CN116121068A - Application system of 3D cell culture perfusion bioreactor and cell culture method - Google Patents

Abstract

The invention discloses an application system and a cell culture method of a 3D cell culture perfusion bioreactor, belonging to the fields of cell biology and tissue engineering. Comprising the following steps: the top surface of the bioreactor is provided with a cavity which is used as a main chamber for culturing cells; the bottom of the bioreactor is provided with a plurality of bottom openings, and the bottom openings are connected with the main chamber; the bioreactor is also provided with a liquid inlet and a liquid outlet which are communicated with the main chamber; a plurality of capillaries are arranged in the main chamber, and the capillaries are arranged at one side close to the bottom opening; the main chamber is also provided with a plurality of supporting members which are used for supporting the capillary tube; a part of capillary longitudinally passes through the supporting member and is connected with the liquid inlet so as to receive liquid; the rest of the capillaries longitudinally penetrate through the supporting member and are connected with the liquid outlet so as to discharge liquid. The invention can more accurately simulate in vivo biological conditions and cell behaviors, and is beneficial to improving the development of medicines.

Description

Application system and cell culture method of 3D cell culture perfusion bioreactor

technical field

The invention belongs to the fields of cell biology and tissue engineering, and in particular relates to an application system of a 3D cell culture perfusion bioreactor and a cell culture method.

Background technique

In recent years, drug development has focused on using in vitro culture models of cells to mimic the in vivo conditions of the human body. The traditional culture method is to seed a specific number or concentration of cells on a microwell plate. Microwell plates are cultured to support cells attached to a two-dimensional (2D) monolayer prior to performing the prescribed assays. While this represents some improvement, growing cells in this 2D fashion is still problematic, at least 2D cell culture models often do not accurately mimic in vivo environmental conditions and/or cell behavior.

For example, in a two-dimensional cell culture model, the loss rate of candidate anticancer drugs is as high as 95%, so that the drug efficacy evaluated in vitro cannot provide guidance for clinical application. There may even be unforeseen toxicity issues. The reasons for this are as follows: First, some extracellular matrix (ECM) components are missing in the 2D cell culture model, resulting in certain cell-cell and cell-matrix interactions not occurring. These extracellular matrix components and interactions are critical for cell differentiation, cell proliferation, and/or cellular functions that normally occur in vivo. Second, it has been found that the environment of two-dimensional cell culture models does not accurately mimic the environment in which living three-dimensional (3D) cancer cells reside because of the absence of hypoxic regions, heterogeneous cell populations, distinct regions of cell proliferation, ECM effects, etc. , soluble signaling gradients, and/or differential nutrient and metabolic waste transport.

3D cell culture models can overcome many of the limitations of 2D cell culture models because 3D cell culture models more closely mimic the characteristics and environments of complex in vivo conditions. Studies have shown that compared with 2D cell culture models, the same tumor cell lines tested in 3D cell culture models are less sensitive to anticancer drugs.

One approach to create three-dimensional cell culture models is to form three-dimensional scaffolds from porous polymers and/or biomaterials. At least one study has identified an optimal pore size of approximately 100-400 μm for 3D scaffolds for cell culture models. (Ref. Han et al., "The Effect of Pore Size on Cell Behavior Using a Melt Electrowriting Scaffold", Frontiers in Bioengineering and Biotechnology, Vol. 9, No. 629270, July 2021.) This result is based on Obtain good cell nutrition, cell growth space and cell-scaffold interactions (results depend on specific cell line).

To achieve reproducible results, high-precision fabrication methods are required to control the size and shape of the pores. For example, the sustained fabrication of pores with a diameter of 100 μm requires fabrication methods with at least 20 μm resolution, such as micro-3D printing. In particular, projection microstereolithography (PμSL) and two-photon polymerization are micro-3D printing techniques capable of consistently generating complex 3D structures. Additionally, these printing techniques can create 3D structures from biocompatible and/or biodegradable polymers such as polyethylene glycol (PEG) and polylactic acid (PLA). After polymerization, these polymers may be hard or soft.

