RU171690U1 - Microfluidic Chip for the Creation of Cellular Models of Mammalian Organs - Google Patents

Abstract

The utility model relates to devices for working with human, animal or plant tissue cells and / or virus cultures and can be used, in particular, for testing drugs, creating cellular models of mammalian tissues and organs, and modeling interorgan interactions. The developed microbioreactor is a a single-use design (biochip), consisting of six cell cells for models of organs (pancreas, liver, intestines, kidneys, as well as muscle and adipose tissue), is connected s microchannels. Cell models of human organs and tissues placed in cells can be represented by spheroids or a membrane insert (for example, Transvel). The cell size is designed in such a way as to ensure the possibility of the required number of cells in a single proportion to the number of cells in the organs (1: 100000). The flow of culture medium through each cell cell is scaled in accordance with the proportion of cardiac output per organ. The cells on top are sealed with plugs fixed in a polycarbonate plate using a threaded connection. The design of the utility model allows for the distribution of the culture medium flow over the cell cells in accordance with the anatomy of the human circulatory system. Flow rates are controlled by placing cells that require high speeds near the pump, and cells that require low speeds away from the pump.

Description

Technical field

The utility model relates to devices for working with human, animal or plant tissue cells and / or virus cultures and can, in particular, be used for testing drugs, creating cellular models of mammalian tissues and organs, and modeling interorgan interactions.

State of the art

According to national regulations, preclinical studies of drug toxicity are performed using laboratory animals. At the same time, it is known that the response of various organisms to the influence of xenobiotics is characterized by species specificity, which determines, among other things, the degree of toxicity of the substance to the body. In this connection, a number of developed medicinal substances and agents are non-toxic for laboratory animals, but are toxic to humans [Johnson J.I. et al. Relationships between drag activity in NCI preclinical in vitro and in vivo models and early clinical trials // British Journal of Cancer. Nature Publishing Group, 2001. Vol. 84, No. 10. P. 1424-1431, Paul S.M. et al. How to improve R&D productivity: the pharmaceutical industry's grand challenge. // Nature reviews. Drug discovery. 2010. Vol. 9, No. 3. P. 203-214]. In this regard, methods are increasingly being developed to assess species-specific toxicity of drugs and their combinations in in vitro studies using cell models of human organs and tissues as an object. Such methodological approaches allow obtaining information on the molecular mechanisms of xenobiotic toxicity, which is problematic in in vivo studies, and are also consistent with ideas about a humane attitude to laboratory animals, the need to reduce experiments with their participation, which is reflected in various international agreements, including Environment Directory of the Organization for Economic Co-operation and Development.

In this connection, the use of human cells as primary or immortalized cell lines as biological test systems is promising. At the same time, it was proved that the in vitro model’s proximity to the real physiological situation is higher when culturing three-dimensional histotypic or organotypic cell models of human organs and tissues, which reproduce the architectonics of cells and their intercellular interactions as accurately as possible.

Difficulties in obtaining reliable reproducible results when working with cell cultures are due to the variable conditions of cell cultivation (depending on the equipment used, media, supplies, etc.). Another limitation of the information content of this approach is the inability to reproduce the systemic response of the body to an exogenous chemical substance, including taking into account its metabolism. It is known that a number of substances in the process of biotransformation in the liver are able to form metabolites more toxic than the original chemical substance. It is impossible to fix this in an experiment on a monoculture of cells.

A promising approach that can mitigate these shortcomings is the use of special equipment - microbioreactors [Esch M.V., King T.L., Shuler M.L. The role of body-on-a-chip devices in drug and toxicity studies. // Annual review of biomedical engineering. 2011. Vol. 13. P. 55-72 .; Sung J.H., Shuler M.L. A micro cell culture analog (microCCA) with 3-D hydrogel culture of multiple cell lines to assess metabolism-dependent cytotoxicity of anti-cancer drugs. // Lab on a Chip. 2009. Vol. 9, No. 10. P. 1385-1394]. In addition to providing standard conditions for the cultivation of cells, a number of such devices allows for the joint cultivation of cell models of several organs and tissues located in different cell cells, which are combined into a single system through microchannels. A nutrient medium that simulates microcirculation circulates through microchannels, as a result of which various cellular models are capable of exerting mutual influence both under the influence of a factor and in its absence. Immortalized cell lines are commonly used as cell models of human organs.

