Vascular salivary gland acinus chip and rapid drug screening detection method thereof

Abstract

The invention belongs to the technical field of biomedicine, and provides a blood vessel salivary gland acinus chip and a rapid drug screening detection method thereof.The blood vessel salivary gland acinus chip is composed of a drug concentration generation chip, a porous membrane, a salivary gland acinus cell sphere layer and a salivary gland acinus sphere generation chip which are sequentially arranged from top to bottom; the aperture of the porous membrane is set to be 0.4-10 [mu] m, and the porous membrane is used for separating the drug concentration generation chip from the salivary gland acinus bulb generation chip; the drug concentration generation chip and the salivary gland acinulceration bulb generation chip can be exchanged with a culture medium; by simulating the physiological anatomical structure of the salivary gland acinus in vitro, high-flux rapid drug screening of new drugs on salivary gland diseases is completed on the basis of a salivary gland cell sphere microfluidic organ chip with a secretion function; according to the constructed blood vessel salivary gland acinus chip, the cell spheres are self-generated in the chip, and the experimental process is saved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, in particular to a blood vessel salivary gland acinus chip and a rapid drug screening detection method thereof. Background Art

[0002] The salivary glands are attacked by autoimmune diseases, resulting in reduced saliva secretion, making it difficult for patients to eat and speak, and increasing the risk of oral infections and tooth decay. The development of existing drugs and treatments often requires a long drug screening process, including animal experiments; In the existing salivary gland chip research, the main structure is to separate the primary tissue into cell spheres and pass them into the constructed spherical chip structure. The cell spheres are fixed in the structure with the help of hydrogel. The formed chip array structure is fixed in a 48-well plate. After 4 days of culture, specific structural immunofluorescence staining of each component of the salivary gland acinus is performed. Subsequently, the effect of new drugs on salivary gland cell spheres is observed through fluorescence experiments such as cell live-death experiments, thereby completing drug screening.

[0003] However, its ethical situation is complicated and time-consuming. Whether the primary salivary glands of healthy people are ethical is an issue that needs to be discussed urgently. The uneven size of primary tissues makes it difficult to homogenize the results of the subsequent characterization of drug effects (such as acinar atrophy), and the drug screening results are unstable. The chip structure of existing studies is not consistent with physiology. The size of normal salivary gland acinar is about 150-200μm, and multiple cell spheres can fall into the structure at the same time. This increases the difficulty for the subsequent characterization of drug screening efficacy. At the same time, it increases the uncertainty of the accuracy of drug screening results; each detection well can only use a single drug and a single concentration test, which is inefficient and cannot meet the research purpose of rapid drug screening; in terms of cell sphere generation, the hanging drop method is applied to the cell sphere pre-preparation process of salivary gland acinar cells. It is found that although the experimental equipment requirements of this technology are low, the size of the prepared cell spheres is inconsistent and does not have long-term culture conditions. It is impossible to achieve gland functionalization, and thus the research purpose of high-throughput rapid drug screening in this experiment cannot be achieved.

[0004] To this end, there is an urgent need for a suitable and efficient detection system to reproduce the physiological function of the salivary glands in vitro, construct a pathological model of Sjögren's syndrome, and then use new drugs for high-throughput and multi-concentration efficacy testing. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a vascular salivary gland acinar chip and a rapid drug screening and detection method thereof to solve the problems raised in the background technology.

[0006] According to the first aspect disclosed, a vascular salivary gland acinar chip is proposed, which is arranged in sequence from top to bottom and consists of a drug concentration generation chip, a porous membrane, a salivary gland acinar cell sphere layer and a salivary gland acinar sphere generation chip.

[0007] Preferably, the pore size of the porous membrane is set to 0.4-10 μm, which is used to separate the drug concentration generation chip and the salivary gland acinar sphere generation chip.

[0008] Preferably, the drug concentration generation chip and the salivary gland acinar sphere generation chip are capable of exchanging culture medium.