Cell culture processes can be described by reaction kinetics. In steady state, the process by which cells consume metabolites (such as cellular nutrients) is usually described by the MichaelisMenten equation:

In the formula, v is the reaction speed, V _max is the maximum absorption rate of metabolites, K _M is the concentration of metabolites when the absorption rate is half of the maximum absorption rate (Michelis constant), and [S] is the concentration of metabolites. In Michaelis-Menten kinetics, the consumption behavior at low concentrations obeys first-order kinetics. This means that the consumption rate is directly proportional to the concentration. As the concentration of metabolites increases, the consumption behavior will gradually change to zero-order kinetics. This means that the consumption rate is close to or equal to the maximal velocity, independent of the metabolite concentration. This is because cells eventually reach saturation, so their uptake of metabolites reaches a plateau. Several embodiments of bioreactors proposed in this project use capillary tubes to transport metabolites to cells within the bioreactor so that even when cells are densely packed, cells can achieve maximum uptake of metabolites for physiological sexual function.

In the in vivo environment, when the cell concentration is on the order of 10 ¹⁰ /ml, the distance between the cell and the capillary is less than 100 μm. In order to provide metabolites to such high density cell populations, a high density of nutrient transport channels (capillaries) is required.

Contents of the invention

In order to solve the above technical problems, the present invention discloses the application system and method of a 3D cell culture perfusion bioreactor, which has a capillary system to promote the interaction between 3D cells and cells and between cells and ECM, As well as facilitating the transport of metabolites and metabolic waste. In order to achieve the purpose of the foregoing invention, the present invention adopts the following technical solutions:

The application system of 3D cell culture perfusion bioreactor, including:

A bioreactor, the top surface of the bioreactor is provided with a cavity, and the cavity is used as a main chamber for culturing cells; the bottom of the bioreactor is provided with a plurality of bottom openings, and the bottom The opening is connected to the main chamber; the bioreactor is also provided with a liquid inlet and a liquid outlet communicating with the main chamber;

Several capillaries are arranged in the main chamber, and the capillaries are arranged on a side close to the bottom opening;

The main chamber is also provided with a plurality of support members, and the support members are used to support the capillary; part of the capillary longitudinally passes through the support member and is connected to the liquid inlet to receive liquid; A part of the capillary passes longitudinally through the support member and is connected to the liquid outlet for discharging liquid.

Further, the width direction of the bioreactor is parallel to the width direction of the main chamber, and the length direction of the bioreactor is parallel to the length direction of the main chamber.

Further, the support member is a support wall, and the support wall intersects and is perpendicular to the capillary to provide structural support for the capillary; the support wall is provided with a plurality of through holes for the two sides of the support wall. cell interaction.

Further, the adjacent capillaries perform liquid exchange through reflux to form a plurality of reflux chambers, the first column of the capillary in the reflux chamber performs liquid exchange with the second column, and the third column and the fourth column perform liquid exchange. Exchange..., the i-th column of the capillary performs liquid exchange with the i+1-th column, and so on;

Wherein, the capillary in the i-th column in the reflux chamber is an arterial capillary, which is used to input liquid for reflux, and the capillary in the i+1th column is a venous capillary, which is used to output liquid;

The arterial capillary is connected with the liquid inlet, and the venous capillary is connected with the liquid outlet.

Further, the bioreactor also includes a liquid inlet chamber and a liquid outlet chamber, the arterial capillary is connected to the liquid inlet through the liquid inlet chamber, and the venous capillary is connected to the outlet through the liquid outlet chamber. Liquid port connection.

Further, the liquid outlet and the liquid inlet are arranged in the same longitudinal direction of the bioreactor, and the liquid outlet chamber is at least partly higher than the liquid inlet chamber to prevent air or other gases from entering the capillary.

Further, it also includes a side pool and an observation window, the side pool is connected with the observation window to form a culture cavity, the culture cavity is provided with a culture room, and the culture room is provided with a culture medium;

The bioreactor is placed in the culture chamber, the observation window at least partially overlaps the bottom opening, and the capillary is submerged in the culture chamber;

An input port is provided on the side of the side tank close to the liquid inlet chamber, and an output port is provided on the side of the side tank close to the liquid outlet chamber; the input port is connected to the liquid inlet, and the distance between the input port and the One end of the liquid inlet is connected to the liquid inlet pump; the output port is connected to the liquid outlet, and the end of the output port far away from the liquid outlet is connected to the liquid outlet pool;

A liquid inlet valve is arranged at the connection between the input port and the liquid inlet; a liquid outlet valve is arranged at the connection between the output port and the liquid outlet.

