ES3037110T3 - Multiorgan-on-a-chip device with integrated microbiosensors, methods and uses thereof - Google Patents

Abstract

This disclosure describes an advanced microfluidic system for the validation and study of nanomaterials, drugs, or mixtures thereof, intended for biomedical and/or pharmaceutical applications. A device for the production of one or more organoid cell cultures is described, comprising: a support base layer; a transducer layer disposed on the base layer; and an intermediate layer, disposed on the transducer layer, comprising several openings to form cavities for organoid cell growth and a network of microchannels for fluid flow; wherein the transducer layer comprises a heating device and at least several sensors in each cavity for cell growth control; wherein each sensor is positioned to measure an organoid cell growth parameter within each cavity; wherein the heating device is located within each cavity; wherein the heating device and the sensors are in direct contact with the organoid cell culture; wherein the sensors are selected from temperature sensors, pH sensors, heaters, or combinations thereof.

Description

DESCRIPTION

Multi-organ device-on-a-chip with integrated micro-sensors, methods and uses thereof

Technical Field

The present invention relates to a device for the production of organoid cell cultures, preferably a multi-organ-on-a-chip (MoC) system, as well as its use.

The present invention relates to an advanced microfluidic system for the validation and study of nanomaterials, drugs, or mixtures thereof, intended for biomedical and/or pharmaceutical applications. The microfluidic system allows for the determination of the toxicological and/or therapeutic effect in three-dimensional (3D) models of human organs, with the integration of micro(bio)sensors directly into the cell models, as well as into the microfluidic system to monitor the feed fluid, allowing for its automatic replacement when necessary.

Background

Driven by advances in nanotechnology, nanomedicine—which uses nanomaterials as multifunctional, stimulus-sensitive drug nanocarriers activated in response to various physicochemical parameters, such as pH and temperature—is developing with great optimism for the treatment and diagnosis of emerging diseases. In particular, cancerous tissues have a more acidic pH than healthy tissues. Therefore, a single nanomaterial can be designed as a pH-dependent drug nanocarrier and simultaneously incorporate other therapeutic properties, such as hyperthermia and gene therapy. However, to date, standard preclinical methodologies for evaluating these nanomaterials fail to replicate the complex physiological microenvironment present in the human body, and thus, these nanosystems have not been clearly validated, leading to shortcomings in the applicability of nanomedicine.

Organ-on-a-chip (OoC) platforms represent a disruptive technology capable of mimicking complex human organ functions and realistic (patho)physiology at the microscopic level. This technology promises to replace animal models in the analysis of the efficacy, toxicity, pharmacokinetics, and pharmacodynamics of active ingredients. More recently, it has also been proposed as a platform for the preclinical evaluation of nanomaterials developed for medical applications, both in diagnosis and therapy (the so-called theranostics).

Despite their potential, the end-use of out-of-cell (OoC) platforms has been limited by the effective integration of multi(bio)functional sensors that robustly capture the complexity of in vivo metabolism, as well as by the combination of multi-organ-on-a-chip (MoC) systems that enable systemic interaction and homeostasis (self-regulation) of various organ models within a single system. Ideally, a MoC should include integrated (bio)multiplexed sensors to monitor various physical and biochemical parameters present in the organ models, such as O2, CO2, temperature, pH, and released biomarkers, among others. This integrated system of multiplexed sensors allows for real-time monitoring of cell viability, growth, and maintenance in the organ models, as well as the evaluation of the toxicological effects of drugs and nanomaterials developed for biomedical applications.

Since the pioneering work in 2010 by the research group led by Donald Ingber, from the Wyss Institute (Boston, MA, USA), where the concept of OoC was introduced with the development of a lung-on-a-chip [Huh et al., “Reconstituting lung functions at the organ level on a chip”, Science, 2010, 328: 1662-1668 (10.1126/science.1188302)], various organ models have been described in the literature, demonstrating the potential of these advanced microfluidic devices for the diagnosis and treatment of human diseases [Rodrigues et al., “Organ-on-a-chip: A preclinical Microfluidic Platform for the Progress of Nanomedicine”, Small, 2020, 2003517 (DOI:10.1002/small.202003517)]. Among the most relevant is the work of Y.S. Zhang et al. [“Organ platform on chips integrated with several sensors for automated and continuous monitoring in situ of organoid behaviors”, p Na S, 2017, 114 (12) E2293-E2302 (DOI: 10.1073/pnas.1612906114)], as the first attempt to integrate an external multi-sensor module to monitor organ models. They describe the implementation of a modular system of physical (pH and temperature) and biochemical (molecular biomarkers) sensors externally connected to an organoid platform to monitor the antitumor effects of drugs. Another important work was published by S. Shin et al. [“Regenerative, unlabeled electrochemical microfluidic biosensors for continuous monitoring of cellular secretomes”, 2017, Adv. Sci. 4: 1600522 (DOI: 10.1002/advs.201600522)], which describes an electrochemical biosensor system for monitoring biomarkers released by organ models. However, these documents describe modular platforms connected, but not integrated, with advanced microfluidic devices. Therefore, in these systems, the response of the organ models to the analyzed parameters is subsequently evaluated externally, which, in addition to being a slow process, is susceptible to contamination and degradation of samples, hindering their viability as a standard, autonomous, and robust preclinical model for laboratory practice.

Patent document WO2013/086486 describes an out-of-context (OoC) system connected to a commercial microclinical analysis sensor chip for glucose, oxygen, lactate, and pH analysis. However, this is a commercial sensor system connected externally to the biological platform for analyzing the culture medium circulating within the device. Therefore, it does not provide physiological information on pH and temperature inside the biological models, which is essential for preclinical evaluation of nanomaterials and/or pharmacological studies.

US patent 10078075B2 describes a MoC system comprised of a microfluidic system with independent microvalves, enabling the control and analysis of drug effects in various organ models. The device consists of a modular microfluidic system, cell models, pressure sensors, and a commercially available microclinical sensor chip for glucose, oxygen, lactate, and pH analysis. However, all the sensors are designed for cell culture medium analysis and not for biological models. Furthermore, this invention does not provide physiological information on pH and cellular temperature in biological models, which is essential for drug and nanomedicine studies.

