Cell microsphere manufacturing and culturing integrated device based on electrospinning membrane and manufacturing and culturing method of cell microsphere manufacturing and culturing integrated device

Abstract

The invention discloses an electrospinning membrane-based cell microsphere manufacturing and culturing integrated device and a manufacturing and culturing method.The device comprises a cell microsphere manufacturing device and a cell microsphere culturing device, the cell microsphere manufacturing device comprises an electrospinning membrane, a microfluid substrate with a surface microstructure and a gel device; the cell microspheres can be rapidly manufactured in batches; the cell microsphere culture device is a microfluid culture device and is used for ensuring dynamic infusion of cell culture fluid. The in-situ culture of the cell microspheres is realized by combining with a process of manufacturing the nanofiber membrane by electrostatic spinning, the device provided by the invention greatly improves the manufacturing efficiency of the cell microspheres, is strong in reproducibility, is easy to realize batch production, facilitates subsequent cell culture, and is suitable for large-scale popularization and application. The device has the advantages of simplicity in operation, high reproducibility, batch production, high cell survival rate and the like, and is suitable for manufacturing and culturing the cell microspheres.

Description

Technical Field

[0001] The invention relates to the technical fields of mechanical device manufacturing and biological manufacturing, and in particular to an integrated device for manufacturing and culturing cell microspheres of an electrospun membrane and a manufacturing and culturing method thereof. Background Art

[0002] The main method currently used to produce nanofibers is electrospinning, which stretches polymers through electric field forces and uses the interaction between the medium and the electric field to produce nanofibers. Electrospinning is a fiber processing technology that is simple to operate but has great potential. The diversity of fiber structures and adjustable preparation designs mean that it is widely used. The advantages can be summarized as simple experimental conditions, low experimental costs, high fiber yields, and high material applicability.

[0003] The traditional technologies currently used to manufacture microspheres include template molding, emulsion polymerization, dispersion polymerization, spray drying, extrusion-based methods, etc., which face inherent challenges in manufacturing materials with good monodispersity, controlled geometry, customized structure and complex composition. In order to meet these challenges, microfluidics, which involves the delicate manipulation of small volumes of liquids in micron-sized channels, has become a promising alternative for preparing microspheres with well-defined characteristics. Microfluidics has become a powerful platform technology for the rational design and manufacture of materials with complex and precisely adjustable sizes, shapes, structures and compositions by appropriately adjusting operating parameters such as flow rate and viscosity. However, in the existing technology, breakthroughs are still needed to successfully produce materials with ideal uniformity and adjustable geometry on a microfluidic platform with a specific chip design. Summary of the invention

[0004] 1. Technical issues to be resolved In view of the shortcomings of the prior art, the present invention provides an integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes, which avoids the steps of collecting and culturing the microspheres separately after manufacturing, so that the cultured cell microspheres can be taken out and used directly.

[0005] (II) Technical solution To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes, comprising a cell microsphere manufacturing device and a cell microsphere culturing device, characterized in that the cell microsphere manufacturing device comprises an electrospinning membrane, a surface microstructure, a microfluidic substrate with a surface microstructure, and a gel device; the microfluidic substrate is used to shape the electrospinning membrane so that the membrane forms a microstructure, thereby manufacturing cell microspheres, and then the material exchangeability of the electrospinning membrane is used to carry out subsequent culture of the cell microspheres.

[0006] Preferably, the electrospinning membrane 1 is made by an electrospinning system, which consists of three basic components: a DC power supply, a capillary and a grounded conductive collector; the capillary is usually a solution container with a spinneret, and natural polymer materials are selected as materials. Other components can be added to the polymer spinning solution to modify it as needed, and the collector is replaced by a microfluid with a microstructure on the surface to obtain a nanofiber membrane with an ordered stacking structure.

[0007] Preferably, the cell microsphere culture device, i.e., a microfluidic culture device, adopts the manufacturing technology used for the above-mentioned microfluidic substrate. A culture device groove is provided in the microfluidic culture device. A through hole is drilled on each side of the culture device groove and penetrates each other to serve as a culture medium inlet and a culture medium outlet respectively. Dynamic perfusion is adopted to maintain sufficient nutrient supply of the culture medium and timely discharge of cell metabolic waste.

[0008] Preferably, it also includes a scraper for scraping off the liquid.

