Biological reaction vessel for in-vitro culture of tissue engineering skin cells

Abstract

The utility model relates to the technical field of cell culture, and discloses a biological reaction vessel for in-vitro culture of tissue engineering skin cells, which comprises a liquid storage tank, the top of the liquid storage tank is fixedly connected with a culture bin through supporting legs, and the culture bin is used for culturing cell samples. A plurality of placing grooves are formed in a supporting ring arranged at the top of a culture bin to place polycarbonate films, so that multiple groups of cells can be conveniently cultured by adopting the same culture solution, and floating rods are arranged on the outer walls of the polycarbonate films, so that the polycarbonate films can float on the liquid surface, and the culture efficiency is improved. In order to facilitate the culture mode of skin cells on a polycarbonate film, gas-liquid interface culture is mainly adopted, the gas-liquid interface culture method can realize the multi-layer growth of the cells, the cells in contact with the liquid level of a culture medium have growth and reproduction capabilities in the culture process, and only the cells in contact with air are keratinized, so that the actual growth environment of the skin cells can be simulated, and the skin cells can be effectively cultured. And the cultured data is more referential.

Description

Technical Field

[0001] This utility model relates to the field of cell culture technology, specifically to a bioreactor for in vitro culture of tissue-engineered skin cells. Background Technology

[0002] A bioreactor for in vitro culture of tissue-engineered skin cells is a special container used to simulate the in vivo environment for culturing tissue-engineered skin cells. Skin consists of two layers: the epidermis and the dermis. The epidermis, located above the dermis, is composed of stratified squamous epithelium, is relatively thin, and lacks blood vessels. It can be divided into five layers from the basal layer to the surface: the basal layer, the spinous layer, the granular layer, the stratum lucidum, and the stratum corneum. The growth environment of skin epithelial cells has a significant impact on their growth, morphological characteristics, and gene expression. In traditional cell culture techniques, skin epithelial cells are seeded at the bottom of a culture dish or flask for cultivation. Traditional cell culture techniques result in short-lived skin and can only cultivate single-layer cells, failing to differentiate into multi-layered three-dimensional structures, which is detrimental to the formation of the stratum corneum. The later-developed three-dimensional cell culture involves constructing skin epithelial tissue cells on a biological scaffold for three-dimensional culture. The culture method for skin epithelial cells is mainly based on gas-liquid interface culture. The gas-liquid interface culture method can achieve multilayer cell growth. During the culture process, cells in contact with the liquid surface of the culture medium have the ability to grow and reproduce, while cells that are only in contact with air undergo keratinization.

[0003] Existing technology, such as Chinese patent "CN215480981U", provides a three-dimensional skin epidermal cell culture device, including at least one cell culture vessel. The cell culture vessel comprises a culture outer shell and a culture inner shell. The size of the culture inner shell is smaller than that of the culture outer shell. The bottom wall of the culture inner shell is formed by a permeable membrane, while the outer wall of the culture inner shell is non-permeable. The top of the culture inner shell is open to the external environment. A cell culture excipient layer is coated on the permeable membrane. A culture solution is contained within the culture outer shell, and the culture inner shell is placed inside the culture outer shell and floats on the culture solution. Using this invention, there is no need to manually adjust the height of the gas-liquid interface; the height of the gas-liquid interface can be automatically adjusted according to cell growth. The culture operation is simple, and it can culture highly realistic artificial epidermal cells in vitro.