US patent document US2014/0302549A1 describes an organ-on-a-chip system that includes at least one biological model and can be connected to other biological models via a capillary network in a microfluidic system. This system allows the integration of one or more sensors, such as temperature, pH, oxygen, and biomarker sensors, connected via microfluidic channels for the analysis of the circulating culture medium. However, this system does not provide direct, individual physiological information on the pH and cellular temperature of the biological models (it only provides data on the cellular medium), which is essential for drug and/or nanomedicine studies.

Patent documents CA2885375A1, WO2014/048637, and PT2712918E describe a MoC system comprising a base layer, an organ layer positioned on the base layer, a connection layer referred to by the authors as the “antral layer” positioned on the organ layer, and an actuator layer. The organ layer accommodates at least one organ model and is configured to include a micro-inlet and a micro-outlet for fluid communication between the biological models and a microfluidic system. The base layer may also include one or more sensors for measuring key homeostasis parameters of the human organoids by monitoring the feeding medium. These sensors may include pH sensors, pO2 sensors, analyte capture sensors, temperature sensors, CO2 sensors, NO sensors, cytokine sensors, ion sensors, pressure sensors, and plasmonic resonance sensors. In particular, the invention aims to include two pO₂ sensors for measuring oxygen in the organ layer fluid; four transepithelial/endothelial electrical resistance (TEER) sensors for detecting leaks in the autonomous circulatory system, two of which are located within the system and two of which are configured and located in representative organ models; and electrical sensors coupled to biological neural nodes. However, this system does not provide direct, individual physiological information on the pH and cellular temperature of the biological models (it only provides data on the cellular environment), which is essential for drug and/or nanomedicine studies.

Since cellular pH and local temperature are essential for monitoring the homeostasis of different organ models in a MoC, existing solutions, which are based on the integration of microsensors into the microfluidic system for culture medium analysis, and are not integrated in situ within the different multi-organ models, fail to provide real-time, direct, and accurate information about the physiological microenvironment present in each organ model.

These facts are set forth in order to illustrate the technical problem addressed by the present invention.

General Description

The invention is defined by a device and its use, as set forth in claims 1 and 15, respectively. The present invention relates to an advanced microfluidic system comprising multiplexed micro(bio)sensors, implemented in situ in conjunction with representative organ models, for monitoring physicochemical parameters in assays with active ingredients and in the preclinical evaluation of nanomaterials. It also includes an optical pH sensor integrated into the microfluidic system for monitoring the feed fluid, allowing for its automatic replacement when necessary, thus ensuring long-term cell culture homeostasis. It further comprises a heating system for simulating hyperthermia treatments.

One aspect of the present invention relates to the insertion of multiplexed micro(bio)sensors in direct contact with the various organ models, enabling direct and individual monitoring of important physicochemical parameters, such as pH and temperature, which can vary depending on the type of organ model. Thus, this invention provides precise information on the individual physiological conditions of each organ, essential for monitoring its viability and toxicological effects, particularly when subjected to trials with drugs and nanomaterials developed for medical applications. Additionally, a heating system is incorporated to simulate hyperthermia treatments, adding a relevant parameter for the preclinical validation of thermoresponsive nanomaterials designed for therapeutic applications.

The present invention differs from other approaches by implementing in situ micro(bio)sensors in contact with representative models of human organs, allowing for the individual monitoring of each organ model, separately and in real time. Furthermore, and complementarily, the device according to the present invention also comprises at least one optical pH sensor system for monitoring the feed fluid circulating in the microfluidic system. In this way, the present invention allows for monitoring the conditions necessary for the culture and growth of the organ models in the advanced microfluidic device, as well as individually evaluating the viability of each organ model subjected to toxicological testing with drugs and/or nanomaterials.

The microfluidic system of the present invention comprises temperature sensors, pH sensors, and a heating system. Therefore, the present invention relates to a device for the production of an organoid cell culture or cultures, preferably a multi-organ-on-a-chip (MoC) device, that mimics the basic functions of one or more organs and/or the organism, with the integration of micro(bio)sensors, including, among others, pH and temperature sensors, inserted into the perfusion chamber of the culture, in direct contact with the human organ models. Furthermore, a heating system is integrated for the local heating of the biological models representing human organs, allowing the simulation of hyperthermia treatment temperatures (41–45 °C). This heating approach adds an important parameter for the preclinical validation of thermo-responsive nanomaterials designed for therapeutic applications, such as magnetic hyperthermia and/or controlled drug release, without the need to integrate electromagnetic systems that could interfere with the monitoring activity of the other (bio)sensors.

The differences of the present invention with respect to the prior art include the insertion of micro(bio)sensors for real-time monitoring of individual organ models and a heating system located in the cell culture perfusion chamber to simulate hyperthermia conditions that allow the preclinical study of thermo-responsive multifunctional nanomaterials developed for biomedical applications.

One aspect of the present invention relates to a device for the production of an organoid cell culture or cultures comprising:

a base support layer;

a transducer layer arranged on the base layer; and

an intermediate layer, arranged over the transducer layer, comprising a plurality of openings to form cavities for the growth of organoid cells and a plurality of slots to form a network of microchannels for fluid flow;

wherein the transducer layer comprises a heating device and at least a plurality of sensors in each cavity for cell control and growth;

where each sensor is located to measure an organoid cell growth parameter inside each cavity, preferably near the desired organoid growth area, allowing the evolution of an organoid with a cell mixture to be studied, for example, tumor cells and healthy cells.

According to the invention, the device further comprises a heating device located inside each cavity.

According to the invention, the heating device and the plurality of sensors are in direct contact with the organoid cell culture or cultures.

According to the invention, the plurality of sensors is selected from temperature sensor, pH sensor, or combinations thereof.

In one embodiment of the device, the sensor can be a temperature sensor, a pH sensor, or a combination thereof. The sensors are distributed uniformly or randomly within the cavity to detect the parameter at different points within the organoid.

In one embodiment of the device, the temperature sensors are resistance temperature detectors.

In one embodiment of the device, the number of temperature sensors in each cavity varies from 1 to 48, preferably from 4 to 16.

In one embodiment of the device, the temperature sensor has a meander-shaped resistor made of a metallic film.

