Perfusion Bioreactor

Abstract

The present invention provides a perfusion bioreactor, which at least includes: a culture chamber, in which a biological scaffold is arranged; a circulation pump, which is connected to the culture chamber; a supply chamber, which is respectively connected to the culture chamber and the circulation pump, and the supply chamber is used to hold a culture medium, and the supply chamber is connected to the outside world; a catheter, and the catheter includes a first catheter, a second catheter, a third catheter and a fourth catheter; when the supply chamber contains a culture medium, one end of the second catheter in the supply chamber is located below the liquid surface of the culture medium, and one end of the fourth catheter in the supply chamber is located above the liquid surface of the culture medium. The perfusion bioreactor of the present invention can simulate the in vivo microenvironment as much as possible during the in vitro construction of organs and tissues, ensure the supply of nutrients to cells and the discharge of excreta, the reactor is a sterile environment, stable, cell growth and vitality are vigorous, and the culture cycle and cost are reduced.

Description

Technical Field

[0001] The invention relates to the field of tissue engineering, in particular to a perfusion bioreactor. Background Art

[0002] Tissue Engineering is a new discipline that has been emerging in recent years. It mainly includes four aspects: seed cells, biomaterials, methods and technologies for constructing tissues and organs, and clinical applications of tissue engineering. The core of tissue engineering is to construct biological scaffold materials loaded with cells, and to build bioreactors through co-culture of cells and scaffold materials to simulate the functions of human organs and tissues.

[0003] Bioreactors include stirred, airlift, membrane and perfusion bioreactors. Stirred bioreactors use impellers and stirrers to rotate the culture fluid to ensure the balanced distribution of oxygen and nutrients in the cell culture fluid. However, since the rotation of the stirrer generates shear stress and small bubbles, it will destroy cells and scaffold materials, so it is not suitable for the cultivation of tissue engineering biomaterials.

[0004] The principle of the airlift bioreactor is to set a gas nozzle at the bottom of the reactor, add air and oxygen into the reactor from the gas nozzle, and achieve the purpose of gas exchange during the rising process. The advantage is that the gas exchange efficiency is high and the oxygen is evenly distributed in the reactor. However, the reactor volume is large and there are disadvantages of poor mixing performance when culturing with high-density microcarriers. In addition, the bubbles generated will damage cells at the moment of rupture, so it is not suitable for the cultivation of tissue engineering materials.

[0005] The main principle of membrane bioreactor is to exchange gas through a dialysis membrane with mass transfer function. The advantage is that it avoids the damage to cells caused by bubbles generated in the reactor. The disadvantage is that because the membrane transfers mass through diffusion, it is easy to cause insufficient oxygen supply when the volume of the culture medium is large or the cell concentration is high. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a perfusion bioreactor.

[0007] The principle of the perfusion bioreactor is to drive the culture fluid through a peristaltic pump, so that the culture medium with fresh nutrients and oxygen can be perfused into the scaffold material, and at the same time, the metabolic waste excreted by the cells can be taken away. According to the different volumes of the reactor and the different cell concentrations, different perfusion rates can be adjusted to ensure the supply of nutrients and oxygen.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides a perfusion bioreactor in a first aspect, characterized in that the perfusion bioreactor at least comprises:

[0009] A culture chamber, wherein a biological scaffold is provided in the culture chamber;

[0010] a circulation pump, connected to the culture chamber;

[0011] A supply cavity, which is connected to the culture cavity and the circulation pump respectively, and is used to hold the culture medium, and is connected to the outside;

[0012] a catheter, the catheter comprising a first catheter, a second catheter, a third catheter, and a fourth catheter;

[0013] The first conduit is used to connect the culture chamber and the circulation pump.

[0014] The second conduit is used to connect the circulation pump and the supply chamber.

[0015] The third conduit is used to connect the supply chamber and the culture chamber.

[0016] The fourth conduit is used to connect the supply cavity with the outside;

[0017] The second conduit and the fourth conduit satisfy the following conditions: when the supply chamber contains culture medium, one end of the second conduit in the supply chamber is located below the liquid surface of the culture medium, and one end of the fourth conduit in the supply chamber is located above the liquid surface of the culture medium.

[0018] A second aspect of the present invention provides use of the aforementioned perfusion bioreactor in cell culture.

