CN101199436A - Three-dimensional three-dimensional bioreactor for culturing hepatocytes - Google Patents

Abstract

A three-dimensional bioreactor for cultivating hepatocytes, comprising a cell culture chamber with a cell inlet and an exhaust port, an inlet chamber with a culture solution inlet and a culture fluid outlet adjacent to the cell culture chamber Outflow chamber, one end of the multiple hollow fibers located in the cell culture chamber communicates with the inflow chamber, and the other end communicates with the outflow chamber, the inflow chamber, outflow chamber and multiple hollow fibers together form the culture fluid through-flow The channel, the culture solution flow channel and the cell culture chamber communicate with each other through the micropores on the hollow fiber, and the feature is that: the cell culture chamber is provided with a three-dimensional porous scaffold material. The invention can provide hepatocytes with growth and culture conditions more in line with the physiological environment, which is conducive to the cultivation of hepatocytes with high activity and high density, and guarantees the liver support and therapeutic effect of the bioartificial liver based on this.

Description

Three-dimensional three-dimensional bioreactor for culturing hepatocytes

technical field

The patent of the present invention relates to a medical device, especially a core device that can be used as a core device of an in vitro bioartificial liver support system, an experimental device or a biological preparation device, and can also be used for experimental research and extraction of cultured liver cells A small production facility for cellular products.

Background technique

The existing clinical bioreactor is a hollow fiber reactor, its structure is exactly the same as that of the hemodialyzer, but the material, pore size and molecular interception of the semipermeable membrane used are different. The hollow fiber reactor is the A bundle (50-100) of hollow fibers is sealed in a transparent organic material shell, which is divided into inner and outer chambers by the hollow fibers, which are used to place liver cells and flow patient blood respectively. With the help of the hollow fiber semi-permeable membrane, Carry out two-way material transmission and immune barrier effect, so as to realize the supporting and therapeutic effect of bioartificial liver.

There are many defects in the practical application of this mode of reactor: 1. In terms of structure, the hollow fibers of the traditional ordinary reactor are arranged in parallel and densely in a bundled column shape, and the culture environment of liver cells is less considered. It is incompatible with material transport and exchange; 2. In terms of cell culture, it can only provide two-dimensional culture for liver cells, but cannot provide a three-dimensional culture microenvironment, which is not conducive to the tissue culture and functional recovery of liver cells in vitro; 3. In cell culture In terms of distribution, when added cells are often deposited at the bottom of the reactor due to the action of gravity, they cannot be evenly distributed among the hollow fibers. Even if the cells can reach the middle of the fiber bundle, they will be inactivated due to insufficient oxygen supply; 4. In clinical liver support However, due to the small amount of cells (generally only 1% to 5% of adult hepatocytes), low viability, and poor function, the traditional reactor is difficult to exert due biological functions and effects.

At present, there is also a perfusion bed scaffold type reactor, which is widely used in bioartificial liver cell culture or bioartificial liver research and animal experiments. Its structure is to seal three-dimensional scaffold materials of different materials or different pore sizes in Inside the shell, a three-dimensional scaffold is used to provide a three-dimensional culture microenvironment for the liver cells. When culturing hepatocytes, the culture medium is continuously circulated from the shell, and the culture medium circulates through the gap between the three-dimensional scaffold and the liver cells to provide rich oxygen and nutrients for the liver cells; when used as a bioartificial liver During research or clinical use, the patient's blood or plasma is continuously passed through the shell, the blood or plasma circulates through the gap between the three-dimensional scaffold and the liver cells, and the normal liver cells detoxify the plasma or blood.

The bioreactor has the following disadvantages: 1. From the structure of the reactor, when it is used as a bioartificial liver for research or clinical use, the patient's blood or plasma directly contacts the liver cells in the three-dimensional scaffold, without any immune barrier effect, no It is beneficial to protect the immune safety of patients, and it is difficult to promote its use in clinical practice; 2. When culturing hepatocytes, in terms of nutrient supply, the nutrient solution flows through the gap between the three-dimensional scaffold and the liver cells. Gradually smaller, the circulating nutrient solution is easily blocked, resulting in poor circulation, uneven perfusion, and limited nutrient supply; 3. The blood, plasma or nutrient solution is circulated under the action of the pump, so that the cells are directly affected by the Larger shear force can easily cause damage to liver cells.