In one embodiment of the device, the meander shape comprises from 5 to 72 turns, preferably from 5 to 24.

In one embodiment of the device, the metallic film of the temperature sensor is selected from a list consisting of platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), gold (Au), aluminum (Al) and their mixtures.

In one embodiment of the device, the heating element configuration is selected from among others: meander, loop, coil. The preferred configuration is a loop.

In one embodiment of the device, the heating system is made of a metal film filament, preferably the metal film is platinum.

In one embodiment of the device, the thickness of the metal film filament varies between 50 nm and 200 nm.

The thickness can be obtained by several different measurement methods; in the present invention it was obtained using a mechanical profilometer.

In one embodiment of the device, the heating system comprises a plurality of heating lines, preferably where the heating line comprises a width of 30 |jm to 500 |jm.

In one embodiment of the device, the heating lines comprise a length of 5000 jm to 50000 jm.

In one embodiment of the device, the spacing between the heating lines varies from 30 jm to 360 jm.

In one embodiment of the device, the transducer layer comprises a plurality of pH sensors.

In one embodiment of the device, the plurality of pH sensors is grouped, where each cavity comprises several groups of a plurality of pH sensors; preferably where the plurality of pH sensors are grouped to form an array. In one embodiment of the device, the groups of a plurality of pH sensors are evenly distributed within the cavity.

In one embodiment of the device, the pH sensors are shaped like a microneedle or a pyramid, preferably a pyramid shape having a flat bottom surface and a sharp point.

In one embodiment of the device, the number of pH sensors in each cavity is from 4 to 200, preferably from 6 to 78.

In one embodiment of the device, the pointed tip of the pyramidal shape comprises eight high-index crystal planes. In another embodiment of the device, the flat bottom surface of the pyramidal shape comprises silicon crystal orientation {100}.

In one embodiment of the device, the aspect ratio of the pyramid height to the lower diameter of the high-index crystal planes is 3:2.

In one embodiment of the device, the pH sensors can be obtained from a silicon wafer comprising crystalline orientation {100} in a plane of the wafer surface, by an anisotropic wet etching process of silicon with potassium hydroxide.

In one embodiment of the device, the method of producing the pH sensors comprises the following steps: obtaining a silicon wafer surface by depositing a layer of silicon dioxide (SiO2) followed by a layer of silicon nitrite (SiNx), using plasma-enhanced chemical vapor deposition;

expose the surface of the silicon wafer to hexamethyldisilazane (HDMS);

patterning the surface of the silicon wafer using optical lithography;

transfer the pattern to an oxide/nitrite bilayer using a reactive ion etching process;

obtain a pyramidal formation by undercutting a convex corner.

In one embodiment of the device, it comprises a plurality of arrays distributed in the transducer layer. In one embodiment of the device, the pH sensor array comprises 3 to 5 mm in length.

In one embodiment of the device, the pH sensor array comprises 3 to 5 mm in width.

In one embodiment of the device, the height of the pH sensor varies from 5 to 1000 |jm.

In one embodiment of the device, the diameter of the pH sensor tip varies from 1 to 50 jm.

In one embodiment of the device, the lower diameter of the pH sensor varies from 3 to 500 jm.

In one embodiment of the device, the pH sensors are made of silicon coated with electrically conductive metals selected from silver, silver chloride, gold, platinum, iridium oxide, aluminum oxide, and coated with biocompatible material selected from silicon oxide, parylene C, polyimide, or combinations thereof.

In one embodiment of the device, it comprises a connection layer comprising connectors for inserting the fluid into the microchannel.

In one embodiment of the device, it further comprises a sealing layer. Preferably, the sealing layer is a top layer for sealing the device. Preferably, the sealing layer is made of polymethyl methacrylate (pMMA) polymer.

In one embodiment of the device, the sealing layer comprises a plurality of tubes and connectors for regulating the pressure and flow of the intermediate layer's microfluidic system.

In one embodiment of the device, the number of cavities varies from 2 to 32.

In one embodiment of the device, the intermediate layer comprises a micro-inlet and a micro-outlet to connect the microfluidic system to the plurality of cavities for cell growth.

In one embodiment of the device, the transducer layer may further comprise at least one of the following sensors: pO2 sensors, CO2 sensors, NO sensors, analyte capture sensors, metabolic sensors, or combinations thereof.

In one embodiment of the device, the transducer layer may further comprise potentiometric sensors, amperometric sensors, impedance sensors, optical sensors, surface acoustic wave sensors, or combinations thereof.

In one embodiment of the device, the intermediate layer is made of glass, silicon oxide, or synthetic polymers, or combinations thereof.

In one embodiment of the device, the synthetic polymer is preferably transparent.

In one embodiment of the device, the synthetic polymer is selected from polystyrene (PS), polycarbonate (PC) or polydimethylsiloxane (PDMS).

In one embodiment of the device, the thickness of the bonding layer is between 200 and 1000 jm.

In one embodiment of the device, the bonding layer is made of a transparent synthetic polymer material, preferably polydimethylsiloxane (PDMS).

In one embodiment of the device, the base layer is made of a material selected from glass or silicon or polymethyl methacrylate (PMMA) polymer or a combination thereof.

In one embodiment of the device, the thickness of the bonding layer is between 200 and 1000 jm.

In one embodiment of the device, the connecting layer is made of a transparent synthetic polymer material, preferably polydimethylsiloxane.

In one embodiment of the device, the base layer and the transducer layer are fused or laminated to form a single layer.

In one embodiment of the device, it also includes a reservoir system.

In one embodiment of the device, the organoids are three-dimensional cell aggregates.

In one embodiment of the device, this is a multi-organ-on-a-chip.