[0004] However, in actual use, the consumption of nutrient solution progressively decreases while the metabolic products continuously increase, which can cause some physiological damage to cells; the two-cycle solution change causes the nutrient composition to fluctuate periodically, which is not conducive to the uniform growth of cells. Utility Model Content

[0005] The purpose of this invention is to provide a bioreactor for in vitro culture of tissue-engineered skin cells in order to solve the above-mentioned problems and overcome the defects of the prior art, as detailed below.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This utility model provides a bioreactor for in vitro culture of tissue-engineered skin cells, including a liquid storage tank, the top of which is fixedly connected to a culture chamber for culturing cell samples via support legs;

[0008] A circulation mechanism is provided below the culture chamber to circulate the culture medium and filter cell metabolites;

[0009] The top of the culture chamber is equipped with an isolation cover to form an independent space for the cultured samples;

[0010] The top of the culture chamber is fixedly connected to a support ring. Five placement slots are respectively opened on both sides of the inner wall of the support ring. A polycarbonate membrane is slidably connected to the inner wall of the placement slot. A floating rod is fixedly connected to the outer wall of the polycarbonate membrane.

[0011] Preferably, there are two sets of isolation covers, which are fixedly connected to the top sides of the culture chamber and cover the placement slots in the corresponding areas on the top sides of the culture chamber.

[0012] Preferably, the isolation cover is made of glass, and air pipes are fixedly connected to both ends of the isolation cover for air circulation.

[0013] Preferably, the circulation mechanism includes a water pump, which is fixedly connected to the storage tank. The water pump outlet is fixedly connected to a water injection pipe, which extends to the top of the storage tank. A slow-flow zone is provided at the top of the water injection pipe, and the slow-flow zone is connected to the interior of the culture chamber through a water inlet pipe. A filter chamber is fixedly connected to one side of the outer wall of the culture chamber, and the filter chamber is connected to the interior of the storage tank.

[0014] Preferably, the bottom of the culture chamber is inclined, and an inclined plate is fixedly connected to the inner wall of the culture chamber above the water inlet pipe. An annular groove is formed at the bottom of the inclined surface of the inclined plate.

[0015] Preferably, the inner wall of the filter chamber is provided with a drawer, and the bottom of the drawer is provided with a filter screen.

[0016] Preferably, the inner wall of the inner ring of the culture chamber is rotatably connected to a rotating ring, and the outer wall of the rotating ring is fixedly connected to a scraper, which is in contact with the top of the inclined plate and the bottom of the inner wall of the culture chamber.

[0017] Preferably, a connecting frame is fixedly connected to the inner wall of the rotating ring, and a large gear is fixedly connected to the bottom of the connecting frame.

[0018] Preferably, a turbine is rotatably connected to the inner wall of the water injection pipe, a rotating shaft is fixedly connected to the top of the turbine, and the rotating shaft extends above the top of the water injection pipe. A sealing gasket is provided on the water injection pipe at the position of the rotating shaft, and a small gear is fixedly connected to the top of the rotating shaft. The small gear meshes with a large gear.

[0019] The beneficial effects are:

[0020] I. This utility model utilizes a support ring at the top of the culture chamber with multiple placement slots to hold polycarbonate membranes, facilitating the culture of multiple cell groups using the same culture medium. The polycarbonate membrane possesses excellent mechanical strength and chemical stability, with selectable pore size, allowing for the exchange of nutrients and metabolites. Furthermore, by incorporating a floating rod on the outer wall of the polycarbonate membrane, it can float on the liquid surface, facilitating the culture of skin cells on the polycarbonate membrane. The culture method primarily employs gas-liquid interface culture, which enables multilayer cell growth. During culture, cells in contact with the culture medium surface exhibit growth and reproduction capabilities, while cells only in contact with air undergo keratinization. This simulates the actual growth environment of skin cells, making the culture data more reliable.

[0021] Second, this utility model isolates the cell samples on the polycarbonate membrane from the outside air by setting an isolation cover over the culture chamber to prevent the cells from being infected by external pathogens. Since the two isolation covers are connected to independent air tubes, different air is introduced into the two isolation covers to simulate a more complex environment. Taking human skin cells as an example, there is a significant difference in surface temperature between the human torso and the limbs. This can be simulated by introducing air at different temperatures.

