Microfluidic devices and methods for determining dose response

Abstract

Novel microfluidic devices and methods for determining dose response are provided. Microfluidic devices that can realize gradients and methods that use these microfluidic devices to determine dose response are described. The microfluidic devices and methods described here enable determination of the desired doses of one or more agents. The desired dose can be including but not limited to an IC50, a minimum dose that results in an effect, a dose that does not result in an effect, a toxic dose, a time it takes for the agent to show an effect, a time an effect lasts, a time it takes for the agent to show no more effects or a combination thereof.

Description

[0001] MICROFLUIDIC DEVICES AND METHODS FOR DETERMINING DOSE RESPONSE

[0002] Technical Field of the Present Invention

[0003] The subject matter described here relates to microfluidic devices and methods for determining dose response.

[0004] Background of the Present Invention

[0005] Classical drug discovery uses 2D cell culture coupled with animal testing for preclinical studies. Neither 2D cell culture nor animal testing truly recapitulate the in vivo microenvironments of cells in a human body. The paradigm shift is to use 3D cell culture in microfluidic devices that can faithfully mimic the in vivo conditions. With this modern drug discovery approach, costs can be reduced ten-fold, results can be achieved ten times faster, animal testing can be significantly reduced and personalized medicine can be realized.

[0006] Microfluidic technology provides precise spatial and temporal control, high-throughput analysis, low fabrication costs and portability. Required material and generated waste volumes can be as low as picoliters. Using small volumes of unknown or toxic materials provides safer experimental study. Moreover, microfluidic technology can provide means to mimic physiological microenvironments. This feature can help us more realistically study cells in both health and disease states and improve drug testing approaches. It can also help reduce animal testing.

[0007] Two important aspects of drug discovery are dose response and combinatorial effects.

[0008] It is important to determine the dose that is effective and at the same time has minimum side effects. It is also important to determine toxic doses as well as doses that do not result in an effect. IC50 is defined as the concentration of an inhibitor where the response is reduced by half. There are different methods to determine IC50 of an agent. Serial dilutions are often used to test different doses of agents. However, this approach is prone to pipetting errors. Another approach is to use microfluidic devices that can generate gradients of agents. Such current devices are complex in structure, require flow and are difficult to fabricate. It is known that using combinations of agents can be synergistic or inhibitory or have no effect compared to using single agents. Combination therapy can be more effective than using single drugs against diseases. Combination therapy can be defined as "treatment in which a patient is given two or more drugs (or other therapeutic agents) for a single disease". Yet, it is possible that one drug can reduce the effectiveness of another when used simultaneously. It is also possible to plan usage of drugs at different points in time. Some drugs can be effective only if they are used after another drug is used first. Such possibilities point to the importance of determining combinatorial and sequential effects of agents when used simultaneously or in a series in time.

[0009] It has been observed that the microfluidic-based apparatus and methods published in academic articles and patents fall into one of the groups (reference 11): 1- In tree shape, modified tree shape methods, different concentration regions are obtained by flowing solutions with and without agents from two different inlets and bringing them together by branching. 2- There is no branching in the Y shape method, two different solutions flow in the common channel, creating a gradient. 3- In the membrane method, the three channels are separated from each other by a semi-permeable structure or material, and a gradient is formed in the middle channel with different solutions in the lower and upper channels. We have used this system for the IC-chip (invasion-chemotaxis) we have developed before. In the pressure balance method, a gradient is created based on the balancing of different pressures. One of the most used methods is the droplet formation method. It is carried out by mixing two materials in different phases with a controlled flow. This method is also used as droplet aggregation and dilution.

[0010] If we list the disadvantages of the existing methods: 1- Almost all of them require flow, so complex and potentially leaky mechanisms have to be used, 2- It is necessary to use pumps in devices that require flow, 3-Due to the tubing used in the devices that require flow, 'dead volumes' are many times greater than the microfluidic volume. 4-Multi-layer production is required in the arrangements where valves are used for flow control 5-Microfluidic designs are complex (complex tree patterns) and therefore difficult to manufacture 6-Microfluidic designs are difficult to reach the sample because they consist of closed channels 7- Microfluidic channel volumes are small and do not provide enough sample for other downstream applications.

[0011] Microfluidic devices have been used to generate gradients of chemicals to be used in life sciences (reference 11). Chemical gradients play important roles in both health and disease states such as embryonic development or cancer metastasis. However, the use of microfluidic devices that can generate gradients for determination of drug doses is almost non-existent because a wide of range of doses need to be tested and current microfluidic devices do not provide such ranges. Multiple microfluidic devices can be used to test wider ranges but this in turn increases time, cost, and labor. Significant limitations of current microfluidic devices used for gradients also include (i) complex designs that require complex fabrication methods, (ii) requirement for bulky and complex fluidic pump systems, (iii) limited gradient range, (iv) limited sample volume, (v) difficulty in collecting samples for follow-up analysis, (vi) need for specialized expensive equipment, and (vii) unhandy usage (e.g. an assembly is required to set up the microfluidic device itself). Furthermore, combination therapies are proving to be more effective than using single drugs. Thus, a microfluidic device that can test doses of two drugs at the same time is sought after. Current microfluidic devices cannot meet this challenge.

[0012] To eliminate these disadvantages, we designed a new microfluidic platform. In one embodiment, there is a continuous interface, the connector, between channels, so the interaction interface is much larger. The material loaded from the top of channels will be able to reach the transition areas between the two channels by capillary transport and be held by hydrostatic pressure differences (references 6-9). After the matrix has polymerized, the culture medium can be loaded. In addition, the channels are open at the top so material is easy to add and remove. As the channel depths are increased, higher volumes of culture medium (~500 pl) can be used. Small volumes and microfluidics are advantageous in terms of cost and ease of use in next-generation testing, including the development of high-output platforms. However, small volumes also have important limitations. One of them is that the amount of sample (for example, tumor tissue) that can be used in these volumes may be insufficient for some final analysis applications (for example, omics analysis), while the other is that it is not possible to create the targeted wide gradient range with small volumes of medium volumes. Therefore, the volumes to contain matrix and culture medium are designed to be small so as not to eliminate advantages such as cost and whole sample analysis, but the middle channel is designed to be larger so that it can provide a wide range of drug gradients. The larger volume microfluidic design was chosen to provide as wide a gradient range as possible. The advantage of the proposed microfluidic device is that a wide range of drug concentrations / doses can be realized on a single chip with a single starting concentration and without the use of flow. Also, the microfluidic devices would be suitable for 3D bioprinting applications when it is open-top and can accommodate larger volumes. Finally, the new microfluidic devices' dimensions are designed to be compatible with automated liquid dispensing robots used with multi-well plates. In addition, a most important difference of the microfluidic device from the existing ones is that a wide range of drug concentrations can be tested simultaneously.