In one embodiment of the device, for producing an organoid cell culture or cultures of the present invention, preferably a MoC device, it may comprise:

- a base layer;

- a transducer layer containing micro(bio)sensors, heaters, photodiodes and microelectronics to read the sensor signals and to actuate the heating systems; the transducer layer being disposed on the base layer; or

Optionally, the base layer and the transducer layer are fused together to form a single layer;

- an intermediate layer arranged over the transducer layer, said intermediate layer comprising the biological models and the autonomous microfluidic system;

- a connecting layer containing connectors for fluid movement and overlaid on the intermediate layer; and

- a sealing layer;

so, the base layer provides solid support for the additional layers;

The transducer layer provides support to house the multiplexed micro(bio)sensor and heating system and the microelectronics for reading, control and actuation;

The intermediate layer is configured to accommodate multiple individual organs, defined by organoid cavities that are representative of an organ. It also comprises a micro-inlet and a micro-outlet in communication with the microfluidic system. Each organ cavity comprises at least one organ configured to represent, but not limited to, the liver, pancreas, spleen, kidney, heart, lungs, brain, and small intestine, which can represent models of healthy and/or diseased organs. The connecting layer is configured to seal and/or stabilize the organ layer, while still allowing fluid exchange between the cavities and organ growth sections and recirculation of the microfluidic system.

The sealing layer is configured to include multiple tubes and connectors that allow for the regulation of pressure and flow within the microfluidic system, or a portion thereof, in an equivalent of selected organs. Furthermore, this layer allows for the incorporation of a screw-nut system, enabling non-permanent sealing of the MoC device and facilitating access to the biological models when necessary. In the present invention, a biological model is an experimental system that recreates aspects of the function or disease of human tissues, namely, an organoid.

Another aspect of the present invention relates to the use of the multi-organ-on-a-chip device as a monitor of long-term organoid cell culture physicochemical parameters.

Further details and embodiments of the invention are defined in the specification below and in the claims.

Brief Description of the Figures

The following figures present embodiments to illustrate the invention and should not be considered as a limitation of the scope of the invention.

Figure 1: Schematic representation of a multi-organ-on-a-chip (MoC) embodiment comprising a base layer1, a transducer layer including micro(bio)sensors, heating and microelectronics2, a layer for the microfluidic system and tissue organ growth3, a connection layer4 and a sealing layer5.

Figure 2: Schematic representation of an embodiment of an advanced organ-on-a-chip (OoC) device with microfluidic system, micro(bio)sensors and heating6, without organoid6A and with organoid6B inside the microfluidic chamber.

Figure 3: Schematic representation of an embodiment of a microfluidic chamber, comprising a temperature sensor7, a heating system8 and an array of pH sensors9.

Figure 4: Top view of a schematic representation of a multi-organ-on-a-chip device6 comprising a temperature sensor7, a heating system8, a pH sensor array9 and a microchannel10.

Figure 5: Schematic detail of an embodiment of the (bio)sensors used in the microfluidic device, showing a platinum resistance temperature detector (RTD)11 of the temperature sensor, and the microneedle12 present in the pH sensor array.

Figure 6: Schematic representation of an embodiment of the microfluidic system10 with pH13 optical sensor, integrated into a 1 mm14 microfluidic chamber.

Figure 7: Schematic representation of an engineering realization of 3D organ models, a diagram of the formation procedure for developing spheroids, b top view of microcuvettes cultured with representative cell lines after 5 days of incubation, c individual and unencapsulated spheroids in cell culture medium after 5 days of incubation in microcuvette structures, d schematic representation of spheroids encapsulated with hydrogel, forming representative and biomimetic 3D organ models.

Figure 8: Representation of different embodiments of temperature microsensors manufactured with different turns (line width and spacing of 10 |jm).

Figure 9: Illustration of the performance of temperature microsensors with 5, 8, 12, 16, 20, and 24 turns. Gray lines represent a temperature increase from 35 to 45°C, and black lines represent a temperature decrease from 45 to 35°C.

Figure 10: Illustration of the temperature sensitivity performance as a function of the number of turns.

Figure 11: Schematic representation of different embodiments for a heating system.

Figure 12: Simulation results of the heating system showing localized heating for each implementation (Fig. 12A and Fig. 12B).

Figure 13: Block diagram illustration of an implementation of an electronic circuit for the control and reading of signals from temperature microsensors.

Figure 14: Transmittance and absorbance values at 560 nm for a cell culture medium in the pH range of 5.0 to 8.5, obtained with a spectrophotometer.

Figure 15: Schematic representation of an embodiment of a microfluidic flow control system,15, connected to the MoC,16.

Figure 16: Representation of an embodiment of heater 1 where a) the heater is without power, therefore no heating, and b) a current of 250 mA flows through it, heating the actuator.

Figure 17: Representation of an implementation of the general geometry of temperature microsensors.

Detailed Description

The present invention relates to a multi-organ-on-a-chip (MoC) device for the long-term production and use of organoid cell cultures. The present invention comprises an advanced microfluidic system—a multi-organ-on-a-chip (MoC) device—for the validation and study of nanomaterials, active ingredients, or mixtures thereof. The described MoC device can be used in biomedical and/or pharmaceutical applications.

The present invention relates to a MoC device for long-term organoid cell culture comprising: a support base layer; a transducer layer disposed on the base layer; an intermediate layer comprising a plurality of cavities for organoid growth and a network of microchannels for fluid circulation; and a connection layer comprising connectors for introducing fluid into the microchannels; wherein the transducer layer comprises a heating device and at least a plurality of sensors in each cavity for monitoring and controlling cell growth; wherein each sensor is configured to measure an organoid cell growth parameter within each cavity.

In one embodiment of the device, Figure 1 shows a schematic representation of a MoC device composed of layers with different functionalities, non-permanently sealed, allowing for better handling and control of the final device.

In one embodiment of the device, the base layer 1 is configured to act as a structural support for the additional layers of the described MoC device. Preferred materials for this base layer comprise glass, silicon, or polymethyl methacrylate (PMMA) polymer, or combinations thereof.

In another embodiment of the device, the base layer 1 can be fused with the transducer layer 2, forming a single layer that serves both as a support and a transducer. Preferably, this base layer is made of a material that supports this dual functionality, such as silicon or glass.

