A device for culturing myocardial cells using transferrin

Abstract

The present invention belongs to the technical field of cell culture equipment, and specifically relates to a device for culturing cardiomyocytes using transferrin, comprising a box body, the box body having an opening at the bottom, a through channel arranged inside, the opening at the bottom of the through channel connected to the opening at the bottom of the box body, a liquid storage box connected to the bottom open of the through channel, a circulation pipe arranged through the through channel, a part of the circulation pipe passing through the through channel, the liquid storage box and a box cover arranged at the top of the box body, and another part arranged outside the box body, a circulation hole is opened in the box body and above the through channel of the circulation pipe, a closed switch that can be opened and closed is arranged at the top of the through channel, a barrier film is laid at the hollow part, and a circulation pump is arranged on the circulation pipe. The present invention provides a device for culturing cardiomyocytes using transferrin, utilizing the property of transferrin that can bind and release iron element, using transferrin as a transport carrier, and avoiding the phenomenon of iron excess or iron deficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture equipment, and in particular relates to a device for culturing myocardial cells using transferrin. Background Art

[0002] Iron is an essential element for cell growth. Iron promotes cell growth and affects the physiological metabolism of cells. Cardiomyocytes are a type of cell that is also affected by iron. Iron has divalent and trivalent forms. Although cells need iron for growth, excessive iron can also cause cell death or inhibit cell growth, especially free iron. Studies have shown that cell free ferrous protoporphyrin (FH) is a decomposition product of ferrous protoporphyrin protein. It has a strong oxidizing effect, leading to cell self-regulatory dysfunction and is directly related to the degree of cell carcinogenesis. It has been used in the detection of cervical cancer, colorectal cancer, urothelial cancer, etc., and has become a causal biomarker factor produced in the process of malignant tumor occurrence (Zhu Shifa, Kong Lingbin. Ferroptosis and the role of cell free ferrous protoporphyrin in tumorigenesis [J]. Advances in Clinical Medicine, 2022, 12(11): 7. DOI: 10.12677 / ACM.2022.12111468.).

[0003] An important function of transferrin (TRF) is to regulate the uptake of iron. Its molecular weight is about 76 kDa. 3+ Transferrin can not only absorb and release iron, but also adjust the absorption and release of iron according to the cell growth and the content of free iron in the nutrient solution. The concentration of TRF is regulated by the iron supply, and TRF increases when iron is deficient. Free iron is harmful to the body, and combining with TRF can prevent iron loss from the kidney. There are also many technologies in the prior art that use transferrin to culture cardiomyocytes, such as Chinese patents 201611252382.2 and 201510046646.8.

[0004] However, cardiomyocyte culture is different from body growth. Cardiomyocyte culture is to place cells in culture medium and grow by adhering to the wall. During cell culture, the nutrient solution does not flow, and the replacement of the culture solution is also regularly replaced at intervals. We set the use time of a culture solution as T, and then replace the culture solution every time T. In the known cardiomyocyte culture solution formula of the prior art, transferrin is quantitative, and the compound iron element in transferrin is also quantitative. However, during the cardiomyocyte culture process, the number of cells continues to increase, and the demand for iron also continues to increase. In the culture time period T of a nutrient solution, the quantitative transferrin in the nutrient solution is either excessive at the beginning, or iron deficiency occurs as the cells proliferate. Whether it is iron excess or iron deficiency, it is not conducive to cardiomyocyte growth. Summary of the invention

[0005] In order to solve the above-mentioned technical problems, and also to give iron in a timely and appropriate amount during the culture process of myocardial cells, the present invention provides a device for culturing myocardial cells using transferrin, utilizing the property that transferrin can bind and release iron, using transferrin as a transport carrier, deeply segmenting the "culture time period T of a nutrient solution" into several shorter time periods T1, and the device of the present invention is also provided with an iron supplement solution, and utilizing transferrin to transport the iron in the iron supplement solution to myocardial cells every T1. In this way, it is possible to increase the iron in a timely and appropriate amount as the cells continue to grow and proliferate, avoiding the phenomenon of excessive iron or iron deficiency, and it is also possible to make full use of the property of transferrin to transport iron, exert its "transport" function, so that transferrin can be reused, without adding excessively high concentrations of transferrin in the nutrient solution formula, saving resources, and being environmentally friendly.