[0013] Summary of the Present Invention

[0014] The present disclosure provides novel microfluidic devices and methods for determining dose response of agents. Microfluidic devices that enable gradients of agents and methods that use these microfluidic devices to determine responses to agents are described.

[0015] The microfluidic devices with the described features are shown in the drawings. The drawings are not necessarily to scale.

[0016] Brief Description of The Technical Drawings

[0017] Accompanying drawings are given solely for the purpose of exemplifying a microfluidic device, whose advantages over prior art were outlined above and will be explained in brief hereinafter.

[0018] The drawings are not meant to delimit the scope of protection as identified in the Claims, nor should they be referred to alone in an effort to interpret the scope identified in said Claims without recourse to the technical disclosure in the description of the present invention.

[0019] FIGS. 1A-1D show a microfluidic device having three channels, wherein width 2 and height 2 of channel 2 increase along length 2 according to the present invention. FIG. 2 shows a microfluidic device having three channels, wherein width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 increase along length 1 and length 3 respectively according to the present invention.

[0020] FIG. 3 shows a microfluidic device having three channels, wherein width 1 and height 1 of channel 1 increase along length 1 and width 3 and height 3 of channel 3 decrease along length 3 according to the present invention.

[0021] FIG. 4 shows a microfluidic device having three channels, wherein width 2 of channel 2 increases along length 2 and width 1 of channel 1 and width 3 of channel 3 decrease along length 1 and length 3 respectively according to the present invention.

[0022] FIG. 5 shows a microfluidic device having three channels, wherein width 2 and height 2 of channel 2 increase along length 2 and width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 decrease along length 1 and length 3 respectively according to the present invention.

[0023] FIG. 6 shows a microfluidic device having three channels, wherein width 1 of channel 1 and width 3 of channel 3 increase and decrease along length 1 and length 3 respectively according to the present invention.

[0024] FIG. 7 shows a microfluidic device having three channels, wherein width 1 of channel 1 and width 3 of channel 3 increase and decrease along length 1 and length 3 respectively and height 1 of channel 1 and height 3 of channel 3 increase and decrease along width 1 and width 3 according to the present invention.

[0025] FIG. 8 shows a microfluidic device having three channels, wherein width 2 and height 2 of channel 2 increase in positive and negative directions along length 2 according to the present invention.

[0026] FIG. 9 shows different profiles of width 1 along length 2 according to the present invention.

[0027] FIG. 10A shows a cross sectional view of a microfluidic device shown in FIGS 1A-1D; FIG. 10B and FIG. IOC show height 2 and width 2 profiles along length 2, respectively according to the present invention. FIG. 11A shows an open-top microfluidic device having three channels; FIG. 11B shows a cross sectional view of said microfluidic device according to the present invention.

[0028] FIG. 12A shows an open-top microfluidic device having five channels; FIG. 12B shows a cross sectional view of said microfluidic device according to the present invention.

[0029] FIG. 13A shows a connector; FIG. 13B shows exemplary profiles of said connector a cross sectional view of said microfluidic device according to the present invention.

[0030] FIGS. 14A-14C show the top view of a microfluidic device, the profile line for intensity and a graph of intensity distribution versus distance for different time points, respectively according to the present invention.

[0031] Detailed Description of the Present Invention

[0032] The present disclosure provides novel microfluidic devices and methods for determining effects of agents. Microfluidic devices that enable gradients of agents and methods that use these microfluidic devices to determine effects of agents are described.

[0033] The words "a" and "an" mean "one or more" unless a clear intent is stated to limit "a" or "an" to "one." Singular claim terms are plural in scope unless there is clear intent to the contrary.

[0034] One definition of an "agent" is "a person or thing that takes an active role or produces a specified effect." An agent can be a drug including but not limited to an anti-cancer drug, an antibiotic, a growth factor, an antibody, a biosimilar, a hormone, a vitamin, culture medium, blood, serum, physiological buffer solution, a biological or chemical molecule, a growth factor, a protein, an enzyme, a peptide, a DNA molecule, an RNA molecule, a lipid molecule, a sugar molecule, a carbohydrate, an amino acid, a soluble or matrix bound agent, a nanoparticle, a micelle, a microbubble, a salt, an acid or a base, a biostimulant, temperature or a combination thereof. An agent can also be a cell because cells can take an active role or produce a specified effect, here an agent can be cells including but not limited to bacteria, archea, eukarya, yeast, fungi, a plant cell, an epithelial tissue cell, a connective tissue cell, a muscle tissue cell, a nervous tissue cell, a cancer cell, an immune cell, a blood cell, a neuron, a cell line cell, a biopsy cell, a spheroid, an organoid or a combination thereof. Cell agents can also be including but not limited to a plant seed, an embryo, a tissue which can be either isolated from an organism such as a mouse or a synthetic tissue, an organ which can be either isolated from an organism such as a mouse or a synthetic organ, a biopsy sample, a blood sample, or a combination thereof. An agent can also be products of cells including but not limited to an exosome, a cytokine, a growth factor, a metabolite, a waste product, or a combination thereof.

[0035] An agent as described here can not only produce an effect, but it can also respond to itself or another agent. Therefore, the methods described can determine a response to an agent whether that agent is itself or another agent. For example: As a response to an anti-cancer drug, cancer cells can have reduced viability. As a response to a cell-secreted growth factor, another cell can have increased proliferation by paracrine or juxtacrine signaling. As a response to a cell-secreted growth factor, same cells that produce the growth factor can have increased cell proliferation by autocrine signaling. As a response to a bacterium, a fungus can produce an antibiotic. As a response to an antibiotic, a bacterium can lose viability. As a response to a growth factor, for example growth factor A, an expression of a gene for example gene B can increase. As a response to the gene B expression, the growth factor A can be produced.