[0022] Thirdly, this utility model, by setting a storage tank below the culture chamber, can hold more culture medium, reducing the frequency of culture medium replacement to some extent. Furthermore, the larger capacity of culture medium can be partially processed in batches, effectively mitigating the impact of periodic fluctuations caused by culture medium replacement. The culture medium is drawn from the storage tank by a water pump, injected into the culture chamber through a water inlet pipe via a slow-flow zone, and then returned to the storage tank through a filter chamber. The constant flow rate from the water pump maintains a stable liquid level in the culture chamber, preventing excessive fluctuations in liquid level that could affect cell growth. Moreover, the design incorporates a relatively spacious... The large slow-flow zone effectively reduces the flow rate of the culture medium. Furthermore, by installing an inclined plate at the height of the inlet pipe in the filter chamber, the chamber is divided into a flowing zone and a relatively still zone. This ensures that the culture medium continues to circulate while the slow flow of the medium in contact with the cells does not affect cell growth. A drawer is installed at the location of the filter chamber to easily intercept and filter some metabolic substances in the nutrient solution, allowing them to accumulate for convenient cleaning. This prevents the continuous accumulation of metabolic products in the nutrient solution from causing physiological damage to the cells. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the overall outer wall structure of this utility model;

[0025] Figure 2 This is a partial sectional view of the present invention;

[0026] Figure 3 This is a diagram illustrating the connection relationship between the polycarbonate membrane and the support ring in this utility model;

[0027] Figure 4 This is a diagram illustrating the connection relationship between the sealing ring and the large gear in this utility model;

[0028] Figure 5 This is a diagram showing the internal structure of the water injection pipe in this utility model;

[0029] Figure 6 This is a schematic diagram of the scraper structure in this utility model;

[0030] Figure 7 This utility model Figure 2 Enlarged view of point A.

[0031] The following are explanations of the reference numerals in the attached diagram: 1. Storage tank; 2. Culture chamber; 20. Support leg; 21. Support ring; 22. Placement trough; 23. Polycarbonate membrane; 24. Floating rod; 25. Inclined plate; 26. Ring groove; 3. Isolation cover; 31. Air pipe; 4. Circulation mechanism; 41. Water pump; 42. Water injection pipe; 43. Filter chamber; 44. Drawer box; 45. Filter screen; 46. Slow flow zone; 47. Water inlet pipe; 48. Turbine; 481. Rotating shaft; 482. Small gear; 483. Sealing gasket; 484. Large gear; 485. Connecting frame; 49. Rotating ring; 491. Scraper. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this utility model clearer, the technical solutions of this utility model will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this utility model, and not all of them. Based on the embodiments of this utility model, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this utility model.

[0033] Example 1

[0034] See Figure 1 - Figure 7 As shown, this utility model provides a bioreactor for in vitro culture of tissue-engineered skin cells, including a storage tank 1. The top of the storage tank 1 is fixedly connected to a culture chamber 2 via a support leg 20 for culturing cell samples. A circulation mechanism 4 is provided below the culture chamber 2 for circulating the culture medium and filtering cell metabolites. An isolation cover 3 is provided on the top of the culture chamber 2 to form an independent space for the cultured samples. A support ring 21 is fixedly connected to the top of the culture chamber 2. Five placement slots 22 are respectively opened on both sides of the inner wall of the support ring 21, and a polycarbonate membrane 23 is slidably connected to the inner wall of the placement slots 22. A floating rod 24 is fixedly connected to the outer wall of the polycarbonate membrane 23.

[0035] In this embodiment, multiple placement slots 22 are opened in the support ring 21 at the top of the culture chamber 2 to place the polycarbonate membrane 23, so as to facilitate the culture of multiple groups of cells under the same culture medium. The polycarbonate membrane 23 has good mechanical strength and chemical stability, and the pore size can be selected to allow the exchange of nutrients and metabolites. By setting a floating rod 24 on the outer wall of the polycarbonate membrane 23, the polycarbonate membrane 23 can float on the liquid surface, so that the skin cells on the polycarbonate membrane 23 are mainly cultured at the gas-liquid interface. The gas-liquid interface culture method can achieve multilayer cell growth. During the culture process, the cells in contact with the liquid surface of the culture medium have the ability to grow and reproduce, while the cells only in contact with the air undergo keratinization, which can simulate the actual growth environment of skin cells, making the culture data more reliable.