[0006] The object of the present invention is to provide a device for culturing cardiomyocytes using transferrin, comprising a box body and a circulation tube, wherein the top of the box body is open, the bottom of the box body has an opening, a through channel is provided inside the box body, the top and bottom of the through channel are both open, and the opening at the bottom of the through channel coincides with and is connected to the edge of the opening at the bottom of the box body;

[0007] The bottom opening of the through-channel is connected with a liquid storage box, the interior of the liquid storage box is communicated with the interior of the through-channel, the circulation pipe is arranged through the through-channel, a part of the circulation pipe is arranged through the through-channel, the liquid storage box and the box cover arranged on the top of the box body, and the other part is arranged outside the box body, the circulation pipe is located in the box body and a flow hole is opened at a position above the through-channel, and a closed switch that can be opened and closed is arranged on the top of the through-channel, and the closed switch includes an open state and a closed state;

[0008] When the closed switch is in a closed state, the closed switch surrounds the flow hole, and the liquid flowing out of the flow hole flows into the through channel; when the closed switch is in an open state, the flow hole is exposed outside the through channel, and the liquid flowing out of the flow hole flows into the through channel and the box body;

[0009] The portion of the circulation tube located in the liquid storage box is hollowed out, and a barrier membrane is provided at the hollowed-out portion. The barrier membrane is used to circulate substances with a molecular weight smaller than transferrin; substances with a molecular weight smaller than transferrin are small molecule nutrients such as iron.

[0010] The circulation pipe is provided with a circulation pump. After the circulation pump is turned on, the flow direction of the liquid inside the box body is from top to bottom.

[0011] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the closed switch comprises a plurality of bendable units, all of which are circumferentially arranged around the circulation tube, one end of the bendable unit is fixed to the open edge of the top of the through-channel, and the other end is a movable end;

[0012] When the closed switch is in a closed state, all the bendable units are stretched out, the edges of adjacent bendable units are in contact, and the bendable units are also in contact with the portion of the circulation tube located above the flow hole;

[0013] When the closing switch is in an open state, all the bendable units are bent, adjacent bendable units are separated, and the flow hole is exposed.

[0014] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the bendable units are stretched, and the top view of the structure after all the bendable units are spliced ​​together is ring-shaped.

[0015] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the bendable unit is fan-shaped when stretched, and after the bendable unit contacts the portion of the circulation tube above the flow hole, the top angle of the fan can be turned outward relative to the through channel, and the remaining portions of adjacent fan-shaped edges contact to surround the flow hole.

[0016] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the liquid storage box includes an iron layer and a water layer, the iron layer is located above the water layer, the iron layer and the water layer are separated by a partition, and the hollow part of the circulation pipe and the barrier membrane are both arranged to penetrate the iron layer and the water layer.

[0017] Preferably, in the above-mentioned device utilizing transferrin to culture myocardial cells, the barrier membrane is a dialysis membrane, and the molecular weight cut-off of the dialysis membrane is 60-75kDa. For example, the molecular weight cut-off of the dialysis membrane is 65kDa, 66kDa, 67kDa, 68kDa, 70kDa, 75kDa. These molecular weights are close to and less than the molecular weight of transferrin, and are used to block transferrin.

[0018] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the space between the outer wall of the through channel and the inner wall of the box body is the place for culturing cardiomyocytes, which is recorded as the culturing space, and the sum of the heights of the through channel and the closed switch is less than the height of the box body.

[0019] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, the liquid storage box is provided with a liquid infusion port at a portion outside the box body, and a first sealing device is provided at the liquid infusion port. When the first sealing device is opened, liquid can be replenished into the liquid storage box through the liquid infusion port. When the first sealing device is closed, the liquid infusion port is in a closed state and liquid does not flow.

