Microfluidic electrical stimulation chip device and system for promoting cells to secrete exosomes by using device

Abstract

The utility model provides a microfluidic electrical stimulation chip device and a system for promoting cells to secrete exosomes by using the device, the microfluidic electrical stimulation chip device comprises an upper substrate, a microfluidic electrical stimulation chip body and a lower substrate, a liquid inlet and a liquid outlet are etched or tapped on the upper substrate in a hollowed-out manner, an upper substrate electrode is embedded in the lower surface of the upper substrate, and a lower substrate electrode is embedded in the lower surface of the lower substrate. A micro-channel liquid inlet section, a micro-channel reaction section and a micro-channel liquid outlet section are engraved on the micro-fluidic electrical stimulation chip body, and a lower substrate electrode is embedded in the upper surface of the lower substrate; the system for promoting the cells to secrete the exosomes by using the device comprises a cell supply bioreactor, a power device, a micro-fluidic electrical stimulation chip device, a power supply device and a cell recovery bioreactor, and can realize on-line electrical stimulation treatment of the cells in a normal suspension culture process of the cells; cell suspensions flowing through a plurality of micro-channels in the micro-fluidic electrical stimulation chip are stimulated through electrical signals, cells are promoted to secrete exosomes, and the yield of the exosomes is increased.

Description

Technical Field

[0001] The utility model relates to the technical field of biomedicine, and in particular to a microfluidic electrical stimulation chip device and a system utilizing the device to promote cell secretion of exosomes. Background Art

[0002] Exosomes are biologically active vesicles released by living cells into the extracellular microenvironment. They are about 100 nm in size, undergo cell membrane invagination, and bud in the formed endocytic vesicles to form multivesicular bodies (MVBs). Then, MVBs fuse with lysosomes or plasma membranes to release exosomes. A variety of cells can secrete exosomes under normal and pathological conditions. Their composition is very complex. They can bind to receptor cell surface receptors through surface bioactive ligands to regulate downstream signal transduction, and they can also be ingested to allow functional contents to act on targets inside receptor cells to regulate their functions.

[0003] Exosomes can protect and transport biologically active complexes, including nucleic acids, proteins, lipids, and carbohydrates, and have multiple medical potentials such as diagnosis, treatment, and drug carriers. However, only exosome diagnostic products are currently on the market, and exosome therapy or drug delivery is still in the clinical research stage. The development of exosomes lags behind liposomes, cell therapy, and biological drugs such as monoclonal antibodies and recombinant proteins. The first reason is that compared with viruses and cells, exosomes themselves do not have the ability to proliferate, and the production of exosomes by cells under natural secretion is low, and the production scale is limited. Therefore, the clinical transformation of exosomes needs to solve the above problems.

[0004] Some studies have shown that cell stress increases exosome secretion, and cells may communicate with neighboring cells by increasing exosome release. With the in-depth study of exosome release conditions, a series of methods to increase exosome production by stimulating release are also being gradually developed. During cell culture, changing culture conditions, applying chemical stimulation or physical stimulation may stimulate cells to produce and secrete more exosomes.

[0005] Adjustment of cell culture conditions includes changing serum, pH, temperature, oxygen content and culture additives, among which the most convenient and commonly used is the reduced serum starvation culture method. Existing research data show that serum-free starvation culture can stimulate cells to produce 2.5 to 4.3 times more extracellular vesicles; adding chemical inducers to change the cell membrane structure and cytoplasmic pressure can also increase vesicle secretion. Such chemical inducers include thiol blockers, calcium ions, cytochalasins, H2O2, etc.; existing literature reports that some physical stimulation methods, such as shear force, ultrasonic treatment, laser irradiation, microgravity and magnetism, can affect the secretion of extracellular vesicles.

[0006] Existing research results have shown that applying electrical stimulation to cells can produce an instantaneous temperature rise at the stimulation site, significantly increase the expression of heat shock proteins in cells, activate the P53-TSAP6 signaling pathway, and at the same time increase the intracellular Ca 2+ The levels increased, which jointly promoted the production of exosomes.

[0007] In US2022243191A1, radio frequency electrical stimulation is applied to cells in a culture chamber to induce exosome secretion, and exosomes are separated from the cells and the culture medium containing the cells. However, the overall electrical stimulation of this large-volume cell culture system is generally not effective in promoting cell secretion of exosomes, and the amount of exosomes secreted by treated HEK293 cells only increased by 2.3 times.

[0008] Compared with larger electrical stimulation devices, more precise electrical stimulation can form focal, reversible cell damage, reduce cell apoptosis, and achieve a higher multiple of exosome production. CN111378685A discloses a method of culturing cells in a substrate with nanochannels, and obtaining a large number of drug-loaded exosomes by electroporation and electrical stimulation. US20200384266A1 regulates the secretion and composition of extracellular vesicles by electrically stimulating target cells. These two methods use smaller and more precise devices to electrically stimulate cells, which can stimulate cells to obtain a higher multiple of exosomes, but both have low cell processing capacity in a single test, making it difficult to process a large number of cells and difficult to scale up, which is not conducive to large-scale industrial production.

[0009] Microfluidics refers to the technology of processing or manipulating tiny fluids using micron-level pipes, which has the advantages of low sample consumption, high precision, high efficiency, and high integration. This technology can be used to achieve online fine processing of sample liquid, including separation, purification, and detection of exosomes. For example, CN116286268 discloses an exosome separation device based on microfluidics technology, which separates and purifies exosomes by applying quantitative electrical stimulation to a microcolumn array. However, there are currently no related research reports on using microfluidics to promote cell secretion of exosomes.

[0010] The utility model provides a microfluidic electrical stimulation chip device combining electrical stimulation and microfluidics, and a system using the device to promote cell secretion of exosomes. Through this device and system, precise and online microfluidic electrical stimulation of cells at the micron level can be achieved, greatly improving the exosome yield and production efficiency, and can be better suitable for industrial production. Utility Model Content

[0011] The utility model provides a microfluidic electrical stimulation chip device and a system using the device to promote cell secretion of exosomes, which is used for close and online microfluidic electrical stimulation of cells during large-scale cell culture, can greatly improve the exosome yield and production efficiency, and is better suitable for large-scale industrial production.

[0012] The utility model provides a microfluidic electrical stimulation chip device 3, comprising an upper substrate 31, a microfluidic electrical stimulation chip body 32 and a lower substrate 33, wherein: the upper substrate 31 is hollowed out or tapped at both ends to form a liquid inlet 311 and a liquid outlet 313, and an upper substrate electrode 312 is embedded on the lower surface; the microfluidic electrical stimulation chip body 32 is engraved with a microfluidic liquid inlet section 321, a microfluidic reaction section 322 and a microfluidic liquid outlet section 323; the microfluidic liquid inlet 311 and the microfluidic reaction section 322 and the microfluidic liquid outlet section 323 are engraved on ...1 and the microfluidic reaction section 322 and the microfluidic reaction section 322 and the microfluidic reaction The microfluidic electrostimulation chip body 32 has a divergent surface etching; the microfluidic reaction section 322 is etched and hollowed out on the microfluidic electrostimulation chip body 32, and the etching depth is equal to the thickness of the microfluidic electrostimulation chip body 32, and is connected to the branch flow channel of the microfluidic inlet section 321; the microfluidic outlet section 323 is etched on the microfluidic electrostimulation chip body 32 in a convergent surface etching; the lower substrate 33 is embedded with a lower substrate electrode 331 on the upper surface;

[0013] in:

[0014] The microfluidic electrical stimulation chip body 32 has a thickness of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, and most preferably 1 mm;

[0015] The microchannel liquid inlet section 321 diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0016] The microfluidic reaction section 322 is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic inlet section 321 is dispersed, each channel has a width of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, an etching depth equal to the thickness of the microfluidic electrical stimulation chip body 32, and a length of 2 to 40 mm, preferably 5 to 10 mm, and most preferably 10 mm;

[0017] The microchannel outlet section 323 is formed by 2 to 8 branch channels corresponding to the microchannel inlet section 321, and the etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0018] The upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip device 3 are rectangular electrode sheets or fence-shaped printed electrodes, preferably rectangular electrodes, and the electrode material can be titanium, platinum or gold, preferably titanium.