In one embodiment of the device, sensors and heating elements can be incorporated into the transducer layer, enabling in situ monitoring of cultured human organ models. The transducer layer may comprise one or more sensors configured and arranged to measure physiological and homeostatic parameters of one or more equivalent organs, including growth sections, complemented by sensors integrated into the microfluidic system to monitor the feed fluid. The sensors that may be used include, but are not limited to, pH sensors, temperature sensors, pO2 sensors, CO2 sensors, NO sensors, analyte capture sensors, metabolic sensors, or combinations thereof. Potentiometric, amperometric, impedance, optical, or surface acoustic wave sensors, or combinations thereof, may also be included. Preferably, the transducer layer comprises at least the following sensor assemblies, Figure 2:

- A set of pH sensors arranged and distributed, preferably in direct contact with the organoid that serves as a human organ model. These sensors have a microneedle or pyramidal geometry, with a tip diameter between 1 and 50 µm, to avoid perforating the organoid models or the hydrogels arranged in a matrix formation.12 Each pyramid has a height between 5 and 1000 µm and a base diameter between 3 and 500 µm, though these dimensions are not limited. The matrix has a length between 3 and 5 mm and a width between 3 and 5 mm. This geometry allows monitoring of the internal pH of organoids representing healthy and/or pathological organs, facilitating the validation and screening of nanomaterials designed for the controlled release of drugs through changes in pH and/or temperature, or for pharmacological studies. The preferred materials for these sensors are silicon coated with conductive metals such as silver (Ag), silver chloride (AgCl), gold (Au), platinum (Pt), iridium oxide (IrO<2>), aluminum oxide (A^O3), among others, coated in turn with biocompatible materials such as silicon oxide, parylene C, polyimide, among others.

- An array of flat temperature microsensors11 arranged in direct contact with the organoid. These temperature microsensors11 are based on resistance temperature detectors (RTDs), which take advantage of the variation of electrical resistance with temperature, providing a resistance value for each absolute temperature. The small size of these RTDs, from 0.076 to 0.38 mm², as well as their ease of integration with microcomponents, makes them suitable for applications in microfluidic devices such as OoC. The geometry of the resistance temperature detectors (RTDs) consists of a specific number of turns (from 5 to 24) in a meander shape, fabricated from a thin metallic film. The width of the RTD lines varies between 10 and 50 jm, the length between 190 and 950 jm, the spacing between lines between 10 and 50 jm, and the film thickness between 50 and 200 nm. The manufacturing process is based on techniques such as physical vapor deposition (PVD), photolithography, and physical or chemical etching. Materials used include platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), gold (Au), and aluminum (Al). Platinum is the preferred material due to its high linearity in thermal response, chemical resistance, mechanical hardness, thermal stability, and biocompatibility.

A heating system (8) allows for the irradiation and delivery of heat to the organoid, simulating hyperthermia temperatures (41–45 °C). This system enables control of the local temperature of the organoid model and the evaluation of the effect of hyperthermia in combination with chemotherapeutic treatments using nanomaterials that act as drug nanocarriers. The system is based on a resistive metallic filament with a meander, spiral, coil, or other geometry. The preferred geometry is spiral. The heating lines of the system (8) have a width of between 30 and 500 µm, a length of between 5,000 and 50,000 µm, a spacing of between 30 and 360 µm, and a film thickness of between 50 and 200 nm. Heat generation occurs via the Joule effect when an electric current is applied, and the temperature is controlled by the intensity of this current. The preferred material for the resistive filament is platinum. Manufacturing the heating system and RTD microsensors together with the same material reduces the complexity of the production process, allowing for simultaneous manufacturing.

In one embodiment of the device, when the transducer layer (2) is made of silicon, it may also comprise an optical sensor system that, through the microfluidic system, allows the measurement and monitoring of homeostasis conditions and/or the effect of drugs (in free form or encapsulated in nanomaterials), such as pH, temperature, O2, CO2, NO, specific biomarkers released by organoids, among other complementary analytes;

Specifically, an optical pH sensor system is envisioned, configured and integrated into the microfluidic system, which allows for the measurement of the pH of the culture medium simulating the blood system, through the analysis of the colorimetric indicator phenol red, commonly added to cell culture media. For this purpose, the optical pH sensor, integrated into the transducer layer and the intermediate layer (Figure 6), is housed in a microfluidic chamber 1 mm long and between 0.4 and 1.0 mm high. This sensor comprises a light-emitting diode (LED) system, which can be white or monochromatic, and a set of photodetectors, typically photodiodes, optimized for the green-blue spectral range, which allow for the measurement of the phenol red indicator at 560 nm by absorbance. This indicator is present in most commercial culture media formulations (Figure 6).

In one embodiment of the device, as illustrated in Figure 14, the optical sensor is capable of continuously measuring pH differences between 5.0 and 8.5 using a CMOS photodiode. The photodiodes can be fabricated using CMOS technology and placed on the same layer as the transducer systems (layer 2), provided that the material of this layer is silicon. Likewise, all the readout electronics, intended for acquiring signals from the sensors, can be integrated on the same silicon layer. Optical filters will be deposited onto the photodiodes using physical vapor deposition (PVD) to optimize the detection spectrum.

In one embodiment of the device, it comprises an intermediate layer (3) positioned on the base layer (2). This intermediate layer comprises one or more growth sections configured to house a representation of a human organ within each organoid cavity. Each organ cavity comprises at least one organ equivalent configured to represent, but is not limited to, organs such as the liver, pancreas, spleen, kidney, heart, lungs, brain, and small intestine, and may represent models of healthy and/or pathological organs. Furthermore, the intermediate layer may include other organ equivalents, such as the skin, circulatory system, immune system, endocrine system, and female and/or male reproductive system. This layer also comprises the microfluidic system implemented for fluid circulation between organs, via micro-inlets and micro-outlets connected by microchannel networks. The organoids described in the present invention are defined as three-dimensional cell aggregates cultured in vitro, with dimensions up to several millimeters, that serve as models of human organs. This method is described in Lee et al., “A Heart-Breast Cancer-on-a-Chip Platform for Disease Modelling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy”, Small, 2020, 2004258 (doi: 10.1002/smll.202004258). Each organoid represents an organ model and is composed of identical, self-sustaining cell lines representative of human organs, and can represent healthy and/or diseased tissues. Organoid models offer an exceptional opportunity for predictive testing of the effects of nanomaterials developed as theranostic nanosystems, as well as of new drugs developed by the pharmaceutical industry for the treatment of human diseases. In the present invention, the MoC device comprises multiple organoid cavities with dimensions ranging from 0.3 to 50 mm³, each representative of a specific organ type, and the size of the organoid model can be adjusted according to the corresponding organ.