[0019] As described above, the perfusion bioreactor of the present invention has the following beneficial effects:

[0020] The perfusion bioreactor of the present invention can simulate the in vivo microenvironment as much as possible during the in vitro construction of organs and tissues, ensure the supply of nutrients to cells and the discharge of excrement, and the reactor is a sterile environment, which is stable, and the cells grow and are vigorous, thereby reducing the culture cycle and cost. According to the different volumes of the reactor and the different concentrations of cells, the speed of the circulation pump can be adjusted to obtain different perfusion rates, thereby fully ensuring the supply of nutrients and oxygen and the discharge of metabolic waste. The device of the present invention does not generate bubbles, thereby preventing cell damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a schematic structural diagram of the perfusion bioreactor of the present invention.

[0022] Component number description

[0023] 1 Culture chamber

[0024] 11 Biological Scaffolds

[0025] 2 Circulation pump

[0026] 3 Supply chamber

[0027] 31 Supply chamber cover

[0028] 32 Supply chamber body

[0029] 4 Catheter

[0030] 41 First catheter

[0031] 42 Second catheter

[0032] 43 Third catheter

[0033] 44 Fourth catheter

[0034] 5. Air filter DETAILED DESCRIPTION

[0035] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0036] See also Figure 1 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0037] The perfusion bioreactor provided by the present invention comprises at least:

[0038] A culture chamber 1, wherein a biological scaffold 11 is provided in the culture chamber 1;

[0039] A circulation pump 2, connected to the culture chamber 1;

[0040] A supply chamber 3 is connected to the culture chamber 1 and the circulation pump 2 respectively. The supply chamber 3 is used to hold the culture medium and is connected to the outside.

[0041] The conduit 4 includes a first conduit 41, a second conduit 42, a third conduit 43 and a fourth conduit 44;

[0042] The first conduit 41 is used to connect the culture chamber 1 and the circulation pump 2.

[0043] The second conduit 42 is used to connect the circulation pump 2 and the supply chamber 3.

[0044] The third conduit 43 is used to connect the supply chamber 3 and the culture chamber 1.

[0045] The fourth conduit 44 is used to connect the supply cavity 3 with the outside;

[0046] The second conduit 42 and the fourth conduit 44 satisfy the following conditions: when the supply chamber 3 contains culture medium, one end of the second conduit 42 in the supply chamber 3 is located below the liquid surface of the culture medium, and one end of the fourth conduit 44 in the supply chamber is located above the liquid surface of the culture medium.

[0047] The third conduit 43 satisfies the condition that one end of the third conduit 43 in the supply chamber is located above the liquid surface of the culture medium.

[0048] The biological scaffold refers to a cell carrier with a three-dimensional porous structure that transports nutrients for cell growth and excretes metabolic products.

[0049] The bioscaffold can be made of natural materials or artificial materials. The natural material is selected from proteins, polysaccharides or inorganic materials, such as collagen, mitogen, silk fibroin or matrigel; such as chitosan, hyaluronic acid or alginate; such as natural coral or hydroxyapatite. The artificial material is selected from polymers, ceramics or metals; such as polycaprolactone (PCL), polyhydroxyethyl methylvinyl acetate (PHEMA), polyglycolic acid (PGA), bioglass, calcium carbonate cement or titanium alloy.

[0050] The circulation pump includes a motor to drive the circulation pump to rotate. According to the different volumes of the reactor and the different concentrations of the cells, the speed of the circulation pump can be adjusted to obtain different perfusion rates, thereby fully ensuring the supply of nutrients and oxygen and the discharge of metabolic waste.

[0051] The power generated by the circulation pump drives the culture medium in the supply chamber to be transported to the culture chamber. The culture medium contains dissolved oxygen, so that the cells on the biological scaffold in the culture chamber are supplied with nutrients and oxygen; and the waste and carbon dioxide generated by cell metabolism are taken out and transferred back to the supply chamber. The carbon dioxide escapes to the outside through the fourth conduit. Oxygen from the outside can also enter the culture chamber and dissolve in the culture medium.

[0052] The bioreactor constructed by this circulating perfusion system can simulate the in vivo microenvironment as much as possible during the in vitro construction of organs and tissues to ensure the supply of nutrients to cells and the discharge of excreta, thereby reducing the culture cycle and cost.

[0053] The inner diameter of the supply cavity can be set as required to ensure sufficient contact area with the air and ensure the oxygen supply.

[0054] In one embodiment, one end of the second conduit 42 in the supply chamber is located at the bottom of the supply chamber and does not contact the bottom of the supply chamber.

[0055] In one embodiment, one end of the third conduit 43 and / or the fourth conduit 44 in the supply cavity is located at the top of the supply cavity.

[0056] In one embodiment, the fourth conduit 44 is provided with an air filter 5 at one end thereof connected to the outside world. The air filter is connected to the air, which not only ensures gas exchange between the culture medium and the air, but also prevents the entry of microorganisms such as bacteria, thereby ensuring a sterile environment in the reactor.