[0019] The utility model also provides a system including the above-mentioned microfluidic electrical stimulation chip device 3, including: a supply cell bioreactor 1, a power device 2, a microfluidic electrical stimulation chip device 3, a power supply device 4, a recovery cell bioreactor 5, and pipes, connectors and wires connecting the modules.

[0020] The system provided by the utility model includes a microfluidic electrical stimulation chip device 3, wherein:

[0021] The cell supply bioreactor 1 is used for normal culture of cells before electrical stimulation, and can be a wave bioreactor, a shaking bioreactor, a stirring bioreactor, or other bioreactors that can suspend and culture cells directly or indirectly through microcarriers;

[0022] The power device 2 is used to drive the cell or cell microcarrier suspension to be continuously output from the supply cell bioreactor 1, flow through the microfluidic electrical stimulation chip device 3 to receive electrical stimulation and then flow to the recovery cell bioreactor 5, and can be a syringe pump, a peristaltic pump, a pressure pump or other devices based on driving forces such as gravity and pressure;

[0023] The microfluidic electrical stimulation chip device 3 comprises an upper substrate 31, a microfluidic electrical stimulation chip body 32 and a lower substrate 33, wherein: the upper substrate 31 is hollowed out or tapped at both ends to form a liquid inlet 311 and a liquid outlet 313, and an upper substrate electrode 312 is embedded on the lower surface; the microfluidic electrical stimulation chip body 32 is engraved with a microfluidic liquid inlet section 321, a microfluidic reaction section 322 and a microfluidic liquid outlet section 323; the microfluidic liquid inlet section 321 The microfluidic electrical stimulation chip body 32 is etched in a divergent manner; the microchannel reaction section 322 is etched and hollowed out on the microfluidic electrical stimulation chip body 32, and the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body 32, and is correspondingly connected to the branch flow channel of the microchannel liquid inlet section 321; the microchannel liquid outlet section 323 is etched in a convergent manner on the microfluidic electrical stimulation chip body 32; the lower substrate 33 is embedded with a lower substrate electrode 331 on the upper surface;

[0024] in:

[0025] The microfluidic electrical stimulation chip body 32 has a thickness of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, and most preferably 1 mm;

[0026] The microchannel liquid inlet section 321 diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0027] The microfluidic reaction section 322 is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic inlet section 321 is dispersed, each channel has a width of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, an etching depth equal to the thickness of the microfluidic electrical stimulation chip body 32, and a length of 2 to 40 mm, preferably 5 to 10 mm, and most preferably 10 mm;

[0028] The microchannel outlet section 323 is formed by 2 to 8 branch channels corresponding to the microchannel inlet section 321, and the etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0029] The upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip device 3 are rectangular electrode sheets or fence-shaped printed electrodes, preferably rectangular electrodes, and the electrode material can be titanium, platinum or gold, preferably titanium;

[0030] The power supply device 4 can provide a frequency of 50 Hz and a voltage of 25 to 50 V. p-p AC power supply, preferably 30-40V p-p AC power supply;

[0031] The cell recovery bioreactor 5 is used for receiving cells after electrical stimulation, subsequent culture, and collection of exosomes in the cell culture supernatant, and can be a wave bioreactor, a shaking bioreactor, a stirring bioreactor, or other bioreactors that can directly or indirectly suspend and culture cells through microcarriers;

[0032] The pipelines between the connecting modules are used to transport cells or cell microcarrier suspensions between the supply cell bioreactor 1 and the power device 2, between the power device 2 and the microfluidic electrical stimulation chip device 3, and between the microfluidic electrical stimulation chip device 3 and the recovery cell bioreactor 5. The inner diameter is 0.8 to 12.7 mm, the outer diameter is 4.0 to 25.4 mm, and the material may be silicone, thermoplastic elastomer or other materials;

[0033] The connector is used for airtight connection between different pipelines, between pipelines and microfluidic electrical stimulation chip devices 3, and between pipelines and bioreactors;

[0034] The wire is used for electrical connection between the electrodes in the microfluidic electrical stimulation chip device 3 and the power supply device 4 .

[0035] The system provided by the utility model may also include a mixing device, which is used for mixing the cell or cell microcarrier suspension before entering the microfluidic electrical stimulation chip device 3, and may be a shaking mixing device, a magnetic stirring device, a vortex mixing device or an oscillating mixing device.

[0036] The utility model provides a method for promoting cell secretion of exosomes by using a system containing a microfluidic electrical stimulation chip device, comprising: a cell or cell microcarrier suspension is suspended and cultured in the supply cell bioreactor 1 to a logarithmic growth phase, flows out of the supply cell bioreactor 1 under the action of a power device 2, flows to the microfluidic electrical stimulation chip device 3 at a stable flow rate, the cell or cell microcarrier suspension enters the microchannel from the liquid inlet 311 of the microfluidic electrical stimulation chip device 3, flows out from the liquid outlet 313 of the microfluidic electrical stimulation chip device 3 after receiving electrical stimulation in the microchannel reaction section 322, collects the cell or cell microcarrier suspension in the recovery cell bioreactor 5, and extracts exosomes after continuing to culture for 24 to 48 hours;

[0037] in:

[0038] The flow rate of the cell or cell microcarrier suspension is 0.05 to 50.00 mL / min, preferably 5 to 10 mL / min;

[0039] The microfluidic electrical stimulation chip device 3 comprises an upper substrate 31, a microfluidic electrical stimulation chip body 32 and a lower substrate 33, wherein: the upper substrate 31 is hollowed out or tapped at both ends to form a liquid inlet 311 and a liquid outlet 313, and an upper substrate electrode 312 is embedded on the lower surface; the microfluidic electrical stimulation chip body 32 is engraved with a microfluidic liquid inlet section 321, a microfluidic reaction section 322 and a microfluidic liquid outlet section 323; the microfluidic liquid inlet section 321 The microfluidic electrical stimulation chip body 32 is etched in a divergent manner; the microchannel reaction section 322 is etched and hollowed out on the microfluidic electrical stimulation chip body 32, and the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body 32, and is correspondingly connected to the branch flow channel of the microchannel liquid inlet section 321; the microchannel liquid outlet section 323 is etched in a convergent manner on the microfluidic electrical stimulation chip body 32; the lower substrate 33 is embedded with a lower substrate electrode 331 on the upper surface;

[0040] in:

[0041] The microfluidic electrical stimulation chip body 32 has a thickness of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, and most preferably 1 mm;

[0042] The microchannel liquid inlet section 321 diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0043] The microfluidic reaction section 322 is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic inlet section 321 is dispersed, each channel has a width of 0.1 to 4.0 mm, preferably 0.5 to 1.0 mm, an etching depth equal to the thickness of the microfluidic electrical stimulation chip body 32, and a length of 2 to 40 mm, preferably 5 to 10 mm, and most preferably 10 mm;

[0044] The microchannel outlet section 323 is formed by 2 to 8 branch channels corresponding to the microchannel inlet section 321, and the etching depth is 0.05 to 2.00 mm, preferably 0.25 to 0.50 mm, and most preferably 0.50 mm;

[0045] The upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip device 3 are rectangular electrode sheets or fence-shaped printed electrodes, preferably rectangular electrodes, and the electrode material can be titanium, platinum or gold, preferably titanium.

[0046] In the microfluidic reaction section 322, the voltage provided by the power supply device 4 forms an electric field perpendicular to the flow direction of the microfluid between the upper substrate electrode 312 and the lower substrate electrode 331, and applies different degrees of electrical stimulation to the cells flowing through by adjusting the voltage and frequency.

