Micro-fluidic chip integrating suspension cell culture and drug concentration generation

Abstract

The utility model provides a suspension cell culture and drug concentration generation integrated micro-fluidic chip, which structurally comprises culture units which are stacked in series, a liquid inlet and a liquid outlet which are mutually independent, each unit comprises a matrix, each culture layer is provided with an independent culture channel, and the micro-channel is modified by bovine serum albumin molecules. A closed loop is formed by vertical overlapping, and cell implantation is realized by a laminar flow phenomenon. And meanwhile, two driving channels are crossed with the culture channel and are separated from the culture channel by an elastic diaphragm. Two ends of the driving channel are communicated with the outside. The liquid inlet and the liquid outlet which are independent from each other automatically form a medicament with a gradient medicament concentration from the sample liquid inlet, the influence of the change of the concentration of the medicament on blood cell culture is researched, the chip suspension culture simulates the real environment of lung cells in vivo, and the bovine serum albumin molecule modification simplifies the cell implantation process. Medicaments with different concentrations are added into different channels, and the optimal concentration range is determined by observing the influence of the medicaments. The method has the advantages of high efficiency and economy.

Description

Technical Field

[0001] This utility model relates to the field of microfluidic chip structure, and more specifically, to a microfluidic chip that integrates suspended cell culture and drug concentration generation. Background Technology

[0002] The early concept of microfluidics can be traced back to the gas chromatographs fabricated on silicon wafers using photolithography in the 1870s. One of the most important characteristics of microfluidics is the unique liquid phenomena in a microscale environment, such as laminar flow and droplets. Utilizing these unique liquid phenomena, microfluidics technology can achieve a series of microfabrications and micromanipulations that are difficult to accomplish using conventional methods. Microfluidic chips are a type of biochip, in which cells are pre-cultured on cell culture units with microchannels. These microchannels are equipped with microvalvees that can control the flow rate of liquids at the nanoliter or picoliter level with extremely high efficiency. Due to their tiny structure, hundreds of microbial culture chambers can be easily integrated onto a single chip, significantly improving the efficiency of microbial experiments. Microfluidic cell chips provide a cell culture microenvironment similar to physiological conditions; however, these chips are mainly used for the culture of adherent cells, which can be cultured simply by injecting culture medium into the chip.

[0003] However, existing chip structures have some shortcomings that require improvement. Firstly, they lack a culture medium mixing structure, making them unsuitable for cells growing in suspension within the human body. Suspension culture, on the other hand, provides multiple culture units, each integrating a pneumatic micropump to drive the circulation of the culture medium and microorganisms within the channels, better simulating the growth environment of non-adherent cells. Furthermore, the concentration of different biological reagents significantly impacts cell culture. Therefore, leveraging the advantages of microfluidic suspension cell culture chips—convenience, speed, efficiency, and timely dispersion of nutrients and metabolites—to study the effects of different reagent concentrations on cells is urgent. This is beneficial not only for exploring the pharmaceutical safety and efficacy of suspension-grown cells such as blood cells (leukocytes, lymphocytes, and tumor cells) but also for exploring treatment methods and therapeutic drugs and dosages for immune system diseases. Therefore, we propose a microfluidic chip integrating suspension cell culture and drug concentration generation. Utility Model Content

[0004] The purpose of this invention is to address the problems identified in the existing background technology. This invention aims to study the relationship between drug efficacy and toxicity and drug concentration in the treatment of hematological diseases, thereby determining the optimal drug concentration range. Utilizing the advantages of microfluidic suspension cell culture chips—convenience, speed, efficiency, and the ability to promptly disperse nutrients and metabolites—this invention addresses the urgent need to study the effects of different reagent concentrations on cells. It is beneficial not only for exploring the pharmaceutical safety range and efficacy of suspension-grown blood cells such as leukocytes, lymphocytes, and tumor cells, but also for researching treatment methods, drugs, and dosages for immune system diseases.

[0005] This invention features an independent inlet that automatically creates a drug concentration gradient, allowing observation of changes in blood cell morphology, activity, proliferation, and apoptosis at different drug concentrations. This method simulates the in vivo environment of blood cells, better helping to determine the optimal drug concentration for treating blood diseases, ensuring the best therapeutic effect while avoiding the toxicity of excessively high concentrations.