[0036] Example 2

[0037] See Figure 1 , Figure 2 As shown, there are two sets of isolation covers 3. The two sets of isolation covers 3 are fixedly connected to the top of the culture chamber 2 on both sides, and respectively cover the placement slots 22 in the corresponding areas on the top of the culture chamber 2. By setting the isolation covers 3 to cover the polycarbonate membrane 23 in the placement slots 22 in the culture chamber 2, the cell samples on the polycarbonate membrane 23 can be isolated from the outside air, avoiding the problem of external cells being infected by external pathogens and affecting cell culture. The isolation covers 3 are made of glass, which facilitates the observation of the cell growth status. The two ends of the isolation covers 3 are fixedly connected to the air tubes 31 for air circulation. By connecting independent air tubes 31 to the two sets of isolation covers 3, different air can be introduced into the two sets of isolation covers 3 to facilitate the simulation of more complex environments.

[0038] In this embodiment, by setting an isolation cover 3 to cover the polycarbonate membrane 23 in the placement tank 22 in the culture chamber 2, the cell samples on the polycarbonate membrane 23 can be isolated from the outside air, avoiding external cells from being infected by external pathogens. Since the two sets of isolation covers 3 are connected to independent air tubes 31, different air is introduced into the two sets of isolation covers 3 to simulate a more complex environment. Taking human skin cells as an example, there is a significant difference in surface temperature between the human torso and the limbs. This can be simulated by introducing air at different temperatures.

[0039] Example 3

[0040] See Figure 1 , Figure 2 , Figure 5 As shown, the circulation mechanism 4 includes a water pump 41, which is fixedly connected to the storage tank 1. The water outlet of the water pump 41 is fixedly connected to a water injection pipe 42, which extends to the top of the storage tank 1. A slow flow zone 46 is provided at the top of the water injection pipe 42. The slow flow zone 46 is connected to the interior of the culture chamber 2 through a water inlet pipe 47. A filter chamber 43 is fixedly connected to one side of the outer wall of the culture chamber 2, and the filter chamber 43 is connected to the interior of the storage tank 1. The bottom of the culture chamber 2 is inclined. An inclined plate 25 is fixedly connected to the inner wall of the culture chamber 2 above the water inlet pipe 47. An annular groove 26 is provided at the bottom of the inclined surface of the inclined plate 25.

[0041] In this embodiment, by setting a storage tank 1 below the culture chamber 2, more culture medium can be contained, which reduces the frequency of culture medium replacement to a certain extent. The larger capacity of culture medium can be partially replaced in batches, which can effectively reduce the impact of periodic fluctuations caused by culture medium replacement. The culture medium in the storage tank 1 is drawn by the water pump 41 and injected into the culture chamber 2 through the water injection pipe 42, the slow flow zone 46, and the water inlet pipe 47. Then it flows back into the storage tank 1 from the filter chamber 43. The constant flow rate output by the water pump 41 can maintain the liquid level in the culture chamber 2, avoiding excessive changes in the liquid level position, which would affect cell growth. The large slow flow zone 46 can effectively reduce the flow rate of the culture medium. On the basis of slowing down the flow rate, the inclined plate 25 is set in the area of ​​the filter chamber 43 at the height of the water inlet pipe 47, dividing the filter chamber 43 into a flowing area and a relatively static area. This ensures that the culture medium is circulated, and the slow flow of the part of the culture medium in contact with the cells does not affect cell growth.

[0042] Example 4

[0043] See Figure 1 , Figure 2 , Figure 7 As shown, the inner wall of the filter chamber 43 is provided with a drawer box 44, and the bottom of the drawer box 44 is provided with a filter screen 45.