Further, at least one of the arterial capillary and the venous capillary is provided with micropores; the density of the capillary is 3-30 capillary/mm ² ; at least part of the bioreactor is made using projection micro-stereolithography and 3D printing by two-photon polymerization.

A cell culture method comprising the steps of:

Place the bioreactor in the culture chamber, where the capillary in the bioreactor is submerged in the culture medium in the culture chamber for cell culture; inoculate the cells in the main chamber, cultivate the cells, and pump the culture medium from the liquid inlet, The metabolic waste produced is output from the liquid outlet into the liquid outlet pool; use a microscope to observe through the observation window;

After seeding cells in a bioreactor, they can be placed in an incubator where environmental conditions can be controlled to promote or prevent cell growth and/or proliferation.

Beneficial effect:

To facilitate 3D cell-to-cell and cell-to-ECM interactions, as well as to facilitate the transport of metabolites and metabolic waste. The invention is suitable for high-density 3D cell culture. This perfusion-based 3D bioreactor can more accurately simulate in vivo biological conditions and cell behavior, helping to improve drug development. Part or all of the structure of the perfusion 3D bioreactor can be realized by PμSL micro 3D printing and two-photon polymerization technology.

Description of drawings

Figure 1 is a schematic diagram of the bioreactor structure.

Figure 2 is a schematic diagram of the bottom structure of the bioreactor.

Figure 3 is a schematic diagram of a typical arrangement of capillaries.

Figure 4 is a cross-sectional view of a bioreactor.

Fig. 5 is a sectional view of the reflux chamber.

Fig. 6 is a side view of the liquid outlet chamber and the liquid inlet chamber.

Fig. 7 is a front view of the liquid outlet chamber and the liquid inlet chamber.

Fig. 8 is a schematic diagram of the positions of the liquid outlet chamber and the liquid inlet chamber.

Fig. 9 is a diagram illustrating an example of an experiment for studying cell biology and/or tissue engineering.

Among them: 10: bioreactor; 20: main chamber; 30: bottom opening; 40: liquid inlet; 50: liquid outlet; 100: capillary; 102: arterial capillary; 104: venous capillary; 110: support wall; 120 : through hole; 130: return flow; 200: return chamber; 210: liquid inlet chamber; 220: liquid outlet chamber; 300: liquid inlet pump; 310: liquid outlet pool; 320: liquid inlet valve; 330: liquid outlet valve; 400 : side pool; 410: observation window; 420: culture room; 500: microscope.

Detailed ways

The technical solutions of the various embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

The present invention will be described in further detail below through specific implementation examples and in conjunction with the accompanying drawings.

Example 1

The application system of 3D cell culture perfusion bioreactor, including:

Bioreactor 10, the top surface of bioreactor 10 is provided with cavity, and cavity is used as main chamber 20, in order to cell is cultivated; The bottom of bioreactor 10 is provided with a plurality of bottom openings 30, and bottom opening 30 and The main chamber 20 is connected; the bioreactor 10 is also provided with a liquid inlet 40 and a liquid outlet 50 communicating with the main chamber 20;

Several capillaries 100 are arranged in the main chamber 20, and the capillaries 100 are arranged on the side close to the bottom opening 30; a plurality of support members are also arranged in the main chamber 20, and the support members are used to support the capillaries 100, and the capillaries 100 pass through the support members longitudinally It is connected with the main chamber 20 and extends from one end of the main chamber 20 to the other end of the main chamber 20; part of the capillary 100 is connected with the liquid inlet 40 for receiving liquid; the remaining part of the capillary 100 is connected with the liquid outlet 50 for Drain the liquid.

Preferably, the main chamber 20 is bowl-shaped, the open top edge of the main chamber 20 is adjacent to the top surface of the bioreactor 10, and the main chamber 20 is approximately 10 mm long, 6 mm wide, and 5 mm deep; the bioreactor The length direction of the bioreactor 10 is parallel to the length direction of the main chamber 20 , and the width direction of the bioreactor 10 is parallel to the width direction of the main chamber 20 .

In this embodiment, five layers of capillary tubes 100 are provided, each layer has 14 independent capillary tubes 100 , 70 capillary tubes in total. In other embodiments, the outer diameter of each capillary 100 is 100 μm, and the inner diameter is 80 μm; the distance between adjacent outer surfaces of the capillary 100 is 0.4 mm; the equivalent cross-sectional density of the capillary 100 is 4 capillary/mm ² .

