Device and method for high-throughput perfusion culture and imaging of pancreatic islets

Abstract

The present invention relates to a microfluidic chip, which is bonded by a substrate and a flow channel layer, wherein the flow channel layer is provided with 2-4 functional units; the functional units are each independently provided with an environmental control module and a plurality of main channels connected to the environmental control module; the environmental control module is sequentially provided with a liquid inlet, a filtration unit, a mixing channel, a buffer tank, a first fence channel and a branch channel; the main channel is sequentially provided with a sample loading inlet, an inflow channel, an islet retention area, an outflow channel and an outlet; the inflow channel of the main channel is connected to the branch channel of the environmental control module at an intersection; wherein the mixing channel is provided with a plurality of staggered fishbone-shaped micro-mixers; the intersection is provided with a second fence channel; the islet retention area includes a step structure and a liquid dredging channel; wherein the step structure has three or four steps. The microfluidic chip of the present invention can be advantageously used for high-throughput perfusion culture of islets, functional imaging and screening of drugs for treating islet-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics, and in particular relates to a device and method for perfusion culture, functional imaging and hormone collection of pancreatic islets or pancreatic islet organoids. Background Art

[0002] As the most important blood sugar regulating organ in the body, the pancreatic islets respond to changes in blood glucose concentration in a rapid, dynamic, and pulsed manner and release hormones to maintain the dynamic balance of glucose in the body. Islet cell dysfunction is one of the main causes of dysglycemia. For patients with type 1 diabetes, insulin injection under strict control has been proven to be the most effective means of controlling blood sugar with the least side effects. However, if the normal β-cell pulsed insulin secretion pattern is not well simulated during actual administration, hypoglycemia often occurs. In addition, in vitro screening of diabetes therapeutic drugs also requires replicating the in vivo microenvironment as much as possible. Therefore, studying the dynamic process of pancreatic islet cell response to glucose and hormone release is crucial to understanding the pathological characteristics of diabetes and screening therapeutic drugs.

[0003] Since the 1970s, macroscopic perfusion systems have been used to study hormone secretion. However, macroscopic perfusion systems are complex to operate and difficult to precisely control flow rates. In recent years, microfluidic chips have been widely used in studies of pancreatic islets at the sub-organ and cellular levels because of their easy adjustment of channel size, ability to achieve dynamic changes in the microenvironment, and ability to accurately simulate rapid diffusion processes in vivo. For example, Mohammed et al. developed a microfluidic chip for quality control of transplanted islets, which can dynamically measure changes in calcium concentration associated with insulin secretion [1]. The microfluidic chip developed by Jun et al. can culture islet cells for a long time and grow them into spherical aggregates similar to islets [2]. The above chips all use microwells to capture islets or islet-like spherical aggregates in the islet retention area. In order to achieve effective fixation, the microwells are often deep, making it difficult to achieve effective diffusion mixing at low flow rates; while higher flow rates can easily flush out smaller islets, making stable observation impossible. As an improvement, a capture area with a trapezoidal cross-section is set on the side of the main channel, and an outlet is added on the opposite side of the main channel [3]. Although optimizing the top and bottom angles of the trapezoid can achieve that most of the capture area captures only a single islet in the horizontal direction, in order to effectively accommodate the islets, the thickness of the capture area is 250-300 μm, and multiple islets may remain in the vertical direction, which is not conducive to imaging. US20220356428 discloses a microfluidic chip for simulating pancreatic function, which separates the islet chamber and epithelial cells through a porous membrane. Although the chip can be used to study the interaction between epithelial cells and islet cells, the simultaneous pressurization and fluid replacement of the upper and lower chambers makes the internal pressure of the system unstable, making it difficult to achieve fixed-point long-term observation.

[0004] How to accurately reconstruct the pancreatic islet microenvironment and effectively control the internal pressure of the chip while achieving a chemical microenvironment similar to that in vivo, so as to simulate the physiological and pathological characteristics of a single pancreatic islet, remains an urgent problem to be solved in this field. Summary of the invention

[0005] In a first aspect, the present invention provides a microfluidic chip, which is bonded by a substrate and a flow channel layer, wherein the flow channel layer is provided with 2-4 functional units; each functional unit is independently provided with an environmental control module and a plurality of main channels connected to the environmental control module; the environmental control module is provided with a liquid inlet, a filtration unit, a mixing channel, a buffer tank, a first fence channel and a branch channel in sequence; the main channel is provided with a sample loading inlet, an inflow channel, an islet retention area, an outflow channel and an outlet in sequence; the inflow channel of the main channel is connected with the branch channel of the environmental control module at an intersection; wherein the mixing channel is provided with a plurality of staggered herringbone-shaped micromixers; a second fence channel is provided at the intersection; the islet retention area comprises a step structure and a liquid drainage channel; wherein the step structure has three or four steps.

[0006] In a second aspect, the present invention provides a method for imaging pancreatic islets using the chip described in the first aspect, the method comprising: injecting a pancreatic islet suspension into the chip through a sample loading inlet; injecting a perfusion solution into the chip through a liquid inlet; and imaging the pancreatic islet retention area.

[0007] In a third aspect, the present invention provides uses of the chip described in the first aspect in perfusion culture of pancreatic islets, functional imaging, and screening of therapeutic drugs for pancreatic islet-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1The chip design according to Example 1. a) The overall design diagram of the chip, the chip flow channel layer includes two completely identical, symmetrically designed functional units, each of which includes an environmental control module and a main channel. In the right functional unit, S1-S4 is the liquid inlet in the environmental control module; F1-F4 is a filtration unit; M is a mixing channel; B is a buffer tank; BL1a and BL1b are first-level branch channels; BL2a, BL2b, BL2c and BL2d are second-level branch channels; BL3a, BL3b, BL3c, BL3d, BL3e, BL3f, BL3g and BL3h are third-level branch channels. As for each main channel controlled by the environmental control module, E1-E8 is the sample loading entrance of the main channel; J1-J8 is an intersection, and the third-level branch channel is connected to the inflow channel of the main channel via the intersection; W1-W8 is the main channel outlet. The scale bar is 1 cm long, and the heights in the channel represented by the color gradient are 40 (light yellow), 70, 100, 130 and 300 μm (red) in sequence. b) is an enlarged view of the liquid inlet end of the environmental control module. The environmental control module is provided with 4 liquid inlets S1-S4, which are connected to the mixing channel M and the buffer tank B via the filter units F1-F4. G1 is the first fence channel. The scale bar is 5 mm long, and the heights in the channel represented by the color gradient are 70 (orange-yellow), 100, 130 and 300 μm (red) in sequence. c) is a partial enlarged view of the mixing channel M. The mixing channel M is provided with 38 groups of 6 staggered herringbone mixers to create turbulence in the channel. The scale bar is 1 mm long, and the heights in the channel represented by the color gradient are 100 μm (orange-yellow) and 130 μm (orange-red) in sequence. d) An enlarged view of the islet retention area of ​​the main channel, where D1-D8 show the four-layer step structure for islet retention. The main channel has a 90-degree turn at the outlet of the islet retention area, so that the flow direction of the fluid changes after flowing through the islet retention area; a 40 μm high fluid diversion channel (DR) is provided on the side of each step structure to prevent the larger islets from completely blocking the channel. The scale bar is 1 mm long, and the cumulative heights represented by the color gradient are 40 (light yellow), 70, 100, 130 and 300 μm (red). e) A side view of the step structure of the islet retention area. With the introduction of the islet suspension, individual islets of different sizes can stay at different positions of the step structure. f) An enlarged view of the intersection J4 where the environmental control module is connected to the main channel. G2 is the second fence channel. E4 is the sample loading inlet. The scale bar is 1 mm long, and the heights in the channel represented by the color gradient are 100 and 300 μm.