[0006] To achieve the aforementioned objectives, this utility model provides the following technical solution: an integrated microfluidic chip for suspension cell culture and drug concentration generation, comprising a bottom layer composed of multiple culture layers stacked in series. Each culture layer has five independent culture channels, and is equipped with separate inlet and outlet ports to prevent cross-interference. The overlapping of the upper and lower layers forms five closed loops, with each culture channel interconnected to form a closed loop, providing a stable liquid environment for blood cell suspension culture.

[0007] The drive system comprises two independent linear drive channels. These channels are connected to the culture channels via an elastic diaphragm. Both ends of the drive channels are open to the outside environment. By introducing pressurized liquid or gas into the drive channels, the elastic diaphragm deforms, generating a driving force that propels the culture medium to circulate, thus suspending blood cells in a manner more closely mimicking their natural growth environment in the human body.

[0008] The microchannel network, with culture channels modified with bovine serum albumin molecules, simplifies cell implantation and the transport of culture medium and drugs. Because of the liquid difference between the inlet and outlet, gravity-driven flow utilizes the laminar flow phenomenon between the liquid streams to simplify cell implantation and the transport of culture medium and drugs.

[0009] The sample inlet and waste outlet system has independent solution inlets and waste outlets, allowing for the simultaneous addition of multiple different concentrations of reagents and effective waste discharge to avoid cross-contamination.

[0010] As a preferred technical solution of this utility model, it further includes a culture unit, the number of culture units is greater than 1, each culture unit includes a substrate, each substrate has two culture channels, and the culture channels are stacked one on top of the other to form a closed loop.

[0011] As a preferred technical solution of this utility model, the microchannel network is modified with bovine serum albumin molecules, and two driving channels are provided, each of which intersects with the culture channel and is separated by an elastic diaphragm.

[0012] As a preferred technical solution of this utility model, the substrate is provided with linear driving channels, and each of the intersecting driving channels is provided with an elastic membrane for isolating the driving channel from the culture channel.

[0013] As a preferred technical solution of this utility model, the independent liquid inlet includes a first independent liquid inlet, a second independent liquid inlet, and a third independent liquid inlet.

[0014] As a preferred technical solution of this utility model, the independent liquid outlet includes a first independent liquid outlet, a second independent liquid outlet, a third independent liquid outlet, and a fourth independent liquid outlet.

[0015] As a preferred technical solution of this utility model, the chip structure includes a first chip layer, a second chip layer, a third chip layer, and a fourth chip layer.

[0016] As a preferred technical solution of this utility model, a first-layer culture channel is provided on the second layer of the chip, and a second-layer culture channel is provided on the third layer of the chip.

[0017] Compared with the prior art, the beneficial effects of this utility model are as follows: In the solution of this utility model:

[0018] 1. Simulates the in vivo environment: It simulates the real environment in which suspended cells are cultured in the body, and the driving channel can simulate the flow of fluid in the body, so that cell growth is closer to the physiological state.

[0019] 2. Simplified cell implantation: The microchannels are modified with bovine serum albumin molecules, which accelerates the cell implantation process.

[0020] 3. Preventing cell aggregation and precipitation: To prevent suspended cells from agglomerating, the drive channel provides mechanical power to the culture unit, enabling cells to be dispersed and cultured in a suspended state, preventing cell precipitation and providing sufficient space for cell growth.

[0021] 4. Facilitates the exchange of nutrients and metabolites: The driving channels keep the nutrients in the cell culture medium in a continuous state of flow, which accelerates the uptake of nutrients by suspended cells and can promptly remove the cell's metabolic products, which is beneficial to cell growth.

[0022] 5. Study the effects of different drug concentrations on cells: Equipped with independent inlet and outlet ports, it can automatically generate different drug concentrations and effectively discharge waste liquid, avoiding cross-contamination and facilitating the study of optimal drug concentrations.