[0009] Preferably, the whole device is assembled by placing the microfluidic culture vessel at the bottom layer, placing the microfluidic substrate with surface microstructure on it, and the electrospinning membrane is placed on the substrate, and the gel device is selected and placed according to the characteristics of the material used for microsphere manufacturing.

[0010] Another technical problem to be solved by the present invention is to provide a method for manufacturing and culturing an integrated device for manufacturing and culturing cell microspheres based on electrospun membranes, comprising the following steps: 1) Fabrication of a microfluidic substrate with a surface microstructure: First, a negative mold is fabricated by photocuring 3D printing, and then a surface microstructure is fabricated by PDMS polydimethylsiloxane soft lithography to obtain a microfluidic substrate; 2) Electrospinning a membrane on a microfluidic substrate: The electrospinning system used is composed of three basic components: a DC power supply, a capillary, and a collector. The collector uses the microfluidic substrate prepared in the previous step, and the spinning material is PAN polyacrylonitrile. A membrane with a microstructure is obtained on the microfluidic substrate.

[0011] 3) Obtaining spherical liquid: Injecting the silk protein solution containing human fibroblasts onto the surface of the electrospinning membrane, waiting for the solution to fall into the microstructure on the membrane surface, and then scraping off the excess liquid with a scraper. The microstructure contains spherical liquid; 4) Cross-linking to form cell microspheres: Use visible light to irradiate the liquid on the surface of the electrospinning membrane, and it can be quickly cross-linked in 1 minute to obtain the desired cell microspheres.

[0012] 5) Introduce cell culture medium: slowly inject DMEM culture medium containing 20% ​​CS and PS through the inlet of the microfluidic culture device until it flows out from the outlet at the other end and the culture medium reaches dynamic equilibrium; 6) To achieve subsequent culture of cell microspheres: remove the microfluidic substrate and place the electrospun membrane on top of the microfluidic culture device so that the bottom of the membrane at the microstructure is in direct contact with the culture medium in the microfluidic culture device. Finally, place the device in a constant temperature incubator with 37.5% CO2 for subsequent culture.

[0013] (III) Beneficial effects Compared with the prior art, the present invention provides an integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes, which has the following beneficial effects: This integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes can easily achieve the purpose of mass manufacturing of cell microspheres, and can basically ensure that a cell microsphere contains only one cell, realizing the integration of manufacturing and subsequent culturing of cell microspheres, avoiding the need to take out the cell microspheres after they are manufactured and transfer them to cell culture fluid for culturing, and avoiding the risk of cell microspheres dying due to improper operation before culturing. The main body of the device designed this time is manufactured by microfluidics and electrospinning technology. These two technologies are not difficult to operate, have strong reproducibility, and are easy to achieve mass production. The integration of manufacturing and subsequent culturing of cell microspheres is achieved by using microfluidics and electrospinning technology. The device has the advantages of simple operation, strong reproducibility, mass production, and high cell survival rate, and is suitable for the manufacture and cultivation of cell microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the structure of the integrated device for manufacturing and culturing cell microspheres provided by the present invention.

[0015] Figure 2 Schematic diagram of electrospinning membrane.

[0016] Figure 3 Schematic diagram of a microfluidic substrate with surface microstructures.

[0017] Figure 4 Schematic diagram of the scraper.

[0018] Figure 5 Schematic diagram of the structure of the microfluidic culture device.

[0019] Figure 6 A simplified process flow chart for the entire device.

[0020] In the figure: 1. electrospinning membrane; 2. microfluidic substrate; 3. surface microstructure; 4. microfluidic culture vessel; 5. scraper; 6. culture vessel groove; 7. culture medium inlet; 8. culture medium outlet. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Embodiment 1: An integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes, including a cell microsphere manufacturing device and a cell microsphere culturing device, wherein the cell microsphere manufacturing device includes an electrospinning membrane 1, a surface microstructure 3, a microfluidic substrate 2 with a surface microstructure 3, and a gel device; the electrospinning membrane is shaped by the microfluidic substrate to form a microstructure of the membrane, thereby manufacturing cell microspheres, and then the material exchangeability of the electrospinning membrane is used to carry out subsequent culture of the cell microspheres.