[0020] Preferably, the above-mentioned device for culturing cardiomyocytes using transferrin is also provided with a protein solution replenishing port running through the liquid storage box and its internal circulation tube, and a second closing device is provided at the protein solution replenishing port. When the second closing device is opened, liquid can be replenished into the liquid storage box through the protein solution replenishing port. When the second closing device is closed, the protein solution replenishing port is in a closed state.

[0021] Preferably, in the above-mentioned device for culturing cardiomyocytes using transferrin, a bracket is arranged at the bottom of the box body, and a circulation tube is laid on the bracket.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention utilizes the property of transferrin that can bind and release iron, uses transferrin as a transport carrier, deeply divides the "one nutrient solution culture time period T" into several shorter time periods T1, sets an iron supplement solution, and uses transferrin to transport iron in the iron supplement solution to myocardial cells every T1, while other elements in the myocardial cell nutrient solution are not supplemented within the total time T. It can achieve timely and appropriate increase of iron as cells continue to grow and proliferate, avoiding the phenomenon of iron excess or iron deficiency.

[0024] More specifically, the present invention makes full use of the property of transferrin in transporting iron and exerts its "transport" function. A barrier membrane is provided, and the molecular weight cutoff of the barrier membrane is 60-75 kDa, which is close to and smaller than the molecular weight of transferrin, and is used to block transferrin. Iron elements, iron compounds and small molecule nutrients with small molecular weight can pass through the barrier membrane, but transferrin cannot. As iron and transferrin are complexed and decomplexed, transferrin can be reused, thereby saving resources and being environmentally friendly.

[0025] More specifically, a circulation pump is provided on the circulation pipe of the present invention. After the circulation pump is turned on, the liquid circulates in the circulation pipe, and in the circulation pipe part inside the box body, the flow direction of the liquid is from top to bottom. As the closed switch is turned on or off, the nutrient solution flows into or does not flow into the culture space, and the circulation pump can control whether to supplement iron to the cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The three-dimensional structure of the device for culturing myocardial cells using transferrin according to the present invention Figure 1 .

[0027] Figure 2 The three-dimensional structure of the device for culturing myocardial cells using transferrin according to the present invention Figure 2 .

[0028] Figure 3 This is a longitudinal section of the device for culturing myocardial cells using transferrin according to the present invention. Figure 1 .

[0029] Figure 4 This is a longitudinal section of the device for culturing myocardial cells using transferrin according to the present invention. Figure 2 .

[0030] Figure 5 It is a structural diagram of the hollowed-out portion of the circulation pipe of the present invention.

[0031] Figure 6 It is a top view of the connection structure of the circulation pipe and the closed switch of the present invention.

[0032] Figure 7 It is a top view of the second connection structure of the circulation pipe and the closed switch of the present invention.

[0033] Figure 8 It is a longitudinal cross-sectional view of the second connection structure of the circulation pipe and the closed switch of the present invention. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0035] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0036] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0037] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features; in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0038] Considering that during the process of cardiomyocyte culture, the number of cells continues to increase and the demand for iron also continues to increase, the transferrin in the nutrient solution is quantitative within the culture time period T of a nutrient solution. Then, when the number of cells is small in the initial state, the iron element is excessive relative to the cardiomyocytes. As the cells continue to proliferate, the number of cells continues to increase, and the demand for iron also increases. Then, in the later stage of culture, the iron element is lacking relative to the cardiomyocytes. Whether it is excess or deficiency of iron, it is not conducive to the growth of cardiomyocytes.

[0039] The present invention provides a device for culturing myocardial cells by using transferrin in order to supplement iron elements to myocardial cells in a timely and appropriate amount. The device uses the property of transferrin that can bind and release iron elements, uses transferrin as a transport carrier, and deeply divides the "culture time period T of a nutrient solution" into several shorter time periods T1. The present invention also provides an iron supplement solution, and uses transferrin to transport iron in the iron supplement solution to myocardial cells every T1, while other elements in the myocardial cell nutrient solution are not supplemented within the total time T. In this way, it is possible to increase the iron element in a timely and appropriate amount as the cells continue to grow and proliferate, avoid the phenomenon of excessive iron or iron deficiency, and fully utilize the property of transferrin to transport iron, exert its "transport" function, so that transferrin can be reused.