[0009] Figure 2To achieve dynamic control of the microenvironment using the chip of Example 1. a) Characterization of the effect of the mixing channel with an interlaced herringbone micromixer. The left figure shows a microscopic fluorescence image (FITC field of view) at the mixing channel in a stable state when 20 μM fluorescein solution and 1 μM fluorescein solution are injected into the two liquid inlets in the environmental control module at a speed of 1600 μL / h. The total flow rate is 3200 μL / h. The right figure shows the fluorescence intensity at 0, 15, and 30 mm of the mixing channel measured according to the microscopic fluorescence image. The positions corresponding to the data points are framed in the left figure. The error bars show the standard deviation of 3 independent repeated experiments. b) The solution perfusion speed ratio through the liquid inlet is controlled by a programmable injection pump to change with time, and square wave, triangle wave, and sinusoidal concentration pulses (upper figure) and step-up concentrations (lower figure) are established in the main channel. The dotted line is the experimentally measured fluorescence intensity at the highest step of the islet retention zone of the 8 main channels, and the black solid line is the average of the above fluorescence intensities; the light blue filling is the theoretical value of the input fluorescence concentration change, and the light red filling is the theoretical value after considering the delay caused by the channel volume. The total flow rate is 3200μL / h (400μL / h for each main channel). c) Bright field micrograph of the islet retention zone after loading the islet suspension. Due to the step structure, islets of different sizes can be stably retained. The scale bar is 1mm. d) The solution perfusion speed through each inlet is adjusted by a programmable injection pump, so that the ratio of the solution perfusion speed changes with time, so that the glucose concentration in the islet retention zone changes with time, and calcium imaging of the islet retention zone is performed by microscopy. The scale bar is 15 minutes. 1, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20 respectively indicate that the glucose concentration in the main channel within the time range is 1 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 10 mM, 12 mM, 15 mM and 20 mM. The vertical axis is the calcium signal intensity characterized by the standardized fluorescence intensity ΔF / F.

[0010] Figure 3The chip of Example 1 is used to screen paracrine factors that are critical for the phase shift of islet glucose dose response. a) Schematic diagram of the experimental process. First, mouse islets are isolated; in vitro culture is performed on a multi-well plate; the isolated and cultured islets are injected into the main channel of the chip of Example 1 through the sample loading inlet, and calcium imaging of a single islet is performed for each islet retention area under a microscope in an environment where the glucose concentration changes over time; the intensity of calcium signals released by islet B cells is calculated by fluorescence values; then, a quantitative analysis is performed based on the calcium activity curves of each group of islets; and statistical results are obtained. b) Fluorescence microscopy is used to obtain a time series diagram of the oscillation of islet calcium signals over time, and the activation threshold and oscillation threshold of islet calcium oscillations are calculated based on this. The figure above shows the experimental results of the change of calcium concentration in pancreatic islet cells over time obtained by fluorescence microscopy when perfusing KRBB solution with a step-by-step increase in glucose concentration over time. The horizontal axis is time and the vertical axis is calcium signal intensity (characterized by the standardized fluorescence intensity ΔF / F). The glucose concentrations given are 1mM (10 minutes), 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 10mM, 12mM, 15mM and 20mM (15 minutes each). The figure below shows the normalized calcium oscillation curve calculated by the timing diagram, with the horizontal axis being the glucose concentration and the vertical axis being the average value of the calcium signal at this concentration. The activation threshold and oscillation threshold of pancreatic calcium oscillation can be calculated from this curve. c) Statistics of activation glucose thresholds of islets treated with different paracrine factors, Gcg for glucagon treatment group; Ins for insulin treatment group; SST for somatostatin treatment group; 8G for blank treatment group; CYN and MK4256 for somatostatin receptor 2 (SSTR2) antagonist treatment group and somatostatin receptor 3 (SSTR3) antagonist treatment group, respectively. The treatment time was 18 to 22 hours. Data were collected from 104 islets from 11 mice. Error bars represent one standard deviation (SD). ****: P<0.0001, *: P<0.05,; ns: p>0.05. d) Quantitative statistics of activation threshold and oscillation threshold of islets in control group and glucagon treatment group. ****: P<0.0001, error bars represent one standard deviation (SD). Data were collected from 19 islets from 3 mice. e) Correlation between the number of α cells in islets and the calcium activation threshold of β cells. The dashed lines represent the 95% confidence interval, R 2 =0.52, P<0.05.

[0011] Figure 4The chip design according to Example 2. The chip flow channel layer includes two symmetrically designed functional units. In the functional unit on the right, S1-S4 are the liquid inlets in the environmental control module; M is the mixing channel; B is the buffer tank; BL1a-BL1d are the branch channels; E1-E4 are the main channel entrances; J1-J4 are the intersections where the branch channels connect to the main channels; W1-W4 are the main channel exits; and P1-P4 are the liquid reservoirs. The scale bar is 1 cm long, and the heights in the channels represented by the color gradients are 40 (light yellow), 70, 100, 130, and 300 μm (red) in sequence.

[0012] Figure 5 The results of the hormone collection experiment using the chip of Example 2. a) A time series diagram of the oscillation of the calcium signal of beta cells in the pancreatic islets under different pretreatment conditions obtained by fluorescence microscopy. The horizontal axis is time, and the scale length is 15min; the vertical axis is the calcium signal intensity (characterized by the standardized fluorescence intensity ΔF / F), and the scale length is 1 times the background fluorescence intensity. The glucose concentrations administered were 3mM, 4mM, 5mM, 6mM, 7mM, 10mM, 12mM, 15mM and 20mM, of which the 10mM step lasted for 60min, and the remaining concentration steps lasted for 15min each. Two repetitions are shown in the figure. b) Dose response curve of insulin secretion caused by different glucose concentrations. c) Dose response curve of glucagon secretion caused by different glucose concentrations.

[0013] Figure 6 The chip design according to Example 3. The chip flow channel layer includes four symmetrically designed functional units. In the functional unit on the upper right side, S1-S4 are the liquid inlets in the environmental control module; M is a mixing channel; B is a buffer tank; BL1a and BL1b are the first-level branch channels, and BL2a-BL2d are the second-level branch channels. E1-E4 are the main channel entrances; J1-J4 are the intersections where the branch channels connect to the main channels; W1-W4 are the main channel exits. The scale length is 1 cm, and the heights in the channels represented by the color gradients are 40 (light yellow), 70, 100, 130, and 300 μm (red), respectively. DETAILED DESCRIPTION

[0014] In the description of the present invention, it should be understood that the terms "center", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, which are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. The terms "first", "second", "third" and "fourth" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first", "second", "third" and "fourth" may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0015] Due to the rapid and pulsed response of islets to blood glucose concentration, research needs to be conducted with high temporal resolution. Building an environment that can rapidly switch glucose concentrations is critical to obtaining the glucose response of islets. In addition, since each islet responds at a different rate, if multiple islets are included in the detection system or the temporal resolution is too low, the observed average may lose characteristic signals. Therefore, accurate measurements of the physiological characteristics of each islet must be made individually to effectively reflect the physiological or pathological characteristics of the islet.