[0023] 6. High efficiency and economy: Combining suspension culture and microfluidic technology for drug concentration generation has the advantages of high efficiency and economy. Attached Figure Description

[0024] Figure 1 A schematic diagram of the layer structure of the culture unit shown in this utility model.

[0025] The image shows:

[0026] 100, Chip Layer 1; 200, Chip Layer 2; 300, Chip Layer 3; 400, Chip Layer 4;

[0027] 1. First independent liquid inlet; 2. Second independent liquid inlet; 3. Third independent liquid inlet;

[0028] 111. First independent liquid outlet; 131. Second independent liquid outlet; 141. Third independent liquid outlet; 151. Fourth independent liquid outlet;

[0029] 112. Drive channel;

[0030] 211. First layer of chip culture channel; 212. Second layer of chip culture channel. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of this utility model clearer, the technical solutions of the embodiments of this utility model will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this utility model.

[0032] Therefore, the following detailed description of the embodiments of this utility model is not intended to limit the scope of the claimed utility model, but merely illustrates some embodiments of this utility model. All other embodiments obtained by those skilled in the art based on the embodiments of this utility model without inventive effort are within the scope of protection of this utility model. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of this utility model can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0033] Example 1: Please refer to Figure 1This microfluidic chip integrates suspension cell culture and drug concentration generation. It consists of a bottom layer composed of multiple culture layers stacked in series. Each culture layer has five independent culture channels, with separate inlet and outlet ports to prevent cross-interference. The overlapping of the upper and lower layers forms five closed loops, with each culture channel interconnected to provide a stable liquid environment for blood cell suspension culture.

[0034] The drive system comprises two independent linear drive channels 112. The drive channels 112 are connected to the culture channels, with an elastic diaphragm between them. Both ends of the drive channels 112 are connected to the outside environment. By introducing a certain pressure of liquid or gas into the drive channels 112, the elastic diaphragm deforms, generating a driving force that propels the culture medium to circulate, thus suspending blood cells in a manner closer to their growth environment in the human body.

[0035] The microchannel network, with culture channels modified with bovine serum albumin molecules, simplifies cell implantation and the transport of culture medium and drugs. Due to the liquid difference between the inlet and outlet, gravity-driven flow utilizes the laminar flow phenomenon to simplify cell implantation and the transport of culture medium and drugs.

[0036] The sample inlet and waste outlet system has independent solution inlets and waste outlets, allowing for the simultaneous addition of multiple different concentrations of reagents and effective waste discharge to avoid cross-contamination.

[0037] The number of culture units is greater than 1. Each culture unit includes a substrate, and each substrate has two culture channels. The culture channels are stacked one on top of the other to form a closed loop.

[0038] The microchannel network is modified with bovine serum albumin molecules. Two drive channels 112 are provided, each of which intersects with the culture channel and is separated by an elastic septum.

[0039] The substrate is provided with linear drive channels 112, and each cross drive channel 112 is provided with an elastic membrane to isolate the drive channel from the culture channel.

[0040] The independent liquid inlets include a first independent liquid inlet 1, a second independent liquid inlet 2, and a third independent liquid inlet 121.

[0041] The independent outlets include a first independent outlet 111, a second independent outlet 131, a third independent outlet 141, and a fourth independent outlet 151. The third independent outlet 141 has a micro-filter pore with a diameter of 50 μm, smaller than the cell to be tested, added inside the circular hole. A membrane switch is provided on the micro-filter pore, and the opening and closing of the third independent outlet 141 is controlled by the membrane switch.

[0042] The chip structure includes a first chip layer 100, a second chip layer 200, a third chip layer 300, and a fourth chip layer 400.

[0043] The second layer 200 of the chip has a first layer culture channel 211, and the third layer 300 of the chip has a second layer culture channel 212.

[0044] The chip comprises one or more culture units, each including a substrate with two culture channels. These are stacked to form a closed loop. The microchannels within the chip are modified with bovine serum albumin molecules to simplify the cell implantation process. Two driving channels 112 are also provided, each intersecting the culture channel and separated by an elastic septum. The substrate has several linear driving channels 112, and each intersecting driving channel is equipped with an elastic membrane to isolate the driving channel from the culture channel. Independent inlet and outlet ports are also provided. A drug concentration gradient is automatically generated from the sample inlet to study the effect of reagent concentration changes on blood cell culture, further understanding the relationship between drug efficacy and toxicity and drug concentration, thereby determining the optimal concentration range.