Therefore, the above defects are one of the important factors that make it difficult to further improve the curative effect of bioartificial liver in the treatment of liver failure.

Contents of the invention

The purpose of the patent of the present invention is to provide a three-dimensional bioreactor with simple structure and convenient use, which can provide growth and culture conditions more in line with the physiological environment for liver cells, and is conducive to the cultivation of liver cells with high activity and high density. Cells, to maximize the biological role, realize liver-specific detoxification function, biosynthesis and transformation metabolism function, realize the perfect combination of hepatocyte culture, immune barrier and material transfer, and ensure the liver support of the bioartificial liver with this as the core and therapeutic effect.

The object of the present invention is achieved by such a technical scheme, which includes a cell culture chamber with a cell inlet and an exhaust port, and an inlet chamber with a culture solution inlet and a culture solution outlet adjacent to the cell culture chamber One end of the multiple hollow fibers located in the cell culture cavity communicates with the inflow cavity, and the other end communicates with the outflow cavity. The inflow cavity, the outflow cavity and the multiple hollow fibers together form a culture solution process. The flow channel, the culture fluid flow channel and the cell culture chamber communicate with each other through the micropores on the hollow fiber, and the feature is that: the cell culture chamber is provided with a three-dimensional porous scaffold material.

In the present invention, the three-dimensional porous scaffold material is a biomaterial referred to in tissue engineering in the field of biomedicine. It is a three-dimensional scaffold on which seed cells live and attach before forming tissues, and it can fix cells in a certain position , providing a place for physiological activities such as growth, reproduction, metabolism and extracellular matrix secretion, and guiding the basic shape of the regenerated tissue. Therefore, it not only has good biocompatibility, but also has a specific shape and a three-dimensional interconnected porous structure, mainly providing support and three-dimensional growth space for cell growth, including degradable and non-degradable three-dimensional scaffold materials, such as chitosan , collagen, fibrin, polylactic acid, polylactic acid-polyglycolate, polyurethane, etc.

The three-dimensional porous scaffold material can be filled in the entire cell culture cavity, and can also be arranged in the cell culture cavity in this way: multi-layer three-dimensional porous scaffold materials are distributed in the cell culture cavity sequentially at a distance from each other, and each layer of three-dimensional porous scaffold material They are all filled with the corresponding cross-section of the cell culture cavity. Certainly, the user or the producer can arbitrarily arrange the three-dimensional porous scaffold material in the cell culture cavity according to actual needs.

The invention can be used for culturing hepatocytes, can also be used for bioartificial liver in vitro, and can also be used for experimental research and preparation of biological preparations.

The method for culturing hepatocytes with the present invention is:

1. Place the patent of the present invention in the cell culture box, and give 95% oxygen, 5% carbon dioxide and 100% humidity conditions.

2. The hepatocytes separated by in vitro digestion technology are inoculated into the three-dimensional porous scaffold material in the cell culture chamber of the present invention by centrifugal inoculation method and circulating perfusion inoculation method, and the specific steps are as follows:

(1), centrifugal inoculation method: the hepatic cell suspension is added in the cell culture cavity through the hepatic cell inlet, and the amount added each time is 30 to 100 grams, and it is centrifuged for 3 minutes, and the order of the present invention is reversed and repeated. Centrifuge 5 times.

(2) Circulating perfusion inoculation method: the hepatocyte inlet is communicated with the exhaust port through a peristaltic pump and a silicone tube to form a circulation channel. The speed per minute circulates in the circulation channel, and after 30 minutes, the hepatocytes are evenly distributed in the three-dimensional porous scaffold material in the hepatocyte culture cavity.

(3) Remove the peristaltic pump and the silicone tube, and close the inlet and outlet of the liver cells.