In one embodiment of the device, at least one of the following models (or organoids) can be produced in the device of the present invention:

• The hepatic organoid, which may represent a hepatic lobe with a volume of 1.6 to 16.0 mmP;

• The pancreatic organoid, which may represent an islet of Langerhans, with a volume of 1.1 to 10.5 mmP;

• The splenic organoid, which may represent white and red pulp tissue with a volume of 0.4 to 4.0 mmP; • The renal organoid, which may represent nephron tissue with a volume of 0.3 to 3.0 mm3;

• The cardiac organoid, which can represent cardiomyocyte tissue with a volume of 0.6 to 5.8 mmP;

• The brain organoid, which can represent cerebral cortex tissue with a volume of 0.5 to 5.0 mmP.

In the present invention, the representation of each organ is carried out by proportionality to the size of each organ in the body, defined by a proportionality factor between 0.0001 (1/10000) and 0.00001 (1/100,000).

In one embodiment of the device, the MoC may comprise in each cavity: 1 hepatic organoid, 1 pancreatic organoid, 1 splenic organoid, 2 renal organoids, 1 cardiac organoid, and 10 cerebral organoids.

In another embodiment of the device, it may comprise multiple organoids, including models of other complementary organs.

In one embodiment of the device, the intermediate layer is preferably made of glass, silicon oxide, or synthetic polymers, or combinations thereof. Among the synthetic polymers, optically transparent ones are preferred, such as polystyrene (PS), polycarbonate (PC), and polydimethylsiloxane (PDMS), with the latter being the preferred material.

In one embodiment of the device, the microfluidic system is connected to the organoid cavities (3) to mimic the vascular system of a higher organism, enabling the delivery of nutrients and oxygen to the organ models, interaction between different models, and the removal of metabolic waste products. This system is a vital component for maintaining the device's homeostasis, monitored by integrated micro(bio)sensors (2). The microfluidic flow control system (15) refers to the fluid circulating through the microchannel system from an external reservoir (Figure 15). This reservoir, connected via micro-inlets and micro-outlets located in the connection layer (4), allows for autonomous fluid replacement or collection without interfering with the MoC's operation. The system can be controlled by external or integrated micropumps, with pneumatic or piezoelectric actuation, and/or by integrated microvalves. It can also include microchannels and bifurcations connecting organs of the digestive, cardiovascular, and reproductive systems, among others. Preferably, the fluid is blood or an equivalent, such as a commercial cell culture medium. These advanced microfluidic architectures allow for the mimicry of essential biological functions, constituting an ideal platform for preclinical trials of nanomaterials developed for biomedical applications and/or drugs developed for the treatment of diseases.

In one embodiment of the device, the connection layer (4), positioned on the intermediate layer (3), allows for the sealing and/or stabilization of the organ equivalents, as well as establishing communication with the sealing layer. Preferably, this layer has a thickness of between 200 and 1000 µm and is made of a transparent synthetic polymeric material, such as PDMS.

In one embodiment of the device, it further comprises a sealing layer (5) disposed over the connection layer (4). This layer allows for non-permanent sealing of the MoC device by means of fastening systems, preferably a screw and nut system, and includes a plurality of inlet/outlet tubes and connectors arranged to regulate the pressure and flow rate applied to the organ models and the microfluidic system, or a part thereof. Preferably, the sealing layer is made of glass or synthetic polymers, with optically transparent polymers such as polycarbonate (PC), polypropylene (PP), or polymethyl methacrylate (PMMA), or combinations thereof, being preferred.

In one embodiment of the device, the present invention relates to a MoC device designed to operate under homeostatic conditions, assisted by an array of micro(bio)sensors integrated in situ with the organoid models, for the preclinical screening and validation of nanomaterials and drugs developed for the treatment of human diseases, and an optical pH sensor integrated into the microfluidic system to monitor the cell culture medium, which can be replaced as needed. The MoC can be applied in different configurations, depending on the architecture of the organoids representing the organs present in the device. Furthermore, the described device allows its application in safety assays, such as immunological, infectious, and/or oncological models.

Example 1:

In one embodiment of the device, the temperature microsensors are fabricated using lift-off techniques, with an area between 0.076 and 0.38 mm², a line width of 10 µm, a line length between 190 and 950 µm, a line spacing of 10 µm, and a film thickness of 200 nm. The temperature microsensor geometry comprises 5 to 24 turns in a meander-shaped resistor configuration of a thin metal film (Figure 8). The following table shows the sensor sensitivity for one embodiment with 5, 8, 12, 16, 20, or 24 turns.

Table I - Number of turns in serpentine (meander) geometry

Number of turns in the shape of Width Length Area (mm2) Sensor sensitivity (mV/°C)

In one embodiment of the device, the manufacturing process begins with the cleaning of a silicon or glass wafer using the PVATepla GIGAbatch 360M, which employs a microwave plasma generated from a mixture of O2 and CF4, operating at 2.45 GHz. A double-layer lift-off process is then performed using standard photolithography and wet etching, with photosensitive resins, developers, and solvents from the MicroChemicals AZ family. A lift-off resin (LOR1A-SB200) and a positive photosensitive resin (AZ4110) are sequentially deposited onto the substrate using spin coating, cured at 200 °C for 3 minutes and 110 °C for 1 minute, respectively. A high-resolution 405 nm laser is used to expose the photosensitive resin using a direct writing system (DWL2000, Heidelberg Instruments), followed by development with AZ400K solvent for 4.5 minutes. Physical vapor deposition is then performed to apply two layers by sputtering: a 5 nm tantalum adhesion layer and a 200 nm platinum layer. The resin is then lifted off using a preheated 60 °C solution of mrREM500 (Microresist Technology GmbH) in an ultrasonic bath to improve the etching rate. This step is performed for 150 minutes. Finally, the resin residue is removed by a high-temperature plasma deashing process for 20 minutes. An electrical insulator with high thermal conductivity is also deposited onto the temperature microsensors to electrically isolate them from the fluid medium, increase their chemical and mechanical resistance, and provide a rapid response to temperature changes. This conformal layer can be either SiO₂ or Al₂O₃, deposited by chemical vapor deposition (CVD) or atomic layer deposition (ALD), respectively. A mask is then transferred to the wafer using a high-resolution mask aligner (MA6BA6 Suss Microtec). The wafer is coated with AZ4110 using spin coating, cured at 110 °C for 1 minute, exposed to a wavelength of 365 nm, and developed with AZ400K solvent. The SiO₂ is then dry-etched using a reactive ion etching system (SPTS APS) or wet-etched with 16:1:2 W/AES aluminum solvent (Fujifilm Electronic Materials), removing the deposited material only in the areas where the electrical contacts will be made. Finally, a plasma deashing step is performed to remove any remaining resin residue. In a further embodiment, temperature measurements were performed using the fabricated sensors. The equipment used included a power supply to power the sensors, a heating plate as a heat source to control the temperature, a multimeter to read the electrical signals from the sensors, and an infrared camera to confirm the temperature changes. The power supply current was 100–500 µA, as higher currents could generate an error known as self-heating.