[0047] The power supply device 4 can provide a frequency of 50 Hz and a voltage of 25 to 50 V. p-p AC power supply, preferably 30-40V p-p AC power supply.

[0048] The method provided by the utility model for promoting cell secretion of exosomes by using a system containing a microfluidic electrical stimulation chip device can also include a mixing process, wherein the mixing is to mix the cell or cell microcarrier suspension before entering the microfluidic electrical stimulation chip body, and the mixing can be performed using a shaker mixing device, a magnetic stirring device, a vortex mixing device or an oscillation mixing device.

[0049] Exosome extraction can be performed by differential ultracentrifugation, tangential flow ultrafiltration concentration, size exclusion chromatography, polymer precipitation, density gradient centrifugation, or immunoaffinity capture.

[0050] After a large number of experimental studies, the inventors found that the factors that affect the secretion of exosomes by cells mainly include the electric field strength acting in the microfluidic reaction section and the time that the cells receive electrical stimulation in the electric field. Therefore, by setting the appropriate voltage and electrode spacing in the microfluidic electrical stimulation chip device, the appropriate electric field strength is obtained, and the appropriate electrical stimulation time is adjusted by adjusting the flow rate of cells or cell microcarriers and the electrode length.

[0051] By using the microfluidic electrical stimulation chip device provided by the utility model and the system using the device to promote cell secretion of exosomes, cells can be subjected to online electrical stimulation during normal cell suspension culture, and cell suspensions flowing through multiple microchannels in the microfluidic electrical stimulation chip can be stimulated by electrical signals to promote cell secretion of exosomes and increase the yield of exosomes. The microchannels in the microfluidic electrical stimulation chip are small in size and short in electrode spacing, and the cell suspension can be controlled to receive electrical stimulation between the reaction electrodes in the form of microfluids at the microliter level. By adjusting the size of the microchannels, cells or cell microcarriers can be continuously, one by one, and sequentially passed through the reaction section microchannels, and more precise electrical stimulation can be performed online. When applied to the preparation and production of exosomes, multiple microfluidic electrical stimulation chip devices provided by the utility model can be connected in parallel, which can achieve a higher multiple of exosome production increase, greatly reduce production costs, improve production efficiency, and be more suitable for industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of microfluidic electrical stimulation chip device 3:

[0053] In the figure: upper substrate 31, liquid inlet 311, fence-type upper substrate electrode 312, liquid outlet 313, microfluidic electrical stimulation chip body 32, microfluidic channel liquid inlet section 321, microfluidic channel reaction section 322, microfluidic channel liquid outlet section 323, lower substrate 33, fence-type lower substrate electrode 331.

[0054] Figure 2 Schematic diagram of microfluidic electrical stimulation chip device 3:

[0055] In the figure: upper substrate 31, liquid inlet 311, rectangular upper substrate electrode 312, liquid outlet 313, microfluidic electrical stimulation chip body 32, microfluidic channel liquid inlet section 321, microfluidic channel reaction section 322, microfluidic channel liquid outlet section 323, lower substrate 33, rectangular lower substrate electrode 331.

[0056] Figure 3 Schematic diagram of the upper substrate 31 of the microfluidic electrical stimulation chip device 3 .

[0057] Figure 4 Schematic diagram of microfluidic electrical stimulation chip body 32.

[0058] Figure 5 Schematic diagram of the lower substrate 33 of the microfluidic electrical stimulation chip device 3 .

[0059] Figure 6 Schematic diagram of a system for promoting cell secretion of exosomes containing a microfluidic electrical stimulation chip device 3:

[0060] In the figure: a supply cell bioreactor 1, a power device 2, a microfluidic electrical stimulation chip device 3, a power supply device 4, and a recovery cell bioreactor 5.

[0061] Figure 7 Particle size distribution diagram of the test sample in Test Example 1.

[0062] Figure 8 Particle size distribution diagram of the control sample in Experimental Example 1.

[0063] Fig. 9 Particle size distribution diagram of the blank sample in Experimental Example 1.

[0064] Fig.10 In Experimental Example 1, Western blot was used to detect exosome marker proteins CD63, CD81, TSG101 and ALIX.

[0065] Fig.11 Particle size distribution diagram of the test sample in Test Example 2.

[0066] Fig.12 Particle size distribution diagram of the control sample in Experimental Example 2.

[0067] Fig.13 Particle size distribution diagram of the blank sample in Experimental Example 2.

[0068] Fig.14 Particle size distribution diagram of the test sample in Test Example 3.

[0069] Fig.15 Particle size distribution diagram of the control sample in Experimental Example 3.

[0070] Fig.16 Particle size distribution diagram of the blank sample in Experimental Example 3.

[0071] Fig.17 Particle size distribution diagram of the test sample in Test Example 8.

[0072] Fig.18 Particle size distribution diagram of the control sample in Test Example 8.

[0073] Fig.19 In Experimental Example 8, Western blot was used to detect exosome marker proteins CD63, CD81, TSG101 and negative protein Calnexin.

[0074] Fig. 20 Overall distribution diagram of gene expression levels in Experimental Example 9.

[0075] Fig.21 Gene expression levels in Experimental Example 9 (top 20 in abundance).

[0076] Fig. 22 Volcano plot of differential gene expression in Experimental Example 9.

[0077] Fig.23Distribution diagram of the expression abundance (top 20 abundance) of each sample after small RNA sequencing in Experimental Example 9.

[0078] Fig.24 Overall distribution diagram of miRNA expression levels in Experimental Example 9.

[0079] Fig.25 Figure 9 shows the differential expression of miRNA detected in Experimental Example 9.

[0080] Fig.26 Protein quantification results in Experimental Example 9 (top 20 in abundance).

[0081] Fig. 27 Diagram of differential expression of proteins detected in Experimental Example 9. DETAILED DESCRIPTION

[0082] The present invention is further described by way of the following examples, which are not intended to limit the present invention in any way.

[0083] The chip specifications of all microfluidic electrical stimulation chip devices involved in the following embodiments and test examples refer to the width×depth×length of each flow channel in the microfluidic reaction section 322, and the unit is millimeter (mm).

[0084] Example 1 Microfluidic electrical stimulation chip device

[0085] like Figures 1 to 5As shown, the microfluidic electrical stimulation chip device 3 provided by the utility model comprises an upper substrate 31, a microfluidic electrical stimulation chip body 32 and a lower substrate 33, wherein: the upper substrate 31 is hollowed out or tapped at both ends to form a liquid inlet 311 and a liquid outlet 313, and an upper substrate electrode 312 is embedded on the lower surface; the microfluidic electrical stimulation chip body 32 has a thickness of 0.1 to 4.0 mm, and is engraved with a microfluidic liquid inlet section 321, a microfluidic reaction section 322 and a microfluidic liquid outlet section 323; the microfluidic liquid inlet section 321 is etched in a divergent manner on the upper surface of the microfluidic electrical stimulation chip body 32, diverging from one flow channel into 2 to 8 branch flow channels, and the etching depth is 0.05 to 2.00 mm; the microfluidic reaction section 322 is a microfluidic electrostimulation chip body 32. 2 to 8 parallel flow channels are etched and hollowed out on the stimulation chip body 32, which are connected to the branch flow channels of the microchannel inlet section 321. The width of each flow channel is 0.1 to 4.0 mm, the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body 32, and the length is 2 to 40 mm; the microchannel outlet section 323 is etched on the microfluidic electrical stimulation chip body 32 in a converging surface, and the 2 to 8 branch flow channels corresponding to the microfluidic reaction section 322 are converged into one flow channel, and the etching depth is 0.05 to 2.00 mm; the lower substrate 33 is embedded with a lower substrate electrode 331 on the upper surface; the upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip device 3 can be rectangular electrode sheets or fence-shaped printed electrodes.