Preferably, arranging the capillary 100 in a row is conducive to observing the cells and/or tissues therein, and using a microscope to observe from the lower bottom opening 30, the lower capillary 100 (near the bottom opening 30) will not block the higher capillary 100 (away from the bottom opening 30) between the horizontal spaces.

In other embodiments, other capillary arrangements, orientations, sizes and densities may be utilized. In other embodiments, some or all of the capillaries 100 may be arranged longitudinally, transversely, and/or diagonally through the main chamber 20; in other embodiments, some or all of the capillaries 100 may be oriented parallel and/or at different angles to each other. orientation; in other embodiments, some or all of the capillary tubes 100 may have different inner diameters, outer diameters, and wall thicknesses; in other embodiments, some or all of the capillary tubes 100 may have different spacings between them; in other embodiments, The cross-sectional density of capillaries may be about 3-30 capillaries/mm ² .

Preferably, the support member is a support wall 110, and the support wall 110 intersects and is perpendicular to the capillary 100 to provide structural support for the capillary 100; the support wall 110 is provided with a plurality of through holes 120 for cell interaction on both sides of the support wall 110 .

In this embodiment, the number of support walls 110 depends on the outer diameter, wall thickness and material of the capillary 100 extending longitudinally through a 10 mm long main chamber, each capillary having an outer diameter of 100 μm and a wall thickness of 10 μm , composed of polyethylene glycol, requires seven equally spaced support walls to adequately support the capillary. In other embodiments, the spacing between adjacent capillaries 100 is approximately an order of magnitude greater than the outer diameter of the capillaries 100, and the capillaries 100 may be supported by a single vertical and/or horizontal support member.

In this embodiment, the diameter of the through holes 120 is 200 μm, and the through holes 120 are circular holes passing through the support wall 110 with uniform spacing. In other embodiments, the through holes 120 may also adopt other pitches and shapes.

Preferably, adjacent capillary tubes 100 perform liquid exchange through reflux to form multiple reflux chambers 200, the first column of capillary tubes 100 in the reflux chamber 200 exchanges liquid with the second column, and the third column exchanges liquid with the fourth column..., The i-th column of the capillary performs liquid exchange with the i+1-th column, and so on;

Wherein, in the reflux chamber 200, the i-th row of capillaries 100 is an arterial capillary 102 for outputting liquid; the i+1th row of capillary 100 is a venous capillary 104 for outputting liquid;

The arterial capillary 102 is connected to the liquid inlet 40 , and the venous capillary 104 is connected to the liquid outlet 50 .

In this embodiment, there are seven flashback chambers between the arterial capillary 102 and the adjacent venous capillary 104, each containing seven flashbacks. In other embodiments, each flashback chamber may consist of a single flashback between a single arterial capillary 102 and a single venous capillary 104 .

Preferably, the bioreactor 10 further includes a liquid inlet chamber 210 and a liquid outlet chamber 220 , the arterial capillary 102 is connected to the liquid inlet 40 through the liquid inlet chamber 210 , and the venous capillary 104 is connected to the liquid outlet 50 through the liquid outlet chamber 220 .

Preferably, the liquid outlet 50 and the liquid inlet 40 are arranged on the same longitudinal direction of the bioreactor 10, and the liquid outlet chamber 220 is at least partly higher than the liquid inlet chamber 210 to prevent air or other gases from entering the capillary; It may be advantageous to connect the uppermost portion of 50 or its vicinity to outlet chamber 220 .

Preferably, it includes a side pool 400 and an observation window 410, the side pool 400 is connected to the observation window 410 to form a culture chamber, a culture chamber 420 is arranged in the culture chamber, and a culture medium is arranged in the culture chamber 420;

The bioreactor 10 is placed in the culture chamber, the observation window 410 at least partially overlaps the bottom opening 30, and the capillary 100 is immersed in the culture chamber 420;

The side tank 400 is provided with an input port near the liquid inlet chamber 210, and the side tank 400 is provided with an output port near the liquid outlet chamber 220; the input port is connected to the liquid inlet port 40, and the end of the input port far away from the liquid inlet port 40 is connected to the liquid inlet port. The pump 300 is connected; the output port is connected to the liquid outlet 50, and the end of the output port away from the liquid outlet 50 is connected to the liquid outlet pool 310;

The connection between the input port and the liquid inlet 40 is provided with a liquid inlet valve 320; the connection between the output port and the liquid outlet 50 is provided with a liquid outlet valve 330; the connection between the input port and the liquid inlet 40 and the output port are connected with the liquid outlet 50 An O-ring is provided to prevent liquid leakage.

