CN119685162A - Microfluidic device and method for cell experiment - Google Patents

Abstract

The invention discloses a microfluidic device and a method for cell experiments, wherein the device comprises a packaging layer, a liquid flow layer and a culture layer, a plurality of groups of culture tanks are arranged on the culture layer, a plurality of groups of liquid injection grooves are arranged on the bottom surface of the liquid flow layer, the liquid injection grooves are arranged above the culture tanks and communicated with the culture tanks, a plurality of groups of middle layer sample adding holes and middle layer drainage holes are vertically arranged at two ends of the liquid injection grooves, a plurality of groups of upper layer sample adding holes and upper layer drainage holes are arranged on the packaging layer, the upper layer sample adding holes and the upper layer drainage holes are respectively and correspondingly arranged above the middle layer sample adding holes and the middle layer drainage holes, the method comprises the steps of S1 packaging the packaging layer, the liquid flow layer and the culture layer from top to bottom, S2 and exhausting, S3 adding experimental cells, S4 connecting the upper layer sample adding holes and the upper layer drainage holes by an automatic liquid adding device, S5 overturning the whole device, and directly settling the cells to the liquid flow layer, and rapidly obtaining the cells by perfusion.

Description

Microfluidic device and method for cell experiment

Technical Field

The invention relates to the technical field of experimental devices, in particular to a microfluidic device and a microfluidic method for cell experiments.

Background

The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed.

Tumor cells (containing tumor stem cells) obtained by separating tumor tissues of a patient by using a tumor organoid are subjected to 3D culture in a micro-environment which is built in a similar body by adding a certain cytokine and a small molecule, so that a miniature tumor model is formed, the miniature tumor model has similar structural characteristics and functional characteristics as those of the tumor tissues from which the tumor tissue is derived, and the miniature tumor model can be stably amplified in an in vitro 3D culture system. Three key points are involved in the tumor organoids, the first is the material drawing, the second is the culture protocol, and the third is the identification protocol.

Chinese patent publication No. CN202211584503.9, a drug screening tumor organoid chip and a drug screening method, discloses a drug screening tumor organoid chip and a drug screening method. The method comprises the steps of microfluidic chip preparation, aqueous two-phase solution preparation, single cell suspension preparation, microfluidic control, single cell loaded microgel generation and high-flux anticancer drug screening of pancreatic cancer organoids. According to the invention, a micro-fluidic chip integrated with a normally-closed pneumatic pump valve is used as a technical platform, single-cell suspension which is incubated by a cross-linking agent in advance is used as a chemical reaction core, water drops in water are used as a molding template, and single-dispersed seed cells are loaded in a micro hydrogel carrier by a one-step method, so that efficient single-cell in-situ loading, pancreatic cancer organoid construction and high-flux drug screening are realized. The method improves the consistency of the organoid culture environment and the starting point, simultaneously has the characteristic of high flux of a liquid drop microfluidic technology, realizes the in-vitro construction of the organoids and the screening of anticancer drugs with high efficiency and high reproducibility, and can play a great role in the fields of basic research, transformation application and the like related to pancreatic cancer organoids.

The inventor discovers in long-term experiments that the structure of the microfluidic device, the size, the height and the like of the culture tank have obvious influence on the culture of tumor organoids.

Disclosure of Invention

The invention overcomes the problems and provides a microfluidic device and a method which can be used for culturing tumor organoids and synchronously carrying out a plurality of groups of experiments.

To achieve the purpose, the invention provides the following technical scheme:

The invention provides a microfluidic device for cell experiments, which comprises a packaging layer, a liquid flow layer and a culture layer from top to bottom, wherein a plurality of groups of culture grooves are formed in the culture layer, each group of culture grooves comprises a plurality of culture holes which are adjacently arranged, a plurality of groups of liquid injection grooves are formed in the bottom surface of the liquid flow layer, the liquid injection grooves are arranged above the culture grooves and are communicated with the culture grooves, a plurality of groups of middle layer sample adding holes and middle layer drainage holes are vertically formed in two ends of the liquid injection grooves, a plurality of groups of upper layer sample adding holes and upper layer drainage holes are formed in the packaging layer, and the upper layer sample adding holes and the upper layer drainage holes are correspondingly arranged above the middle layer sample adding holes and the middle layer drainage holes respectively.