[0016] The chip of the present invention is provided with a step structure with 3-4 layers of different heights in the islet retention area, so that the main channel has a height difference in the islet retention area, so that when a suspension containing multiple islets is injected into the main channel from the sample loading inlet, the islets can stay in the islet retention area in the form of a single layer. In a preferred embodiment, the width of the inflow channel and the outflow channel of the main channel is 250-350 μm; the total length is 8-14 mm. In a preferred embodiment, the width of the islet retention area is 350-450 μm, so that the islet retention area can capture a single islet. Each islet retention area includes a step structure portion and a liquid drainage channel; the height difference of each layer of steps is independently 30-40 μm. That is, at the step structure, the first step has a first height of 30-40 μm; the second step has a second height of 60-80 μm; the third step has a third height of 90-120 μm; the step structure may also include an optional fourth step, which has a fourth height of 120-150 μm. The height of the main channel is the fifth height, 250-350μm. By adopting an optimized step structure size, islets with large volume differences (diameter of 50-300μm) can stay in the islet retention area for a long time in the form of a single islet, so that the rapid and pulsed response of a single isolated islet can be effectively observed. On both sides of the step structure, there is a liquid drainage channel with a width of 40-50μm and a height of the first height. The drainage channel is parallel to the flow direction of the perfusion fluid, which can prevent larger islets from blocking the entire channel and causing uneven flow rate. The drainage channel has a first height, so that the flow resistance of the step structure part and the drainage channel is equivalent, ensuring sufficient contact between the perfusion fluid and the islets. It can be understood by those skilled in the art that the term "step structure" used in this article refers to the top of the microfluidic channel formed by bonding the glass substrate and the flow channel layer of polydimethylsiloxane material with multiple heights ( Figure 1d, 1e). The height of the step structure refers to the height relative to the base. In a preferred embodiment, the length of each step is 80-300 μm, so that most islets can at least enter the highest step. The total length of the main channel at the islet retention area is 500-1000 μm and the width is 350-450 μm. The design of this size minimizes the impact of the main channel fluid on the islets during perfusion and fluid replacement under the premise of comprehensively considering the volume of the islets and the 10x field of view of the microscope, and reduces the displacement of the islets during the observation process, so as to achieve long-term automatic tracking of the islets and their markers. In a preferred embodiment, each islet retention area is arranged in parallel to maximize the use of the field of view of the microscope. Since the focal plane can be predicted, the live cell imaging method can be applied to the chip of the present invention to achieve automatic imaging. In other embodiments, the fifth height can be set to no more than 500 μm, and the width of the main channel and the islet retention area can be set to no more than 1 mm to accommodate the perfusion and imaging of larger diabetic model mouse islets, other species (such as human) islets, human islet organoids, etc. In the present invention, the term "inflow channel" refers to the fluid channel between the main channel sample loading inlet and the islet retention area; the term "outflow channel" refers to the fluid channel between the islet retention area and the outlet. It can be understood by those skilled in the art that, with respect to the chip of the present invention, the main channel sample loading inlet, the main channel outlet (or liquid reservoir) and each liquid inlet in the environmental control module are connected to the outside world.

[0017] In a preferred embodiment, a liquid reservoir ( Figure 4 ), a microsyringe can be used to collect the outflow solution of each channel and measure the hormone concentration therein. In fact, in applications such as islet transplantation evaluation, in addition to measuring the calcium signal (GSIS) released by the islets in response to glucose, it is also necessary to perform biochemical measurements of the hormones secreted by the islets to control the quality of the transplanted organs. However, since the amount of hormones secreted by a single islet is small, how to minimize the measurement volume and improve the detection accuracy has always been a difficult problem in this field. By setting a liquid storage tank at the outlet end, the concentration of insulin, glucagon or other small molecules secreted by a single islet can be measured. It is easy to maintain a small total liquid volume under the microfluidic system, avoiding excessive dilution of hormones. In addition, the micromixer structure in the environmental control module helps to shorten the fluid channel and reduce delays.

[0018] In a preferred embodiment, the reservoir is an open circular reservoir with a diameter of 4-6 mm and is located 5-8 mm away from the main channel outlet. During the perfusion process, the effluent accumulates in the reservoir, and a microsyringe is used to draw the effluent from the outlet and collect it for testing at the time point where it needs to be recorded. Since the reservoir has a large volume and provides a buffer, the flow rate of the liquid in the islet retention area will not be affected when the effluent is drawn, so the captured islets will not undergo obvious position changes, which is convenient for automatic imaging. In addition, the added reservoir structure can make the pressure inside the channel more stable.

[0019] On the other hand, the inventors found in experiments that the systematic errors between batches produced during the islet isolation process are large, and only quantitative comparisons between parallel experiments or repeated experiments of the same batch have biological significance. Therefore, in the microfluidic chip of the present invention, 2-4 functional units are symmetrically arranged in the flow channel layer, and each functional unit is provided with an environmental control module and a plurality of main channels connected to the environmental control module. The number of main channels in each functional unit may be 4-8. In the functional unit, each main channel is directly connected to the environmental control module through an intersection ( Figure 1 a, 1f), so it is possible to switch the type and / or concentration of the solution in the main channel at the desired time point through the environmental control module, and observe the response of the single islets staying in each islet retention area to the change in the type and / or concentration of the solution. Since the flow channel layer is provided with 2-4 independent functional units. Different environmental conditions can be applied in each functional unit, so that more than 4 repeated experimental group and control group experiments can be carried out on the same chip. In some embodiments, the single isolated islets loaded into each functional unit are all from more than two animals. For example, the experimental group concentration can be given in the first functional unit, and the control group concentration can be given in the second functional unit, so as to obtain the responses of islets from the same source to different stimuli and the individual response differences to the same stimulus in the same batch of experiments, thereby avoiding the systematic errors caused by the differences in pretreatment operations of different batches. In the present invention, a group of channels interconnected are referred to as "functional units".

[0020] In an embodiment of the present invention, the environmental control module includes a liquid inlet, a filtration unit, a mixing channel, a buffer tank, a first fence channel and a branch channel arranged in sequence. Since all the main channels in the same functional unit are connected to the environmental control module through an intersection, the perfusion fluid is transported to multiple main channels in parallel with the environmental control module, so that the real-time control and regulation of the environment of the islet retention area can be achieved. The design of parallel branch channels can achieve independent and synchronous perfusion and imaging for multiple islets with different pretreatment conditions or different donors. This greatly improves the throughput and parallelism of the islet calcium imaging experiment, helps to increase the number of conditions and independent biological repetitions in the same group of experiments under research scenarios such as condition screening and pharmacological mechanism verification, and improves the experimental efficiency and the reliability of the experimental results. Compared with the prior art, the microfluidic chip of the present invention has obvious advantages in environmental uniformity, environmental switching speed, organoid morphology controllability and high-throughput analysis.

[0021] Specifically, the filter unit disposed between the liquid inlet and the mixing channel can prevent the chip from being blocked ( Figure 1 b). In a preferred embodiment, the filter unit is provided with more than two inlet channels, a filter array and a confluence channel. The filter array is a cylinder with a cross section of any shape, and the cross section may be square, triangular, or circular, but is not limited thereto. In some embodiments, the cross section of the cylinder is a square with a side length of 80-100 μm. There is a gap d between adjacent cylinders, and the size of the gap d is defined as the distance between the two closest points on the structure of two adjacent cylinders. The filter array is preferably a cylinder with more than three columns. In some embodiments, the cylinders are evenly arranged, and the gap d is 20-30 μm. In other embodiments, the gap d between cylinders in different layers of arrays decreases step by step from top to bottom. In a preferred embodiment, the filter unit comprises three inlet channels, and the ratio of the total width of the inlet channel, the maximum width of the filter array, and the width of the confluence channel is preferably 2:16:1. In a preferred embodiment, in order to reduce the jet effect, the ratio of the inlet channel to the cross-sectional area of ​​the filter unit is 1:8 to 1:5. Most preferably, the filter cross-sectional width is 1.6 mm and the length is 0.8 mm. The optimization of the filter unit size can ensure the solution switching speed under the conditions required for calcium imaging experiments.

[0022] In an embodiment of the present invention, the number of liquid inlets of the environmental control module in each functional unit is preferably 2-8, so as to achieve rapid switching of solute types (such as glucose and drugs), or to use solutions of multiple concentration orders of magnitude to achieve cross-order concentration gradient output, and to construct a concentration time gradient in the islet retention area. This design also reflects the scalability of the present invention. In addition, the provision of a spare liquid inlet provides redundancy, which can avoid internal pressure instability caused by debris blockage during the PDMS drilling process.