3. The present invention is connected as follows: the culture solution pool is communicated with the culture solution inlet of the present invention through a silicone tube, a peristaltic pump, and an oxygenator, and the culture solution outlet of the present invention is connected to the culture solution pool through a silicone tube, so that the The invented culture fluid flow channel forms a culture fluid circulation channel together with an oxygenator, a peristaltic pump and a culture fluid pool.

4. Under the action of the peristaltic pump, the nutrient solution circulates as follows: the nutrient solution pool flows into the nutrient solution inlet of the present invention through the oxygenator, then flows through the nutrient solution flow channel, and then flows from the nutrient solution outlet of the present invention. Return to the storage tank; the speed of the circulation of the culture medium is controlled at about 30ml/min. During the circulating flow of the culture medium, the micropores on the hollow fibers evenly provide rich oxygen and nutrients to the hepatocytes in the cell culture chamber, so that the hepatocytes grow three-dimensionally in the three-dimensional porous scaffold material, thereby cultivating A large number, high activity, and high density of liver cells, which have excellent biological functions.

After using the present invention to cultivate a large amount of hepatocytes according to the above-mentioned method, the present invention can be used for the bioartificial liver in vitro. The normal liver cells in the culture chamber detoxify the patient's blood or plasma through the micropores of the hollow fiber, and at the same time, prevent the blood or plasma from directly contacting the liver cells, which plays an immune barrier role, thus exerting better artificial liver support and therapeutic effect. Of course, the present invention can also be used in experimental research, which can meet the physiological requirements of liver cells; when the present invention is used to prepare biological preparations, more cell products can be extracted.

Due to the adoption of the above technical solution, the patent of the present invention has the following advantages:

1. The structure is reasonable, and the three-dimensional porous scaffold material and hollow fiber are combined and applied to the bioreactor, which can more effectively meet the requirements of the artificial liver bioreactor for hepatocyte culture, material transmission, and immune barrier.

2. The use of hollow fibers can effectively carry out the material transmission of the culture medium or the patient's blood, and at the same time play the role of immune barrier, and significantly reduce the influence of the shear force of the culture medium on the liver cells.

3. The three-dimensional porous scaffold material is used to provide three-dimensional support for the liver cells, so that the liver cells grow three-dimensionally in the three-dimensional porous scaffold material, which is easy to organize, and the density of liver cells is high, the number is large, and the function is strong.

4. The small-diameter micropores in the three-dimensional porous scaffold material can make the hepatocytes evenly distributed in the three-dimensional porous scaffold material, avoiding the deposition of hepatocytes at the bottom of the cell culture chamber, and effectively overcoming the difficulty of cultivating hepatocytes efficiently in the original hollow fiber reactor lack of.

5. The culture medium uniformly provides oxygen and nutrients to the liver cells in two ways of radial flow and diffusion, which significantly increases the supply distance of nutrients and oxygen, breaks through the distance limit of 100 μm to 200 μm in the diffusion effect, and enables the liver cells to obtain Oxygen and nutrients are more abundant.

6. The hepatocyte metabolites flow radially from the micropores on the cellulose isolation membrane into the radial cavity, which can effectively reduce the toxic effect of the metabolites on the hepatocytes.

Description of drawings

The accompanying drawings of the present invention are as follows:

Fig. 1 is the structural representation of first kind of embodiment of the present invention;

Fig. 2 is the structural representation of the second embodiment of the present invention;

Fig. 3 is A-A sectional view among Fig. 2;

Fig. 4 is the structural representation of the third embodiment of the present invention;

Fig. 5 is B-B sectional view among Fig. 4;

Fig. 6 is a connection relationship diagram of the use status of the third embodiment of the present invention.

In the figure: 1. Shell; 2. Inflow chamber; 3. Outflow chamber; 4. Cellulose isolation membrane; 5. Cell culture chamber; 6. Radial flow chamber; 7. Three-dimensional porous scaffold material; 8. Hollow fiber ; 9. Culture fluid flow channel; 10. Culture fluid inlet; 11. Culture fluid outlet; 12. Radiation fluid outlet; 13. Cell addition port; 17. Culture liquid pool; 18. Silicone tube; 19. Peristaltic pump; 20. Oxygenator; 21. Peristaltic pump; 22. Silicone tube; 23. Waste liquid pool.