Preferably, the height of the culture well is 0.2-0.3mm.

In the invention, the height of the culture hole is 0.2-0.3mm, which can promote the aggregation of tumor organoids and can not settle to the bottom of the culture hole. The height of the culture hole is 0.2-0.3mm, and the replacement of the liquid medicine or the culture solution is not affected. The liquid medicine or the culture solution can be replaced by utilizing uninterrupted gas-liquid exchange and scouring of mechanical shearing force, and the aggregation of tumor organoids in the culture holes is not influenced.

Preferably, the height of the liquid injection groove is 0.3-0.8mm.

In the invention, the height of the liquid injection groove has a great influence on the generated gas-liquid exchange and the magnitude of mechanical shearing force. The height of the liquid injection groove is 0.3-0.8mm, which can just simulate the growth environment of cells in vivo and promote the normal growth of cells.

Preferably, the aperture of the culture well is 0.2mm.

Preferably, the surfaces of the culture tank and the liquid injection groove are provided with a hydrophobic coating.

Preferably, the encapsulation layer, the fluid flow layer and the culture layer are all transparent materials.

Preferably, the culture tanks are not less than 6 groups.

In the invention, the culture tank is not less than 6 groups, and each group is provided with independent sample adding holes and drainage holes, so that experiments can be independently carried out. In particular, in the drug concentration screening experiment, the drug concentration can be adjusted according to the experimental progress.

In a second aspect of the invention, there is provided a method of cell assay comprising the steps of:

S1, packaging a packaging layer, a liquid flow layer and a culture layer from top to bottom;

S2, after sterilization, exhausting by using a vacuum filtration method;

S3, respectively adding experimental cells into a plurality of groups of culture tanks of the culture layer;

S4, two ends of the automatic liquid adding device are respectively connected with the upper layer sample adding hole and the upper layer drainage hole, and the liquid flow layer can realize uninterrupted gas-liquid exchange and scouring of mechanical shearing force through the automatic liquid adding device, so that a more similar environment with the body is provided for cell growth;

s5, turning over the whole device, and enabling the cells to directly settle to a liquid flow layer, so that the cells can be rapidly obtained through perfusion.

Preferably, the automatic liquid adding device comprises a peristaltic pump.

Preferably, the cell assay comprises rapid in situ expansion of tumor organoid cells, rapid assessment of multiple drug treatment modalities, and cell acquisition following assessment, with tumor organoids co-cultured with immune cells.

The liquid flow layer is characterized in that a plurality of groups of culture grooves are arranged on the culture layer, each group of culture grooves comprises a plurality of culture holes which are adjacently arranged, a plurality of groups of liquid injection grooves are arranged on the bottom surface of the liquid flow layer, the liquid injection grooves are arranged above the culture grooves and are communicated with the culture grooves, a plurality of groups of middle layer sample adding holes and middle layer drainage holes are vertically arranged at two ends of the liquid injection grooves, a plurality of groups of upper layer sample adding holes and upper layer drainage holes are arranged on the packaging layer, and the upper layer sample adding holes and the upper layer drainage holes are correspondingly arranged above the middle layer sample adding holes and the middle layer drainage holes respectively.

Preferably, the height of the culture well is 0.2-0.3mm.

Preferably, the height of the liquid injection groove is 0.3-0.8mm.

Preferably, the aperture of the culture well is 0.2mm.

Preferably, the surfaces of the culture tank and the liquid injection groove are provided with a hydrophobic coating.

Preferably, the encapsulation layer, the fluid flow layer and the culture layer are all transparent materials.

Preferably, the culture tanks are not less than 6 groups.