[0036] One definition of "dose" is "a quantity of a medicine or drug taken or recommended to be taken at a particular time." The microfluidic devices and methods described here enable determination of the desired doses of one or more agents. The desire dose can be including but not limited to an IC50, a minimum dose that results in an effect, a dose that does not result in an effect, a toxic dose, a time it takes for the agent to show an effect, a time an effect lasts, a time it takes for the agent to show no more effects or a combination thereof. An effect can be including but not limited to a decrease or an increase in mechanical properties such as stiffness, elasticity, viscosity, a decrease or an increase in expression of one or more genes, RNA molecules or proteins, a decrease or an increase in toxicity, apoptosis, autophagy, cell viability, cell division, cell proliferation, cell differentiation, cell death, cell adhesion, cell motility, invasion, angiogenesis, intravasation, extravasation, cell migration, cell-to-cell adhesion, signal transduction, a decrease or an increase in uptake of non-cell agents by cell agents, a decrease or increase in production of non-cell agents by cell agents, or a combination thereof. A decrease can be such that the end point is 'zero' for example no cell viability, no cell adhesion, etc. An increase can be such that the beginning point is 'zero' for example no cell differentiation, no invasion, etc.

[0037] Sensors, probes, electrodes, or a combination thereof can be incorporated in the microfluidic devices described here; including but not limited to a temperature sensor, a gas sensor, a light sensor, a conductivity sensor, a fluorescence sensor, a mechanical sensor, a stiffness sensor, or a combination thereof.

[0038] The microfluidic devices described here or their fabrication masters can be fabricated in whole or in parts, for example individual channels or membranes or inlets and / or outlets or reservoirs or their fabrication masters, by including but not limited to 3D printing, UV lithography, electron beam lithography, ion beam lithography, hot embossing, microinjection molding, injection molding, xurography, or a combination thereof. Microfluidic devices themselves or their parts can be lego like meaning parts can be assembled from existing parts including but not limited to channels, membranes and / or reservoirs. Parts can be combined using included but not limited to PSA bonding, chemical bonding, permanent bonding, transient bonding, electrostatic bonding, hot bonding, UV / ozone treatment, plasma bonding or a combination thereof.

[0039] The channels of the microfluidic device can share a common base and / or each channel can have a separate base. A base can be a solid plane or a solid volume or a membrane, posts, interface structures, phase guides or connectors or a combination thereof. A base can be larger than the total projected area of the channels. The thickness of the base can be from 1 nanometer to 5 centimeters.

[0040] The material of the microfluidic devices described here or their fabrication masters can be including but not limited to silicon, glass, a metal, a thermoplastic, a thermoset, an elastomer, a paper, a shape memory polymer, an electrospun fiber, a natural or synthetic hydrogel, an agarose, an agar, a natural or synthetic polysaccharide, a resin for 3D printing, an SU-8, polydi methyl siloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyethylene (PE), a cyclic olefin copolymer (COC), cyclic olefin polymer (COP), poly methyl pentene (PMP), poly (1-trimethylsilyl-l-propyne) (PTMSP), a poly-methylated polymer, such as (diphenylacetylene) methylated polymer, Poly(methyl methacrylate) (PMMA), or a combination thereof. The material of the microfluidic devices described here or their fabrication masters can also be one of the matrix materials described below:

[0041] A matrix to provide a 2.5D and / or a 3D environment can be used in the microfluidic devices. Cell agents can be embedded or placed on a matrix. Non-cell agents can be incorporated into a matrix. The matrix to be used in the microfluidic devices described here can be including but not limited to hydrogels, natural matrices such as matrigel, a collagen, a laminin, a gelatin, an agarose, an agar, synthetic matrices such as puramatrix, biogelx, a natural or synthetic polysaccharide, a polylactic acid, a polyglycolic acid, a poly(lysine), a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine) (PAM), a poly(acrylate), a polyester, polyhydroxybuty rate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, a poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly(vinylpyrrolidone), a copolymer including one or more of an alginate or a derivative thereof, a natural or synthetic polysaccharide, a poly lactic acid, a polyglycolic acid, poly(lysine), a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine) (PAM), a poly(acrylate), a polyester, polyhydroxybutyrate and polyepsilon caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, poly(4- aminomethylstyrene), a pluronic, a pluronic polyol, a polyoxamer, a poly(uronic 5 acid), and a poly(vinylpyrrolidone), a poly-2-hydroxyethyl methacrylate, or a combination thereof.

[0042] The channels in the microfluidic devices can be separated by a separating means, wherein said separating means is chosen from a group containing membranes, posts, interface structures, phase guides, connectors, and a combination thereof. One definition of "membrane" is a thin pliable sheet of material forming a barrier or lining. The thickness of the membranes can be as small as 1 nanometer and as large as a 10 millimeters. Here the membrane is porous, in the sense that it is permeable. The sizes of the pores in the membranes can be as small as 1 nanometer and as large as a 10 millimeters. The sizes of the pores in the membranes can be homogenous or heterogeneous. Posts can be as previously defined (references 1-3). Phase guides can be as previously defined (references 4 and 5).

[0043] Connectors are defined as structures between channels that can have a depth / height smaller than the adjacent channels and enable flow control by capillary forces (references 6-9). The connector can have a flat structure, or it can have an angle such that the connecting region is narrow at one side and wide at the other side (FIGS. 13A-13B).

[0044] The separating means such as membranes, posts, interface structures, phase guides and / or connectors can be of the same as or different than the materials of the other parts of the microfluidic device.

[0045] One definition of "dimension" is a "measurable extent of a particular kind, such as length, breadth, depth, or height". Each channel in the microfluidic device has three dimensions: a length, a width, and a height. For a microfluidic device with three channels, there are nine dimensions. For a microfluidic device with four channels, there are twelve dimensions. The microfluidic devices described here can have three or more channels. The channels of the microfluidic device can be arranged in different ways: For example, channels can be next to each other, or two channels can be on top of one channel, or one channel can be on top of two channels, and so on. Length, width and / or height of a channel does not have to be the same as length, width and / or height of another channel.

[0046] The dimensions of the channels of the microfluidic devices described here can be from a 50 nanometers up to 50 centimeters.

[0047] Of all the dimensions of the channels of the microfluidic devices, at least two dimensions are not the same or constant along another dimension, i.e. the profiles of at least two dimensions have a gradient, i.e. at least two dimensions are variable along another dimension. Profile corresponds to how the dimension looks when a cross-section is taken for that dimension. For example, a height profile shows how the height changes along a length. For example, a width profile shows how the width changes along a length. Sample profiles are shown in FIG.