[0044] In this embodiment, a drawer 44 is provided at the position of the filter chamber 43 to facilitate the interception and filtration of some metabolic substances in the nutrient solution, so that they accumulate in the drawer 44 for easy centralized cleaning, thereby avoiding the problem that the continuous increase of metabolic products in the nutrient solution will cause some physiological damage to the cells.

[0045] Example 5

[0046] See Figure 2 , Figure 4 , Figure 5 , Figure 6 As shown, a rotating ring 49 is rotatably connected to the inner wall of the inner ring of the culture chamber 2. A scraper 491 is fixedly connected to the outer wall of the rotating ring 49. The scraper 491 is in contact with the top of the inclined plate 25 and the bottom of the inner wall of the culture chamber 2. A connecting frame 485 is fixedly connected to the inner wall of the rotating ring 49. A large gear 484 is fixedly connected to the bottom of the connecting frame 485. A turbine 48 is rotatably connected to the inner wall of the water injection pipe 42. A rotating shaft 481 is fixedly connected to the top of the turbine 48, and the rotating shaft 481 extends above the top of the water injection pipe 42. A sealing gasket 483 is provided on the water injection pipe 42 at the position of the rotating shaft 481. A small gear 482 is fixedly connected to the top of the rotating shaft 481, and the small gear 482 meshes with the large gear 484.

[0047] In this embodiment, when the culture medium flows through the turbine 48 inside the water injection pipe 42, the potential energy of the culture medium flow will drive the turbine 48 to rotate, thereby driving the small gear 482 at the top of the rotating shaft 481 connected to the turbine 48 to rotate, so as to facilitate the rotation of the large gear 484 meshing with the small gear 482. This causes the connecting frame 485 at the top of the large gear 484 to drive the rotating ring 49 to rotate slowly, causing the scraper 491 connected to the rotating ring 49 to rotate together. Since the scraper 491 is in contact with the top of the inclined plate 25 and the bottom of the inner wall of the culture chamber 2, the metabolic deposits at the contact position of the scraper 491 will be scraped off and pushed to the filter chamber 43 during the rotation of the scraper 491.

[0048] Working principle

[0049] In use, multiple placement slots 22 are provided on the support ring 21 at the top of the culture chamber 2 to place the polycarbonate membrane 23, facilitating the culture of multiple groups of cells using the same culture medium. A floating rod 24 is provided on the outer wall of the polycarbonate membrane 23 to allow it to float on the liquid surface, facilitating the culture of skin cells on the polycarbonate membrane 23. The culture method primarily utilizes the gas-liquid interface, which enables multilayer cell growth. The culture medium in the storage tank 1 is drawn by activating the water pump 41. Water is injected into the culture chamber 2 via the inlet pipe 47 through the slow-flow zone 46, and then flows back into the storage tank 1 through the filter chamber 43. A constant flow rate from the water pump 41 maintains the liquid level in the culture chamber 2, preventing excessive fluctuations in liquid level that could affect cell growth. The relatively large slow-flow zone 46 effectively reduces the flow rate of the culture medium. Furthermore, by installing an inclined plate 25 at the height of the inlet pipe 47 within the filter chamber 43, the chamber is divided into a flowing area and a relatively static area. This design ensures that the culture medium maintains circulation while allowing for slow flow in the portion in contact with the cells, without affecting cell growth. When the culture medium flows through the turbine 48 within the water injection pipe 42, the potential energy of the flowing medium drives the turbine 48 to rotate, which in turn drives the small gear 482 at the top of the shaft 481 connected to the turbine 48 to rotate. This facilitates the rotation of the large gear 484, which meshes with the small gear 482. The connecting frame 485 at the top of the large gear 484 drives the rotating ring 49 to rotate slowly, causing the scraper 491 connected to the rotating ring 49 to rotate as well. Because the scraper 491 is in contact with the top of the inclined plate 25 and the bottom of the inner wall of the culture chamber 2, the rotation of the scraper 491 removes metabolic deposits at the contact points and pushes them to the filter chamber 43. A drawer 44 is installed in the filter chamber 43 to intercept and filter some metabolic substances in the nutrient solution, allowing them to accumulate in the drawer 44 for easy cleaning. This prevents the continuous increase of metabolic products in the nutrient solution from causing physiological damage to the cells.