Compared with the prior art, the invention has the beneficial effects and remarkable progress that:

1. According to the microfluidic device for the cell experiment, through the unique structural design and the adjustment of parameters such as the height of the culture hole, the height of the liquid injection groove and the like, the microfluidic device for the cell experiment can simulate a normal in-vivo environment, and an optimal experiment environment is created for the culture and experiment of cells, particularly tumor organoids;

2. experiments prove that the microfluidic device for cell experiments can be used for drug experiments with different concentrations, cell culture and acquisition experiments and the like.

Drawings

In order to more clearly illustrate the technical solution of the present invention, a brief description will be given below of the drawings that are required to be used for the embodiments of the present invention.

It is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and that other drawings may be obtained from these drawings without inventive faculty for a person skilled in the art, but these other drawings also fall within the drawings required for the embodiments of the present invention.

FIG. 1 is an exploded view of a microfluidic device for cell experiments according to example 1 of the present invention;

FIG. 2 is a top view of a microfluidic device for cell experiments according to example 1 of the present invention;

FIG. 3 is a side view of a microfluidic device for cell experiments according to example 1 of the present invention;

FIG. 4 is an enlarged view of FIG. 2 at A;

FIG. 5 is a perspective view of a microfluidic device for cell experiments according to example 1 of the present invention;

FIG. 6 is a graph of cell growth within the microfluidic device of example 2 of the present invention;

FIG. 7 shows the results of a cell drug sensitivity assay in a microfluidic device according to example 3 of the present invention;

FIG. 8 shows the results of a cell drug sensitivity assay in a microplate of example 3 of the present invention;

FIG. 9 shows the experimental results of killing tumor organoids by chemotherapeutic agents in the microfluidic device co-culture system of example 5 of the present invention;

Fig. 10 shows the experimental results of the combined treatment of a chemotherapeutic agent and an immune checkpoint inhibitor agent in a microfluidic device co-culture system according to example 5 of the present invention.

In the figure, 1, a packaging layer, 2, a liquid flow layer, 3, a culture layer, 1.1, an upper layer sample adding hole, 1.2, an upper layer drainage hole, 2.1, a middle layer sample adding hole, 2.2, a middle layer drainage hole, 2.3, a liquid injection groove, 3.1 and a culture tank.

Detailed Description

In order to make the objects, technical solutions, beneficial effects and significant improvements of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings provided in the embodiments of the present invention.

It is evident that all of these described embodiments are only some, but not all, embodiments of the present invention, and that all other embodiments, based on the embodiments of the present invention, that a person of ordinary skill in the art would achieve without inventive effort are within the scope of the present invention.

It should be noted that the terms "first," "second," and "third" (if any) in the description and claims of the present invention and the drawings of the embodiments of the present invention are used merely for distinguishing between different objects and not for describing a particular sequential order. Furthermore, the terms "comprise," "include," and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed steps or elements but may include other steps or elements not listed or inherent to such process, method, article, or apparatus.

It is to be understood that:

In the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected or movably connected, or integrally formed, directly connected, indirectly connected or intangible signal connected through an intermediary, or even optically connected, in communication with each other, or in an interaction relationship between two elements, unless otherwise explicitly specified.

The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.

It should also be noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments.

The following describes the technical scheme of the present invention in detail by using specific examples.

Example 1

As shown in figures 1-5, the microfluidic device for cell experiments comprises a packaging layer 1, a liquid flow layer 2 and a culture layer 3 from top to bottom, wherein a plurality of groups of culture grooves 3.1 are formed in the culture layer 3, each group of culture grooves 3.1 comprises a plurality of adjacent culture holes 3.2, a plurality of groups of liquid injection grooves 2.3 are formed in the bottom surface of the liquid flow layer 2, the liquid injection grooves 2.3 are arranged above the culture grooves 3.1, the liquid injection grooves 2.3 are communicated with the culture grooves 3.1, a plurality of groups of middle layer sample adding holes 2.1 and middle layer drainage holes 2.2 are vertically formed in two ends of the liquid injection grooves 2.3, a plurality of groups of upper layer sample adding holes 1.1 and upper layer drainage holes 1.2 are formed in the packaging layer 1, and the upper layer sample adding holes 1.1 and the upper layer drainage holes 1.2 are respectively and correspondingly arranged above the middle layer sample adding holes 2.1 and the middle layer drainage holes 2.2.