[0040] The device for culturing myocardial cells using transferrin provided by the present invention is shown in Figure 1, including a box body 1, the top of the box body 1 is open, the bottom of the box body 1 has an opening, the box body 1 is provided with a through channel 2 inside, the top and bottom of the through channel 2 are open, and the opening at the bottom of the through channel 2 is matched and connected with the opening edge at the bottom of the box body 1, and the matching part does not leak. The through channel 2 is convex relative to the bottom of the box body 1, and the space between the outer wall of the through channel 2 and the inner wall of the box body 1 and the bottom wall of the box body 1 is a place for culturing cardiomyocytes, which is recorded as a culture space 21, and the longitudinal section of the culture space 21 is U-shaped. The bottom of the through channel 2 is connected to a liquid storage box 5, and the bottom opening of the through channel 2 is connected to the inside of the liquid storage box 5, and the inside of the through channel 2 is also connected to the liquid storage box 5, and the iron element supplement liquid is placed in the liquid storage box 5. A circulation pipe 3 is arranged in the through channel 2, and the circulation pipe 3 is annular. A part of the circulation pipe 3 is arranged through the through channel 2, the liquid storage box 5 and the box cover 11 arranged on the top of the box body 1, and the other part of the circulation pipe 3 is arranged outside the box body 1. The circulation tube 3 is located in the box body 1 and is provided with a flow hole 31 at a position above the through-channel 2. Liquid can pass through the flow hole 31, such as nutrient solution liquid can pass through the flow hole 31. A closed switch 6 that can be opened or closed is provided at the top of the through-channel 2. When the closed switch 6 is closed, the flow hole 31 is surrounded and the top opening of the through-channel 2 is separated from the box body 1. Liquid can only flow to the through-channel 2. When the flow hole 31 is opened, the flow hole 31 is exposed to the outside. Liquid can flow to the through-channel 2 or to the culture space 21. A liquid replenishment port 51 is provided at the part of the liquid storage box 5 located outside the box body 1. A protein liquid replenishment port 52 is also provided at the through-liquid storage box 5 and the circulation tube 3 inside it. The part of the circulation tube 3 located in the liquid storage box 5 is hollow, and the nutrient solution component substances with a molecular weight less than transferrin can freely shuttle in the hollow part. A barrier film 53 is laid at the hollow part, and the nutrient solution component substances with a molecular weight less than transferrin can pass through the barrier film 53, and transferrin cannot pass through the barrier film. A circulation pump 32 is provided on the circulation pipe 3. After the circulation pump is turned on, the liquid circulates in the circulation pipe 3, and in the part of the circulation pipe 3 inside the box body 1, the flow direction of the liquid is from top to bottom.

[0041] When using the device of the present invention, the circulation pump 32 is not turned on first, and the nutrient solution without transferrin is added into the culture space 21 to inoculate the cardiomyocytes. The liquid level of the nutrient solution is lower than the through channel 2. The nutrient solution adopts the existing technology, such as the nutrient solution components of Chinese patents 201611252382.2 and 201510046646.8, and then the iron-containing nutrient solution or the pure iron compound solution or the iron-containing suspension is added into the liquid storage box 5 through the liquid replenishing port 51, and then the protein solution is added into the circulation tube 3 through the protein solution replenishing port 52. The position of the barrier membrane 53 is set to supplement the transferrin solution, so that the iron element passes through the barrier membrane 53 and is compounded with the transferrin. Then the closed switch 6 is closed and the circulation pump 32 is turned on. As the transferrin circulates continuously in the circulation tube 3, or is stored in the space between the through channel 2 and the closed switch 6 through the flow hole 31, due to the continuous circulation of the liquid, it is also continuously exchanged with the liquid between the through channel 2 and the closed switch 6. After the iron and transferrin are fully compounded, the closed switch 6 is turned on, and the transferrin solution flows into the culture space 21 to supplement the iron for the cardiomyocytes. The above-mentioned working process is carried out once within the time of T1. After repeated cycles of the T1 time period several times, the sum of the several times of T1 is equal to the culture time T of the nutrient solution once, that is, after the time period of T, all the nutrient solutions are replaced, and the cardiomyocytes are passaged. Ultimately, it will be possible to increase the amount of iron in a timely manner as cells continue to grow and proliferate, thus avoiding iron excess or iron deficiency, and to fully utilize the iron transport properties of transferrin to exert its "transport" function, so that transferrin can be reused without adding excessively high concentrations of transferrin to the nutrient solution formula, thus saving resources and being environmentally friendly.