In one embodiment of the device, the performance of temperature microsensors with 5, 8, 12, 16, 20, and 24 turns was evaluated, considering their sensitivity and linearity (Figure 9). The results showed a linear increase in sensitivity with the number of turns (Figure 10).

In one embodiment of the device, Figure 9 shows the response of the resistance temperature detectors. A high degree of linearity between temperature and electrical potential is observed. Furthermore, as the number of turns increases, the sensitivity also increases linearly (Figure 10). The experimental sensitivity of the sensors ranges from 0.0719 to 0.3542 mV/°C for sensors with 5 to 24 turns. In one embodiment, since the highest sensitivity is obtained with the 24-turn sensor (approximately 35 V per 0.1 °C increment), this geometry was selected for integration into the organ-on-a-chip device.

In one embodiment of the device, the heating system is manufactured simultaneously with the temperature sensors, using the same fabrication process. The heaters consist of a thin resistive metal film shaped like a meander, spiral, coil, or other configuration (Figure 11). Numerical simulations of five resistive microheaters were performed, and their temperature distributions were analyzed (Figures 12A and 12B). All of them show potential for generating a heating system in the 35–45 °C range, but heater 1 provides greater uniformity in the thermal distribution, with a variation of less than 2 °C between the periphery and the central region (Figures 12A and 12B). Table 1 shows the simulation results of the temperature obtained for each heater with different electrical voltages. Figure 16 shows the experimental thermal profile of heater 1, obtained with an infrared camera: a) without power, without heating; and b) with a current of 250 mA, heating the actuator.

In one embodiment of the device, the temperature sensors include a power supply and an electronic circuit for their control and reading (Figure 13). An adjustable current source (e.g., the LM134) was used to provide a precise current of 100–500 µA to excite the sensors. This component was chosen for its ability to adjust the current between 1 µA and 10 mA with high accuracy and low noise. Any fluctuation in the excitation current generates a variation in the sensor's electrical potential, so the accuracy of the current source is crucial for the performance of the temperature measurements, especially when high resolution (0.1 °C) is required. A high-sensitivity, low-noise electronic reading circuit was implemented, preferably based on a 4-wire or Wheatstone bridge configuration, connected to an amplifier (e.g., the Texas Instruments INA126), to amplify the sensor signals and convert them into a voltage level usable by the microcontroller's analog-to-digital converter (ADC), and subsequently store them on an SD card or display them on a screen.

In one embodiment of the device, Figure 17 schematically represents the meander-shaped structure of one of the temperature microsensors.

In one embodiment of the device, the geometry of the temperature detector studied consists of meander-shaped turns (from 5 to 24) of a 200 nm platinum thin film, with a width and line spacing of 10 jm. The total width was fixed at 400 jm and the length varies depending on the number of turns.

In one embodiment of the device, these microsensors were designed with a four-wire configuration, where excitation is applied through the external contacts and the electrical potential is measured at the internal contacts. This configuration allows for an accurate reading of the electrical potential without interference from the excitation wires.

Table II shows the simulation results of the average temperature obtained in the heater using different electrical voltages.

Table II - Results of the average temperature obtained in the heater using various electrical voltages.

In one embodiment of the device, simulations and experimental results show that one of the best geometries for the RTD temperature sensor is a platinum meander shape with 24 turns, a platinum film thickness of 200 nm, a line width and spacing of 10 µm, and a total RTD area of 0.38 mm². Furthermore, the microsensor exhibits high sensitivity to temperature changes, approximately 0.3542 mV/°C. The small size of each sensor allows for the integration of multiple sensors into a microfluidic chamber for highly accurate, real-time temperature measurements. In one embodiment of the device, the sensor also allows for the evaluation of the microenvironment temperature, providing essential information for assessing the efficiency of moderate-temperature-activated drug nanocarriers within the organ-on-a-chip device.

The pH sensor was manufactured using the following method. In one embodiment of the device, the pyramidal microstructures for the pH sensors are obtained from a silicon wafer with {100} crystal orientation in the surface plane, using an anisotropic wet etching process with potassium hydroxide (KOH).

In one embodiment of the device, the fabrication process begins with the deposition of the materials used as a wet-etching mask onto the surface of the silicon wafer. A layer of silicon dioxide (SiO2) is deposited, followed by a layer of silicon nitride (SiNx), using plasma-assisted chemical vapor deposition (PECVD). The wafer is then exposed to hexamethyldisilazane (HDMS) at 150 °C for 5 minutes to remove moisture and promote the adhesion of the photosensitive resin. Next, it is coated with a positive photosensitive resin using spin coating and baked onto the wafer surface, followed by optical lithography. After development to remove the exposed resin, the mask pattern is transferred to the oxide/nitride bilayer using reactive ion etching (RIE). Finally, the resin mask is removed by oxygen plasma incineration, and wet etching is performed with an aqueous KOH solution to define the silicon microstructures.

In one embodiment of the device, pyramid formation relies on undercutting convex corners, revealing locally fast-etched crystal planes. The process begins with the formation of silicon {111} crystal planes. After reaching a certain etching depth, these {111} planes are removed by faster-etching planes, forming an octagonal base. The pyramidal shape is completed when eight high-index crystal planes, revealed as {312} planes, converge at the top of the trunnion, creating a sharp point. At this stage, the remaining mask is removed. Once the desired pyramid height is reached, the wafer is rinsed with deionized water to stop the etching process and dried with nitrogen.