[0009] Preferably, the in vitro construction process of the vascular salivary gland acinar chip is: S1. Perform low adsorption treatment on the salivary gland acinar sphere generation chip; S2, introducing acinar cells into the salivary gland acinar sphere generation chip after low adsorption treatment; S3, acinar cells complete self-generation of cell spheres; S4, maintaining the growth of cell spheres by regularly changing the medium in the microfluidic channel; S5. After the size of the cell sphere reaches the physiological size in vivo, the drug concentration generation chip is introduced into the vascular endothelial cells to construct the capillary barrier layer; S6. Complete the construction of the self-generated cell ball vascular salivary gland acinar chip detection system.

[0010] According to the second aspect of the present disclosure, a rapid drug screening detection method is proposed, which applies the vascular salivary gland acinar chip of the first aspect, simulates the physiological and anatomical structure of the salivary gland acinar in vitro, and conducts high-throughput rapid drug screening of new drugs for salivary gland diseases based on the vascular salivary gland acinar chip with secretory function.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention simulates the physiological and anatomical structure of salivary gland acinus in vitro, and realizes high-throughput rapid drug screening of new drugs for salivary gland diseases based on the vascular salivary gland acinus chip with secretory function.

[0012] 2. The present invention uses the constructed vascular salivary gland acinar chip to enable cell spheres to be self-generated in the chip, saving experimental steps; after the cell suspension is introduced into the drug concentration generation chip and the salivary gland acinar sphere generation chip, the culture medium is replaced to construct the system.

[0013] 3. The present invention observes the fluorescence intensity of the cell spheroids under a fluorescence microscope to statistically analyze the effects of the developed drug on cell viability, acinar water secretion function, and reactive oxygen release levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A three-dimensional diagram of the upper and lower chip structures of the vascular salivary gland acinus of the present invention; Figure 2 This is a physical picture of the cell sphere self-generating chip structure of the present invention; Figure 3 It is a simulation diagram of the upper concentration chip of the present invention; Figure 4 Construct a flow chart for the chip of the present invention; Figure 5 This is a real picture of the surface treatment of the self-generated structure of the cell sphere of the present invention; Figure 5 (a) is a cross-sectional view of the self-generated structure of the cell sphere before surface treatment. Figure 5 (b) is a cross-sectional view of the self-generated structure of the cell sphere after surface treatment; Figure 6 This is a schematic diagram of the morphology of the self-generated structure of the cell sphere observed under a scanning electron microscope before and after surface treatment of the present invention; Figure 7 This is a picture showing the live and dead immunofluorescence double staining results of the cell spheres of the present invention; Figure 8 It is a statistical chart for monitoring the cell sphere growth curve of the present invention; Fig. 9 This is a schematic diagram of the structure of the salivary gland acinar rapid drug screening chip of the present invention; Fig.10 This is a schematic diagram of the rapid drug screening efficiency of the present invention. DETAILED DESCRIPTION

[0015] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0016] Embodiment 1: A vascular salivary gland acinus chip is proposed, as shown in the attached Figure 1 As shown, the chip is arranged from top to bottom, and is composed of a drug concentration generation chip, a porous membrane, a salivary gland acinar cell sphere layer, and a salivary gland acinar sphere generation chip. The chip generated in this three-dimensional view shows the upper concentration gradient chip, the lower cell sphere self-generated structure chip structure, and the combination relationship between the two.

[0017] The pore size of the porous membrane is set to 3 μm, which is used to separate the drug concentration generation chip and the salivary gland acinar sphere generation chip.

[0018] The drug concentration generation chip and salivary gland acinar sphere generation chip can exchange culture medium, and cells will not cross-link due to size restrictions.

[0019] The in vitro construction process of the vascular salivary gland acinar chip is as follows: S1. Perform low adsorption treatment on the salivary gland acinar sphere generation chip; S2, introducing acinar cells into the salivary gland acinar sphere generation chip after low adsorption treatment; S3, acinar cells complete self-generation of cell spheres; S4, maintaining the growth of cell spheres by regularly changing the medium in the microfluidic channel; S5. After the size of the cell sphere reaches the physiological size in vivo, the drug concentration generation chip is introduced into the vascular endothelial cells to construct the capillary barrier layer; S6. Complete the construction of the self-generated cell ball vascular salivary gland acinar chip detection system.