[0023] In an embodiment of the present invention, a mixing channel M is provided downstream of the confluence channel of the filtration unit. In order to reduce the jet effect, the width ratio of the confluence channel of the filtration unit to the mixing channel is 1:2 to 3:5. In order to maintain a precisely controllable microenvironment for a long time under the microfluidic system, the mixing channel is provided with a plurality of staggered herringbone-shaped micromixers [4]( Figure 1 c). Each staggered herringbone micromixer includes a plurality of grooves. This structure can achieve rapid mixing and uniform distribution of the solution at a small volume and low flow rate by introducing turbulence into the solution in the channel, and can achieve a preset concentration change even when the solution is quickly replaced, while reducing the damage of the islet cells to the shear force, maintaining sufficient oxygen and nutrient supply, and prolonging the time for the islets to maintain physiological activity. In a preferred embodiment, each staggered herringbone micromixer includes 5-10 grooves. In a preferred embodiment, the width of the mixing channel is 150-200 μm, each groove has a width of 30-40 μm, and the interval between two adjacent grooves is 40-50 μm. In some embodiments, the height of the mixing channel is 80-120 μm, and the height difference between the groove and the interval is preferably 30 μm. In a preferred embodiment, the plurality of staggered herringbone micromixers are stacked in the channel, and the patterns of two adjacent opposing staggered herringbone micromixers are mirror-symmetrical along the stacking direction of the plurality of staggered herringbone micromixers. In a preferred embodiment, in order to achieve effective mixing, the total length of the mixing channel is 30mm-38mm. This length can ensure more than 95% terminal mixing efficiency while avoiding the large perfusion delay and fluid resistance problems caused by longer channels. According to experimental measurements, under the above parameters, the minimum solution switching cycle (the minimum length of each step) when the solution mixing zone outputs a step-like concentration change can reach the order of 30 seconds. As mentioned above, this design can also optimize the use of imaging space, thereby rationally arranging the islet retention area.

[0024] In an embodiment of the present invention, a buffer pool B is provided at the outlet of the mixing channel, and a first fence channel is provided between the buffer pool and the branch channel, so that when bubbles exist in the solution, the bubbles can be captured by the buffer pool to avoid causing a drastic change in pressure in the environmental control module. In a preferred embodiment, the buffer pool has a radius of 0.7-0.8 mm and a volume of 0.4 μL-0.6 μL; the height of the buffer pool is the fifth height. The first fence channel can prevent bubbles captured in the buffer pool from entering the downstream branch channel to maintain the stability of the perfusion system. The first fence channel is composed of no less than 3 connecting channels with a length of 100-300 μm and a width of 60 μm. In some embodiments, the height of the first fence channel is 100-110 μm.

[0025] In an embodiment of the present invention, by a first stage ( Figure 4 )、Level II( Figure 6 ) or level 3 ( Figure 1 ) branches into 4 or 8 parallel branch channels with the same flow resistance, thereby ensuring that the flow resistance of the islet retention areas of each main channel controlled by the environmental control module is substantially the same. In a preferred embodiment, the total length of the branch channel is 20-35 mm and the width is 100-150 μm. In some embodiments, the height of the branch channel is 100-110 μm.

[0026] In an embodiment of the present invention, a second fence channel ( Figure 1 e), thereby preventing the islets from flowing back into the branch channel along with the liquid. In a preferred embodiment, the second fence channel is composed of no less than 2 connecting channels with a length of 100-300 μm and a width of 35 μm. In some embodiments, the height of the second fence channel is 100-110 μm.

[0027] In a preferred embodiment, for the purpose of simple preparation, the inlet channel and the filter array of the filter unit in the environmental control module have a second height. In the environmental control module, the confluence channel, the mixing channel, the first fence channel, the branch channel and the second fence channel of the filter unit have a third height. The groove of the staggered herringbone micro-mixer in the environmental control module has a fourth height. The main channel and the buffer tank have a fifth height. This design can reduce the number of alignments in template preparation and improve the repeatability of the structure preparation process. The first to fifth heights are given in other parts of this article.

[0028] In summary, the present invention can avoid the generation of free-moving bubbles in the solution to the greatest extent by setting multiple liquid inlets, setting a filtering unit containing microcolumns at the liquid inlet, setting a buffer tank downstream of the mixing channel, and setting a fence channel at the outlet of the buffer tank and the connection between the branch channel and the main channel, and avoid the debris falling off the chip punching place from entering the perfusion area, maintain a stable and reliable perfusion state for a long time, and meet the requirements of perfusion imaging and culture systems. In terms of size, the chip of the present invention is based on the principle of fluid mechanics, comprehensively considering the volume of the islet, the magnification of the microscope, the liquid replacement speed, etc., to select the width and height of each structure. By optimizing the size of the first mixing zone, the second mixing zone and the parallel branch channel and the number of herringbone-shaped micromixers, it is not only possible to reduce the delay of the stimulation signal and realize the preset waveform while maintaining a faster liquid replacement speed, but also to effectively utilize the imaging space, rationally layout the sample injection area and the observation area, and realize up to 16 parallel processes on a single chip.

[0029] The chip of the present invention provides a dynamically changing environment with controllable frequency and waveform through an environmental control module. Since it can realize rapid and multiple waveform switching of the influent concentration, it can provide a microenvironment that matches the physiological state for the pancreatic islets, and can quantitatively collect the dynamic response signals (such as calcium concentration, endogenous hormones) of the pancreatic islets to the concentrations of nutrients (such as glucose, amino acids) and drugs (such as hormones, chemical genetics induction factors) in real time, so as to facilitate the study of highly dynamic changes in the pancreatic islets and oscillatory secretion patterns. Since the chip of the present invention is simple to operate, has strong stability and good repeatability, it avoids the problem that the isolated cells in the 2D in vitro model are difficult to reconstruct the sub-organ morphology and function, and can ensure high experimental throughput and parallelism. Compared with the traditional isolated islet perfusion tool, it has significant advantages and can be used as a standardized platform for studying the physiological and pathological characteristics of the pancreatic islets, the pathogenesis of diabetes, and the screening of targeted drugs and treatment methods.

[0030] The present invention also provides a method for imaging pancreatic islets using the chip of the present invention, the method comprising: injecting a pancreatic islet suspension into the chip through a sample loading inlet to load the pancreatic islets; injecting a perfusion solution into the chip through a liquid inlet; providing an environment in which the concentration of the solution changes over time in the chip; and imaging the pancreatic islet retention area. During the imaging process, the concentration ratio of the solution perfused by each liquid inlet is controlled in real time, thereby providing an environment that changes over time for the pancreatic islets, so as to observe the response of the pancreatic islets to the environment. By controlling the perfusion speed, a mechanical force that is compatible with physiological conditions can be provided without damaging the tissue, sufficient nutrition can be provided, and insulin and glucagon can be taken away for further detection. In a preferred embodiment, the chip can be vented before sample loading, and the sample loading inlet can be closed after sample loading. In an embodiment in which a liquid reservoir is provided, the effluent can be sucked out from the liquid reservoir to measure the hormone concentration therein.

[0031] Since the method of the present invention can reconstruct the physiological environment of pancreatic islets in vitro and simulate pancreatic islet functions, the system and method of the present invention can be advantageously used to develop treatment methods for pancreatic islet-related diseases such as diabetes and pancreatic cancer, especially personalized treatment methods.