In this example, the height of the culture well 3.2 was 0.2mm.

In this embodiment, the height of the liquid filling groove 2.3 is 0.5mm.

In this example, the pore diameter of the culture well 3.2 was 0.2mm.

In this embodiment, the surfaces of the culture tank 3.1 and the liquid filling groove 2.3 are provided with a hydrophobic coating.

In this embodiment, the encapsulation layer 1, the fluid flow layer 2 and the culture layer 3 are all transparent materials.

In this example, the number of culture tanks 3.1 was not less than 6.

The method for using the microfluidic device for cell experiments of the embodiment comprises the following steps:

S1, packaging a packaging layer 1, a liquid flow layer 2 and a culture layer 3 from top to bottom;

S2, after sterilization, exhausting by using a vacuum filtration method;

S3, respectively adding experimental cells into a plurality of groups of culture tanks 3.1 of the culture layer 3;

S4, two ends of the automatic liquid adding device are respectively connected with the upper layer sample adding hole 1.1 and the upper layer drainage hole 1.2, and the liquid flow layer 2 can realize uninterrupted gas-liquid exchange and scouring of mechanical shearing force through the automatic liquid adding device, so that a more similar environment with the in-vivo environment is provided for cell growth;

s5, turning over the whole device, and enabling the cells to directly settle to the liquid flow layer 2, and obtaining the cells rapidly through perfusion.

In this embodiment, the automatic liquid feeding device comprises a peristaltic pump.

In this example, the cell experiments included rapid in situ expansion of human tumor organoid cells, rapid assessment of multiple drug multiple treatment modes, and cell acquisition after assessment.

Comparative example 1

The device of the comparative example comprises a packaging layer, a liquid flow layer and a culture layer from top to bottom, wherein a plurality of groups of culture grooves are formed in the culture layer, each group of culture grooves comprises a plurality of culture holes which are adjacently arranged, a plurality of groups of liquid injection grooves are formed in the bottom surface of the liquid flow layer and are arranged above the culture grooves, the liquid injection grooves are communicated with the culture grooves, a plurality of groups of middle layer sample adding holes and middle layer drainage holes are vertically formed in two ends of the liquid injection grooves, a plurality of groups of upper layer sample adding holes and upper layer drainage holes are formed in the packaging layer, and the upper layer sample adding holes and the upper layer drainage holes are correspondingly arranged above the middle layer sample adding holes and the middle layer drainage holes respectively.

In this example, the height of the culture well was 0.5mm.

In this embodiment, the height of the liquid injection groove is 1mm.

In this example, the diameter of the culture well was 0.5mm.

In this embodiment, the surfaces of the culture tank and the liquid injection groove are provided with a hydrophobic coating.

In this embodiment, the encapsulation layer, the fluidic layer, and the culture layer are all transparent materials.

In this example, the number of culture tanks is not less than 6.

Example 2 Rapid in situ expansion and acquisition of human tumor organoid cells

The microfluidic device for cell experiments of example 1 was used for cell expansion and harvesting of human tumor organoid cells. The amplification method is briefly summarized as follows:

Step 1, packaging a packaging layer, a liquid flow layer and a culture layer from top to bottom;

step 2, after sterilization, exhausting by using a vacuum filtration method;

step 3, adding human tumor organoid cells into a plurality of groups of culture tanks of the culture layer respectively;

Step 4, two ends of the automatic liquid adding device are respectively connected with the upper layer sample adding hole and the upper layer drainage hole, and a liquid flow layer can realize uninterrupted gas-liquid exchange and scouring of mechanical shearing force through the automatic liquid adding device so as to provide a more similar environment for cell growth in vivo;

And 5, turning over the whole device, and enabling cells to directly settle to a liquid flow layer, so as to rapidly obtain the human tumor organoid cells through perfusion.