[0045] The preparation method of the integrated microfluidic chip structure of suspension culture and reagent concentration responsiveness of this utility model includes the following steps: Step 1. Design stage: Use computer-aided design software to design and draw the microstructure and microchannel pattern of each layer of the microfluidic chip, accurately plan the layout and size of culture channel, drive channel 112, sample inlet and waste liquid discharge hole, etc., to ensure that the functions of each part are realized in a coordinated manner.

[0046] Step 2. Microfabrication stage: Using microfabrication techniques such as laser etching, LIGA technology, molding, hot pressing, and soft etching, the required microstructures and microchannels, including culture channels, drive channels 112, liquid inlets, and liquid outlets, are fabricated on the selected chip substrate surface and elastic diaphragm.

[0047] Step 3. Surface modification stage: Bovine serum albumin solution is injected into the microchannel for surface modification and fixation, simplifying the blood cell implantation process while ensuring that the modification process does not affect other functions of the chip.

[0048] Step 4. Assembly stage: Using a double-layer adhesive film, the chip layers are precisely aligned, tightly bonded, and pressure-sealed to form an integrated microfluidic chip, ensuring the sealing between layers and the connectivity of microchannels, while preventing liquid leakage and channel blockage.

[0049] This invention comprises multiple culture layers stacked in series at the bottom. Each culture layer has five independent culture channels, with separate inlet and outlet ports to prevent cross-interference. The overlapping of the upper and lower layers forms five closed loops, and the interconnected culture channels provide a stable liquid environment for blood cell suspension culture.

[0050] The drive system comprises two independent linear drive channels 112. The drive channels 112 are connected to the culture channels, with an elastic diaphragm between them. Both ends of the drive channels 112 are connected to the outside environment. By introducing a certain pressure of liquid or gas into the drive channels 112, the elastic diaphragm deforms, generating a driving force that propels the culture medium to circulate, thus suspending blood cells in a manner closer to their growth environment in the human body.

[0051] The culture channels of the microchannel network are modified with bovine serum albumin molecules, simplifying blood cell implantation and the transport of culture medium and drugs. Because of the liquid difference between the inlet and outlet, gravity-driven flow utilizes the laminar flow phenomenon between the liquid streams to simplify cell implantation and the transport of culture medium and drugs.

[0052] The sample inlet and waste outlet system has independent solution inlets and waste outlets, allowing for the simultaneous addition of multiple different concentrations of reagents and effective waste discharge to avoid cross-contamination.

[0053] This novel chip includes one or more culture units, each including a substrate with two culture channels. These are stacked to form a closed loop. The microchannels within the chip are modified with bovine serum albumin molecules to simplify the cell implantation process. Two driving channels 112 are also provided, each intersecting the culture channel and separated by an elastic septum. The substrate has several linear driving channels 112, and each intersecting driving channel is equipped with an elastic membrane to isolate the driving channel from the culture channel. Independent inlet and outlet ports are also provided. A drug concentration gradient is automatically formed from the sample inlet port to study the effect of reagent concentration changes on blood cell culture, further understanding the relationship between drug efficacy and toxicity and drug concentration, thereby determining the optimal concentration range.

[0054] This chip-based suspension culture simulates the real environment of lung cells in vivo. Modification with bovine serum albumin simplifies the cell implantation process. Different concentrations of drugs are added to different channels, and their effects are observed to determine the optimal concentration range. It offers advantages of high efficiency and cost-effectiveness.