[0086] Example 2 System for Promoting Cell Secretion of Exosomes Using a Microfluidic Electrical Stimulation Chip Device

[0087] like Figure 6As shown, the system for promoting cell secretion of exosomes by using a microfluidic electrical stimulation chip device provided by the utility model comprises a supply cell bioreactor 1, a power device 2, a microfluidic electrical stimulation chip device 3, a power device 4, a recovery cell bioreactor 5, and pipes, connectors and wires connecting the modules, wherein: the supply cell bioreactor 1 is connected to the power device 2 by a pipe, the power device 2 is connected to the microfluidic electrical stimulation chip device 3 by a pipe and a connector, the microfluidic electrical stimulation chip device 3 is connected to the recovery cell bioreactor 5 by a pipe and a connector, and the recovery cell bioreactor 5 is connected to the power device 2 by a pipe. The cell bioreactor 5 is connected by pipelines and joints, and the microfluidic electrical stimulation chip device 3 and the power supply device 4 are connected by wires; the microfluidic electrical stimulation chip device 3 includes an upper substrate 31, a microfluidic electrical stimulation chip body 32 and a lower substrate 33; the upper substrate 31 is hollowed out or tapped at both ends to form a liquid inlet 311 and a liquid outlet 313, and an upper substrate electrode 312 is embedded on the lower surface; the microfluidic electrical stimulation chip body 32 has a thickness of 0.1 to 4.0 mm, and is engraved with a microfluidic liquid inlet section 321, a microfluidic reaction section 32 2 and the microfluidic outlet section 323, the microfluidic inlet section 321 is etched in a divergent manner on the upper surface of the microfluidic electrical stimulation chip body 32, and diverges from one channel into 2 to 8 branch channels, and the etching depth is 0.05 to 2.00 mm. The microfluidic reaction section 322 is 2 to 8 parallel channels etched and hollowed out on the microfluidic electrical stimulation chip body 32, and is connected to the branch channels of the microfluidic inlet section 321, and each channel has a width of 0.1 to 4.0 mm, and the etching depth is equal to the microfluidic electrical stimulation chip The thickness of the body 32 is 2 to 40 mm in length; the microchannel outlet section 323 is etched on the microfluidic electrical stimulation chip body 32 in a convergent surface manner, and 2 to 8 branch channels corresponding to the microfluidic reaction section 322 converge into one channel, and the etching depth is 0.05 to 2.00 mm; the lower substrate 33 is embedded with a lower substrate electrode 331 on the upper surface; the upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip device 3 can be rectangular electrode sheets or fence-shaped printed electrodes.

[0088] Experimental Example 1 Promoting exosome secretion from 293F cells using a system containing a microfluidic electrical stimulation chip device

[0089] The system containing the microfluidic electrical stimulation chip device is:

[0090] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump (Lange Constant Flow Pump Co., Ltd., model: L100-1S-1), the microfluidic electrical stimulation chip device 3 is: chip specifications 1.0×1.0×10.0mm, the upper substrate electrode 312 and the lower substrate electrode 331 are fence-shaped gold-sprayed electrodes, the microfluidic electrical stimulation chip body 32 is 1.0mm thick, the microchannel inlet section 321 and the microchannel outlet section 323 in the microfluidic electrical stimulation chip body 32 have a channel depth of 0.5mm, the microchannel reaction section 322 is 5 parallel channels, each channel has a width of 1.0mm, a depth of 1.0mm, and a length of 10.0mm, the power supply device 4 is an AC voltage regulating power supply (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), the voltage is set to 30V, and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0091] Suspension 293F cells: Thermo Fisher Scientific (China), catalog number: A14527.

[0092] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0093] Test sample:

[0094] Take 20 ml of suspended 293F cells for experiment and stir them at 70 rpm in a stirred bioreactor. Turn on the peristaltic pump and AC voltage regulating power supply, set the cell flow rate to 5 mL / min to transport the cell suspension, and the cell suspension flows through the microfluidic electrical stimulation chip device 3 and then is recovered into the cell culture shake flask. Continue to culture in a carbon dioxide shaking incubator for 48 hours, and collect the cell suspension for exosome extraction. The specific extraction method is as follows: the collected cell suspension is first centrifuged at a centrifugal force of 300 g for 10 minutes to remove the cell precipitate, then centrifuged at a centrifugal force of 2000 g for 10 minutes to remove the cell debris precipitate, and then centrifuged at a centrifugal force of 10000 g for 20 minutes to remove the microvesicles, apoptotic bodies and other precipitates, and the supernatant is collected; the supernatant is filtered through a 0.2 um sterile hydrophilic polyethersulfone filter membrane and the filtrate is collected; the filtrate is centrifuged at a centrifugal force of 100000 g for 70 minutes, and the supernatant is discarded, and the precipitate is filtered with 35 mL The cells were resuspended in PBS and centrifuged again at 100,000 g for 70 min, the supernatant was discarded, and the precipitate was collected with 100 μL PBS to obtain 293F cell exosomes.

[0095] Control sample:

[0096] Take 20 ml of suspended 293F cells for the experiment and continue to culture them in a carbon dioxide shaking incubator for 48 hours, collect the cell suspension for exosome extraction, and the specific extraction method is as follows: the collected cell suspension is first centrifuged at 300g for 10 minutes to remove the cell precipitate, then centrifuged at 2000g for 10 minutes to remove the cell debris precipitate, and then centrifuged at 10000g for 20 minutes to remove microvesicles, apoptotic bodies and other precipitates, and the supernatant is collected; the supernatant is filtered through a 0.2um sterile hydrophilic polyethersulfone filter membrane and the filtrate is collected; the filtrate is centrifuged at 100000g for 70 minutes, the supernatant is discarded, the precipitate is resuspended with 35mL PBS, and the supernatant is discarded after centrifugation at 100000g for 70 minutes, and the precipitate is collected with 100μL PBS to obtain 293F cell exosomes.

[0097] Blank sample:

[0098] Take 20 ml of suspended 293F cells for the experiment, centrifuge to remove the cells, and continue to culture the supernatant in a carbon dioxide shaking incubator for 48 hours. Collect the supernatant for exosome extraction. The specific extraction method is as follows: the collected supernatant is first centrifuged at 300g for 10 minutes to remove the cell precipitate, then centrifuged at 2000g for 10 minutes to remove the cell debris precipitate, and then centrifuged at 10000g for 20 minutes to remove microvesicles, apoptotic bodies and other precipitates, and collect the supernatant; the supernatant is filtered through a 0.2um sterile hydrophilic polyethersulfone filter membrane and the filtrate is collected; the filtrate is centrifuged at 100000g for 70 minutes, the supernatant is discarded, the precipitate is resuspended with 35mL PBS, and the supernatant is discarded after centrifugation at 100000g for 70 minutes, and the precipitate is collected with 100μL PBS to obtain 293F cell exosomes.

[0099] The exosomes obtained from the test sample, control sample and blank sample were compared and characterized in terms of quantity, particle size and marker protein identification:

[0100] The number and size of exosomes were determined using a nanoparticle tracking analyzer (Malvern Panalytical, NS300): Take an appropriate amount of exosomes and dilute them 1000 to 10000 times to obtain a sample with a particle concentration of about 5e+10particles / mL, turn on the nanotracking analyzer, draw 1mL of sample with a 1mL sterile syringe and slowly inject it into the injector, observe the camera screen, adjust the brightness and focus, install the syringe into the syringe pump, set the parameters and start the injection measurement. The nanoparticle tracking analyzer records the number of nanoparticles and the Brownian motion trajectory by capturing the scattered light of nanoscale particles in the sample, and calculates the particle size by the Stokes-Einstein equation after obtaining the Brownian motion rate of the nanoparticles. The final exosome particle size, concentration and increase factor are listed in Tables 1 and 2. The exosome particle size distribution diagrams obtained for the experimental sample, control sample and blank sample are shown in Tables 1 and 2, respectively. Figure 7-Figure 9 listed.