[0048] 9. The y axis is labeled as width 1 but it can also be width 2, width 3, height 1, height 2 or height 3. The x axis is labeled as length 2 but it can also be length 1 or length 3. The x and y axes can be any one of the dimensions of the microfluidic device. As an example, a microfluidic device cut along its channel 2 is shown in FIG. 10A. The profiles of height 2 and width 2 along length 2 are shown in FIG. 10B and FIG. IOC, respectively.

[0049] Some definitions of a "gradient" are: (i) An inclined part of a road or railway; a slope, (ii) The degree of a slope (steepness, angle, slant, slope, inclination, leaning), (iii) The degree of steepness of a graph at any point, (iv) An increase or decrease in the magnitude of a property (e.g. temperature, pressure, or concentration) observed in passing from one point or moment to another, (v) The rate of such a change. The minimum definition of a gradient here is that the profile is not the same or constant. The profile of a gradient can be including but not limited to linearly increasing, linearly decreasing, logarithmic, exponential, polynomial, cosine, sine, concave, convex, wave like or a combination thereof. The changes in the profile of a dimension can be continuous or in steps. For example, along a channel, part of the channel can have large width and the rest can have narrow width. For another example, width of a channel can continuously increase along its length. The changes in the widths or heights of channels along their lengths can be positive, i.e. increasing, or negative, i.e. decreasing, or a combination thereof. For example, the height of a channel can first increase along its length and then decrease along its length.

[0050] The microfluidic devices described here can enable gradients of agents such that in one or more channels the agent can be at low and / or high levels at different positions in the channels. The changes in agent levels can be continuous or in steps. For example, along a channel, part of the channel can have high levels of agent and the rest can have low levels of agent. When the agent is loaded into a channel, the initial density or concentration will be homogenous. In time, due to diffusion or active transport or flow or other means, an agent can move in the channel it was originally loaded and it can also pass to neighboring channels where it was not present before. Then the agent can have different levels in different parts of the original channel it was loaded and in the other parts of the microfluidic device. However, due to diffusion, in long enough time, the differences in the levels of the agent can diminish and the level of the agent can be the same in everywhere in the microfluidic device. Yet, if the agent is added to a channel next to a channel of interest (Ci) the dimensions of which change, i.e. are not constant, the diffusion into this channel can result in a gradient in this channel and can last for a long enough time for various experiments to be carried out. The gradient in the channel of interest (Ci) can also be dynamic enough in time for certain applications (FIGS. 14A-14C).

[0051] Of all the dimensions of the channels of the microfluidic devices, at least two dimensions are not the same or constant along another dimension, i.e. the profiles of at least two dimensions have a gradient, i.e. at least two dimensions are variable along another dimension. For example, if the microfluidic device has three channels, it has nine dimensions: width 1, height 1, length 1, width 2, height 2, length 2, width 3, height 3, length 3. "gradient," "changing" and "variable" are used interchangeably. A few examples of gradient or changing dimensions are: (i) the width 1 can be changing along length 1 and width 3 changing along length 3; (ii) the width 1 and height 1 can be both changing along length 1 (iii) width 1, width 2, width 3 can be all changing along length 2 (iv) width 1, width 3, height 1 and height 3 can be all changing along length 2. A change in one dimension can be different than the change in another dimension or the changes can be the same. The different combinations of a changing or gradient dimension can be including but not limited to the ones shown in Table 1 below. In Table 1 widths and heights change along their respective lengths, i.e. for example width 1 changes along length 1, width 2 changes along length 2 and so on. The dimension is shown with a "C" if it is changing.

[0052] Table 1 The different combinations of a changing or gradient dimension in a microfluidic device having three channels. In one embodiment, (FIGS. 1A-1D) the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The width 2 and height 2 both increase from one side of the channel 2 to the other side, i.e. along length 2. Width 1, height 1, width 3 and height 3 are constant along their respective lengths. Here, channel 2 is the channel of interest (Ci). Here and in other embodiments, the height of one channel can be same as or different from the height of other channels.

[0053] In another embodiment, the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The width 1 and width 3 both increase from one side of their respective channels to the other side, i.e. along length 1 and length 3, both increasing in the same direction. Width 2, height 1, height 2 and height 3 are constant along their respective lengths. In another embodiment of this configuration, height 1 and height 3 can also increase with their respective widths (FIG. 2). Here, channels 1 and 3 are the channels of interest (Ci).

[0054] In another embodiment (FIG. 3), the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The width 1 and width 3 both increase from one side of their respective channels to the other side, i.e. along length 1 and length 3, each increasing in the opposite direction. Width 2, height 1, height 2 and height 3 are constant along their respective lengths. In another embodiment of this configuration, height 1 and height 3 can also increase with their respective widths. Here, channels 1 and 3 are the channels of interest (Ci).

[0055] In another embodiment (FIG. 4), the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The width 1, width 2 and width 3 all increase from one side of their respective channels to the other side, i.e. along length 1, length 2 and length 3, where width 1 and width 3 increase in the same direction while width 2 increases in the opposite direction. Height 1, height 2 and height 3 are constant along their respective lengths. Here, all channels can act as the channels of interest (Ci). In another embodiment of this configuration, height 1, height 2 and height 3 can also increase with their respective widths (FIG. 5). Here, again all channels can act as the channel of interest (Ci).

[0056] In another embodiment (FIG. 6), the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The channel 2 can have constant height and width along its length. The width 1 of channel 1 and width 3 of channel 3 can first increase and then decrease along the length of the channel 2 such that the maxima of width 1 and width 3 are at the middle of the channel 2. Here, channels 1 and 3 are the channels of interest (Ci). In another embodiment of this configuration, height 1 and height 3 can also increase and decrease with their respective width 1 and width 3 (FIG. 7). Here, channels 1 and 3 are the channels of interest (Ci).

[0057] In another embodiment (FIG. 8), the microfluidic device has three channels next to each other. The channel 1 has dimensions width 1, height 1, length 1, the channel 2 has dimensions width 2, height 2, length 2, the channel 3 has dimensions width 3, height 3, length 3. The channel 2 is between channel 1 and channel 3. The widths are along the x- axis, the lengths are along the y-axis and the heights are along the z-axis. The width 2 and height 2 both increase from one end of the channel 2 to the other end, i.e. along length 2 such that when the middle point of height 2 is at z=0, the maximum point of height 2 is at a z>0, i.e. a positive z and higher than the maximum z of either height 1 or height 3; and the minimum point of height 2 is at a z<0, i.e. a negative z and lower than the minimum z of either height 1 or height 3. For example, height 2 can be 10 mm where it is -5 mm and +5 mm from the middle of the height 2 while the height 1 and height 3 are 0.2 mm. Width 1, height 1, width 3 and height 3 are constant along their respective lengths. Here, channel 2 is the channel of interest (Ci).