In other embodiments, the outlet tank 310 may be connected to a waste treatment device for separating metabolic waste from nutrients, and may transport valuable metabolites to the inlet 40 for recycling. The waste treatment unit transports the recovered metabolites to a recovery repository from which the recovered nutrients can be extracted by pumping or gravity. In the case of pumping, the intake pump 300 may transport the recovered and/or reprocessed metabolites from the recovery reservoir to the intake port 40, or a second pump may transport the recovered and/or reprocessed metabolites The food is transported to the liquid inlet 40 or fresh storage.

Preferably, at least one of the arterial capillary 102 and the venous capillary 104 is provided with micropores.

The permeability of a solute across a membrane (such as the wall of a capillary) is directly proportional to the concentration gradient of the solute across the membrane. Accordingly, the wall thickness and/or material of the capillary 100 is selected to allow certain solutes, including metabolites and metabolic wastes, to enter and exit the capillary 100 at an appropriate rate. If the concentration of metabolic waste in the main chamber 20 is too high (eg, about 100 μmol/L), the health of the cells in the main chamber 20 may be affected. Metabolic waste products may include toxins such as nitrogen compounds. Likewise, if the concentration of nutrients in the main chamber 20 is too low, the cells may not be able to proliferate and/or grow, or the cells may starve and/or die. Therefore, it is important to provide the main chamber 20 with sufficient nutrients and to constantly remove metabolic wastes, which can be increased by adding micropores to the walls of the capillary 100 (for either or both metabolites and metabolic wastes). diffusion.

In this embodiment, micropores with a diameter of approximately 5-15 μm can be added to the wall of the capillary 100 to significantly increase the diffusion rate (s).

In other embodiments, the diameter and/or density (spacing) of micropores added to arterial capillary 102 may be different than the diameter and/or density (spacing) of micropores added to venous capillary 104 .

In other embodiments, micropores may be added only to the arterial capillary 102 or only to the venous capillary 104, or only to selected portions thereof.

As the medium is transported within the capillary, metabolites may diffuse out through the walls of the capillary 100 where they may be consumed by cells within the main chamber. Thus, cells that consume metabolites may generate and release metabolic waste products that may enter and be transported by diffusion through capillary walls. The arterial capillary 102 transports fresh medium from the inlet chamber 210 to the return chamber 200 , and the venous capillary 104 transports the medium from the return chamber 200 to the outlet chamber 220 . Thus, metabolic waste that diffuses into the capillary 100 is transported to the outflow chamber 220 therein, while metabolic waste that diffuses into the arterial capillary 102 is transported to the reflux chamber where it will enter the capillary 100 and then be transported to the outflow chamber 220 . The flow velocity in the capillary 100 is on the order of several millimeters per second.

Preferably, at least part of the bioreactor 10 in this embodiment is 3D printed using projection microstereolithography and two-photon polymerization.

In other embodiments, the bioreactor is indexed or printed from a Class I or higher biocompatible material, such as polyethylene glycol (PEG, molecular weight 575). Some materials may require surface treatment to promote cell adhesion, which may be important for cell culture experiments that require model cells to attach to and proliferate on the surface of a bioreactor. The poly-l-lysine solution can be used to coat one or more surfaces of the bioreactor, covering one or more surfaces of the main chamber 20 .

Example 2

Cell culture method, adopt the application system of the 3D cell culture perfusion bioreactor that embodiment 1 provides, this method comprises:

The bioreactor 10 is placed in the culture chamber, wherein the capillary 100 in the bioreactor 10 is submerged in the culture medium of the culture chamber 420 for cell culture; the cells are inoculated in the main chamber 20, the cells are cultivated, and the culture medium is removed from the The liquid inlet is pumped in, and the metabolic waste produced is output from the liquid outlet 50 into the liquid outlet pool 310; use a microscope to observe through the observation window;

After seeding cells in bioreactor 10, they can be placed in an incubator where environmental conditions can be controlled to promote or prevent cell growth and/or proliferation.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (9)