[0057] The air filter 5 is made of plastic material, and a filter core is arranged inside the air filter 5 to filter bacteria and other microorganisms in the air. The air filter is a commercially available product.

[0058] In one embodiment, a first filter screen 12 is provided at one end of the bio-scaffold 11 close to the circulation pump 2 for filtering impurities.

[0059] The first filter screen 12 and the second filter screen 13 are plastic filter screens that can filter solid impurities in the circulating culture liquid. The first filter screen and the second filter screen are commercially available products.

[0060] In one embodiment, a second filter screen 13 is provided at one end of the bio-scaffold 11 close to the supply cavity 3 for filtering impurities and fixing the bio-scaffold.

[0061] The first filter screen and the second filter screen can remove impurities in the pipeline to prevent them from affecting the growth and vitality of cells on the bio-scaffold.

[0062] In one embodiment, the supply chamber 3 includes a supply chamber cover 31 and a supply chamber body 32 , and the second conduit 42 , the third conduit 43 and the fourth conduit 44 are respectively disposed on the supply chamber cover 31 .

[0063] In one embodiment, the circulation pump 2 is selected from a peristaltic pump.

[0064] When the device of the present invention is used to perform a bioscaffold perfusion operation, the bioscaffold 11 is first placed between the first filter screen 12 and the second filter screen 13 in the culture chamber 1. The supply chamber cover 31 is opened, and a suitable amount of culture medium is placed in the supply chamber body, so that the liquid level of the culture medium is higher than the lower edge of one end of the second conduit 42 located in the supply chamber, and lower than the lower edge of one end of the third conduit 43 and the fourth conduit 44 located in the supply chamber. The peristaltic pump 2 is started, so that the culture medium is sucked into the second conduit 42, and then sequentially passes through the peristaltic pump 2 and the first conduit 41 to reach the culture chamber 1. The culture medium that reaches the culture chamber is filtered by the first filter screen 12 to immerse the bioscaffold 11 and provide nutrients and oxygen. Then, after being filtered by the second filter screen 13, the culture medium returns to the supply chamber 3 through the third conduit 43.

[0065] The present invention also provides use of the above-mentioned perfusion bioreactor in cell culture.

[0066] Furthermore, the cell culture is the culture of a biological scaffold material loaded with cells.

[0067] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A perfusion bioreactor, characterized in that: The perfusion bioreactor comprises at least: A culture chamber (1), wherein a biological scaffold (11) is provided in the culture chamber (1); A circulation pump (2) connected to the culture chamber (1); A supply chamber (3) is connected to the culture chamber (1) and the circulation pump (2) respectively, and the supply chamber (3) is used to hold the culture medium and is connected to the outside; The conduit (4) comprises a first conduit (41), a second conduit (42), a third conduit (43) and a fourth conduit (44); the first conduit (41) is used to connect the culture chamber (1) and the circulation pump (2), The second conduit (42) is used to connect the circulation pump (2) and the supply chamber (3). The third conduit (43) is used to connect the supply chamber (3) and the culture chamber (1). The fourth conduit (44) is used to connect the supply chamber (3) with the outside world, and an air filter (5) is provided at one end of the fourth conduit (44) that is connected with the outside world; The second conduit (42) and the fourth conduit (44) satisfy the following conditions: when the supply chamber (3) contains culture medium, one end of the second conduit (42) in the supply chamber (3) is located below the liquid surface of the culture medium, and one end of the fourth conduit (44) in the supply chamber is located above the liquid surface of the culture medium; one end of the bio-scaffold (11) close to the circulation pump (2) is provided with a first filter net (12) for filtering impurities; and one end of the bio-scaffold (11) close to the supply chamber (3) is provided with a second filter net (13) for filtering impurities and fixing the bio-scaffold; One end of the third conduit (43) in the supply chamber is located above the liquid surface of the culture medium, and one end of the second conduit (42) in the supply chamber is located at the bottom of the supply chamber and does not contact the bottom of the supply chamber; one end of the third conduit (43) and / or the fourth conduit (44) in the supply chamber is located at the top of the supply chamber.

2. The perfusion bioreactor according to claim 1, characterized in that: The supply chamber (3) comprises a supply chamber cover (31) and a supply chamber body (32), and the second conduit (42), the third conduit (43) and the fourth conduit (44) are respectively arranged on the supply chamber cover (31).

3. The perfusion bioreactor according to claim 1, characterized in that: The circulation pump (2) is selected from a peristaltic pump.

4. Use of the perfusion bioreactor according to any one of claims 1 to 3 in cell culture.

5. The use according to claim 4, characterized in that The cell culture is the culture of a biological scaffold material loaded with cells.

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