Detailed ways

Below in conjunction with accompanying drawing and embodiment the present invention will be further described:

As shown in Figures 1, 2, and 3, the present invention includes a cell culture chamber 5 with a cell inlet 13 and an exhaust port 14, and an inflow chamber 2 with a culture solution inlet 10 is arranged next to the cell culture chamber 5. Outflow chamber 3 with culture solution outlet 12, one end of a plurality of hollow fibers 8 located in cell culture chamber 5 communicates with inflow chamber 2, and the other end communicates with outflow chamber 3, inflow chamber 2, outflow chamber The chamber 3 and a plurality of hollow fibers 8 together form a culture fluid flow channel 9, and the culture fluid flow channel 9 and the cell culture chamber 5 communicate with each other through the micropores on the hollow fibers 8, and it is characterized in that: the cell culture chamber 5 A three-dimensional porous scaffold material 7 is provided.

Embodiment 1: As shown in FIG. 1 , the three-dimensional porous scaffold material 7 can fill the entire cell culture cavity 5 . Embodiment 2: As shown in Figures 2 and 3, the three-dimensional porous scaffold material 7 can also be arranged in the cell culture chamber 5 in this way: the multilayer three-dimensional porous scaffold materials 7 are distributed in the cell culture chamber 5 sequentially at a distance from each other, And each layer of three-dimensional porous scaffold material 7 is filled with the corresponding cross-section of the cell culture cavity 5 .

The invention can be used for culturing hepatocytes, can also be used for bioartificial liver in vitro, and can also be used for experimental research and preparation of biological preparations.

The method for culturing hepatocytes with the present invention is:

1. Place the patent of the present invention in the cell culture box, and give 95% oxygen, 5% carbon dioxide and 100% humidity conditions.

2. The hepatocytes separated by in vitro digestion technology are inoculated into the three-dimensional porous scaffold material 7 in the cell culture cavity 5 of the present invention by centrifugal inoculation method and circulating perfusion inoculation method, and the specific steps are as follows:

(1), centrifugal inoculation method: the hepatic cell suspension is added in the cell culture chamber 5 through the hepatic cell inlet, and the amount added each time is 30 to 100 grams, and centrifuged for 3 minutes, and the order of the present invention is reversed, Repeat centrifugation 5 times.

(2), circulating perfusion inoculation method: the hepatic cell inlet 13 communicates with the exhaust port 14 through a peristaltic pump and a silicone tube to form a circulation channel, and under the action of the peristaltic pump, the hepatic cell suspension in the hepatic cell culture Circulating flow in the circulation channel at a speed of 1 ml/min. After 30 minutes, the hepatocytes are uniformly distributed in the three-dimensional porous scaffold material 7 in the hepatocyte culture chamber.

(3) Remove the peristaltic pump and the silicone tube, and close the hepatic cell inlet 13 and exhaust port 14 .

3. The present invention is connected as follows: the culture solution pool 17 is communicated with the culture solution inlet 10 of the present invention through the silica gel tube 18, the peristaltic pump 19, the oxygenator 20, and the culture solution outlet 11 of the present invention is passed through the silica gel tube 18 to The culture fluid pool 17, so that the culture fluid flow channel 9 of the present invention together with the oxygenator 20, the peristaltic pump 19, and the culture fluid pool 17 form a culture fluid circulation channel, as can be seen with reference to FIG. 6 .