The application system of 1.3D cell culture perfusion bioreactor is characterized in that, comprising: The top surface of the bioreactor is provided with a cavity, and the cavity is used as a main chamber for culturing cells; the bottom of the bioreactor is provided with a plurality of bottom openings, and the bottom openings are connected to the main chamber. connected; the bioreactor is also provided with a liquid inlet and a liquid outlet communicated with the main chamber; Several capillaries are arranged in the main chamber, and the capillaries are arranged on a side close to the bottom opening; The main chamber is also provided with a plurality of support members, and the support members are used to support the capillary; part of the capillary longitudinally passes through the support member and is connected to the liquid inlet to receive liquid; A part of the capillary passes longitudinally through the support member and is connected to the liquid outlet for discharging liquid. 2. the application system of 3D cell culture perfusion bioreactor as claimed in claim 1, is characterized in that, the width direction of described bioreactor is arranged in parallel with the width direction of described main chamber, and the length of described bioreactor The direction is set parallel to the length direction of the main chamber. 3. The application system of 3D cell culture perfusion bioreactor as claimed in claim 1, characterized in that, the support member is a support wall, and the support wall intersects and is perpendicular to the capillary to provide support for the capillary. Structural support; the support wall is provided with a plurality of through holes for cell interaction on both sides of the support wall. 4. The application system of 3D cell culture perfusion bioreactor as claimed in claim 1, characterized in that, the adjacent said capillaries carry out liquid exchange by reflux to form a plurality of reflux chambers, and said capillaries in said reflux chambers The first column of the capillary performs liquid exchange with the second column, the third column performs liquid exchange with the fourth column..., the i-th column of the capillary tube performs liquid exchange with the i+1-th column, and so on; Wherein, the capillary in the i-th column in the reflux chamber is an arterial capillary, which is used to input liquid for reflux, and the capillary in the i+1th column is a venous capillary, which is used to output liquid; The arterial capillary is connected with the liquid inlet, and the venous capillary is connected with the liquid outlet. 5. The application system of the 3D cell culture perfusion bioreactor as claimed in claim 4, wherein the bioreactor also includes an inlet chamber and an outlet chamber, and the arterial capillary passes through the inlet chamber and the outlet chamber. The liquid inlet is connected, and the venous capillary is connected with the liquid outlet through the liquid outlet chamber. 6. The application system of 3D cell culture perfusion bioreactor as claimed in claim 5, characterized in that, the liquid outlet and the liquid inlet are arranged in the same longitudinal direction of the bioreactor, and the outlet The liquid chamber is at least partially higher than the inlet chamber to prevent air or other gases from entering the capillary. 7. The application system of 3D cell culture perfusion bioreactor as claimed in claim 6, further comprising a side pool and an observation window, the side pool is connected with the observation window to form a culture chamber, and the culture chamber A culture chamber is arranged inside, and a culture medium is arranged in the culture chamber; The bioreactor is placed in the culture chamber, the observation window at least partially overlaps the bottom opening, and the capillary is submerged in the culture chamber; An input port is provided on the side of the side tank close to the liquid inlet chamber, and an output port is provided on the side of the side tank close to the liquid outlet chamber; the input port is connected to the liquid inlet, and the distance between the input port and the One end of the liquid inlet is connected to the liquid inlet pump; the output port is connected to the liquid outlet, and the end of the output port far away from the liquid outlet is connected to the liquid outlet pool; A liquid inlet valve is arranged at the connection between the input port and the liquid inlet; a liquid outlet valve is arranged at the connection between the output port and the liquid outlet. 8. The application system of 3D cell culture perfusion bioreactor as claimed in claim 7, wherein at least one of the arterial capillary and the venous capillary is provided with micropores; the density of the capillary is 3-30 Capillaries/mm ² ; at least part of the bioreactor was 3D printed using projection microstereolithography and two-photon polymerization. 9. the method for cell culture, is characterized in that, adopts the application system of the 3D cell culture perfusion bioreactor described in any one of claim 1-8, described method comprises the following steps: Place the bioreactor in the culture chamber, where the capillary in the bioreactor is submerged in the culture medium in the culture chamber for cell culture; inoculate the cells in the main chamber, cultivate the cells, and pump the culture medium from the inlet, The metabolic waste produced is output from the liquid outlet into the liquid outlet pool; use a microscope to observe through the observation window; After seeding cells in a bioreactor, they can be placed in an incubator where environmental conditions can be controlled to promote or inhibit cell growth and/or proliferation.

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