[0058] In another embodiment, the microfluidic device has three or more channels next to each other such that each channel has a pie shape. The bottoms of the channels can be curved. In other embodiments the bottoms are not curved but can be straight and the channels are more triangular than pie shaped. The tops of the channels meet in the middle of the pie and share an outlet. The microfluidic device does not have to be a full circle, i.e. the total projection area of the channels does not have to be a full circle. In another embodiment of this configuration, one or more of the channels can be on top or at the bottom of one or more of the other channels instead of being next to each other.

[0059] Two or more of the microfluidic devices described here can be connected to each other in series and / or in parallel or a combination thereof. In one embodiment, different tissues can be mimicked in each microfluidic device. For example, one microfluidic device with three channels can have liver cells embedded in a matrix in the channel 2. One side channel for example the channel 1 can have a drug molecule as non-cell agent that is metabolized by liver cells. The other side channel for example the channel 3 can be connected to another microfluidic device and the resulting metabolite can diffuse to both side channels so it can be advantageous to connect the originally drug free channel to another microfluidic device. In addition, the channel that originally has drug can be connected to another microfluidic device with cells of a different tissue and drug responses can be determined.

[0060] The concept of channels with varying dimensions can be multiplexed for example: A device with five channels (FIGS. 12A-12B). All channels are connected to neighboring channels via connectors. Channels are numbered from left to right 1 to 5. Each channel has dimensions of height, width, length. Channel 1 has same height and width along its length as does channel 3 and channel 5. Channel 2 and channel 4 have height and width increasing along their lengths. Channel 2 and channel 4 have cells, channel 1, channel 3, channel 5 have different drugs. Thus, cells in channel 2 will experience the combination effect of drugs in channel 1 and channel 3; cells in channel 4 will experience the combination effect of drugs in channel 3 and channel 5. Two or more of the microfluidic devices described here can be fabricated on a common substrate and / or base. If the common substrate and / or base has dimensions of a multi-well plate, the multi-microfluidic device can be examined or manipulated in equipments that can accommodate multi-well plates including but not limited to plate readers or automated liquid handling systems / robots.

[0061] A previous invention (reference 3) has a middle channel the width of which changes. However, in that invention, the microfluidic device has a total of five channels / reservoirs and only the width of the middle channel changes. The current invention has at least two dimensions changing.

[0062] Each channel in the microfluidic devices described here can have its own inlet and / or outlet or channels can share inlets and / or outlets or the microfluidic device can have a single inlet and / or outlet. The inlets and / or outlets can be connected to the channels via connecting channels that are narrower or same or wider in width and shallower or same or higher in height than the corresponding channels or they can be a direct part of the channels such that a hole at one end and / or side and / or a corner of a channel serves as an inlet and / or outlet or a combination thereof. The inlets and / or outlets can be on the top, at the bottom, at one of the corners or on one of the sides of a channel. The inlets and / or outlets can be kept open or closed with tape or plugs. The inlets and / or outlets can be small openings for example with a diameter of 1mm in the material of the microfluidic device or large reservoirs for example with a diameter of 10 mm or a combination thereof. The dimensions of the inlets and / or outlets can be from 1 pm to 10 cm. External reservoirs can be added and / or combined with and / or plugged-in to inlets and / or outlets.

[0063] The height of the inlets and / or outlets can be smaller than, equal to or higher than the heights of the channels. The differences in the heights of the fluids in the channels and inlets and / or outlets can be adjusted by changing the dimensions of the channels and inlet and / or outlets to minimize, equalize or maximize pressure differences due to potential energy and / or flow according to Bernoulli principle. For example, if the height of an inlet and / or outlet is less than the height of a channel, there can be flow from the channel towards the inlet and / or outlet. The channels in the microfluidic devices described here can also be "open-top", i.e. the top plane of a channel will be partially or completely absent. Instead of or together with an inlet and / or outlet for a channel, the channel top can be wide open enough to allow entry of including but not limited to a large pipet or an electrode or such that the channel becomes more like a well like that in a multi-well plate or like a petri dish. Here the top plane of the channel can be completely absent or there can be some plane present either straight or like an overhang along one, two, three or four sides or select parts / areas / regions of the top can be absent (FIGS. 11A-11B). Such an overhang will guide the matrix to be loaded into the channel to have varying height along the channel.

[0064] The microfluidic devices described here can be used under static conditions or one or more of the channels can have flow. Here flow can be generated by including but not limited to a pressure driven pump, a syringe pump, a peristaltic pump, a piezoelectric pump, a micropump or by gravity induced flow including but not limited to repeatedly moving the microfluidic device at an angle (reference 10) or a combination thereof.

[0065] The microfluidic devices described here can be used in including but not limited to life sciences, drug discovery, clinical research, diagnostics, therapy such as combination therapy, chemical reactions, chemical synthesis, diffusion, interstitial flow.

[0066] Examples for methods that use the microfluidic devices described here are provided below:

[0067] Cells can be cultured in one or more channels of the microfluidic devices in 2D, either directly on the surface of the microfluidic device or a coated surface of the microfluidic device. The coating can be promoting cell adhesion or inhibiting cell adhesion including but not limited to extracellular matrix proteins like fibronectin, laminin or poly-L-lysine or poly-D-lysine, or a matrix as described above or poly-2-hydroxyethyl methacrylate, pluronic, or a combination thereof.

[0068] Cells can be cultured in one or more channels of the microfluidic devices in 2.5D such that a channel is filled partially with a matrix as described above and cells are introduced onto the matrix. Cells can be cultured in one or more channels of the microfluidic devices in 3D such that a channel is filled with cells mixed and / or embedded in a matrix as described above.

[0069] A microfluidic device can have cells cultured in 2D, 2.5D or 3D or a combination thereof.

[0070] To determine the concentration of a non-cell agent in the microfluidic device, fluorescent probes with different sizes, electrochemical and / or hydrodynamic properties can be used. At different time points, the microfluidic device can be imaged to calibrate fluorescence intensity with non-cell agent concentration. Other light properties such as absorbance, transmittance, or electrical properties such as conductivity or impedance can also be used for calibration.