[0042] Specifically, the present invention includes the following embodiments.

[0043] Example 1

[0044] A device for culturing cardiomyocytes using transferrin, see Figure 1 and Figure 3 , including a box body 1, the top of the box body 1 is open, and a box cover 11 is detachably provided at the open top of the box body 1. The box body 1 can be cylindrical, elliptical, cubic or truncated cone-shaped, etc., and the purpose is to contain myocardial cells and nutrient solution. The box body 1 is preferably made of transparent material to facilitate observation of the growth status of myocardial cells and the height of the nutrient solution. For experiments requiring light protection, the outside of the box body 1 is wrapped with light-shielding cloth, aluminum foil or other light-shielding material components, and then light-shielding culture is performed. At this time, the box cover 11 can be opened to observe the internal situation of the box body 1.

[0045] The box body 1 has an opening at the bottom, and a through channel 2 is provided inside the box body 1. The top and bottom of the through channel 2 are both open, and the open bottom of the through channel 2 is matched and connected with the edge of the opening at the bottom of the box body 1, and the matching part does not leak. A mold can also be designed to produce the box body 1 and the through channel 2 in one piece. The through channel 2 is convex relative to the bottom of the box body 1. The space between the outer wall of the through channel 2 and the inner wall of the box body 1 and the inner bottom wall of the box body 1 is a place for culturing cardiomyocytes, which is recorded as the culture space 21. The cardiomyocytes are inoculated in the culture space 21, and the nutrient solution is also added in the culture space 21. The main function of the through channel 2 is to flow liquids such as transferrin solution, so there is no specific restriction on its shape, such as the through channel 2 is set to a cylindrical, elliptical, cubic or truncated cone shape. The sum of the height of the through channel 2 and the closed switch must be lower than the height of the box body 1, so the liquid overflowing from the through channel 2 enters the culture space 21, and the nutrients therein can be used by the cardiomyocytes to make the cardiomyocytes grow and proliferate.

[0046] Please continue to refer to Figure 1 and Figure 3 , a liquid storage box 5 is connected to the bottom opening of the through-channel 2, and the inside of the through-channel 2 is connected to the inside of the liquid storage box 5. An iron supplement is placed in the liquid storage box 5, such as iron-containing compounds such as divalent iron or trivalent iron, as long as the iron can be compounded with transferrin. However, it should be noted that no matter how many valent irons there are, it is necessary to convert them into iron that can be compounded with transferrin under suitable pH and enzyme conditions. Then, the iron supplement needs to add substances that adjust pH, corresponding enzymes, etc. to convert the iron valence state, and it should be noted that the molecular weight of these components is less than that of transferrin; or, substances that adjust pH, corresponding enzymes, etc. are added in the culture space 21 to convert the iron valence state. When transferrin contacts myocardial cells, under suitable pH and enzyme conditions, iron is converted into an iron valence state that is easily absorbed by cells. Exemplarily, the iron supplement is set as a trivalent iron solution, which is easy for transferrin to compound, and then a reducing agent is added to the culture space 21 to reduce the trivalent iron to divalent iron, which is easily absorbed by myocardial cells.