[0101] The prepared exosomes were further identified and characterized by Western blot: the results showed that both the experimental samples and the control samples contained exosome membrane marker proteins CD63 and CD81 as well as membrane marker proteins TSG101 and ALIX. Fig.10 shown.

[0102] Table 1 Exosome size and concentration

[0103]

[0104] The exosome production increase factor was calculated according to the ratio of the number of exosomes secreted by the test sample and the control sample, as shown in Table 2: wherein the corrected exosome concentration was the exosome concentration in the cell suspension of the test sample or the control sample minus the exosome concentration in the cell suspension of the blank sample, so as to deduct the exosomes secreted by the cells before the experiment; the number of exosomes secreted by the cells was the corrected exosome concentration divided by the cell concentration.

[0105] Table 2 Increased production of exosomes

[0106]

[0107] in conclusion:

[0108] From the experimental data, we can see that the exosome concentration of the test sample is much higher than that of the control sample, and the exosome amplification factor can reach 25.0 times, which can greatly increase the exosome production secreted by 293F cells.

[0109] Experimental Example 2 Promoting exosome secretion from 293F cells using a system containing a microfluidic electrical stimulation chip device

[0110] The system containing the microfluidic electrical stimulation chip device is:

[0111] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump (Lange Constant Flow Pump Co., Ltd., model: L100-1S-1), the microfluidic electrical stimulation chip device 3 is: chip specifications 0.5×0.5×8.0mm, the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular titanium metal electrodes, the microfluidic electrical stimulation chip body 32 is 0.5mm thick, the microchannel inlet section 321 and the microchannel outlet section 323 in the microfluidic electrical stimulation chip body 32 have a channel depth of 0.25mm, the microchannel reaction section 322 is 5 parallel channels, each channel has a width of 0.5mm, a depth of 0.5mm, and a length of 8.0mm, the power supply device 4 is an AC voltage regulating power supply (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), the voltage is set to 40V, and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0112] Suspension 293F cells: Thermo Fisher Scientific (China), catalog number: A14527.

[0113] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0114] The preparation of the test samples, control samples and blank samples, as well as the determination and calculation methods of exosomes were the same as those in Experimental Example 1. The final exosome particle size, concentration and increase multiple are listed in Tables 3 and 4. The exosome particle size distribution diagrams obtained for the test samples, control samples and blank samples are shown in Tables 3 and 4. Figure 11-13 listed.

[0115] Table 3 Exosome size and concentration

[0116]

[0117] Table 4 Increased production of exosomes

[0118]

[0119] in conclusion:

[0120] Experimental data show that the exosome concentration of the test sample is much higher than that of the control sample, and the exosome amplification factor can reach 74.3 times, greatly increasing the exosome production secreted by 293F cells.

[0121] Experimental Example 3 Promoting exosome secretion from 293F cells using a system containing a microfluidic electrical stimulation chip device

[0122] The system containing the microfluidic electrical stimulation chip device is:

[0123] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump, the microfluidic electrical stimulation chip device 3 is: chip specifications are 0.5×1.0×8.0mm, the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular titanium metal electrodes, the microfluidic electrical stimulation chip body 32 is 1.0mm thick, the microfluidic channel inlet section 321 and the microfluidic channel outlet section 323 in the microfluidic electrical stimulation chip body 32 have a channel depth of 0.5mm, the microfluidic channel reaction section 322 is 5 parallel channels, each channel has a width of 0.5mm, a depth of 1.0mm, and a length of 8.0mm, the power supply device 4 is an AC voltage regulating power supply, and the voltage is set to 40V (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0124] Suspension 293F cells: Thermo Fisher Scientific (China), catalog number: A14527.

[0125] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0126] The preparation of the test samples, control samples and blank samples, as well as the determination and calculation of exosomes were the same as in Experimental Example 1. The final exosome particle size, concentration and increase factor are listed in Tables 5 and 6. The exosome particle size distribution diagrams obtained for the test samples, control samples and blank samples are shown in Tables 5 and 6. Figure 14-16 listed.

[0127] Table 5 Particle size and concentration of exosomes

[0128]

[0129] Table 6 Exosome production increase

[0130]

[0131] in conclusion:

[0132] Experimental data show that the exosome concentration of the test sample is much higher than that of the control sample, and the exosome amplification factor can reach 59.7 times, greatly increasing the exosome production secreted by 293F cells.

[0133] Experimental Example 4 Effect of different power types on the production of exosomes in 293F cells

[0134] The system containing the microfluidic electrical stimulation chip device is:

[0135] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump with a set flow rate of 5 mL / min, the microfluidic electrical stimulation chip device 3 is as follows: the chip specification is 0.5×1.0×8.0 mm, the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular copper metal electrodes, the thickness of the microfluidic electrical stimulation chip body 32 is 1.0 mm, the flow channel depths of the microfluidic inlet section 321 and the microfluidic outlet section 323 in the microfluidic electrical stimulation chip body 32 are 0.5 mm, the microfluidic reaction section 322 is 5 parallel flow channels, each flow channel has a width of 0.5 mm, a depth of 1.0 mm, and a length of 8.0 mm, and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0136] Suspension 293F cells: Thermo Fisher Scientific (China), catalog number: A14527.

[0137] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0138] The power supply device 4 respectively selected a 20Vp-p signal generator or a 20V AC voltage regulating power supply or a 20V DC voltage regulating power supply (Shenzhen Guce Electronic Technology Co., Ltd., KPS2003D) to investigate the different effects of electrical stimulation generated by different power supply devices on the increase in cell exosome production. The sample preparation method, determination method and calculation method were the same as those in Experimental Example 1. The exosome production increase multiples of 293F cells under electrical stimulation conditions of different power supply devices are shown in Table 7.

[0139] Table 7 Exosome production increase of 293F cells under different power supply device electrical stimulation conditions

[0140] Power supply unit Exosome production multiple Signal Generator 1.1 AC regulated power supply 21.9 DC regulated power supply 2.1

[0141] in conclusion:

[0142] Experimental data show that compared with signal generators and DC regulated power supplies, using AC regulated power supplies to stimulate cells can promote 293F cells to secrete more exosomes.

[0143] Experimental Example 5 Effect of different electrode materials on the production of exosomes in 293F cells

[0144] The system containing the microfluidic electrical stimulation chip device is:

[0145] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump with a set flow rate of 5 mL / min, the microfluidic electrical stimulation chip device 3 is as follows: the chip specification is 0.5×1.0×8.0 mm, the thickness of the microfluidic electrical stimulation chip body 32 is 1.0 mm, the flow channel depths of the microfluidic inlet section 321 and the microfluidic outlet section 323 in the microfluidic electrical stimulation chip body 32 are 0.5 mm, the microfluidic channel reaction section 322 is 5 parallel flow channels, each flow channel has a width of 0.5 mm, a depth of 1.0 mm, and a length of 8.0 mm, the power supply device 4 is an AC voltage regulating power supply with a voltage set to 25 V (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0146] Suspension 293F cells: Thermo Fisher Scientific (China), catalog number: A14527.

[0147] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0148] Rectangular copper, titanium, platinum and gold were selected as metal electrodes for the upper substrate electrode 312 and the lower substrate electrode 331 in the microfluidic electrical stimulation chip 3, respectively, to investigate the effect of electrodes of different materials on the increased production of exosomes in 293F cells. The sample preparation method, determination method and calculation method were the same as those in Experimental Example 1. The increased production multiples of exosomes in 293F cells obtained by electrodes of different materials are shown in Table 8.