[0071] Non-cell agents can be loaded into the channels in solution or attached and / or linked and / or associated with a matrix or polymer that can provide controlled release.

[0072] Detection of effects of cell and non-cell agents can utilize the sensors, probes, electrodes, or a combination thereof that can be incorporated into the microfluidic devices.

[0073] Detection of effects of cell and non-cell agents, i.e. response to cell and non-cell agents can be performed using including but not limited to microscopy, spectroscopy, electrical measurements, mechanical measurements, temperature measurements, pressure measurements, pH measurements, oxygen level measurements, chemical detection, colorimetry, visual inspection by eye, inspection and / or recording by a camera or a combination thereof.

[0074] In one method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have its width 2 and height 2 increasing from one end to the other end along its length 2 and the height 1, height 3, width 1 and width 3 of the channel 1 and channel 3 do not change along length 1, length 2 and length 3, dose response can be determined. A non-cell agent such as an anti-cancer drug can be loaded into channel 1. If desired, multiple non-cell agents can also be loaded into channel 1 to determine their combined effects. The channel 2 can be loaded with a cell agent such as cancer cells embedded in a matrix; the channel 3 can be loaded with just cell culture medium. In time, the non-cell agent can diffuse in or move from the channel 1 to the channel 2. There will be more non-cell agent in the channel 2 where its width and / or height are smaller corresponding to e.g. higher fluorescence intensity for a fluorescent non-cell agent (FIGS. 14A-14C). Here assessment of response to the non-cell agent can be determined at different places in the channel 2, for example by staining for dead cells. Here at a desired time point such as fortyeight hours, the fluid in the channel 1 and channel 3 can be exchanged with a dye that stains dead cells. Thus, the dose effect of the agent on cancer cells can be determined. In another embodiment of this method, the channel 2 can also be coated with endothelial cells such that that the channel 2 can mimic a blood vessel dimensions of which are changing. Other embodiments described can also have one or more channels coated with endothelial cells to mimic a blood vessel or its interface. Instead of endothelial cells, other cells including but not limited to lung epithelial or kidney epithelial cells or brain cells can be used to mimic the relevant interfaces that are found in vivo. In another embodiment for this microfluidic device configuration, the channel 2 can be loaded with bacteria embedded or under a matrix and the channel 1 and channel 3 can be loaded with a different (combination effect) or same antibiotic; or only channel 1 or only channel 3 can be loaded with an antibiotic. Effects of antibiotics on viability of bacteria can be determined by microbiological or microscopic or spectroscopic assays. In another embodiment for this microfluidic device configuration, the channel 1 or channel 3 here can have only culture medium at first, and the toxic chemical or vitamin can later be added in sequence or simultaneously, or vice versa to examine time effects. In yet another embodiment of this method, plant seeds can be placed in channel 2; channel 1 and channel 3 can be loaded with different or same agents such as high salt medium, biostimulant for plant growth, etc. In another embodiment of this method, the channel 1 or channel 3 here can have only culture medium at first, and the high salt solution or biostimulant can later be added in sequence or simultaneously, or vice versa to examine time effects. In this method, the agent can also be loaded into both channel 1 and channel 3 such that diffusion from both into channel 2 can provide an increase in concentration. This can be useful for agents that diffuse slowly.

[0075] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have its width 2 and height 2 increasing from one end to the other end along its length 2 and the height 1, height 3, width 1 and width 3 of the channel 1 and channel 3 do not change along length 1, length 2 and length 3, dose response can be determined. A non-cell agent such as an anti-cancer drug can be loaded into channel 1. The channel 2 can be loaded with a cell agent such as cancer cells embedded in a matrix; the channel 3 can be loaded with another non-cell agent such as another drug. In time, the noncell agents can diffuse in or move from the channel 1 and channel 3 to the channel 2. There will be more non-cell agents in the channel 2 where its width and / or height are smaller; there will be areas in channel 2 where (i) both of the non-cell agents are present, (ii) only one of the non-cell agents is present, (iii) none of the non-cell agents are present. Here, the combinatorial effects of two different non-cell agents can be determined on, for example cell death by using live or dead cell fluorescent dyes and microscopy at a desired time point.

[0076] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have its width 2 and height 2 increasing from one end to the other end along its length 2, and the height 1, height 3, width 1 and width 3 of the channel 1 and channel 3 do not change along length 1, length 2 and length 3, dose response can be determined in a different way: A non-cell agent such as an anti-cancer drug can be loaded into channel 1. The channel 2 can be loaded with a cell-free matrix, the channel 3 can be loaded with first a cell agent such as cancer cells embedded in matrix and then filled with cell culture medium. In time, the non-cell agent can diffuse in or move from the channel 1 to the channel 2 and to the channel 3. There will be more non-cell agent in the channel 3 where the width and / or height of channel 2 are smaller. Here assessment of response to the non-cell agent can be determined at different places in the channel 3, for example by staining for dead cells. Compared to the method where cells were in channel 2, here cells would experience the gradient later, which can be useful for agents that diffuse faster.

[0077] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have constant height 2 and width 2 along its length 2. The channel 1 can have height 1 and width 1 both increasing along length 2. The channel 3 can have height 3 and width 3 both increasing along length 2 and in the same direction as the channel 1. A cell agent such as cancer cells mixed with a matrix can be loaded into the channel 1. A non-cell agent such as a toxic chemical can be loaded into the channel 2. Another cell agent such as normal cells mixed with a matrix can be loaded into the channel 3. In time, the amount of agents reaching the channel 1 will be different along the length 2 of the channel 2: There will be more non-cell agent delivered to the channel 1 and channel 3 where the dimensions of the channel 1 and channel 3 are smaller. Here assessment of response to non-cell agents can be determined at different places in the channel 1 and channel 3 for example by assessment of cell membrane integrity using fluorescent probes.

[0078] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have constant height 2 and width 2 along its length 2. The channel 1 can have height 1 and width 1 both increasing along length 2. The channel 3 can have height 3 and width 3 both increasing along length 2 in the same direction as the channel 1. A cell agent such as neurons mixed with a matrix can be loaded into the channel 1. A non-cell agent such as a growth factor can be loaded into the channel 2. A cell agent such as glial cells can be loaded into the channel 3. An assessment of the effect of the non- cell agent on two different cell types such neurons and glia in this example, can be performed.

