized nature of the renal epithelium and can discriminate between basal and apical uptake processes. The PTOC was used to determine the impact of cellular polarization in ASO uptake in the kidney and elucidate which receptors predominantly facilitated uptake. In the PTOC the uptake occurred predominantly from the basolateral side and was extensively inhibited using the selective epidermal growth factor receptor (EGFR) antagonist cetuximab. These results demonstrate that ASO uptake in a physiologically relevant model is predominately mediated by the EGFR and takes place from the basolateral side of the proximal tubules. In comparison to the 2D culture, PTOC could differentiate between distinct ASO uptake routes mediated by either Megalin or EGFR, which proper membrane expression is highly dependent on cellular polarity. Our results highlight the limitations of renal 2D models and demonstrate how an organ on a chip model can fill the physiological gap and is a powerful tool to study ASO, which have intricate transport mechanisms that ultimately impact both their efficacy and safety.

Tumor-on-a-chip models are rapidly coming to the fore as a promising platform to accurately mimic tumor biology. These models overcome ethical concerns of animal usage in research, and are of particular use in the field of immuno-oncology, as there are substantial interspecies differences in how cells of the immune system operate. Additionally, they allow a human-centered investigation of novel immunotherapeutic approaches. Here, we report a new application of a microfluidic tumor-on-a-chip system and show its utility by investigating macrophage responses in the context of a promising therapeutic approach that combines anti-epidermal growth factor receptor (EGFR) IgA with an anti-CD47 innate immune checkpoint inhibitor. We report a novel on-chip microscopy-based antibody-dependent cellular phagocytosis (ADCP) assay with human M1-like pro- or M2-like anti-inflammatory macrophages and tumor cells in a collagen matrix. The tumor microenvironment was further characterized by ELISA for secreted factors in the culture medium and through endpoint analyses of gene expression by RT-qPCR. Employing the novel on-chip assay, we show for the first time that the combination of anti-EGFR IgA and a CD47 checkpoint inhibitor synergistically activate macrophage phagocytic function to specifically kill cancer cells, especially for M2-like macrophages. We further demonstrate that the checkpoint inhibition is responsible for elevated secretion of inflammatory cytokines such as TNFα and IL-6, and tends to elevate expression of genes regulating both inflammation (IL-1β) and phagocytic function (CD209), particularly in M2-like macrophages. Taken together, we demonstrate a novel on-chip ADCP assay compatible with multi-parameter characterization of the tumor microenvironment on-chip and demonstrate its utility for yielding novel insights regarding innate immunotherapy combinations.

Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but the combination of patterned hydrogels with organs-on-chip to generate organized 3D cultures under microfluidic perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥10⁷/mL) did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. To generate the desired pattern on-chip, the cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask. The on-chip 3D cultures were self-standing and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 to 900 μm in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells were patterned on-chip with high regional specificity. Viability remained high (>85%) after 12-hr culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D co-cultures to mimic organ-like structures that were previously difficult to obtain.

For over a decade, we have seen significant strides in the microfluidics field that have led to the concept of microphysiological systems. These systems emerged in the early 2010s as versatile in vitro platforms that allowed researchers to mimic tissue complexity in vitro. Early models focused on showing the advantages of fluid physics at the microscale and demonstrating proof-of-concept experiments. As the technology evolved, microfluidic models became more complex and showed their capacity to mimic complex biological responses at an organ level, coining the concept of organ-on-a-chip platforms. Gathered under the banner of “microphysiological systems”, current platforms evaluate complex dynamics that involve numerous cell types in highly organized scenarios. Recent models have leveraged advanced imaging and multi-omics techniques to study a large variety of cellular and molecular processes, from cancer and strokes to reproductive biology and infectious diseases. In this piece, we highlight the main hallmarks of each of these periods and outline current and upcoming trends in the field of microphysiological systems.

The integration of vasculature at physiological scales within 3D cultures of cell-laden hydrogels for the delivery of spatiotemporal mass transport, chemical and mechanical cues, is a stepping-stone towards building in vitro tissue models that recapitulate in vivo cues. To address this challenge, we present a versatile method to micropattern adjoining hydrogel shells with a perfusable channel or lumen core, for enabling facile integration with fluidic control systems, on one hand, and to cell-laden biomaterial interfaces, on the other hand. This microfluidic imprint lithography methodology utilizes the high tolerance and reversible nature of the bond alignment process to lithographically position multiple layers of imprints within a microfluidic device for sequential filling and patterning of hydrogel lumen structures with single or multiple shells. Through fluidic interfacing of the structures, the ability to deliver physiologically relevant mechanical cues for recapitulating cyclical stretch on the hydrogel shell and shear stress on endothelial cells in the lumen are validated. We envision application of this platform for recapitulation of the bio-functionality and topology of micro-vasculatures, alongside the ability to deliver transport and mechanical cues, as needed for 3D culture to construct in vitro tissue models.