[0149] Table 8 Exosome production increase of 293F cells obtained from electrodes of different materials

[0150]

[0151]

[0152] in conclusion:

[0153] According to experimental data, when copper is used as the metal electrode, the exosome production is stimulated to increase by the lowest multiple, and copper metal is easily hydrolyzed and oxidized in this microfluidic device, releasing a large amount of copper ions and affecting cell growth. Therefore, the electrode material is selected from titanium, platinum or gold, preferably titanium.

[0154] Experimental Example 6 Effect of different voltage electrical stimulation conditions on the production of exosomes in 293F cells

[0155] The system containing the microfluidic electrical stimulation chip device is:

[0156] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump with a set flow rate of 5 mL / min, the microfluidic electrical stimulation chip device 3 is as follows: the chip specification is 0.5×0.5×8.0 mm, the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular titanium metal electrodes, the thickness of the microfluidic electrical stimulation chip body 32 is 1.0 mm, the flow channel depths of the microfluidic inlet section 321 and the microfluidic outlet section 323 in the microfluidic electrical stimulation chip body 32 are 0.25 mm, the microfluidic channel reaction section 322 is 5 parallel flow channels, each flow channel is 0.5 mm wide, 0.5 mm deep, and 8.0 mm long, the power supply device 4 is an AC voltage regulating power supply (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0157] Suspension 293F cells: Thermo Fisher Scientific (China), Catalog No.: A14527

[0158] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0159] The voltage of the AC voltage regulating power supply was set to 20V, 25V, 30V, 40V, 45V, 50V and 55V respectively to investigate the effect of different voltage electrical stimulation on the increase in exosome production of 293F cells. The sample preparation method, determination method and calculation method were the same as those in Experimental Example 1. The exosome production increase multiples of 293F cells obtained under different voltage electrical stimulation are shown in Table 9.

[0160] Table 9 Exosome production increase of 293F cells obtained under different voltage electrical stimulation

[0161] Voltage Exosome production multiple 20V 3.0 25V 15.6 30V 35.3 40V 74.3 45V 40.1 50V 18.5 55V ——

[0162] in conclusion:

[0163] According to experiments, the exosome production increase is low when the voltage is 20V. When the voltage is higher than 50V, electric sparks will be generated, which is dangerous and may interrupt the experimental operation. Therefore, the voltage is selected to be 25V-50V.

[0164] Experimental Example 7 Effect of different specifications of microfluidic electrical stimulation chips on the production of exosomes in 293F cells

[0165] The system containing the microfluidic electrical stimulation chip device is:

[0166] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump with a set flow rate of 10 mL / min, the microfluidic electrical stimulation chip device 3 is: the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular titanium metal electrodes, the microfluidic reaction section 322 is 5 parallel flow channels, the power supply device 4 is an AC voltage-regulated power supply with a set voltage of 40 V (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), and the recovery cell bioreactor 5 is a carbon dioxide oscillation incubator (Shanghai Zhichu Instrument Co., Ltd., model: ZCZY-AS8E).

[0167] Suspension 293F cells: Thermo Fisher Scientific (China), Catalog No.: A14527

[0168] Suspension 293F cells for the experiment: 200 mL of suspension 293F cells in the logarithmic growth phase were taken, with a concentration of 2.0×10 6 cells / mL, diluted to 1.5×10 cells / mL using chemically defined medium (Situofan, catalog number: SH31037). 6 cells / mL.

[0169] The specifications of the microfluidic electrical stimulation chip device 3 are listed in Table 10 below. The effects of microfluidic electrical stimulation chip devices of different specifications on the increased production of exosomes in 293F cells were investigated. The sample preparation method, determination method and calculation method were the same as those in Experimental Example 1. The increased production multiples of exosomes in 293F cells obtained from microfluidic electrical stimulation chips of different specifications are shown in Table 10.

[0170] Table 10 Exosome production increase of 293F cells obtained by microfluidic electrical stimulation chips of different specifications

[0171] Chip size (mm) 322 Width (mm) 322 Depth (mm) 322 Length (mm) Exosome production multiple 0.5×0.5×8 0.5 0.5 8 23.1 1.0×1.0×10 1.0 1.0 10 19.1 1.0×0.5×10 1.0 0.5 10 48.2 1.0×1.5×10 1.0 1.5 10 84.8 1.0×1.0×5 1.0 1.0 5 15.0 1.0×1.0×20 1.0 1.0 20 78.6 2.0×1.0×10 2.0 1.0 10 27.1

[0172] in conclusion:

[0173] Experimental data show that under the experimental conditions of 40V and 10mL / min, microfluidic electrical stimulation chips of different specifications can achieve a high exosome production increase multiple. Among them, when the chip specification is 1.0×1.5×10mm, it can stimulate 293F cells to secrete more exosomes.

[0174] Experimental Example 8 Promoting MSC Cells to Secrete Exosomes Using a System Containing a Microfluidic Electrical Stimulation Chip Device

[0175] The system containing the microfluidic electrical stimulation chip device is:

[0176] The cell supply bioreactor 1 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN), the power device 2 is a peristaltic pump, and the flow rate is set to 5 mL / min. The microfluidic electrical stimulation chip device 3 has the following specifications: 1.0×1.0×10.0 mm, the upper substrate electrode 312 and the lower substrate electrode 331 are rectangular titanium metal electrodes, the thickness of the microfluidic electrical stimulation chip body 32 is 1.0 mm, and the flow channel depths of the microchannel inlet section 321 and the microchannel outlet section 323 in the microfluidic electrical stimulation chip body 32 are 0.5 mm. The microchannel reaction section 322 is 5 parallel flow channels, each of which has a width of 1.0 mm, a depth of 1.0 mm, and a length of 10.0 mm. The power supply device 4 is an AC voltage regulating power supply, and the voltage is set to 30 V (Shanghai Delixi Switch Co., Ltd., model: TDGC3-500W), and the recovery cell bioreactor 5 is a stirred bioreactor (Beijing Huakan Biotechnology Co., Ltd., model: 3D miniSPIN).

[0177] Umbilical cord mesenchymal stem cells (MSC): sourced from Shandong Qilu Stem Cell Engineering Co., Ltd.

[0178] MSC cells for the experiment: MSC cells in the logarithmic growth phase were inoculated onto microcarriers (Beijing Huakan Biotechnology Co., Ltd., W01-200), and cultured in a stirred bioreactor until the third day to obtain a suspension of MSC cells in microcarriers for the experiment.

[0179] 100 mL of the experimental MSC cell microcarrier suspension was stirred at 50 rpm in a stirred bioreactor, and the peristaltic pump and AC voltage regulating power supply were turned on to transport the cell microcarrier suspension. The cell microcarrier suspension flowed through the microfluidic electrical stimulation chip device 3 and then was recovered into the stirred bioreactor. The suspension was cultured in a carbon dioxide shaking incubator at a stirring speed of 33 rpm for 48 hours, and the cell microcarrier suspension was collected for exosome extraction. The exosome extraction method was the same as that in Experimental Example 1. Control sample:

[0180] 100 ml of the experimental MSC cell microcarrier suspension was added to a stirred bioreactor and cultured in a carbon dioxide shaking incubator at a stirring speed of 33 rpm for 48 hours. The cell microcarrier suspension was collected for exosome extraction. The exosome extraction method was the same as that in Experimental Example 1.

[0181] The determination and calculation methods of exosomes are the same as those in Experimental Example 1. The final exosome particle size, concentration and increase multiple are listed in Tables 11 and 12. The exosome particle size distribution diagrams obtained for the test samples and the control samples are shown in Figure 17-Figure 18 listed.

[0182] Western blot was used to further identify and characterize the prepared exosomes: the results showed that both the test sample and the control sample contained the exosome membrane marker proteins CD63 and CD81 and the membrane marker protein TSG101, but did not contain the negative protein Calnexin. Fig.19 shown.

[0183] Table 11 Exosome particle size and concentration

[0184]

[0185] Table 12 Exosome production increase

[0186]

[0187] in conclusion:

[0188] According to the experimental data, the exosome concentration of the test sample is much higher than that of the control sample, and the exosome amplification multiple can reach 27.7 times, which can greatly increase the exosome production secreted by MSC cells.