[0032] Example

[0033] Example 1

[0034] 1. Preparation of Microfluidic Chip

[0035] Utilize multilayer SU8 photolithography to make the template of chip of the present invention. First utilize acetone and isopropanol to clean silicon wafer, and use nitrogen drying. Then expose silicon wafer to oxygen plasma, oxidize remaining organic matter. Spin-coat SU83025 for 30 seconds with the rotating speed of 2200 rev / min, obtain 40 μ m thickness, pre-bake 15 minutes on 95 ℃ hot plate, carry out suitable exposure time according to the technical parameters of photoresist manufacturer, and then post-bake 5 minutes on 95 ℃ hot plate. For making process simplicity, after coating and exposure of each layer, development is temporarily not carried out, but coating and exposure of next layer are continued, and development is carried out uniformly after all layers are completed. Spin-coat SU83025 for 30 seconds with the rotating speed of 3100 rev / min in a similar manner, obtain three layers of 30 μ m thickness and expose, and the pre-bake and post-bake time of these three steps are the same as the first step. Finally, SU83050 was spin-coated at 800 rpm for 30 seconds to obtain a thickness of 170 μm (300 μm in total), pre-baked for 60 minutes, exposed, and post-baked for 15 minutes. The template was developed with SU8 developer to remove the uncross-linked photoresist. The above production process was completed in a yellow light room to avoid unwanted photocross-linking. The template structure obtained is as follows Figure 1 In this design, the chip contains two functional units that are symmetrical on the left and right.

[0036] A chip with a microfluidic channel pattern was prepared using a standard polydimethylsiloxane (PDMS) soft lithography method. PDMS glue A and glue B were mixed in a ratio of 8:1 and poured onto the template. The mixture was placed in a vacuum oven and evacuated for 10 minutes to remove bubbles. The mixture was then placed in a forced air drying oven and cured at 70°C for 1 to 2 hours. The cured PDMS was peeled off from the template, the chip body was cut out along the edge of the chip, and holes were punched at the inlet and outlet of the chip using a 0.8 mm or 1 mm diameter circular puncher, and the debris was removed using Scotch tape. The PDMS pattern surface and the glass surface were treated with oxygen plasma, and the two were bonded by extrusion. Before imaging, the chip and all solutions were evacuated for 5 minutes using a vacuum pump.

[0037] 2. Characterization of Microfluidic Chip

[0038] The chip perfusion efficiency was characterized by injecting fluorescein (Rhodamine 6G) solution and performing microscopic imaging.

[0039] The injection pump (TS-1B, Langer Constant Flow Pump Co., Ltd.) was controlled by Matlab program to introduce 20 μM and 1 μM fluorescein solutions at different flow rate ratios from two of the liquid inlets of the chip (the other two spare liquid inlets were blocked with plastic nails during the test), to produce square waves, triangle waves, sine waves and step-like concentration changes over time, and record the changes in fluorescence signals in each islet retention area.

[0040] Figure 2 b shows the concentration changes at a total flow rate of 3200 μL / h using a program-controlled injection pump to output a square wave with a period of 12 min, a triangle wave with a period of 20 min, or a sine wave with a period of 5 min or 20 min (the sampling frequency of the flow rate change signal output by the injection pump control program is 1 Hz). Figure 2 In b, the dotted line is the experimentally measured fluorescence intensity of the highest step in the islet retention area of ​​the 8 main channels, and the black solid line is the average of the above fluorescence intensities; the light blue filling is the theoretical value of the change in fluorescein concentration, which is determined according to the input of the injection pump control program, and the light red filling is the theoretical value after considering the delay caused by the channel volume. The dotted line and the black solid line basically overlap, indicating that the solution is mixed evenly after passing through the mixing zone, and the perfusion concentration consistency between channels is high. Therefore, there is no substantial difference in the fluorescence intensity (fluorescein concentration) of each islet retention area, and the change of the fluorescence intensity (fluorescein concentration) in the islet retention area over time is basically consistent with the theoretical value (the error is within 1%). In addition, the total flow rate was controlled to 1600, 2400 or 3200 μL / h, and the program was used to achieve a step-like concentration change of 1 μM-4 μM-5 μM-6 μM-7 μM-8 μM-10 μM-12 μM-15 μM-20 μM. At each concentration value (corresponding to the flow rate ratio of the two pumps) with a residence time of 4 min, 5 min or 6 min, the change in fluorescence intensity (fluorescein concentration) in the islet residence area over time was basically consistent with the theoretical value, with a maximum error of less than 0.5 μM (corresponding to 2.5% of the highest concentration).

[0041] In the above tests, the experimentally measured delay of concentration change relative to the switching of the injection pump (mainly caused by the volume of the channel in the environmental control module) was 1.2 min, which is basically consistent with the theoretical calculated value (0.6 min). The changes in the fluorescence signals of each channel over time are basically consistent and consistent with the theoretical value. The above results show that the chip of the present invention can achieve a concentration gradient of any shape in time with acceptable time resolution and accuracy, and achieve good concentration consistency in each islet retention area, and can be used to implement parallel experiments.

[0042] In addition, we tested the effect of the micromixer. Figure 2 As shown in a, due to the limited diffusion effect of laminar flow, the difference in fluorescence intensity on both sides of the same position at the entrance of the mixing channel can reach 60%-70%; after the fluid passes through the herringbone micromixer, the unevenness of the fluorescence intensity in the mixing channel is reduced to less than 5%. For a smooth channel of the same size without a micromixer, the diffusion coefficient of the fluorescein aqueous solution is taken as D = 400μm 2 / s

[12] , it can be calculated that the channel length required for uniform mixing is of the order of magnitude L~(vL 2 / D) ~ 1000mm, which is much larger than the actual mixing channel length used, confirming the effectiveness of the micromixer.

[0043] The structural parameters and performance parameters of the chip involved in Example 1 are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] 3. Isolation and Culture of Pancreatic Islets

[0048] Labeling of mice with Ins1-Cre using a fluorescent probe specific for calcium ions in β cells + ;GCaMP6f fl+

[11] Calcium imaging of pancreatic islets was performed. Mice aged 8-20 weeks were euthanized and the pancreas was perfused through the common bile duct with freshly prepared collagenase P solution (0.5 mg / ml). The perfused pancreas was digested at 37°C for 17 minutes and the islets were isolated under a stereoscope. The time of isolation was defined as day 0. After isolation, the islets were transferred to RPMI 1640 medium supplemented with 10% fetal bovine serum (10099141C, Gibco), 5.5 mM D-glucose, 100 units / ml penicillin and 100 mg / ml streptomycin and cultured overnight in an incubator at 37°C and 5% carbon dioxide; imaging was performed on days 1 and 2 (10 am-10 pm). The time between islet isolation and experiment was usually 17h-53h.

[0049] 4. Perfusion and Imaging

[0050] Before use, the microfluidic chip was perfused with KRBB solution (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1.2 mM MgCl2, 1 mM L-glutamine, 25 mM HEPES and 0.1% BSA, pH 7.4) containing 3 mM D-glucose. The vented solution was injected into the microfluidic chip from the PA single-lumen tube (0.45 × 0.8 mm, Shenzhen Kaisite Medical Technology Co., Ltd.) through the inlet S1 and S4 using a 10 mL syringe (needle 25G × 20 mm). If necessary, the solution was injected from each opening of the chip to exhaust bubbles or use the spare inlet S2 or S3. Under a stereoscope, the cultured single islet was aspirated into a 10 μL pipette and from Figure 1The sample loading inlet of each main channel in a is injected into the chip. Since the diameter of a single islet is generally 50μm-300μm, the islet will stay in the islet retention area with a step structure at the top. After injection, the sample loading inlet is closed. The sophisticated design of the chip of the present invention can minimize the pressure change in the chip during the liquid switching process. Therefore, during the imaging process, the islet can remain stable for a long time, and the liquid replacement will not cause the position of the islet to change.

[0051] During the imaging process, the mouse islets in the microfluidic chip were kept at 37°C on the microscope stage, and the KRBB buffer containing 1mM and 20mM glucose was automatically pumped into the microfluidic chip from the inlet of the environmental control module at a program-set flow rate ratio, with a total flow rate of 1600μL / h (the solution was distributed to 8 independent channels, and the flow rate was 200μL / h for each islet). Wide-field calcium imaging of the islets was performed using a Nikon Plan Apo 10x objective (NA=0.45) with an Andor Dragonfly spinning disk confocal system using the 488-GFP channel.