[0047] See also Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5, the liquid storage box 5 is connected to the through-channel 2, and a sealing component is provided at the connection seam between the liquid storage box 5 and the through-channel 2 to prevent leakage. A circulation pipe 3 is provided through the through-channel 2. The circulation pipe 3 is annular. A part of the circulation pipe 3 passes through the through-channel 2, the liquid storage box 5 and the box cover 11 provided on the top of the box body 1. The other part of the circulation pipe 3 is provided outside the box body 1. When the circulation pipe 3 passes through the bottom of the liquid storage box 5, a sealing component is provided at the connection seam with the circulation pipe 3 to prevent leakage. A circulation hole 31 is provided at the position of the circulation pipe 3 located inside the box body 1 and above the through-channel 2, and the nutrient solution can pass through the circulation hole 31. A closed switch 6 that can be opened and closed is provided at the top of the through-channel 2.

[0048] The closed switch 6 includes an open state and a closed state. When the closed switch 6 is in the closed state, the circulation hole 31 is wrapped, and the liquid can only flow to the through channel 2. When the closed switch 6 is in the open state, the circulation hole 31 is exposed to the outside, and the liquid can flow to the through channel 2 and the culture space 21. The liquid storage box 5 is provided with a liquid replenishing port 51 at the position outside the box body 1. The liquid replenishing port 51 is provided with a first closing device at the outside of the liquid storage box 5. When the first closing device is opened, the liquid can be replenished into the liquid storage box 5 through the liquid replenishing port 51. When the first closing device is closed, the liquid replenishing port 51 is in a closed state and the liquid is not circulated. A protein liquid replenishing port 52 is also provided at the liquid storage box 5 and its internal circulation pipe 3. The protein liquid replenishing port 52 is provided with a second closing device at the outside of the liquid storage box 5. When the second closing device is opened, the liquid can be replenished into the liquid storage box 5 through the protein liquid replenishing port 52. When the second closing device is closed, the protein liquid replenishing port 52 is in a closed state. The part of the circulation pipe 3 located in the liquid storage box 5 is hollowed out, and the nutrient solution components with a molecular weight less than transferrin can freely shuttle through the hollowed out part. The barrier film 53 is laid in the hollowed out part. The function of the barrier film 53 is to only allow the iron element and the nutrient solution components with a molecular weight less than transferrin to flow through, but not transferrin. A circulation pump 32 is provided on the circulation pipe 3. After the circulation pump is turned on, the liquid circulates in the circulation pipe 3, and in the circulation pipe 3 part inside the box body 1, the flow direction of the liquid is from top to bottom.

[0049] Exemplarily, the first closing device and the second closing device are both valves.

[0050] Exemplarily, the closed switch 6 is a bendable unit 61 arranged around the circulation pipe 3, for example, memory metal is used, one end of the bendable unit 61 is fixed to the open edge of the top of the through channel 2, and the other end of the bendable unit 61 is a movable end. When the closed switch 6 is closed, each bendable unit 61 stretches and the edges of adjacent bendable units 61 contact, and the bendable unit 61 also contacts the part of the circulation pipe 3 located above the circulation hole 31; when the closed switch 6 is opened, the bendable unit 61 bends, and adjacent bendable units 61 separate to expose the circulation hole 31.

[0051] Exemplarily, a sealing strip is provided on the edge of the bendable unit 61. When the edges of adjacent bendable units 61 are in contact, the sealing strip acts as a seal to prevent leakage.

[0052] For example, see Figure 6 The top view of the structure after the bendable units 61 are stretched and spliced ​​is a ring.

[0053] For example, see Figure 7 and Figure 8 The bendable unit 61 is fan-shaped after being stretched. After it contacts the part of the circulation pipe 3 above the flow hole 31, the top angle of the fan is turned outward relative to the through channel 2, and the remaining parts of the fan-shaped edges of the adjacent bendable units 61 contact to surround and block the flow hole 31.

[0054] Exemplarily, the molecular weight cutoff of the barrier membrane is 60-75 kDa, which is close to but smaller than the molecular weight of transferrin. Iron elements, iron compounds and small molecule nutrients with small molecular weight can pass through the barrier membrane, but transferrin cannot. As the iron and transferrin are complexed and decomplexed, the transferrin can be reused.