Known application US 2012/0214189 A1, which describes a microfluidic chip for culturing cell models. The device of the chip contains four cells for the cultivation of cell models: the cell "liver", the cell "lung", the cell "fat" and the cell "other tissues".

The disadvantage of this device is the inability to simulate, for example, type 2 diabetes mellitus, which requires the presence of at least six cell cells for models of organs (pancreas, liver, intestines, kidneys, as well as muscle and adipose tissue). In addition, the above construction is not anatomically correct.

Utility Model Essence

The technical result of the claimed utility model is to provide optimal living conditions for co-cultured cells.

The microfluidic chip for creating cellular models of mammalian organs contains a plate on which a layer of polydimethylsiloxane is molded, in which there is a microfluidic system sealed from below with a glass slide, where the microfluidic system consists of six typical cells united by a system of microfluidic channels for the simultaneous cultivation of cellular models of mammalian tissues and organs in flow or closed modes, designed for models of the pancreas, liver, intestines, kidneys, and the same muscle and adipose tissue, at least four distribution channels located on the side of the entrance to the standard cells, at least four mixing channels located on the side of the exit from the standard cells, and a bypass channel for the cell model of the intestine, two of four distribution and mixing channels are made with the possibility of pairing typical cells with the formation of two closed loops, which are parallel connected to the microfluidic chip input channel through the input side the third and fourth distribution channels, from the output side through the remaining third and fourth mixing channels, are connected to the output channel of the microfluidic chip, the output channel of the liver model cell passes into the fourth mixing channel, and the fourth distribution channel has two branches, the first of which is designed to connect to third distribution channel. The maximum channel width is 1 mm, the minimum 0.2 mm. The channel width at the pump connection points is 0.6 mm, which ensures the same range of shear stresses throughout the circuit. The minimum radius is 0.2 mm. The minimum distance between the structural elements is 0.4 mm. The damper chamber consists of a PDMS membrane located in front of the cell cells relative to the flow. A measuring cell designed to measure electrochemically the pH of the culture medium is located after the cell cells relative to the flow. The Y-shaped branching of the channels in front of the cell cells relative to the flow is intended to reduce the effects of sedimentation. It is possible to partially replace the culture medium in microchannels. The volume of the culture medium is 50.1 mm ³ .

Brief Description of the Drawings

The utility model is illustrated by drawings, where in FIG. 1 is a schematic representation of the inventive device.

In FIG. 2 shows the distribution of speed in the inventive device for the case of a stationary flow and a pressure difference of 30 kPa.

The positions in the drawings indicate: 1 - pump chambers, 2 damper chamber, 3 - kidney cells, 4 - intestinal cell, 5 - liver cell 6 - measuring cell, 7 - adipose tissue cell, 8 - cell "muscle".

Description of the specific implementation of the utility model.

The developed microbioreactor is a single-use design (biochip) consisting of six cell cells for models of organs (pancreas, liver, intestines, kidneys, as well as muscle and adipose tissue) connected by microchannels. Cells are compatible with Transwell type membrane inserts. The size of the biochip is limited by the size of a standard slide (26 mm2 × 76 mm2). The circulation of the culture medium in the channels is provided by a micropump consisting of two micro valves and a working chamber. The micropump and valves are connected to the control unit of the BAVP.422522.001 microbioreactor. The cell size is designed in such a way as to ensure the possibility of the required number of cells in a single proportion to the number of cells in the organs (1: 100000). The flow of culture medium through each cell cell is scaled in accordance with the proportion of cardiac output per organ.

The biochip is made of a layer of polydimethylsiloxane (PDMS) with a height of 2 mm, containing microchannels, a pump, valves and openings for cell cells. The side containing the microchannels is covered with glass, the other side is connected to the polycarbonate layer, which provides mechanical strength of the biochip, connecting the pneumatic control outputs to pumps and valves, and sealing the cell cells with covers. The height of the channels is 0.1 mm.

Flow rates are controlled by placing cells that require high speeds near the pump, and cells that require low speeds away from the pump. This is due to the fact that large distances to the cell cells lead to large hydraulic losses. Fine tuning of the flow rates was carried out by adjusting the length and width of the channels in those parts that correspond to specific cells. The width of the channels cannot be less than 0.2 mm for technological reasons and more than 1 mm to ensure the stability of the geometry of the channels.