[0052] The above method was used to culture Ins1-Cre in vitro under different conditions + ;GCaMP6f fl+ Calcium imaging was performed on pancreatic islets of transgenic mice, with 16 islets imaged simultaneously. Figure 2 c shows the microscope bright field image after the islet suspension is introduced into the main channel. Due to the step structure, islets of different sizes can stay stably, and only a single islet stays in each islet retention area. In order to match the movement speed of the microscope translation stage, the time resolution of the imaging was set to 4.5 seconds. This sampling rate is sufficient to capture various modes of calcium oscillation signals of a single islet (the minimum oscillation period is about 15 seconds). As a pretreatment, the control group was given 8mM glucose solution on the chip for 21 hours (8G21h) before the step-up stimulation; the low-sugar group was perfused with 1mM glucose solution for 21 hours (1G21h) before stimulation; and the high-sugar group was perfused with 20mM glucose solution for 21 hours (20G21h). As shown Figure 2As shown in Figure d, in response to the step-up of glucose concentration (1mM-3mM-4mM-5mM-6mM-7mM-8mM-10mM-12mM-15mM-20mM, each concentration lasted 15min), the calcium activity of each islet clearly showed a sequence of rest-activation, synchronous oscillation-sustained activation. For islets that have undergone different pretreatments, the activation threshold and oscillation threshold for switching between the rest-activation state and the synchronous oscillation-sustained activation state are very different. The activation threshold and oscillation threshold of pancreatic β cells cultured in a long-term low-concentration glucose environment have significantly shifted to the right compared with the untreated control group (increased by 1-2mM, corresponding to the decreased glucose sensitivity of pancreatic β cells), while the islets cultured in a long-term high-concentration glucose environment have shifted to the left in the opposite direction (decreased by 2-4mM, corresponding to the increased glucose sensitivity of pancreatic β cells). This is consistent with the results reported in previous literature. Using this chip, we were able to perform synchronous imaging of multiple islets processed in the same batch, reproducing the results that previously required multiple batches of repetitions in a low-throughput perfusion imaging system; this experimental method reduced the batch effect, making the observed intra-group and inter-group differences more reliable, while greatly improving the experimental efficiency. The glucose response translation signal obtained by the chip of the present invention is clearer and has a high signal-to-noise ratio, so that the oscillation threshold and activation threshold can be used as characteristic parameters of the glucose response to achieve quantification of the research system. The above tests confirmed the reliability and efficiency of the experimental system.

[0053] 5. Screening of key paracrine factors in the regulation of pancreatic islet glucose dose-response movement

[0054] Under physiological and pathological conditions, the glucose dose-response curve of pancreatic β-cell secretion activity is dynamically regulated by the external long-term nutritional environment, showing changes in glucose sensitivity [8-10]. This regulatory process is an important part of the body's long-term blood glucose regulation mechanism and is closely related to the compensation and failure of β-cells in the development of type 2 diabetes. Previous studies on the mechanism of this regulatory process have focused on the intrinsic mechanism of β-cells themselves, lacking systematic studies that consider the long-term effects of paracrine interactions within the pancreas from the perspective of the pancreas as a whole.

[0055] In this context, we designed an automated experimental process to screen for paracrine factors that contribute to the shift in the islet glucose dose-response curve. Figure 3 As shown in a, for the isolated Ins1-Cre + ;GCaMP6f fl+Mouse islets were cultured for a long time (18 to 22 hours) in a culture medium containing different high concentrations of paracrine factors, and then the in vitro cultured islets were loaded onto the chip. As a pretreatment, before giving a step-up stimulus, a 3mM glucose solution was given on the chip for 15 minutes to balance the islet calcium activity to a resting state, and then calcium imaging was performed under perfusion of a glucose solution with a step-up concentration to obtain the average calcium activity curve of the islets, quantify and count the offset of the activation threshold and oscillation threshold relative to the control group. For the overall calcium signal curve of the pancreatic β cells obtained from the calcium imaging image, preprocessing was first performed, and the signal was subtracted from the minimum value (background fluorescence value) and divided by the minimum value for standardization to exclude the influence of the fluorescent protein expression and relatively accurately reflect the changes in the intracellular calcium ion concentration, and obtain the calcium signal ΔF / F curve. Figure 3 b The figure above shows the pre-treatment calcium signal curve of pancreatic β cells after culture under 8mM glucose concentration without additional paracrine factors. On this basis, the signal of each perfusion glucose concentration interval was intercepted, and after deducting the first 25% (which may include a sudden response that did not reach a stable state when the glucose concentration increased), the least squares quadratic polynomial fitting line of the interval signal was subtracted (using MATLAB's detrend tool) to remove the overall signal trend caused by factors such as fluorescent protein quenching, and obtain a relatively stable calcium oscillation signal. The final calculated difference between the maximum and minimum values ​​of the signal is the overall amplitude of pancreatic β cells at this glucose concentration. Figure 3 b The figure below shows the change of the overall amplitude with glucose concentration. For each islet, the corresponding glucose concentration value when the calcium signal amplitude first reaches 25% of the maximum value (linear interpolation is used between actual glucose concentration points) is defined as the activation threshold of the β cells in the islet, and the corresponding glucose concentration value when the calcium signal amplitude first drops below 60% of the maximum value after reaching the maximum value (linear interpolation is used between actual glucose concentration points) is defined as the oscillation threshold of the β cells in the islet. This image processing and data quantification process can be completed automatically by writing a program.

[0056] The paracrine factors we screened included hormones secreted by the three major neuroendocrine cells in the pancreas (insulin, glucagon, somatostatin) and competitive inhibitors of the two somatostatin receptors SSTR2 (mainly expressed in α cells) and SSTR3 (mainly expressed in β cells). Taking into account the individual differences of different mice, each group of conditions was set up with at least 3 independent replicates of mice and at least 2 technical replicates of each mouse. The screening results showed that the only pancreatic hormone among the three islet hormones that caused a significant shift in the islet glucose response curve was glucagon (Glucagon, Figure 3 c), under the action of long-term high-concentration glucagon, the activation threshold and oscillation threshold of β cells in the pancreatic islet shifted significantly to the right (decreased glucose sensitivity). Figure 3c. In the experiment using CYN 154806 (hereinafter referred to as CYN), a specific inhibitor of SSTR2, and MK 4256, a specific inhibitor of SSTR3, the CYN group also had a relatively obvious threshold shift to the right, while the MK 4256 group was relatively unobvious. This shows that relieving the inhibition of pancreatic δ cells on the secretion activity of pancreatic α cells can also play a similar role as the addition of glucagon, verifying the long-term effect of glucagon.

[0057] Table 2 Activation threshold shift and oscillation threshold shift of islets cultured with various paracrine factors

[0058]

[0059] In the table, n represents the number of mice independently repeated, and NS means no significance.

[0060] Combined with the short-term paracrine effect of glucagon reported in previous literature (activating the cAMP-PKA pathway through GCGR and GLP1-R to promote insulin secretion from β cells) [4-7], the long-term effect of glucagon found in this experiment that shifts the β-cell glucose dose response to the right (relatively reduces sensitivity) is a relatively counterintuitive phenomenon. Repeated experiments and quantification results showed that the activation threshold of islets cultured in the medium containing 100nM glucagon for 21 hours shifted to the right by 1.2mM ( Figure 3 d, left); the mean oscillation threshold also shifted rightward by 2.4 mM, but this was not statistically significant ( Figure 3 d, right side), which may be partly due to the fact that the concentration steps taken at high sugar concentrations are relatively sparse, so the interpolation results have large errors.