4, under the effect of peristaltic pump 19, culture solution carries out following circulation flow: flow into culture solution inlet 10 of the present invention through oxygenator 20 by culture solution pool 17, then flow through culture solution flow channel 9, then from this The inventive culture solution outlet 11 returns to the culture solution pool 17; the speed of the culture solution circulating flow is controlled at about 30ml/min. During the circulating flow of the culture medium, the micropores on the hollow fiber 8 evenly provide abundant oxygen and nutrients to the hepatocytes in the cell culture cavity 5, so that the hepatocytes grow three-dimensionally in the three-dimensional porous scaffold material 7, In this way, a large number, high activity, and high density of hepatocytes are cultivated, and the hepatocytes have excellent biological functions.

After using the present invention to cultivate a large number of hepatocytes according to the above method, the present invention can be used for in vitro bioartificial liver. At this time, what circulates in the culture fluid flow channel 9 is not the culture fluid, but the patient's blood or plasma. , the normal hepatocytes in the cell culture cavity 5 detoxify the patient's blood or plasma through the micropores of the hollow fiber 8, and at the same time, prevent the blood or plasma from directly contacting the hepatocytes, which plays an immune barrier role, thereby playing a better role. artificial liver support and treatment. Of course, the present invention can also be used in experimental research, which can meet the physiological requirements of liver cells; when the present invention is used to prepare biological preparations, more cell products can be extracted.

Embodiment 3: Outside the cell culture chamber 5, a radial flow chamber 6 covering the cell culture chamber 5 is provided. The walls of the cell culture chamber 5 are all made of cellulose isolation membrane 4. 6 communicate with each other through micropores on the cellulose isolation membrane 4 , and a radial liquid outlet 12 communicating with the radial flow chamber 6 is provided on the body of the radial flow chamber 6 . Realize the concrete structure of this embodiment as shown in Fig. 4, 5, and it is to be respectively provided with inflow cavity 2 and outflow cavity 3 on the left and right opposite sides in casing 1, between inflow cavity 1 and outflow cavity 3 A cell culture chamber 5 is formed by the inner wall of the barrel-shaped cellulose isolation membrane 4, the outer wall of the inlet cavity 2, and the outer wall of the outlet cavity 3, the outer wall of the cellulose isolation membrane 4 and the inner wall of the housing 1, the inlet cavity 2 The outer wall and the outer wall of the outflow chamber 3 together form a radial flow chamber 6, the cell culture chamber 5 is filled with a three-dimensional porous scaffold material 7, and one end of a plurality of hollow fibers 8 located in the cell culture chamber 5 communicates with the inflow chamber 2, The other ends are connected to the outflow cavity 3, the inflow cavity 2, the outflow cavity 3 and a plurality of hollow fibers 8 together form the culture fluid flow channel 9, and the culture fluid flow channel 9 and the cell culture cavity 5 are passed through the hollow fiber The micropores on 8 communicate with each other, the cell culture chamber 5 and the radial flow chamber 6 communicate with each other through the micropores on the cellulose isolation membrane 4, and the housing 1 is provided with a culture solution inlet 10 communicated with the inflow chamber 2, A culture solution outlet 11 communicating with the outflow chamber 3 is also provided, a radial liquid outlet 12 communicating with the radial chamber 6 is provided on the housing 1, and a cell inlet port communicating with the cell culture chamber 5 is provided on the housing 1 13 and exhaust port 14.

As shown in Figure 6, it is a connection diagram of the use state of Embodiment 3 of the present invention. In the figure, the radial liquid outlet 12 of the radial flow chamber 6 is connected to the culture liquid pool 17 and the waste liquid through the peristaltic pump 21 and the silicone tube 22. Pool 23. The connection relationship of other parts is the same as that of the foregoing embodiments 1 and 2.