[0079] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have constant height 2 and width 2 along its length 2. The channel 1 can have height 1 and width 1 both increasing along length 2. The channel 3 can have height 3 and width 3 both increasing along length 2 but in the opposite direction of the channel 1. A cell agent such as cells expressing a FRET biosensor mixed with a matrix can be loaded into the channel 1. A non-cell agent such as a drug can be loaded into the channel 2. Another cell agent with the same or different FRET biosensor can be loaded into the channel 3. In time, the amount of agents reaching the channel 1 will be different along the length 1 of the channel 1: There will be more non-cell agent delivered to the channel 1 where the dimensions of the channel 1 and channel 3 are smaller where the profiles are opposite, one increasing while the other one is decreasing. Here assessment of response to non-cell agents can be determined at different places in the channel 1 and channel 3 including but not limited to by assessment of for example gene expression using the biosensors in the cells in the channel 1. In another embodiment of this method, the channel 2 here can have only culture medium at first, and a drug or hormone can later be added in sequence or simultaneously, or vice versa to examine time effects. In yet another embodiment of this method embryos at different stages of development can be loaded into channel 1 and channel 3. In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have constant height 2 and width 2 along its length 2. The channel 1 can have height 1 and width 1 both increasing along length 2. The channel 3 can have height 3 and width 3 both increasing along length 2 but in the opposite direction of the channel 1. A cell agent can be loaded into the channel 1, a non-cell agent such as a growth factor that promotes cell proliferation can be loaded into the channel 3 and culture medium without that growth factor can be loaded into the channel 2. Here the gradient of the non-cell agent will be emphasized, i.e. it will be sharper due to the channel 1 and channel 2 acting like a sink and due to opposite gradients in the dimensions of the channel 1 and channel 3. An assessment, including but not limited to determination of cell proliferation by observing cells under a microscope continuously or at different time points, can be performed.

[0080] In another method, using a microfluidic device with the channel 2 between channel 1 and channel 3 where the channel 2 can have constant height 2 and width 2 along its length 2. The width 1 and width 3 of the channel 1 and channel 3 can first increase and then decrease along the length 2 of the channel 2 such that the maxima of width 1 and width 3 are at the middle of the channel 2. Here a non-cell agent such as a chemical that inhibits cell motility can be loaded into the channel 2, different types of cells can be loaded in a matrix into each of the channel 1 and channel 3. The cell number in channel 1 and channel 3 will be maximum where the channel 1 and channel 3 meet the channel 2 and minimum at further away from the channel 2. Here effect of the non-cell agent on different cell types and different cell masses can be determined by including but not limited to determining changes in cell motility. Here, tissues with small or large tumor masses are mimicked and drug effects on including but not limited to cell viability can be determined using including but not limited to microscopy or spectroscopy.

[0081] The microfluidic devices can be held or maintained at various orientations depending on the experiment including but not limited to upside down, vertical, horizontal, a wide part at the bottom or on top or at an angle of 1 to 90 degrees, etc. or a combination thereof. During incubation of cells in a matrix, the microfluidic device can be rotated constantly or at certain intervals to ensure homogenous distribution of cells in the hydrogel or the microfluidic device can be intentionally held for example vertical or upside down or another position to concentrate cells at a desired side of the microfluidic device using gravity and / or centrifugation force.

[0082] It will be readily apparent to persons skilled in the relevant arts that various modifications and improvements may be made to the foregoing embodiments, in addition to those already described, without departing from the basic inventive concepts of the present invention. For example, the microfluidic devices in the described embodiments are each provided with three sets of microfluidic channels. However, the chips can be custom-designed to incorporate any desired number of channels and in a variety of configurations. The methods using the described microfluidic devices present examples for various cell and non-cell agents but can be customized for a variety of configurations. Therefore, it will be appreciated that the scope of the present invention is not limited to the specific embodiments described.

[0083] In a nutshell, the present invention proposes a microfluidic device comprising three or more fluid channels, wherein said channels are separated from each other by separating means, and each channel (channel n) have a volume defined by three dimensions comprising a length n, a width n, and a height n, and wherein at least two dimensions are variable along the direction of another dimension.

[0084] In one variation of the present invention, said at least two dimensions are variable along the direction of another dimension in the manner of linearly increasing, linearly decreasing, logarithmic, exponential, polynomial, cosine, sine, concave, convex, wave like, or a combination thereof.

[0085] In a further variation of the present invention, said at least two dimensions are variable along the direction of another dimension in an identical manner.

[0086] In a further variation of the present invention, said at least two dimensions are variable along the direction of another dimension in different manners.

[0087] In a further variation of the present invention, said at least two dimensions are variable along the direction of another dimension in a continuous manner, a stepwise manner, or a combination thereof. In a further variation of the present invention, said separating means are chosen from a group containing membranes, posts, interface structures, phase guides, connectors, and a combination thereof.

[0088] In a further variation of the present invention, said microfluidic device comprises at least one sensor, at least one probe, at least one electrode, or a combination thereof incorporated therein.

[0089] In a further variation of the present invention, said sensor is chosen from a group containing a temperature sensor, a gas sensor, a light sensor, a conductivity sensor, a fluorescence sensor, a mechanical sensor, and a stiffness sensor.

[0090] In a further variation of the present invention, said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively.

[0091] In a further variation of the present invention, at least one channel of said microfluidic device has an open-top structure, said channel comprises at least one overhang structure projecting from at least one side wall towards the center of said channel so as to provide a partial cover for said channel, and the position of said overhang structure on said side wall is variable over the length of said side wall.

[0092] The present invention further proposes a microfluidic device system comprising at least two microfluidic devices as described in Claim 1 connected to each other in series, in parallel or in a combination thereof.

[0093] In a further variation of the present invention, said at least two microfluidic devices are fabricated on a common base having the dimensions of a multi-well plate.

[0094] The present invention further proposes a method for determining the dose effect of an agent on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cells embedded in a matrix into channel 2, c) Loading cell culture medium into channel 3, d) Incubating said microfluidic device at appropriate cell culture conditions, and e) Determining cell death and / or viability and / or motility.

[0095] The present invention further proposes a method for determining the combinatorial effect of two agents on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cells embedded in a matrix into channel 2, c) Loading another agent in cell culture medium into channel 3, d) Incubating said microfluidic device at appropriate cell culture conditions, and e) Determining cell death and / or viability and / or motility.