[0023] The cell microsphere manufacturing device includes an electrospinning membrane 1, a microfluidic substrate 2 with a surface microstructure 3 and a gel device; the electrospinning membrane is prepared using a typical electrospinning system, and the system consists of three basic components: a DC power supply, a capillary and a grounded conductive collector; the capillary is usually a solution container with a spinneret, and natural polymer materials are selected, and other components are added to the polymer spinning solution to modify it, and the collector is replaced by a microfluid with a microstructure on the surface to obtain a nanofiber membrane with an ordered stacking structure.

[0024] The microfluidic substrate 2 with the surface microstructure 3 can be designed in advance by 3D modeling software to design the surface microstructure 3 and the overall shape, and then printed out by 3D printing technology such as light-curing 3D printing technology; or the structural master mold is made by soft lithography technology, and then the microfluidic substrate 2 with the surface microstructure 3 is obtained by material casting.

[0025] The gel device is designed according to the properties of the material used to manufacture the microspheres, such as common thermal gel properties, photo-crosslinking properties, and ionic crosslinking properties.

[0026] The cell microsphere culture device, i.e., the microfluidic culture device 4, adopts the manufacturing technology used in the above-mentioned microfluidic substrate 2. It is shaped like a hollow structure similar to a culture dish. A through hole is punched on each side, which penetrates each other and serves as the inlet and outlet of the culture medium respectively. A dynamic perfusion method is adopted to maintain sufficient nutrient supply of the culture medium and timely discharge of cell metabolic waste.

[0027] The cell microsphere culture device, namely the microfluidic culture device 4, adopts the manufacturing technology used in the above-mentioned microfluidic substrate 2. A culture device groove 6 is provided in the microfluidic culture device 4. A through hole is punched on each side of the culture device groove 6 and penetrates each other to serve as a culture medium inlet 7 and a culture medium outlet 8 respectively. Dynamic perfusion is adopted to maintain sufficient nutrient supply of the culture medium and timely discharge of cell metabolic waste.

[0028] The whole device is assembled by placing the microfluidic culture vessel 4 at the bottom layer, placing the microfluidic substrate 2 with the surface microstructure 3 thereon, and the electrospinning membrane 1 on the substrate, while the gel device is selected and placed according to the characteristics of the material used for the manufacture of the microspheres.

[0029] 1) Manufacturing a microfluidic substrate 2 with a surface microstructure 3: firstly, a negative mold is manufactured by photocuring 3D printing, and then a surface microstructure 3 is manufactured by PDMS polydimethylsiloxane soft lithography to obtain a microfluidic substrate 2; 2) Electrospinning a membrane on the microfluidic substrate 2: The electrospinning system used is composed of three basic components: a DC power supply, a capillary and a collector. The collector uses the microfluidic substrate 2 prepared in the previous step, and the spinning material is PAN polyacrylonitrile. A membrane with a microstructure is obtained on the microfluidic substrate 2; 3) Obtaining spherical liquid: injecting the silk protein solution containing human fibroblasts onto the surface of the electrospinning membrane 1, and after the solution falls into the microstructure 3 on the membrane surface, scraping off the excess liquid with a scraper 5, the microstructure contains spherical liquid; 4) Cross-linking to form cell microspheres: Use visible light to irradiate the liquid on the surface of the electrospinning membrane 1, and it can be quickly cross-linked in 1 minute to obtain the desired cell microspheres; 5) Introducing cell culture medium: slowly inject DMEM culture medium containing 20% ​​CS and PS through the inlet of the microfluidic culture device (4) until it flows out from the outlet at the other end and the culture medium reaches dynamic equilibrium; 6) To achieve subsequent culture of cell microspheres: remove the microfluidic substrate 2, place the electrospinning membrane 1 above the microfluidic culture device 4, so that the bottom of the membrane at the microstructure is in direct contact with the culture fluid in the microfluidic culture device 4, and finally place the device in a constant temperature incubator with 37.5% CO2 for subsequent culture.

[0030] The beneficial effects of the present invention are as follows: the integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes can easily achieve the purpose of mass manufacturing of cell microspheres, and can basically ensure that a cell microsphere contains only one cell, realizing the integration of manufacturing and subsequent culturing of cell microspheres, avoiding the step of taking out the cell microspheres after manufacturing and transferring them to cell culture fluid for culturing, and avoiding the risk of cell microspheres dying due to improper operation before culturing. The main body of the device designed this time is manufactured by microfluidic technology and electrospinning technology. The operation difficulty of these two technologies is not high, the reproducibility is strong, and mass production is easy to achieve. The integration of manufacturing and subsequent culturing of cell microspheres is achieved by using microfluidic technology and electrospinning technology. The device has the advantages of simple operation, strong reproducibility, mass production, high cell survival rate, etc., and is suitable for the manufacture and cultivation of cell microspheres.