The present invention is placed in the cell culture box, and 95% of oxygen, 5% of carbon dioxide and 100% of humidity conditions are given, and it cultivates hepatocytes in this way: the hepatocytes separated by in vitro digestion technology are subjected to centrifugal inoculation and Inoculate into the three-dimensional porous scaffold material 7 in the cell culture cavity 5 of the patent of the present invention by circulating perfusion inoculation method, and use appropriate centrifugal force and circulating perfusion to make the liver cells evenly distributed in the three-dimensional porous scaffold material 7; The culture fluid circulates through the channel 9, and a certain pressure is applied to form a pressure difference between the culture fluid flow channel 9 and the radial flow chamber 6. Under the action of this pressure difference, part of the culture fluid flows from the culture fluid flow channel The hollow fiber 8 in 9 passes through the cell culture chamber 5 and flows radially to the radial flow chamber 6. During the radial flow of the culture medium, on the one hand, the liver cells are evenly provided with rich oxygen and nutrients, and on the other hand, the liver cells are also taken away. Metabolites enter the radial flow cavity 6 and are discharged through the radial liquid outlet 12. This process conforms to the hepatic physiological environment and blood environment of the normal liver. Therefore, a large number of highly active and high-density hepatocytes can be cultivated, and the hepatocytes have excellent biological functions.

In order to prevent impurities in the nutrient solution from flowing into the cell culture chamber 5 , as shown in FIG. 1 , filter screens 15 corresponding to the ports of the plurality of hollow fibers 8 may be provided in the inflow chamber 2 . Likewise, as shown in FIG. 2 , filter screens 16 corresponding to the ports of the plurality of hollow fibers 8 may be provided in the outflow cavity 3 . As shown in FIG. 4 , filter screens 15 and 16 corresponding to the ports of the plurality of hollow fibers 8 may be arranged in the inlet chamber 2 and the outlet chamber 3 .

Claims (7)

1. A three-dimensional bioreactor for cultivating hepatocytes, comprising a cell culture chamber (5) with a cell addition port (13) and an exhaust port (14), and a cell culture chamber (5) adjacent to the cell culture chamber (5) is provided with An inflow chamber (2) with a culture solution inlet (10) and an outflow chamber (3) with a culture solution outlet (11), one end of a plurality of hollow fibers (8) located in the cell culture chamber (5) are connected to The inflow cavity (2) is connected, and the other end is connected with the outflow cavity (3). The inflow cavity (2), the outflow cavity (3) and a plurality of hollow fibers (8) together form a culture solution flow channel (9 ), the culture solution flow channel (9) and the cell culture cavity (5) communicate with each other through the micropores on the hollow fiber (8), and it is characterized in that: the cell culture cavity (5) is provided with a three-dimensional porous scaffold material ( 7). 2. The three-dimensional bioreactor for culturing hepatocytes according to claim 1, characterized in that: the three-dimensional porous scaffold material (7) fills the entire cell culture chamber (5). 3. The three-dimensional bioreactor for culturing hepatocytes as claimed in claim 1, characterized in that: the three-dimensional porous support material (7) is arranged in the cell culture cavity (5) like this: multilayer three-dimensional porous support material (7) are distributed sequentially in the cell culture chamber (5) at a distance from each other, and each layer of three-dimensional porous scaffold material (7) is filled with the corresponding cross-section of the cell culture chamber (5). 4. The three-dimensional bioreactor for culturing hepatocytes according to claim 1, 2 or 3, characterized in that: outside the cell culture chamber (5), a radial flow covering the cell culture chamber (5) is provided Cavity (6), cell culture chamber (5) chamber walls are made of cellulose isolation membrane (4), between the cell culture chamber (5) and radial flow chamber (6) through the cellulose isolation membrane (4) The micropores communicate with each other, and the cavity of the radial flow chamber (6) is provided with a radial liquid outlet (12) communicating with the radial flow chamber (6). 5. The three-dimensional bioreactor for cultivating hepatocytes as claimed in claim 4, characterized in that: the filter screen (15) corresponding to the port of a plurality of hollow fibers (8) is arranged in the inflow chamber (2) ). 6. The three-dimensional bioreactor for culturing hepatocytes as claimed in claim 4, characterized in that: a filter screen (16) corresponding to the ports of a plurality of hollow fibers (8) is provided in the outflow chamber (3) ). 7. The three-dimensional bioreactor for culturing hepatocytes as claimed in claim 4, characterized in that: the inflow chamber (2), the outflow chamber (3) are all provided with a plurality of hollow fibers (8) Port corresponding filter screen (15, 16).

Images