[0061] It is difficult to process and experimentally measure directly in vivo, but the difference in glucose dose response between islets with different cell composition and structure in the same individual can be measured by the chip of the present invention, which can indirectly prove the effect of long-term paracrine action on the glucose dose response of pancreatic β cells. We infer that in the same mouse, the long-term level of glucagon in the paracrine environment of islets with high α-cell content is correspondingly higher. Therefore, based on previous results, for the pancreatic islets of the same mouse, the activation and oscillation threshold of β-cell calcium activity should be positively correlated with the number of α-cells in the islets. By transplanting mice Glu-Cre specifically expressing the red fluorescent marker protein tdTomato in pancreatic α-cells + ; tdTomato f / f mice were mated with fully labeled GCaMP mice to obtain α-tdt GCaMP mice that can simultaneously perform calcium imaging and statistics on the number and distribution of α cells. The high-throughput calcium imaging and activation threshold quantification results of this mouse preliminarily confirmed this inference ( Figure 3e), providing evidence for the existence of a long-term role of glucagon in regulating the glucose dose response of β cells under physiological conditions in vivo.

[0062] 6. Imaging under different glass thicknesses

[0063] Considering the difference in working distance of objective lenses with different magnifications, we used slides of different thicknesses (Xinxiang Weike Science and Education Instrument Co., Ltd.) to prepare the above chip and tested the imaging effect. The basic design uses a 1mm thick slide, and a Nikon Plan Apo 10x objective lens (NA = 0.45) can be used with the Andor Dragonfly spinning disk confocal system for wide-field imaging; when a 0.2mm thick slide is used, Nikon Plan Apo 10x objective lens (NA = 0.45), 40x objective lens (NA = 0.85), and 63x oil objective lens (NA = 1.40) can all be imaged normally.

[0064] 7. Imaging of Hormone Secretion Using Fluorescent Membrane Probes

[0065] In addition to calcium imaging, our array can also be used to image mouse islets with genetically encoded membrane probes, allowing high-throughput measurement of islet hormone secretion activity. fl+ ;EIIa-Cre+;ATP-sensor fl+ ; Transgenic mouse islets can simultaneously image the calcium activity and extracellular ATP release of pancreatic β cells (corresponding to the release of insulin vesicles). In this experiment, the chip we used used a 0.2mm thick slide, a Nikon Plan Apo 40x objective (NA=0.85) and an Andor Dragonfly spinning disk confocal system to simultaneously image the calcium activity and secretion activity of 4-8 islets at a time resolution of 3 seconds, and obtain the temporal and spatial distribution information of pancreatic hormone secretion.

[0066] Example 2

[0067] 1. Preparation of Microfluidic Chip for Hormone Collection

[0068] The template of the chip of the present invention is made by using multi-layer SU8 photolithography technology. The basic manufacturing process is the same as that described in Example 1. The obtained template structure is as follows: Figure 4 In this design, the chip contains two functional units that are symmetrical on the left and right, and each functional unit contains four liquid inlets and four islet retention areas.

[0069] A chip with a microfluidic channel pattern was prepared using a standard polydimethylsiloxane (PDMS) soft lithography method. PDMS glue A and glue B were mixed in a ratio of 8:1 and poured onto the template. The mixture was placed in a vacuum kettle and evacuated for 10 minutes to remove bubbles. The mixture was then placed in a forced air drying oven and cured at 70°C for 1 to 2 hours. The cured PDMS was peeled off from the template, and the chip body was cut out along the edge of the chip. A 0.8mm or 1mm diameter circular puncher was used to punch holes at the inlet and outlet of the chip. A 4mm or 6mm diameter puncher was used to punch holes at 5-8mm (0.6mm) from the outlet of each liquid outlet. Figure 4 The reservoir structure was obtained by punching holes (at the P position shown), and the debris was removed with Scotch tape. The PDMS pattern surface and the glass surface were treated with oxygen plasma and bonded by extrusion. Before imaging, the chip and all solutions were evacuated for 5 minutes with a vacuum pump.

[0070] 2. Characterization of Microfluidic Chips for Hormone Collection

[0071] The solution mixing efficiency, switching efficiency and uniformity of this design were tested using the same method as in Example 1. The injection pump was controlled by the Matlab program to pass 20 μM and 1 μM fluorescein solutions from two of the inlets of the chip (the other two spare inlets were blocked with plastic nails in the test) at different flow rate ratios. At a total flow rate of 1600, 2400 and 3200 μL / h, the program was used to control the step-like concentration change of 1 μM-4 μM-5 μM-6 μM-7 μM-8 μM-10 μM-12 μM-15 μM-20 μM. At each concentration value (corresponding to the flow rate ratio of the two pumps) when the residence time was 4 min, 5 min or 6 min, the change in the fluorescence intensity (fluorescein concentration) of the islet residence area over time was basically consistent with the theoretical value, and the maximum error was less than 0.5 μM (corresponding to 2.5% of the highest concentration).

[0072] The structural parameters and performance parameters of the chip involved in Example 2 are shown in Table 3.

[0073] Table 3

[0074]

[0075]

[0076] 3. Simultaneous determination of pancreatic islet calcium activity and hormone secretion activity

[0077] The pancreatic β-cell-specific calcium ion fluorescent probe Ins1-Cre was isolated and cultured using the same method as in Example 1. + ;GCaMP6f fl+Transgenic mouse islets. The chip was perfused and loaded with islets in the same manner as in Example 1, and a 30 cm long PA single lumen tube (0.45×0.8 mm, Shenzhen Kaisite Medical Technology Co., Ltd.) was inserted into the 8 reservoirs for the purpose of aspirating the outflow fluid.

[0078] During the imaging process, the mouse islets in the microfluidic chip were kept at 37°C on the microscope stage. KRBB buffer containing 1mM and 20mM glucose was automatically pumped into the microfluidic chip from the inlet at a programmed flow rate ratio by a TS-1B syringe pump (LongerPump), with a total flow rate of 1600μL / h (the flow rate was 400μL / h for each islet). The glucose concentration ladder used in the experiment was 3mM-4mM-5mM-6mM-7mM-10mM-12mM-15mM-20mM. Except for the time corresponding to 10mM glucose, which was 60min, the time spent at each concentration was 15min. Wide-field calcium imaging of the islets was performed using a Nikon Plan Apo 10x objective (NA=0.45) with an Andor Dragonfly spinning disk confocal system using the 488-GFP channel at a time resolution of 3s. Every 15 minutes, 100 μL of the outflow liquid in this period was drawn from the previously inserted outlet channel using a microsyringe, collected in a 150 μL centrifuge tube (Eppendorf), and temporarily stored on ice. After the experiment, the collected outflow liquid was stored at -80°C for future use.

[0079] For the effluent of each group and time point, the concentrations of the two hormones were measured according to the manufacturer's protocol using the glucagon AlphaLISA immunoassay kit (PerkinElmer) and the human / mouse insulin shared AlphaLISA immunoassay kit (PerkinElmer) to obtain the insulin and glucagon secretion of each islet in each glucose concentration stimulation section. The above hormone secretion data were divided by the total cell volume of the corresponding islet (estimated by the 3 / 2 power of the maximum cross-sectional area of ​​the fluorescent image) for standardization to obtain the average hormone secretion rate per unit cell volume of each islet in each glucose stimulation section, that is, the glucose dose response curve of the two hormone secretion. Figure 5As shown, for the control group islets cultured at a moderate glucose concentration (8mM) and the islets cultured at a high glucose concentration (20mM) for a long time (~40h), the high glucose concentration culture group showed a consistent hypersensitive phenotype in the glucose dose response of calcium activity and insulin secretion activity (the dose response curve shifted to the left). On the other hand, the glucagon secretion rate per unit cell volume of the two groups of islets showed a similar downward trend with the increase of glucose concentration. Among them, the glucagon secretion of the 8G group showed a slight rebound when the external glucose concentration reached 10mM or above, and this phenomenon was not obvious in the 20G group. The slight rebound in the secretion rate under 8G conditions is consistent with literature reports. The above results show that with the help of the microfluidic chip of this embodiment, it is possible to achieve stable and reliable synchronous monitoring of the calcium activity and secretion activity of the islets, and capture the functional differences of the islets under different treatment conditions and different physiological states.