The adipose tissue is a metabolically active endocrine organ with a dynamic secretome that is known to be implicated in metabolic disorders. Various studies have demonstrated detrimental downstream endocrinal effects of dysfunctional adipose tissue on other metabolic tissues, such as skeletal muscle and liver. In vitro ‘Adipose-on-Chip’ (AOC) models have been developed as an animal-alternative experimental platform to mimic adipose dysfunction in metabolic diseases. However, existing AOCs have not modeled both overtime lipid accumulation and inflammation of adipocytes in the presence of excess circulating free fatty acids (FFA), which are hallmarks of dysfunctional adipose tissue in obesity. This study reports for the first time, the establishment of a physiologically-relevant AOC disease model, which mimics adipose tissue pathophysiology in obesity via excessive FFA loading. The AOC model supports 3D perfusion culture of human bone marrow mesenchymal stem cell (BMMSC) differentiated adipocytes with improved adipogenic phenotypes as compared to conventional 2D well-plate cultures. Adipocytes in the AOC can be induced into a diseased phenotype on-chip, where they become both hypertrophic and inflamed when treated with an FFA mixture. This AOC disease model provides a more physiological experimental system to study the effects of adipose tissue dysfunction on downstream tissues for mechanistic investigations into obesity-related metabolic diseases.

The Aerobic intestinal Host – Anaerobic Microbiota (AHAM) interface is an important tissue barrier in our intestine where the microbiota resides in close proximity and in symbiosis with ourselves: the host. A disturbance in this delicate balance between our cells and the commensal microorganisms is associated with effects on the host's health and/or the microbiota. These host-microbiota interactions are believed to be influenced by several factors, which hampers the study of the effect of a single element exclusively. Organ-on-chips (OoCs), microengineered in vitro cell culture models, aim to mimic the physiologically relevant microenvironment of organs. These OoCs can be used to mimic the AHAM interface and study the host-microbiota interactions in a well-controlled environment. In this review, we summarize existing models for (components of) the AHAM interface and provide an overview of four different AHAM-on-chip systems. Furthermore, we defined challenges that need to be taken in consideration when designing or using an AHAM-on-chip, such as the importance of oxygen modulation, sensors and choice of chip material. It is essential to achieve a balance between the accuracy of representing the in vivo interface and the (technical) attainability of the in vitro AHAM-on-chip. The technological and biological aspects make an AHAM-on-chip extremely complex, which emphasizes the need for a multi-disciplinary team. We believe that standardization and higher throughput systems are crucial to accelerate the development of OoC technology.

Cancer is the second cause of death worldwide after heart disease. Despite the still developing anticancer therapies, the main challenge in cancer research is the establishing the appropriate predictive in vitro tumor model. Standard 2D and the increasingly used 3D cultures, as well as animal models suffer from numerous morphological and physiological differences. Current in vitro models often do not accurately predict toxicity due to non-linear dose-toxicity relationships, unclear mechanisms, non-organ specific toxicity as well as adverse side effects. In contrast, animal models do not always reflect human toxicity due to differences in physiology and interspecies metabolic capacity. In response to these, microfluidic Tumor-on-chip systems and their connections with other Organ-on-chip models (multi-Organ-on-Chip) have become a promising tool in cancer research. This type of tools are able to highly reproduce the dynamic microenvironment of the tumor and other organs. With on-chip approach is possible to observe and understand the mechanism and the changes taking place in metastases. In addition, multi-Organ-on-chip systems enable an assessment of the impact of anti-cancer therapies (outside the human body) directly on cancer, but also on surrounding organs, which brings new hope in personalized medicine.

Here, we report a testis-on-chip platform for the ex vivo culture of seminiferous tubules isolated from human and non-human primate testis. Tissues are cultured in a dedicated chamber with continuous perfusion via a vascular-like channel. The platform is fabricated from PDMS using a 3D printed mold, after design has been optimized, e.g., for the barrier between the culture chamber and the perfusion channel. COMSOL modeling revealed no direct negative impact of the flow on the tissues for the applied flowrate in the device, shear rate remaining in the physiological range. Culture experiments were performed using adult human seminiferous tubules from gender dysphoria patients and prepubertal seminiferous tubules from a 6-month-old marmoset for up to 11 and 9 days, respectively. First, microscopic, and live imaging revealed the presence of viable cell populations in both types of samples. Next, marmoset tissues were exposed to stimulatory conditions through perfusion of gonadotropins at different doses. The tissue response was characterized by histological analysis after their recovery from the device and testosterone and estradiol analysis in the effluent. Histological observations suggested improved maintenance of marmoset testicular tissues under stimulatory conditions, which similarly resulted in an increase in both testosterone and estradiol production, with yet different patterns for the low-dose and high-dose stimulation. The herein reported testis-on-chip platform shows great promise to evaluate endocrine and toxic effects on the testis.