[0189] Experimental Example 9 Multi-omics analysis of exosomes prepared in Experimental Example 3

[0190] 1. Gene Expression Analysis

[0191] Nucleic acid was extracted from each sample in Experimental Example 3 and compared with the reference genome sequence. mRNA was obtained based on the comparison results. A total of 10046 mRNAs were detected. The expression of each RNA was quantified and differentially analyzed. The overall distribution of gene expression is shown in Fig. 20 As shown, the gene expression levels (the top 20 in abundance) are shown in Fig.21 The differential expression volcano plot is shown in Fig. 22 shown.

[0192] 2. miRNA Analysis

[0193] The samples in Experimental Example 3 were subjected to small RNA sequencing, and 389 miRNAs were detected. The miRNAs in each sample were quantified, and their expression abundance (the top 20 in abundance) was shown in Fig.23 The overall distribution of miRNA expression is shown in Fig.24 As shown, the differentially expressed miRNAs detected are shown in Tables 13 and Fig.25 As shown,

[0194] Table 13 Differentially expressed miRNAs detected

[0195] Number of differentially expressed miRNAs Number of upregulated miRNAs Downregulation of miRNA number 44 26 18

[0196] Hisashi et al.'s study on signaling pathways showed that miR-491 specifically inhibits the phosphoinositide-3 kinase / threonine protein kinase (PI3K / AKT) pathway. Inhibiting the PI3K / AKT pathway and weakening the phosphorylation of AKT can inhibit the inflammatory response and accelerate the transition from the inflammatory stage to the tissue regeneration stage.

[0197] Zhou et al. demonstrated that the miR-23 family (miR-23a / b / c) activated angiogenic signals by targeting Sprouty2 and Sema6A, thereby enhancing angiogenesis; Karan et al. found that miR23c could regulate diabetic foot wound healing by targeting stromal cell-derived factor-1α (SDF-1α / CXCL12); Fang et al. found in a full-thickness skin defect mouse model that miR-23 carried by UBMSC-Exos played a key role in myofibroblast differentiation during scar formation by inhibiting TGF-β / SMAD2 stimulation.

[0198] The expression levels of miR-491-5p and miR-23c in the test samples were significantly increased (difference fold FC>4, P value ≤0.05), suggesting that the exosomes prepared using the microfluidic electrical stimulation chip system provided by the present invention may have higher potential for anti-inflammatory, promoting angiogenesis, promoting wound healing and reducing scar tissue formation.

[0199] 3. Protein Analysis

[0200] The samples in Experimental Example 3 were purified and enriched for exosomes, and exosome proteins were extracted and quantitatively analyzed.

[0201] The statistical table of the number of identified proteins is shown in Table 14 below, and the protein quantification results (the top 20 in abundance) are shown in Fig.26 As shown:

[0202] Table 14 Protein quantity statistics

[0203] Sample name Protein quantity Test samples 1495 Control samples 1472

[0204] The exosomes of both the control sample and the test sample expressed abundant proteins, which is consistent with the multiple biological functions of mesenchymal stem cell exosomes, such as anti-inflammatory, regeneration promotion, and immune regulation. Compared with the control sample, the expression of some proteins in the exosomes of the test sample was upregulated or downregulated. By detecting and analyzing these differentially expressed proteins, the number of differentially expressed proteins was detected as shown in Tables 15 and Fig. 27 The main differentially expressed proteins are shown in Table 16 below:

[0205] Table 15 Number of differentially expressed proteins

[0206] Number of differentially expressed proteins Number of upregulated proteins Number of down-regulated proteins 416 227 189

[0207] Table 16 Main differentially expressed proteins

[0208] Differentially expressed proteins Control samples Test samples PValue FDR log2FC regulated NUCB1 1.61E+05 4.02E+07 9.50E-08 0.000127 7.96706 up NCL 2.59E+05 3.25E+07 3.21E-06 0.004273 6.971089 up F11 1.98E+05 1.96E+07 9.54E-06 0.012702 6.634002 up SERPINA7 4.38E+05 2.43E+07 0.000117 0.155493 5.790824 up NPM1 7.42E+05 3.97E+07 0.000134 0.178175 5.741465 up SYNCRIP 3.27E+05 1.28E+07 0.000445 0.590497 5.291083 up ILF3 1.01E+05 3.51E+06 0.000696 0.922606 5.113526 up CA2 6.02E+05 2.06E+07 0.000728 0.963843 5.095613 up CCN2 3.89E+06 1.11E+08 0.001353 1 4.839884 up SERPINA3 9.33E+06 2.66E+08 0.001369 1 4.834849 up KRT19 7.68E+05 2.17E+07 0.001415 1 4.820773 up IGLC2 1.45E+07 3.83E+08 0.001765 1 4.726306 up CCDC80 5.92E+05 1.51E+07 0.001997 1 4.672807 up PPM1F 1.38E+05 3.42E+06 0.002182 1 4.634216 up HRG 8.81E+07 2.19E+09 0.002183 1 4.633942 up IGLV5-39 2.38E+05 5.78E+06 0.002353 1 4.600983 up PEBP1 8.39E+05 1.92E+07 0.002825 1 4.519526 up PSMD4 2.10E+05 4.76E+06 0.002952 1 4.499743 up CCN1 1.89E+06 4.23E+07 0.003043 1 4.486101 up BASP1 1.12E+06 2.27E+07 0.004206 1 4.33781 up MAPK8IP2 0.00E+00 9.08E+07 / / / up PNP 0.00E+00 7.11E+07 / / / up IGKV3D-15 0.00E+00 6.81E+07 / / / up TFPI2 0.00E+00 5.07E+07 / / / up CRP 0.00E+00 3.94E+07 / / / up S100A4 0.00E+00 2.67E+07 / / / up IGKV2-29 0.00E+00 1.89E+07 / / / up ARPC5L 0.00E+00 1.61E+07 / / / up IGKV6-21 0.00E+00 1.56E+07 / / / up RPL23A 0.00E+00 1.34E+07 / / / up

[0209] The most significant difference was in nuclear binding protein 1 (NUCB1), which increased 250-fold in exosomes prepared using the microfluidic electrical stimulation chip system. NUCB1 is the main calcium-binding protein in the Golgi apparatus and is involved in Golgi calcium homeostasis and Ca 2+ Regulate signal transduction. Lin et al. demonstrated that in cells overexpressing NUCB1, Ca 2+ The storage capacity can be increased by 2 to 3 times. 2+ Homeostasis is very important in cell secretion and other cellular activities. The amount of exosome release is related to intracellular Ca 2+ Therefore, the method of promoting cell secretion of exosomes by using the system containing a microfluidic electrical stimulation device provided by the utility model may increase intracellular Ca by overexpressing NUCB1. 2+ level, thereby promoting cells to secrete more exosomes.

[0210] In the exosomes of the test samples, a variety of proteins regulating cell functions were significantly upregulated compared to those in the control samples. It can be inferred that the exosomes prepared by the system including the microfluidic electrical stimulation chip device provided by the utility model may have enhanced therapeutic potential in certain biological functions and certain diseases.

[0211] The mitogen-activated protein kinase (MAPK) signaling pathway is one of the most widely studied signal transduction pathways. As one of the core members of the MAPK family, the c-Jun NH2-terminal kinase (JNK) pathway helps regulate cell migration, differentiation, proliferation, and apoptosis. Mitogen-activated protein kinase 8 interacting protein 2 (MAPK8IP2, also known as JIP2) is located on chromosome 22 (22q13) and is involved in the regulation of JNK and P38 MAPK signaling pathways, enhancing the activation of JNK by mixed lineage protein kinases, and plays a key role in the MAPK signaling pathway. The expression of MAPK8IP2 was significantly increased in the experimental samples (MAPK8IP2 was not detected in the control samples, and the relative expression in the experimental samples was as high as 9.08E+7).