[0020] From the above, we can see that based on the physiological structure of the salivary glands, the mechanism of salivary gland autoimmune diseases and the route of administration, the results of the vascular salivary gland acinar chip reproduced the "vascular-salivary gland" administration route. The chip function realizes the functional reproduction of salivary gland acinar cells in vitro, and the chip structure enables the cell spheres to be self-generated and uniform in size.

[0021] As attached Figure 2 As shown, using the 3D printing equipment Prism MP-00-6L, according to the designed processing drawings and 3D models, using the light yellow BIO resin material with a curing wavelength of 405nm, the chip mold was 3D printed with a precision of 6μm. The picture shows the actual mold of the self-generated chip structure of the lower cell sphere. The upper right corner of the picture shows the enlarged cell sphere self-generated array combination structure for observation.

[0022] As attached Figure 3 As shown in the figure, the general computational fluid dynamics Fluent software was used to simulate the fluid of the concentration gradient of the upper concentration chip. The results show that the molar fraction of the terminal channel gradually changes from 0 to 1, indicating that the chip can generate extreme concentrations from pure solvent to pure solute. The simulation results verify the accuracy of the fluid dynamics model, indicating that a uniform concentration gradient is successfully formed inside the chip, indicating that the upper chip can effectively generate the required concentration gradient, providing an ideal high-throughput drug screening platform for subsequent simulation drug screening and further rapid detection experiments.

[0023] As attached Figure 4 As shown, Figure 4The chip construction flow chart is shown below. The chip design and STL files of the upper and lower chips are completed by computer. Polydimethylsiloxane (PDMS) mixed with prepolymer and cross-linker in a ratio of 10:1 is poured into the chip mold and heated and cured at 65°C for 5 hours to complete the PDMS curing. After cooling at room temperature, the PDMS chip is demolded. The upper chip construction is completed. Among them, the surface treatment of the lower cell sphere self-generated chip requires dripping 20: 1 (prepolymer: cross-linker) thin PDMS on the surface of the chip cell sphere self-generated structure to complete the chip surface treatment. It is cured at 65°C for 2 hours again, and the chip is packaged after cooling at room temperature to complete the construction of the lower chip.

[0024] As attached Figure 5 As shown, Figure 5 This is a real picture of the surface treatment of the self-generated structure of the cell sphere; Figure 5 (a) is a cross-sectional view of the self-generated structure of the cell sphere before surface treatment. Figure 5 (b) is a cross-sectional view of the self-generated structure of the cell sphere after surface treatment; The above results show that after the surface pretreatment of the cell spheroid self-generated structure, the surface of the cell spheroid self-generated structure changes from a step-like shape to a smooth concave surface. After measurement, the structure can accommodate cell spheroids with a maximum diameter of 200±25μm. This result is consistent with the design concept of this patent.

[0025] As attached Figure 6 As shown in the figure, in order to further understand the high-resolution surface morphology and microstructure of the cell sphere self-generated chip, the morphology of the chip before and after surface treatment was collected using a scanning electron microscope (SEM). After the chip was pre-treated with gold spraying (to improve conductivity), electron microscope images of the chip cell sphere self-generated structure were collected at 30 times, 100 times and 150 times magnification.

[0026] The results show that the cell sphere self-generated array structure on the chip is uniform and stable, and the surface is flat. After surface treatment, the circular step contour of the cell sphere self-generated structure changes from clear and sharp to blurred and smooth. Compared with the actual chip image, it can be seen that the blurriness of the cell sphere self-generated structure is negatively correlated with the distance from the center of the circle, that is, the closer to the center of the circle, the blurrier the step contour. The above chip morphology changes observed under a scanning electron microscope are consistent with Figure 5 (a) and (b) are consistent, further verifying the effectiveness and good uniformity of chip pretreatment.