Cell growth in the microfluidic device is shown in fig. 6, and it can be seen that cells are growing in clusters and are not growing in adherence. In summary, the microfluidic device of the invention realizes non-adherent culture of primary tumor cells, thus realizing rapid amplification of a small amount of humanized tumor primary cells in the microfluidic device. In addition, the upper part of the microfluidic device is provided with an uninterrupted liquid flow system, so that the rapid gas-liquid exchange of a culture system can be realized, and the defects of the traditional chip, such as narrow gap space, poor cell growth state and the like, are overcome. In addition, after the cells are cultured or processed, after the micro-fluidic device body is turned over, organoid cells can be directly deposited to an intermediate liquid flow layer, and the target cells can be rapidly obtained through perfusion and used for subsequent experiments, so that the phenotype screening and molecular difference analysis of the same cell sample are realized, and the smooth development of a subsequent drug resistance mechanism is ensured.

In this example, the device of comparative example 1 was also used to expand and obtain human tumor organoid cells, and the specific experimental procedure was as above. As a result, cells were collected at the bottom of the culture layer, and the cell growth state was poor.

EXAMPLE 3 multiple drug multiple modes of treatment

Human tumor organoid cell expansion was performed using the microfluidic device and microplate (commercially available) for cell experiments of example 1. The amplification method is briefly summarized as follows:

step 1, respectively culturing cell lines in a micro-pore plate and a micro-fluidic device;

step 2, using the same medicine and medicine concentration to stimulate,

And step 3, detecting the cell activity by using ATP or AO/PI method on the micro-porous plate, detecting the cell activity by using fluorescence quantitative method on the micro-fluidic device drug sensitive detection platform, and comparing the difference of the two platforms.

The results are shown in fig. 7 and 8, wherein fig. 7 shows the results of cell drug sensitivity experiments in the microfluidic device, and fig. 8 shows the results of cell drug sensitivity experiments in the microplate. Therefore, the microfluidic device can synchronously perform multiple groups of experiments, and experimental cells grow in a clustered mode and grow in a non-adherent mode. In summary, the microfluidic device of the invention retains the sample adding hole and the drainage hole, and can realize the combination screening of a plurality of medicines under the same culture condition by externally designing a multichannel system, so that the cell demand is small, the medicine concentration can be adjusted at any time according to the reaction of cells to medicines, the medicine sensitivity detection period is shortened, the medicine sensitivity detection efficiency is improved, and the medium-and-long-term medicine administration guidance, the sequential medicine administration scheme and the continuous evaluation of the curative effect are realized.

EXAMPLE 4 drug sensitive detection of body fluid samples

Drug sensitive detection of body fluid samples was performed using the microfluidic device for cell experiments of example 1, the experimental procedure being briefly summarized as follows:

step 1, after a body fluid sample is selectively cultured, cell micro-clusters are injected into a microfluidic device,

Step 2, realizing automatic cyclic drug delivery and providing nutritional ingredients by using an automatic liquid feeding device;

And 3, detecting the cell activity difference in each channel under various medicines and medicine concentrations.

EXAMPLE 5 Co-culture of tumor organoids and immune cells

The microfluidic device and microplate (commercially available) for cell experiments of example 1 were used for co-culture of tumor organoids with immune cells and target-immune and chemoimmune drug-responsive assays were performed using co-culture systems.

Step 1, respectively culturing organoids in a micro-pore plate and a micro-fluidic device;

step 2, adding immune cells marked by fluorescent dye into the micro-fluidic chip according to a certain target cell proportion after organoids are formed, and constructing a co-culture system;

And 3, adding a targeting drug, an immune drug or both into the co-culture system for treatment, and adding PI dye to detect the activity of organoid cells after the drug treatment for 4-7 days.

The results are shown in fig. 9 and 10, fig. 9 shows the experimental results of killing tumor organoids by the chemotherapeutic drugs in the micro-fluidic device co-culture system, and fig. 10 shows the experimental results of combined treatment of tumor organoids by the chemotherapeutic drugs and the immune checkpoint inhibitor drugs in the micro-fluidic device co-culture system. FIG. 9 shows that immune cells can enhance the killing effect of low-dose chemotherapeutics on tumor organoids, and FIG. 10 shows that the microfluidic device of the invention can be used for evaluating the killing effect of immune checkpoint inhibitors in combination with other drugs on tumor organoids.