[0055] This novel chip combines suspension culture with reagent concentration-responsive microfluidic technology. The chip structure includes an inlet, an outlet, culture channels, drive channels, and an elastic membrane. Cells and cell culture medium enter through the inlet of the first layer of the chip, passing through microchannels whose surfaces are modified with bovine serum albumin. Due to the liquid difference between the inlet and outlet, gravity is generated, simplifying the cell implantation process within the microchannels. This is a publicly disclosed patent in this field, as shown in patent CN103060197A. The chip also features serially stacked culture units, each with its own culture channel. The overlapping of upper and lower culture layers forms a closed loop. Each culture channel has its own inlet and outlet, preventing cross-contamination. Furthermore, the fourth layer of the chip has two drive channels parallel to the culture channels. Furthermore, an elastic membrane exists between the fourth driving channel and the culture layer. By introducing liquid or gas at a certain pressure into the driving channel at a specific time sequence, a certain force is generated to deform the elastic membrane, thereby dispersing microorganisms or agents in the liquid environment. This is a well-known technology in the field, as disclosed in the chip in patent CN102234614B. The driving force facilitates the exchange of nutrients and metabolites: the driving channel keeps the nutrients in the cell culture medium in a continuous flow state, accelerating the uptake of nutrients by suspended cells and promptly removing cell metabolites, which is beneficial to cell growth. The driving force prevents cell aggregation and precipitation: to prevent suspended cells from agglomerating, the driving channel provides mechanical power to the culture unit, enabling cells to be dispersed and kept in suspension, preventing cell precipitation and providing sufficient growth space for cells. The driving force can simulate the real environment of suspended cells in vivo, mimicking the flow of liquid in vivo, making cell growth closer to the physiological state. Furthermore, by adding 100% concentrated drug solution and physiological saline through two independent inlets on the first layer of the chip, a concentration gradient can be automatically formed. This allows observation of changes in cell morphology, cell activity, cell proliferation, or apoptosis at different drug concentrations. This method simulates the cellular environment in vivo, better helping to determine the optimal drug concentration for treating infectious diseases, ensuring the best therapeutic effect while avoiding the toxicity caused by excessively high concentrations.

[0056] The above embodiments are only used to illustrate the present utility model and are not intended to limit the technical solutions described in the present utility model. Although the present utility model has been described in detail with reference to the above embodiments, the present utility model is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present utility model, and all technical solutions and improvements that do not depart from the spirit and scope of the utility model, are covered within the scope of the claims of the present utility model.

Claims

1. A microfluidic chip integrating suspension cell culture and drug concentration generation, characterized in that, The system includes a bottom layer, a drive system, a microchannel network, a sample inlet, and a waste outlet system. The bottom layer consists of stacked culture layers connected in series. Each culture layer has five independent culture channels, and each has an independent inlet and an independent outlet. The two layers overlap to form five closed loops, and the culture channels are interconnected to form closed loops. The drive system includes two independent linear drive channels (112). The drive channels (112) are connected to the culture channels, and an elastic diaphragm is provided between the drive channels (112) and the culture channels. The two ends of the drive channels (112) are connected to the outside. Pressurized liquid or gas is introduced into the drive channels (112), and the elastic diaphragm provides the driving force. The sample inlet and the waste outlet system have independent solution inlets and waste outlets.

2. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 1, characterized in that, It also includes culture units, the number of which is greater than 1. Each culture unit includes a substrate, and each substrate has two culture channels, which are stacked one on top of the other to form a closed loop.

3. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 2, characterized in that, The microchannel network is modified with bovine serum albumin molecules, and two driving channels (112) are provided. Each driving channel (112) intersects with the culture channel and is separated by an elastic diaphragm.

4. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 3, characterized in that, The substrate is provided with linear drive channels (112), and each drive channel (112) is provided with an elastic membrane to isolate the drive channel from the culture channel.

5. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 4, characterized in that, The independent liquid inlets include a first independent liquid inlet (1), a second independent liquid inlet (2), and a third independent liquid inlet (121).

6. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 5, characterized in that, The independent liquid outlets include a first independent liquid outlet (111), a second independent liquid outlet (131), a third independent liquid outlet (141), and a fourth independent liquid outlet (151).

7. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 6, characterized in that, It also includes a chip structure, which includes a first chip layer (100), a second chip layer (200), a third chip layer (300), and a fourth chip layer (400).

8. The integrated microfluidic chip for suspension cell culture and drug concentration generation according to claim 7, characterized in that, The second layer (200) of the chip has a first layer culture channel (211), and the third layer (300) of the chip has a second layer culture channel (212).

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