[0212] Interleukin enhancer binding factor 3 (ILF3) is an RNA binding protein that participates in various cellular functions, such as mRNA stabilization, translational repression, and noncoding RNA biogenesis, promoting cell proliferation and migration. Cell communication network factor (CCN) is a cysteine-rich secreted matrix cell protein that is highly expressed in mesenchymal cells under various types of mechanical stress (e.g., tension, compression, and shear stress). CCN2 highly interacts with a variety of molecules, such as growth factors, heparan sulfate proteoglycans, and integrins, and can promote cell differentiation, migration, adhesion, and extracellular matrix formation. It also regulates the stability of the mRNA of the mechanosensitive ion channel TRPV4 in chondrocytes and plays an important role in promoting damage repair in multiple organs and tissues. Metal-dependent protein phosphatase 1F (PPM1F) is involved in regulating various cellular functions, such as cell cycle control, cell differentiation, immune response, and cell metabolism. The expression levels of ILF3, CCN2 and PPM1F in the test samples were significantly increased (log2(FC)>4, P value≤0.05), indicating that the exosomes prepared by the system containing the microfluidic electrical stimulation chip device provided by the utility model may have more advantages in tissue regeneration and injury repair by promoting cell differentiation, proliferation, migration and adhesion.

[0213] In addition, the exosomes prepared by the system containing the microfluidic electrical stimulation chip device provided by the utility model overexpress some active proteins related to disease treatment, and when used as a drug delivery carrier, they can also play a certain therapeutic effect. Synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) has multiple functions in controlling RNA metabolism by recognizing multiple sequences and regulating pre-mRNA splicing, translation, transport and degradation. For example, SYNCRIP regulates the selective splicing of SMN2 transcripts, which can compensate for the loss of SMN1 in patients with spinal muscular atrophy; Santangelo et al. also demonstrated that SYNCRIP can widely recognize mature miRNAs containing hEXO sequences (GGCU / A) and mediate the enrichment of miRNAs in exosomes. After testing, the expression of SYNCRIP protein in the test samples was significantly increased (log2(FC)=5.29, P=0.0004).

[0214] In summary, the utility model provides a microfluidic electrical stimulation chip device and a system containing the device, which promotes cell secretion of exosomes through the system, can perform online electrical stimulation of cells during normal cell suspension culture, stimulate the cell suspension flowing through multiple microchannels in the microfluidic electrical stimulation chip device through electrical signals, promote cell secretion of exosomes, increase the exosome yield to 10 to 80 times, and reduce the production cost by dozens of times, greatly improve the production efficiency, and is very suitable for industrial mass production. At the same time, the system provided by the utility model invention can also be used to perform cell transfection by electroporation while electrically stimulating cells, and can realize large-scale exosome drug loading by donor cell transfection method, and prepare exosomes containing endogenous transcribed mRNA or translated protein of exogenous genes.

Claims

1. A microfluidic electrical stimulation chip device, comprising an upper substrate (31), a microfluidic electrical stimulation chip body (32) and a lower substrate (33), wherein: The upper substrate (31) is hollowed out or tapped at both ends to form a liquid inlet (311) and a liquid outlet (313), and an upper substrate electrode (312) is embedded on the lower surface; the microfluidic electrical stimulation chip body (32) is engraved with a microfluidic liquid inlet section (321), a microfluidic reaction section (322) and a microfluidic liquid outlet section (323); the microfluidic liquid inlet section (321) is engraved on the microfluidic electrical stimulation chip body (32) in a divergent surface. The microfluidic reaction section (322) is etched and hollowed out on the microfluidic electrical stimulation chip body (32), the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body (32), and is correspondingly connected to the branch flow channel of the microfluidic liquid inlet section (321); the microfluidic liquid outlet section (323) is etched on the microfluidic electrical stimulation chip body (32) in a convergent surface manner; and the lower substrate (33) is embedded with a lower substrate electrode (331) on the upper surface.

2. The microfluidic electrical stimulation chip device according to claim 1, characterized in that: The thickness of the microfluidic electrical stimulation chip body (32) is 0.1 to 4.0 mm; the microfluidic channel inlet section (321) diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.05 to 2.00 mm; the microfluidic channel reaction section (322) is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic channel inlet section (321) is dispersed, the width of each channel is 0.1 to 4.0 mm, the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body (32), and the length is 2 to 40 mm; the microfluidic channel outlet section (323) is formed by the 2 to 8 branch channels corresponding to the microfluidic channel inlet section (321) converging into one channel, and the etching depth is 0.05 to 2.00 mm; the upper substrate electrode (312) and the lower substrate electrode (331) in the microfluidic electrical stimulation chip device are rectangular electrodes or fence-shaped printed electrodes, and the electrode material is titanium, platinum or gold.

3. The microfluidic electrical stimulation chip device according to claim 2, characterized in that: The microfluidic electrical stimulation chip body (32) has a thickness of 0.5 to 1.0 mm; the microfluidic channel inlet section (321) diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.25 to 0.50 mm; the microfluidic channel reaction section (322) is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic channel inlet section (321) is dispersed, each channel has a width of 0.5 to 1.0 mm, the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body (32), and the length is 5 to 10 mm; the microfluidic channel outlet section (323) is formed by the 2 to 8 branch channels corresponding to the microfluidic channel inlet section (321) converging into one channel, and the etching depth is 0.25 to 0.50 mm; the upper substrate electrode (312) and the lower substrate electrode (331) in the microfluidic electrical stimulation chip device are rectangular electrodes, and the electrode material is titanium, platinum or gold.

4. The microfluidic electrical stimulation chip device according to claim 3, characterized in that: The microfluidic electrical stimulation chip body (32) has a thickness of 1 mm; the microfluidic channel inlet section (321) diverges from one channel into 2 to 8 branch channels, and the upper surface etching depth is 0.50 mm; the microfluidic channel reaction section (322) is 2 to 8 parallel channels, corresponding to the number of branch channels after the microfluidic channel inlet section (321) is dispersed, each channel has a width of 0.5 to 1.0 mm, the etching depth is equal to the thickness of the microfluidic electrical stimulation chip body (32), and the length is 10 mm; the microfluidic channel outlet section (323) is formed by the 2 to 8 branch channels corresponding to the microfluidic channel inlet section (321) converging into one channel, and the etching depth is 0.50 mm; the upper substrate electrode (312) and the lower substrate electrode (331) in the microfluidic electrical stimulation chip device are rectangular electrodes, and the electrode material is titanium.

5. A system for promoting cell secretion of exosomes using a microfluidic electrical stimulation chip device, comprising: A supply cell bioreactor (1), a power device (2), a microfluidic electrical stimulation chip device (3), a power supply device (4), a recovery cell bioreactor (5), and pipes, connectors and wires connecting the modules, characterized in that the microfluidic electrical stimulation chip device (3) is the microfluidic electrical stimulation chip device described in any one of claims 1-4.

6. The system according to claim 5, characterized in that: The supply cell bioreactor (1) and the recovery cell bioreactor (5) are wave-type bioreactors, shaking-type bioreactors, stirring-type bioreactors, or bioreactors for suspending and culturing cells directly or indirectly through microcarriers.

7. The system according to claim 5, characterized in that: The power device (2) is an injection pump, a peristaltic pump or a pressure pump.

8. The system of claim 5, wherein: The power supply device (4) is an AC power supply with a frequency of 50 Hz and a voltage of 25 to 50 Vp-p.

9. The system of claim 5, wherein: The pipeline between the connection modules has an inner diameter of 0.8 to 12.7 mm and an outer diameter of 4.0 to 25.4 mm, and is made of silicone or thermoplastic elastomer.

10. The system according to any one of claims 5 to 9, characterized in that: Also includes a mixing device.

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