[0031] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An integrated device for manufacturing and culturing cell microspheres based on electrospinning membranes, comprising a cell microsphere manufacturing device and a cell microsphere culturing device, characterized in that: The cell microsphere manufacturing device comprises an electrospinning membrane (1), a surface microstructure (3), a microfluidic substrate (2) having the surface microstructure (3), and a gel device; the electrospinning membrane (1) is shaped by the microfluidic substrate (2) so that the membrane forms a microstructure, thereby manufacturing cell microspheres, and then the material exchangeability of the electrospinning membrane (1) is used to carry out subsequent cultivation of the cell microspheres.

2. The integrated device for manufacturing and culturing cell microspheres based on electrospun membrane according to claim 1, characterized in that: The electrospinning membrane (1) is produced by an electrospinning system, which consists of three basic components: a DC power supply, a capillary and a grounded conductive collector; the capillary is a solution container with a spinneret, and a natural polymer is selected and incorporated into the polymer spinning solution. The collector is replaced by a microfluid with a microstructure on the surface to produce a nanofiber membrane with an ordered stacking structure.

3. The integrated device for manufacturing and culturing cell microspheres based on electrospun membrane according to claim 1, characterized in that: The cell microsphere culture device, i.e., a microfluidic culture device (4), adopts the manufacturing technology used in the above-mentioned microfluidic substrate (2). A culture device groove (6) is provided in the microfluidic culture device (4). A through hole is drilled on each side of the culture device groove (6) and penetrates each other to serve as a culture medium inlet (7) and a culture medium outlet (8), respectively. Dynamic perfusion is adopted to maintain sufficient nutrient supply of the culture medium and timely discharge of cell metabolic waste.

4. The method for manufacturing and culturing an integrated device for manufacturing and culturing cell microspheres based on electrospun membranes according to claim 1, characterized in that: It also includes a scraper (5) for scraping off the liquid.

5. According to claim 1, the integrated device for manufacturing and culturing cell microspheres based on electrospun membranes is assembled by placing the microfluidic culture device (4) at the bottom layer, placing the microfluidic substrate (2) with a surface microstructure (3) thereon, and placing the electrospun membrane (1) on the substrate (2), while the gel device is selected and placed according to the characteristics of the material used for manufacturing the microspheres.

6. A method for manufacturing and culturing an integrated device for manufacturing and culturing cell microspheres based on electrospun membranes, characterized in that: The following steps are involved: 1) Manufacturing a microfluidic substrate (2) with a surface microstructure (3): firstly, a negative mold is manufactured by photocuring 3D printing, and then the surface microstructure (3) is manufactured by PDMS polydimethylsiloxane soft lithography to obtain a microfluidic substrate (2); 2) Electrospinning a membrane on a microfluidic substrate (2): The electrospinning system used is composed of three basic components: a DC power supply, a capillary and a collector. The collector uses the microfluidic substrate (2) prepared in the previous step, and the spinning material is PAN polyacrylonitrile. A membrane with a microstructure is obtained on the microfluidic substrate (2); 3) Obtaining spherical liquid: injecting a silk protein solution containing human fibroblasts onto the surface of the electrospinning membrane (1), waiting for the solution to fall into the microstructure (3) on the membrane surface, and then scraping off the excess liquid with a scraper (5), so that the microstructure contains spherical liquid; 4) Cross-linking to form cell microspheres: Using visible light to irradiate the liquid on the surface of the electrospun membrane (1), rapid cross-linking can be achieved within 1 minute to obtain the desired cell microspheres; 5) Introducing cell culture medium: slowly inject DMEM culture medium containing 20% ​​CS and PS through the inlet of the microfluidic culture device (4) until it flows out from the outlet at the other end and the culture medium reaches dynamic equilibrium; 6) To achieve subsequent culture of cell microspheres: remove the microfluidic substrate (2), place the electrospun membrane (1) on top of the microfluidic culture device (4), so that the bottom of the membrane at the microstructure is in direct contact with the culture medium in the microfluidic culture device (4), and finally place the device in a constant temperature incubator with 37.5% CO2 for subsequent culture.

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