[0080] Example 3

[0081] Fabrication and characterization of a four-functional unit microfluidic chip

[0082] The template of the chip of the present invention is made by using multi-layer SU8 photolithography technology. The basic manufacturing process is the same as that described in Example 1. The obtained template structure is as follows: Figure 6 As shown. In this design, the chip contains a total of 4 symmetrical functional units, each of which contains 4 liquid inlets and 4 islet retention areas. The chip with a microfluidic channel pattern was prepared using the same soft lithography method as in Example 1 and characterized. The structural parameters and performance parameters of the chip involved in Example 3 are shown in Table 4.

[0083] Table 4

[0084]

[0085]

[0086] The chip can realize repeated experiments under four experimental conditions and four times under each condition. The chip of Example 1 or the chip of Example 3 can be selected independently according to the required number of repetitions and experimental conditions.

[0087] References

[0088] 1.Lab Chip, 2009, 9, 97-106, https: / / doi.org / 10.1039 / B809590F

[0089] 2.Sci.Adv.2019,5,11,DOI:10.1126 / sciadv.aax4520

[0090] 3.Lab Chip,2016,16,1466-1472,DOI https: / / doi.org / 10.1039 / C5LC01173F

[0091] 4.Science,2002,295,5555,DOI:10.1126 / science.1066238

[0092] 5.J Endocrinol,1995,147,87-93,doi:DOI 10.1677 / joe.0.1470087.

[0093] 6.Diabetologia,2002,43,1012-1019,doi:DOI 10.1007 / s001250051484.

[0094] 7.JCI Insight,2019,4,doi:10.1172 / jci.insight.126742.

[0095] 8.Diabetes,1992,41,792-806.

[0096] 9.Am J Physiol-Endoc M,1995,268,E21-E27,doi:DOI10.1152 / ajpendo.1995.268.1.E21(1995).

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[0099] 12.Talanta,2002,56(2),365-373,doi:10.1016 / S0039-9140(01)00602-6。

Claims

1. A microfluidic chip, the microfluidic chip is formed by bonding a substrate and a flow channel layer, wherein: The flow channel layer is provided with 2-4 functional units; each of the functional units is independently provided with an environmental control module and a plurality of main channels connected to the environmental control module; the environmental control module is provided with a liquid inlet, a filtration unit, a mixing channel, a buffer tank, a first fence channel and a branch channel in sequence; the main channel is provided with a sample loading inlet, an inflow channel, an islet retention area, an outflow channel and an outlet in sequence; the inflow channel of the main channel is connected with the branch channel of the environmental control module at an intersection; wherein the mixing channel is provided with a plurality of staggered herringbone-shaped micro-mixers; the intersection is provided with a second fence channel; the islet retention area includes a step structure and a liquid drainage channel; The islet retention area comprises a four-layer step structure, wherein the first step, the second step, the third step and the fourth step of the step structure have a first height, a second height, a third height and a fourth height, respectively, and the first height, the second height, the third height and the fourth height are 30-40µm, 60-80µm, 90-120µm and 120-150µm, respectively; the inflow channel and the outflow channel have a fifth height, and the fifth height is 250-350µm; or The islet retention area comprises a three-layer step structure, wherein the first step, the second step and the third step of the step structure have a first height, a second height and a third height, respectively, and the first height, the second height and the third height are 30-40µm, 60-80µm and 90-120µm, respectively; the inflow channel and the outflow channel have a fifth height, and the fifth height is 250-350µm; Wherein, the liquid drainage channels are arranged on both sides of the step structure and are parallel to the flow direction of the perfusion liquid, and each of the liquid drainage channels has a width of 40-50 μm and a height of the first height; Wherein, the total length of the main channel at the islet retention area is 500-1000 μm, and the width is 350-450 μm.

2. The microfluidic chip according to claim 1, wherein: The flow channel layer is provided with two functional units; each of the functional units is independently provided with an environmental control module and eight main channels connected to the environmental control module; in the environmental control module, the branch channels include three-level branch channels.

3. The microfluidic chip according to claim 1, wherein: The flow channel layer is provided with four functional units; each of the functional units is independently provided with an environmental control module and four main channels connected to the environmental control module; in the environmental control module, the branch channels include two levels of branch channels.

4. The microfluidic chip according to claim 1, wherein: The flow channel layer is provided with two functional units; each of the functional units is independently provided with an environmental control module and four main channels connected to the environmental control module; in the environmental control module, the branch channel includes a primary branch channel; each main channel is further provided with a liquid reservoir, and the liquid reservoir is located downstream of the islet retention area.

5. The microfluidic chip as claimed in claim 4, wherein: The liquid storage tank is arranged at a distance of 5-8 mm from the outlet of the main channel.

6. The microfluidic chip according to claim 4, wherein: The diameter of the liquid reservoir is 4-6 mm.

7. The microfluidic chip according to claim 1, wherein: The total length of the inflow channel and the outflow channel of each main channel is 8-14 mm.

8. The microfluidic chip according to claim 1, wherein: The filtration unit is provided with more than two inlet channels, a filtration array and a merging channel.

9. The microfluidic chip according to claim 8, wherein: The inlet channel and the filter array have a second height; the merging channel has a third height.

10. The microfluidic chip according to claim 8, wherein: The ratio of the total width of the inlet channel, the maximum width of the filter array and the width of the confluence channel is 2:16:

1.

11. The microfluidic chip according to claim 8, wherein: The ratio of the width of the merging channel of the filtration unit to the width of the mixing channel is 1:2 to 3:

5.

12. The microfluidic chip according to claim 1, wherein: The mixing channel is provided with 30-40 staggered herringbone-shaped micromixers; each of the staggered herringbone-shaped micromixers independently comprises 5-10 grooves.

13. The microfluidic chip according to claim 12, wherein: The mixing channel has the third height and the slot has the fourth height.

14. The microfluidic chip according to claim 12, wherein: The mixing channel has a width of 150-200 μm, the grooves each independently have a width of 30-40 μm, and adjacent grooves each independently have a spacing of 40-50 μm.

15. The microfluidic chip according to claim 1, wherein: The buffer pool has a radius of 0.7-0.8 mm and a volume of 0.4 μL-0.6 μL; and the buffer pool has the fifth height.

16. The microfluidic chip according to claim 1, wherein: The first fence channel consists of more than three connecting channels.

17. The microfluidic chip according to claim 16, wherein: The connecting channel in the first fence channel has a length of 100-300 μm and a width of 60 μm; the first fence channel has the third height.

18. The microfluidic chip according to claim 1, wherein: The branch channel has the third height.

19. The microfluidic chip according to claim 1, wherein: The second fence channel is composed of more than two connecting channels.

20. The microfluidic chip according to claim 19, wherein: The length of the connecting channel in the second fence channel is 100-300 μm and the width is 35 μm; the second fence channel has the third height.

21. The microfluidic chip according to any one of claims 1 to 20, wherein: The base layer is a glass slide, wherein the thickness of the glass slide is 1 mm or 0.2 mm.

22. A method for imaging pancreatic islets using the chip according to any one of claims 1 to 21, the method comprising: injecting the islet suspension into the chip through the sample loading inlet; Injecting a perfusion solution into the chip through a liquid inlet; Providing an environment in the chip where the solution concentration changes over time; as well as The islet retention area is imaged.

23. A method for imaging pancreatic islets using the chip of claim 4, the method comprising: injecting the islet suspension into the chip through the sample loading inlet; Injecting a perfusion solution into the chip through a liquid inlet; Providing an environment in the chip where the solution concentration changes over time; imaging the islet retention area; and The effluent was aspirated from the reservoir and the hormone concentration therein was determined.

24. Use of the microfluidic chip according to any one of claims 1 to 21 or the method according to any one of claims 22 to 23 in perfusion culture of pancreatic islets, functional imaging, and screening of therapeutic drugs for pancreatic islet-related diseases.

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