Decoupling shear stress and pressure effects in the biomechanics of autosomal dominant polycystic kidney disease using a perfused kidney-on-chip.

Kidney tubular cells are submitted to two distinct mechanical forces generated by the urine flow: shear stress and hydrostatic pressure. In addition, the mechanical properties of the surrounding extracellular matrix modulate tubule deformation under constraints. These mechanical factors likely play a role in the pathophysiology of kidney diseases as exemplified by autosomal dominant polycystic kidney disease, in which pressure, flow and matrix stiffness have been proposed to modulate the cystic dilation of tubules with PKD1 mutations. The lack of in vitro systems recapitulating the mechanical environment of kidney tubules impedes our ability to dissect the role of these mechanical factors. Here we describe a perfused kidney-on-chip with tunable extracellular matrix mechanical properties and hydrodynamic constraints, that allows a decoupling of shear stress and flow. We used this system to dissect how these mechanical cues affect Pkd1 -/- tubule dilation. Our results show two distinct mechanisms leading to tubular dilation. For PCT cells (proximal tubule), overproliferation mechanically leads to tubular dilation, regardless of the mechanical context. For mIMCD-3 cells (collecting duct), tube dilation is associated with a squamous cell morphology but not with overproliferation and is highly sensitive to extracellular matrix properties and hydrodynamic constraints. Surprisingly, flow alone suppressed Pkd1 -/- mIMCD-3 tubule dilation observed in static conditions, while the addition of luminal pressure restored it. Our in vitro model emulating nephron geometrical and mechanical organization sheds light on the roles of mechanical constraints in ADPKD and demonstrates the importance of controlling intraluminal pressure in kidney tubule models.

Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Construction of a Multitubular Perfusable Kidney-on-Chip for the Study of Renal Diseases.

The organ-on-chip model offers versatility and modularity of in vitro models while approaching the biological fidelity of in vivo models. We propose a method to build a perfusable kidney-on-chip aiming at reproducing key features of the densely packed segments of nephrons in vitro; such as their geometry, their extracellular matrix, and their mechanical properties. The core of the chip is made of parallel tubular channels molded into collagen I that are as small as 80 µm in diameter and as close as 100 µm apart. These channels can further be coated with basement membrane components and seeded by perfusion of a suspension of cells originating from a given segment of the nephron. We optimized the design of our microfluidic device to achieve high reproducibility regarding the seeding density of the channels and high fluidic control of the channels. This chip was designed as a versatile tool to study nephropathies in general, contributing to building ever better in vitro models. It could be particularly interesting for pathologies such as polycystic kidney diseases where mechanotransduction of the cells and their interaction with adjacent extracellular matrix and nephrons may play a key role.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples.

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Precise and fast control of the dissolved oxygen level for tumor-on-chip.

In vitro cell cultures are most often performed in unphysiological hyperoxia since the oxygen partial pressure of conventional incubators is set at 141 mmHg (18.6%, close to ambient air oxygen 20.1%). This value is higher than human tissue oxygen levels, as the in vivo oxygen partial pressures range from 104 mmHg (lung alveoli) to 8 mmHg (skin epidermis). Importantly, under pathological conditions such as cancer, cells can experience oxygen pressure lower than the healthy tissue. Although hypoxic incubators can regulate gas oxygen, they do not take into account the dissolved oxygen concentration in the cell culture medium. In the context of organ on chip and micro-physiological system development, we present here a new system, called Oxalis (OXygen ALImentation System) that allows fine control of the dissolved oxygen level in the cell culture medium. Oxalis regulates simultaneously the gas composition and the inlet reservoir pressure by modulating the pneumatic valve opening. This dual regulation allows both the pressure driven liquid flowrate and the level of oxygen dissolved in the chip to be controlled independently. Oxalis offers unprecedented features such as an oxygen equilibration time lower than 3 minutes and an accuracy of 3 mmHg. These performances can be reached for chip perfusion flow as low as 1 µL min-1. This low flow rate allows the shear stress experienced by the cells in the chip to be accurately controlled. In addition, the system enables modulation of the pH in the cell culture medium through the modulation of CO2. The fine control and monitoring of both O2 and pH pave the way for new precise investigations on physiological and pathological biological processes. Using Oxalis in the context of tumor-on-chip, we demonstrate the capacity of the system to recapitulate hypoxia-induced gene expression, offering an innovative strategy for future studies on the role of hypoxia in malignant progression and drug resistance.

A Multitubular Kidney-on-Chip to Decipher Pathophysiological Mechanisms in Renal Cystic Diseases.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.