[0027] As attached Figure 7As shown, in order to verify the viability of the cell spheres formed on the chip, the cell spheres were double-stained with live and dead fluorescence using Calcein-AM and Propidium Iodide (PI). Calcein-AM can penetrate the cell membrane and remove the AM group after being hydrolyzed by esterase in living cells to generate Calcein, which can emit strong green fluorescence, so living cells show green fluorescence under a fluorescence microscope. PI is used to stain dead cells with damaged cell membranes and emit red fluorescence. Using a laser confocal microscope, cell sphere images were collected in bright field and fluorescence channels under 488nm and 573nm excitation light conditions. The multi-channel fusion image in the lower right corner shows that the cell spheres are uniformly spherical, most of the surface area shows green fluorescence, and sporadic point-like red fluorescence is visible. The results show that the cell spheres have good vitality, meet the design requirements of this patent, and verify the vitality of the cell spheres formed on the chip.

[0028] As attached Figure 8 As shown, a gradient experiment was conducted with different numbers of inoculated cells. The cell sphere microscope bright field images were collected immediately after inoculation and 1-7 days in sequence, and the cell sphere diameter was measured. Through the above cell sphere growth curve monitoring, it was determined that 1000 cells per cell sphere generation unit can stably generate cell spheres with a diameter of 200μm. The experimental window period can last from 2 days to 7 days, thus determining the cell sphere experimental window period.

[0029] As attached Figures 9 and 10 As shown: Example 2: A rapid drug screening detection method is proposed, which uses the vascular salivary gland acinar chip in Example 1 to simulate the physiological and anatomical structure of the salivary gland acinar chip in vitro, and conducts high-throughput rapid drug screening of new drugs for salivary gland diseases based on the vascular salivary gland acinar chip with secretory function.

[0030] By observing the fluorescence intensity of the cell spheres in the lower salivary gland acinar chip under a fluorescence microscope, the effects of the developed drug on cell viability, acinar water secretion function, and reactive oxygen release levels can be statistically analyzed to complete rapid screening of drug efficacy.

[0031] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in the application. Without departing from the scope of the present invention, other replacements, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications still falling within the scope of the appended claims.

[0032] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment may not be described (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those features that are not relevant to implementing the invention).

[0033] It should be understood that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but for those of ordinary skill having the benefit of this disclosure, without undue experimentation, the development effort will be a routine task of design, fabrication, and production.

[0034] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A vascular salivary gland acinar chip, characterized in that: The device is arranged sequentially from top to bottom and is composed of a drug concentration generation chip, a porous membrane, a salivary gland acinar cell sphere layer and a salivary gland acinar sphere generation chip.

2. A vascular salivary gland acinar chip as claimed in claim 1, characterized in that: The pore size of the porous membrane is set to 0.4-10 μm, and is used to separate the drug concentration generation chip and the salivary gland acinar sphere generation chip.

3. The vascular salivary gland acinar chip according to claim 1, characterized in that: The drug concentration generation chip and the salivary gland acinar sphere generation chip are capable of exchanging culture medium.

4. The vascular salivary gland acinar chip according to claim 1, characterized in that: The in vitro construction process of the vascular salivary gland acinar chip is as follows: S1. Perform low adsorption treatment on the salivary gland acinar sphere generation chip; S2, introducing acinar cells into the salivary gland acinar sphere generation chip after low adsorption treatment; S3, acinar cells complete self-generation of cell spheres; S4, maintaining the growth of cell spheres by regularly changing the medium in the microfluidic channel; S5. After the size of the cell sphere reaches the physiological size in vivo, the drug concentration generation chip is introduced into the vascular endothelial cells to construct the capillary barrier layer; S6. Complete the construction of the self-generated cell ball vascular salivary gland acinar chip detection system.

5. A rapid drug screening detection method, using the vascular salivary gland acinar chip according to claims 1-4, characterized in that: By simulating the physiological and anatomical structure of salivary gland acinar cells in vitro, high-throughput rapid drug screening of new drugs for salivary gland diseases is carried out based on the vascular salivary gland acinar chip with secretory function.

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