[0096] The present invention further proposes a method for determining the dose effect of an agent on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cell-free matrix into channel 2, c) Loading cells embedded in a matrix into channel 3 so as to at least partially fill said channel 3, d) Loading cell culture medium into channel 3, e) Incubating said microfluidic device at appropriate cell culture conditions, and f) Determining cell death and / or viability and / or motility.

[0097] In a further variation of the present invention, wherein said cells are chosen from a group consisting of bacteria, archea, eukarya, yeast, fungi, plant cells, epithelial tissue cells, connective tissue cells, muscle tissue cells, nervous tissue cells, cancer cells, immune cells, blood cells, neuron cells, cell line cells, biopsy cells, spheroids, organoids, or a combination thereof.

[0098] References

[0099] Reference 1: (W02006052223) CELL CULTURE DEVICE

[0100] Reference 2: (W02012050981) DEVICE FOR HIGH THROUGHPUT INVESTIGATIONS OF CELLULAR INTERACTIONS

[0101] Reference 3: (W02015052034) MICROFLUIDIC DEVICE FOR INVESTIGATION OF DISTANCE DEPENDENT INTERACTIONS IN CELL BIOLOGY

[0102] Reference 4: (EP2213364) PHASE GUIDE PATTERNS FOR LIQUID MANIPULATION

[0103] Reference 5: (US20180169656) MICROFLUIDIC PLATE

[0104] Reference 6: (US4271119) CAPLLARY TRANSPORT DEVICE HAVING CONNECTED TRANSPORT ZONES

[0105] Reference 7: (US4426451) MULTI-ZONED REACTION VESSEL HAVING PRESSURE- ACTUATABLE CONTROL MEANS BETWEENZONES

[0106] Reference 8: (US4618476) CAPILLARY TRANSPORT DEVICE HAVING SPEED AND MENSCUS CONTROL MEANS

[0107] Reference 9: (US5230866) CAPILLARY STOP-FLOW JUNCTION HAVING IMPROVED STABILITY AGAINST ACC DENTAL FLUID FLOW

[0108] Reference 10: Sung, Jong Hwan, Carrie Kam, and Michael L. Shuler. "A microfluidic device for a pharmacokinetic-pharmacodynamic (PK-PD) model on a chip." Lab on a Chip 10.4 (2010): 446-455. DOI: 10.1039 / b917763a

[0109] Reference 11: Wang, Xiang, Zhaomiao Liu, and Yan Pang. "Concentration gradient generation methods based on microfluidic systems." RSC advances 7.48 (2017): 29966- 29984. D01: 10.1039 / C7RA04494A

Claims

CLAIMS1) A microfluidic device comprising three or more fluid channels, wherein said channels are separated from each other by separating means, and each channel (channel n) have a volume defined by three dimensions comprising a length n, a width n, and a height n, and wherein at least two dimensions are variable along the direction of another dimension.2) A microfluidic device as set forth in Claim 1, wherein said at least two dimensions are variable along the direction of another dimension in the manner of linearly increasing, linearly decreasing, logarithmic, exponential, polynomial, cosine, sine, concave, convex, wave like, or a combination thereof.3) A microfluidic device as set forth in Claim 1 or 2, wherein said at least two dimensions are variable along the direction of another dimension in an identical manner.4) A microfluidic device as set forth in Claim 1 or 2, wherein said at least two dimensions are variable along the direction of another dimension in different manners.5) A microfluidic device as set forth in any one of Claims 1 to 4, wherein said at least two dimensions are variable along the direction of another dimension in a continuous manner, a stepwise manner, or a combination thereof.6) A microfluidic device as set forth in any preceding Claim, wherein said separating means are chosen from a group containing membranes, posts, interface structures, phase guides, connectors, and a combination thereof.7) A microfluidic device as set forth in any previous Claim, wherein said microfluidic device comprises at least one sensor, at least one probe, at least one electrode, or a combination thereof incorporated therein.8) A microfluidic device as set forth in Claim 7, wherein said sensor is chosen from a group containing a temperature sensor, a gas sensor, a light sensor, a conductivity sensor, a fluorescence sensor, a mechanical sensor, and a stiffness sensor.9) A microfluidic device as set forth in any previous Claim, wherein said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively.10) A microfluidic device as set forth in any previous Claim, wherein at least one channel of said microfluidic device has an open-top structure, said channel comprises at least one overhang structure projecting from at least one side wall towards the center of said channel so as to provide a partial cover for said channel, and the position of said overhang structure on said side wall is variable over the length of said side wall.11) A microfluidic device system comprising at least two microfluidic devices as described in Claim 1 connected to each other in series, in parallel or in a combination thereof.12) A microfluidic device system as set forth in Claim 11, wherein said at least two microfluidic devices are fabricated on a common base having the dimensions of a multi-well plate.13) A method for determining the dose effect of an agent on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cells embedded in a matrix into channel 2, c) Loading cell culture medium into channel 3, d) Incubating said microfluidic device at appropriate cell culture conditions, and e) Determining cell death and / or viability and / or motility.14) A method for determining the combinatorial effect of two agents on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises threechannels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cells embedded in a matrix into channel 2, c) Loading another agent in cell culture medium into channel 3, d) Incubating said microfluidic device at appropriate cell culture conditions, and e) Determining cell death and / or viability and / or motility.15) A method for determining the dose effect of an agent on cells using a microfluidic device as described in Claim 1, where said microfluidic device comprises three channels positioned side by side along their lengths, width 2 and height 2 of channel 2 increases in the direction of length 2, width 1 and height 1 of channel 1 and width 3 and height 3 of channel 3 are constant in the direction of length 1 and length 3 respectively, said method comprising the steps of: a) Loading an agent in cell culture medium into channel 1, b) Loading cell-free matrix into channel 2, c) Loading cells embedded in a matrix into channel 3 so as to at least partially fill said channel 3, d) Loading cell culture medium into channel 3, e) Incubating said microfluidic device at appropriate cell culture conditions, and f) Determining cell death and / or viability and / or motility.16) A method as set forth in Claim 13, 14 or 15, wherein said cells are chosen from a group consisting of bacteria, archea, eukarya, yeast, fungi, plant cells, epithelial tissue cells, connective tissue cells, muscle tissue cells, nervous tissue cells, cancer cells, immune cells, blood cells, neuron cells, cell line cells, biopsy cells, spheroids, organoids, or a combination thereof.

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