In conclusion, the microfluidic device can be used for constructing a tumor organoid immune microenvironment by adding immune cells, so that the microfluidic device can be applied to multi-dimensional drug combination therapy efficacy evaluation, and the application scene of the microfluidic chip for guiding clinical drug selection is enriched.

In the description of the above specification:

The terms "this embodiment," an embodiment of the invention, "" such as "further," "further improved technical solutions," etc., mean that a particular feature, structure, material, or characteristic described in this embodiment or example is included in at least one embodiment or example of the invention, that a schematic representation of the above terms in this specification is not necessarily for the same embodiment or example, and that the particular feature, structure, material, or characteristic described, etc., may be combined or combined in any one or more embodiments or examples in a suitable manner, and that, furthermore, different embodiments or examples and features of different embodiments or examples described in this specification may be combined or combined by persons of ordinary skill in the art without creating contradictions.

Finally, it should be noted that:

the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting thereof;

Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some or all of the technical features thereof, without departing from the spirit of the technical solutions of the embodiments of the present invention, and that insubstantial improvements and modifications or substitutions by one skilled in the art from the disclosure herein are within the scope of the invention as claimed.

Claims (10)

1. A microfluidic device for cell experiments is characterized by comprising an encapsulation layer, a liquid flow layer and a culture layer from top to bottom,

A plurality of groups of culture tanks are arranged on the culture layer, and each group of culture tanks comprises a plurality of culture holes which are adjacently arranged;

The bottom surface of the liquid flow layer is provided with a plurality of groups of liquid injection grooves, the liquid injection grooves are arranged above the culture tank and are communicated with the culture tank, and two ends of the liquid injection grooves are vertically provided with a plurality of groups of middle layer sample adding holes and middle layer drainage holes;

The packaging layer is provided with a plurality of groups of upper layer sample adding holes and upper layer drainage holes, and the upper layer sample adding holes and the upper layer drainage holes are respectively and correspondingly arranged above the middle layer sample adding holes and the middle layer drainage holes.

2. A microfluidic device for cell experiments according to claim 1 wherein the culture wells have a height of 0.2-0.3mm.

3. A microfluidic device for cell experiments according to claim 1 wherein the height of the liquid injection groove is 0.3-0.8mm.

4. A microfluidic device for cell experiments according to claim 1 wherein the culture well has a pore size of 0.2mm.

5. The microfluidic device for cell experiments according to claim 1, wherein the surfaces of the culture tank and the liquid injection groove are provided with a hydrophobic coating.

6. The microfluidic device for cell experiments of claim 1 wherein said encapsulation layer, said fluid flow layer and said culture layer are all transparent materials.

7. A microfluidic device for cell experiments according to claim 1 wherein the culture tanks are not less than 6 groups.

8. A method of cell testing comprising the steps of:

S1, packaging a packaging layer, a liquid flow layer and a culture layer from top to bottom;

S2, after sterilization, exhausting by using a vacuum filtration method;

S3, respectively adding experimental cells into a plurality of groups of culture tanks of the culture layer;

S4, two ends of the automatic liquid adding device are respectively connected with the upper layer sample adding hole and the upper layer drainage hole, and the liquid flow layer can realize uninterrupted gas-liquid exchange and scouring of mechanical shearing force through the automatic liquid adding device, so that a more similar environment with the body is provided for cell growth;

s5, turning over the whole device, and enabling the cells to directly settle to a liquid flow layer, so that the cells can be rapidly obtained through perfusion.

9. A method of cell assay according to claim 8, wherein said automated liquid feeding device comprises a peristaltic pump.

10. The method of claim 8, wherein the cell assay comprises rapid in situ expansion of tumor organoid cells, rapid assessment of multiple drug multiple treatment modes, and cell acquisition after assessment, the tumor organoid being co-cultured with immune cells.