In one embodiment of the device, the aspect ratio between the pyramid's height and its base diameter, defined by high-index crystallographic planes, is 3:2. With a detailed understanding of the convex corner undercutting process for pyramid formation, it is possible to calculate the size of the masks to achieve any desired height. Preferably, both the KOH concentration (between 5% and 50% by weight) and the temperature should be carefully selected to achieve more precise control of the etching speeds in the various crystal orientations. This fabrication process allows for obtaining pyramidal microstructures on a flat surface defined by the {100} crystal orientation. In subsequent stages, a physical vapor deposition process can be employed to deposit electrically conductive metals, such as, but not limited to, silver, silver chloride, gold, platinum, iridium oxide, or aluminum oxide. Finally, a layer of biocompatible material, such as silicon oxide, parylene C, or polyimide, can be applied by chemical vapor deposition or a spin coating process.

The integration of the sensors into the microfluidic device is described below.

In one embodiment of the device, the fabrication process used for the pyramidal microstructures yields a relatively flat underside, defined by planes with a {100} crystal orientation. With appropriate selection of the KOH concentration and temperature, the resulting surface roughness will allow for the monolithic integration of the metal heaters and temperature sensors. After fabricating the pH sensors and metal structures on the silicon substrate, all these devices can be integrated into a microfluidic platform by creating microfluidic channels using an SU-8 lithography process, or by coupling them to a pre-molded PDMS microfluidic layer.

In one embodiment of the device, a low surface roughness (~10 nm) can be achieved on silicon surfaces with {100} crystal orientation if relatively high KOH concentrations (>30%) and high temperatures (>70 °C) are used, allowing for subsequent monolithic integration of the metal heaters and temperature sensors.

The present invention comprises an advanced microfluidic system for the validation and study of nanomaterials, drugs, or mixtures thereof, intended for use in biomedical and/or pharmaceutical applications. The present invention relates to a device for the production of one or more organoid cell cultures comprising: a base support layer; a transducer layer disposed on the base layer; an intermediate layer disposed on the transducer layer, comprising a plurality of openings to form cavities for organoid growth and a plurality of slots to form a microchannel network for fluid flow; wherein the transducer layer comprises a heating device and at least a plurality of sensors in each cavity for cell growth monitoring; wherein each sensor is arranged to measure an organoid cell growth parameter within each cavity.

Claims (15)

1. A device for the production of an organoid cell culture or cultures, comprising: a base layer (1) of support; a transducer layer (2) disposed on the base layer (1); and an intermediate layer (3), arranged over the transducer layer (2), comprising a plurality of openings to form cavities intended for the growth of organoids and a network of microchannels for fluid flow; wherein the transducer layer (2) comprises a heating device (8) and at least a plurality of sensors in each cavity for cell control and growth; where each sensor is arranged to measure an organoid cell growth parameter inside each cavity; wherein the heating device (8) is located inside each cavity; wherein the heating device (8) and the plurality of sensors are in direct contact with the organoid cell culture or cultures; where the plurality of sensors is selected from temperature sensor (7), pH sensor (9) or combinations thereof. 2. The device according to claim 1, wherein the temperature sensors (7) are resistance temperature detectors. 3. The device according to any of the preceding claims, wherein the number of temperature sensors (7) in each cavity varies between 1 and 48, preferably between 4 and 16. 4. The device according to any of the preceding claims, wherein the temperature sensor (7) comprises a meander-shaped resistor of a metallic film, preferably comprising the meander shape of between 5 and 72 turns, more preferably between 5 and 24. 5. The device according to any of the preceding claims, wherein the configuration of the heating device (8) is selected from meander, spiral or coil; preferably a spiral and/or wherein the heating device is made of a metal film filament, preferably the metal film being platinum. 6. The device according to any of the preceding claims, wherein the plurality of pH sensors (9) is grouped, and wherein each cavity comprises several groups of a plurality of pH sensors (9), preferably wherein the plurality of pH sensors (9) is grouped to form an array, more preferably the groups of a plurality of pH sensors (9) being evenly distributed in the cavity. 7. The device according to any of the preceding claims, wherein the shape of the pH sensors (9) comprises a microneedle shape (12) or a pyramidal shape, preferably a pyramidal shape with a flat base and a sharp point. 8. The device according to claim 7, wherein the sharp tip of the pyramidal shape comprises 8 high-index crystal planes and/or wherein the flat base comprises silicon crystal orientation {100} and/or wherein the aspect ratio between the height of the pyramid and the diameter of the base is 3:2. 9. The device according to any of the preceding claims, wherein the pH sensors (9) comprise silicon coated with an electrically conductive metal, wherein the metal is selected from a list consisting of: silver, silver chloride, gold, platinum, iridium oxide, aluminum oxide, and coated with a biocompatible material selected from silicon oxide, parylene C, polyimide, and combinations thereof. 10. The device according to any of the preceding claims, comprising a connection layer (4) including connectors for introducing the fluid into the microchannel and/or a sealing layer (5). 11. The device according to claim 10, wherein the sealing layer (5) comprises a plurality of tubes and connectors for regulating the pressure and flow of the microfluidic system of the intermediate layer. 12. The device according to any of the preceding claims, wherein the intermediate layer (3) comprises a micro-inlet and a micro-outlet for connecting a microfluidic system of the microchannel network (10) with the plurality of organoid cavities for cell growth. 13. Device according to any of the preceding claims, wherein the transducer layer (2) further comprises at least one pO2 sensor, or CO2 sensors, or NO sensors, or analyte capture sensors, or metabolic sensors, or combinations thereof; and/or wherein the transducer layer (2) further comprises potentiometric sensors, or amperometric sensors, or impedance sensors, or optical sensors, or surface acoustic wave sensors, or combinations thereof. 14. The device according to any of the preceding claims, wherein the device is a multi-organ-on-a-chip. 15. Use of the device according to any of claims 1 to 14 as a monitor of physicochemical parameters of long-term organoid cell cultures.