Table 1 provides an overview of the parameters of the simulated organs. The share of cardiac output per organ is shown, and the relative share of the flow of culture medium in a multi-organ biochip. All cell cells, except for that intended for intestinal cells, are supposed to be made using membrane inserts from 96-well Trans well plates. The intestine occupies a significantly larger area [Multi-organ-chip with improved life time and homoeostasis: Pat. EP 2712918 A1 European Patent Application: C12M 3/00 Marx. U .; applicant Technische Universitat Berlin. - No. 12186550.5; filled 09/28/2012; publ. 04/02/2014, Bulletin 2014/14.], Therefore, it is proposed to use one insert from a 12-hole plate for it.

The design of the microfluidic chip (microbioreactor) is based on a three-layer structure, the top layer of which is formed by a plate made of polycarbonate 10 mm thick, on which a layer of polydimethylsiloxane (PDMS) is cast, containing a system of microfluidic channels of rectangular cross section and cells sealed from below with a layer formed by a standard slide . The use of channels of constant cross-section made it possible to provide an acceptable manufacturability of the mold for casting a PDMS layer.

The microfluidic chip contains at least six cells combined by microfluidic channels, designed for simultaneous co-cultivation of the cellular models of human organs and tissues located in them in flowing or closed modes. Models can be represented by spheroids or a membrane insert (e.g., Transvel). The cells on top are sealed with plugs fixed in a polycarbonate plate using a threaded connection.

A microchannel system, the circuit diagram of which is shown in FIG. 1, has the following characteristics:

- The maximum channel width is 1 mm.

- The minimum channel width is 0.2 mm.

- The channel width at the pump connection points is 0.6 mm, which ensures the same range of shear stresses in the entire circuit.

- The minimum radius is 0.2 mm.

- The minimum distance between the structural elements is 0.4 mm.

- The damper chamber consists of a PDMS membrane located in front of the cell cells relative to the flow.

- A measuring cell designed to measure electrochemically the pH of the culture medium is located after the cell cells relative to the flow.

- The Y-shaped branching of the channels in front of the cell cells relative to the flow is intended to reduce the effects of sedimentation.

- It is possible to partially replace the culture medium in microchannels.

- The volume of the culture medium is 50.1 mm ³ .

The design of the utility model allows to ensure the distribution of the flow of the culture medium among the cell cells in accordance with the anatomy of the human circulatory system (Table 2).

Thus, the utility model allows you to create physiological conditions for the cultivation of cell lines:

- the flow of culture medium is distributed over the cell cells in accordance with the anatomy of the human circulatory system;

- the average flow rate of the culture medium is sufficient so that the cells receive a sufficient amount of oxygen and nutrients;

- under membrane inserts containing cells, the flow is uniform over the surface of the membranes;

- tangential stresses correspond to physiological norms;

- the ratio of the volumes of the culture medium and cells should be close to the ratio in vivo.

Claims (2)

1. A microfluidic chip for creating cellular models of mammalian organs, including a polydimethylsiloxane layer placed therein, in which a microfluidic system is placed, sealed from below with a glass slide, where the microfluidic system consists of six typical cells combined by a microfluidic channel system for the simultaneous cultivation of cellular models of mammalian tissues and organs in flow or closed modes, designed for models of the pancreas, liver, intestines, kidneys, as well as m tissue and adipose tissue, four distribution channels located on the input side of the typical cells, four mixing channels located on the output side of the typical cells, and a bypass channel for the intestinal model cell, two of the four distribution and mixing channels being made with the possibility of ensure pairing of typical cells with the formation of two closed loops, which are parallel connected to the microfluidic chip input channel through the remaining third and fourth distribution dye channels, on the output side, through the remaining third and fourth mixing channels, they are connected to the output channel of the microfluidic chip, the output channel of the liver model cell passes into the fourth mixing channel, and the fourth distribution channel has two branches, the first of which is designed to connect to the third distribution channel, the maximum channel width is 1 mm, the minimum 0.2 mm. 2. The microfluidic chip according to claim 1, characterized in that the volume of the culture medium is 50.1 mm ³ .

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