[0050] The above description is merely a specific embodiment of this utility model, but the protection scope of this utility model is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this utility model should be included within the protection scope of this utility model. Therefore, the protection scope of this utility model should be determined by the protection scope of the claims.

Claims

1. A bioreactor for in vitro culture of tissue-engineered skin cells, characterized in that, Includes a storage tank (1), the top of which is fixedly connected to a culture chamber (2) via a support leg (20) for culturing cell samples; The culture chamber (2) is equipped with a circulation mechanism (4) for circulating the culture medium and filtering cell metabolites. The top of the culture chamber (2) is provided with an isolation cover (3) to form an independent space for the cultured sample; The top of the culture chamber (2) is fixedly connected to a support ring (21). Five placement slots (22) are respectively opened on both sides of the inner wall of the support ring (21). A polycarbonate membrane (23) is slidably connected to the inner wall of the placement slot (22). A floating rod (24) is fixedly connected to the outer wall of the polycarbonate membrane (23).

2. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 1, characterized in that: The isolation cover (3) consists of two sets, which are fixedly connected to the top two sides of the culture chamber (2) and cover the placement slots (22) of the corresponding areas on the top two sides of the culture chamber (2).

3. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 2, characterized in that: The isolation cover (3) is made of glass, and air pipes (31) are fixedly connected to both ends of the isolation cover (3) for air circulation.

4. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 1, characterized in that: The circulation mechanism (4) includes a water pump (41), which is fixedly connected to the storage tank (1). The water outlet of the water pump (41) is fixedly connected to a water injection pipe (42), which extends to the top of the storage tank (1). A slow flow zone (46) is provided at the top of the water injection pipe (42). The slow flow zone (46) is connected to the interior of the culture chamber (2) through a water inlet pipe (47). A filter chamber (43) is fixedly connected to one side of the outer wall of the culture chamber (2), and the filter chamber (43) is connected to the interior of the storage tank (1).

5. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 4, characterized in that: The bottom of the culture chamber (2) is inclined, and an inclined plate (25) is fixedly connected to the inner wall of the culture chamber (2) above the water inlet pipe (47). An annular groove (26) is opened at the bottom of the inclined surface of the inclined plate (25).

6. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 4, characterized in that: The inner wall of the filter chamber (43) is provided with a drawer box (44), and the bottom of the drawer box (44) is provided with a filter screen (45).

7. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 5, characterized in that: The inner wall of the inner ring of the culture chamber (2) is rotatably connected to a rotating ring (49), and the outer wall of the rotating ring (49) is fixedly connected to a scraper (491). The scraper (491) is attached to the top of the inclined plate (25) and the bottom of the inner wall of the culture chamber (2).

8. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 7, characterized in that: The inner wall of the rotating ring (49) is fixedly connected to a connecting frame (485), and the bottom of the connecting frame (485) is fixedly connected to a large gear (484).

9. The bioreactor for in vitro culture of tissue-engineered skin cells according to claim 8, characterized in that: A turbine (48) is rotatably connected to the inner wall of the water injection pipe (42). A rotating shaft (481) is fixedly connected to the top of the turbine (48), and the rotating shaft (481) extends above the top of the water injection pipe (42). A sealing gasket (483) is provided on the water injection pipe (42) at the position of the rotating shaft (481). A small gear (482) is fixedly connected to the top of the rotating shaft (481), and the small gear (482) meshes with a large gear (484).

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