[0055] Exemplarily, a support 4 is provided at the bottom of the box body 1, and a circulation pipe 3 is laid on the support 4. For example, the support 4 is fixed to the bottom of the side wall of the box body 1, the bottom outer wall, or the box body 1 is placed on a boss provided by the support 4.

[0056] The workflow of this embodiment is as follows:

[0057] S1, without starting the circulation pump 32, add the nutrient solution without transferrin into the culture space 21, inoculate the cardiomyocytes, the liquid level of the nutrient solution is lower than the through channel 2, and the nutrient solution component is the nutrient solution component of Chinese patent 201611252382.2 or 201510046646.8. The liquid level is lower than the height of the culture space 21, such as Figure 3 The midpoint dashed line is the liquid level.

[0058] S2, adding an iron-containing nutrient solution, or a pure iron compound solution or an iron-containing suspension into the liquid storage box 5 through the liquid replenishing port 51.

[0059] S3, the transferrin liquid is replenished to the position where the barrier membrane 53 is set in the circulation tube 3 through the protein liquid replenishing port 52, and the iron element passes through the barrier membrane 53 and is compounded with the transferrin. Under the barrier effect of the barrier membrane 53, the transferrin is located inside the through channel 2 and cannot enter the liquid storage box 5. However, the iron in the liquid storage box 5 and the substances with a molecular weight smaller than that of transferrin can enter the through channel 2.

[0060] S4, close the closed switch 6, start the circulation pump 32, as the transferrin circulates continuously in the circulation tube 3, or is stored in the space between the through channel 2 and the closed switch 6 through the flow hole 31, see Figure 3 The dashed line with an arrow is the direction of liquid flow when the closed switch 6 is closed, after the iron and transferrin are fully complexed.

[0061] Open the closed switch 6, and the transferrin solution flows into the culture space 21. Figure 4 The dotted line in the culture space 21 indicates the height of the liquid level compared to Figure 3 There has been an increase, Figure 4 The dashed line with an arrow is the direction in which the liquid flows when the closed switch 6 is turned on, and the transferrin liquid flows to supplement iron to the myocardial cells.

[0062] The aforementioned working process S1-S4 is performed once within the time period T1. After several cycles of the time period T1, that is, after the time period T, all the nutrient solutions are replaced and the cardiomyocytes are passaged.

[0063] The above working process can not only achieve timely and appropriate increase of iron elements as cells continue to grow and proliferate to avoid iron excess or iron deficiency, but also make full use of the property of transferrin to transport iron and exert its "transport" function, so that transferrin can be reused without adding too high a concentration of transferrin to the nutrient solution formula, thus saving resources and being environmentally friendly.

[0064] In order to reduce the probability of free iron entering the culture space 21 and avoid free iron from damaging myocardial cells, we set the liquid storage box 5 to an iron layer 54 and a water layer 55, wherein the iron layer 54 is located above the water layer 55, and the iron layer 54 and the water layer 55 are separated by a partition 56. The hollow part of the circulation tube 3 and the barrier film 53 are all set through the iron layer 54 and the water layer 55, and the liquid replenishment port 51 and the protein liquid replenishment port 52 are all located at the iron layer 54. When transferrin flows from the iron layer 54 to the water layer 55, the deionized water or ultrapure water in the water layer 55 exchanges the free iron, and the free iron enters the water layer 55, so that the amount of free iron in the circulation tube 3 is reduced. Of course, the water layer 55 can also be set to be fluid, and clean deionized water or ultrapure water is continuously added to the water layer 55 to achieve the effect of maximally removing free iron.

[0065] It should be noted that the sealing component of the present invention is a sealant or a sealing sleeve.

[0066] It should be noted that in order to prevent myocardial cells from being contaminated by bacteria or other impurities, the device must be sterilized and disinfected before it can be used to culture myocardial cells.

[0067] It should be noted that the component connection relationships not specifically mentioned in the present invention are assumed to adopt the existing technology. Since they do not involve the invention point and are widely used in the existing technology, the structural connection relationships are not described in detail.

[0068] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A device for culturing cardiomyocytes using transferrin, characterized in that: The invention comprises a box body (1) and a circulation pipe (3), wherein the top of the box body (1) is open, the bottom of the box body (1) has an opening, a through channel (2) is provided inside the box body (1), the top and bottom of the through channel (2) are both open, and the opening at the bottom of the through channel (2) is connected to the opening at the bottom of the box body (1); The bottom opening of the through-channel (2) is also connected to a liquid storage box (5); a portion of the circulation pipe (3) penetrates the through-channel (2), the liquid storage box (5) and a box cover (11) arranged on the top of the box body (1); the other portion is arranged outside the box body (1); the circulation pipe (3) is located inside the box body (1) and is provided with a flow hole (31) at a position above the through-channel (2); a closed switch (6) is provided at the top of the through-channel (2); and the closed switch (6) includes an open state and a closed state; When the closed switch (6) is in a closed state, it surrounds the flow hole (31), and the liquid flowing out of the flow hole (31) flows into the through-channel (2); when the closed switch (6) is in an open state, the flow hole (31) is exposed outside the through-channel (2), and the liquid flowing out of the flow hole (31) flows into the through-channel (2) and the box body (1); The portion of the circulation tube (3) located in the liquid storage box (5) is hollowed out, and a barrier film (53) is provided at the hollowed-out portion, and the barrier film (53) is used to circulate substances with a molecular weight smaller than that of transferrin; The circulation pipe (3) is provided with a circulation pump (32), and after the circulation pump (32) is turned on, the flow direction of the liquid inside the box body (1) is from top to bottom; The sealing switch (6) comprises a plurality of bendable units (61), all of which are arranged circumferentially around the circulation pipe (3), one end of the bendable unit (61) is fixed to the top open edge of the through-channel (2), and the other end is a movable end; When the closed switch (6) is in a closed state, all the bendable units (61) are stretched out, the edges of adjacent bendable units (61) are in contact, and the bendable units (61) are also in contact with the portion of the circulation pipe (3) located above the flow hole (31); When the closed switch (6) is in an open state, all the bendable units (61) are bent, adjacent bendable units (61) are separated, and the flow hole (31) is exposed; The top view of the structure after the bendable unit (61) is stretched and spliced ​​is a ring; or, the bendable unit (61) is stretched to be a fan-shaped, and after the bendable unit (61) contacts the portion of the circulation pipe (3) located above the flow hole (31), the vertex angle of the fan-shaped can be turned outward relative to the through channel (2), and the remaining portions of the adjacent fan-shaped edges are in contact to surround the flow hole (31); The sum of the heights of the through channel (2) and the closed switch (6) is less than the height of the box body (1).

2. The device for culturing cardiomyocytes using transferrin according to claim 1, characterized in that: The liquid storage box (5) comprises an iron layer (54) and a water layer (55), wherein the iron layer (54) is located above the water layer (55), and the iron layer (54) and the water layer (55) are separated by a partition (56), and the hollow part of the circulation pipe (3) and the barrier film (53) are both arranged to penetrate the iron layer (54) and the water layer (55).

3. The device for culturing cardiomyocytes using transferrin according to claim 2, characterized in that: The barrier membrane (53) is a dialysis membrane, and the molecular weight cut-off of the dialysis membrane is 60-75 kDa.

4. The device for culturing cardiomyocytes using transferrin according to claim 1, characterized in that: The liquid storage box (5) is provided with a liquid replenishing port (51) at a position outside the box body (1).

5. The device for culturing cardiomyocytes using transferrin according to claim 4, characterized in that: The liquid storage box (5) is also provided with a protein liquid replenishing port (52) at a position outside the box body (1), and the protein liquid replenishing port (52) is arranged through the liquid storage box (5) and the internal circulation pipe (3) thereof.

6. The device for culturing cardiomyocytes using transferrin according to claim 1, characterized in that: A support (4) is arranged at the bottom of the box body (1), and the circulation pipe (3) is arranged and laid on the support (4).

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