Temperature-sensitive adherent cell culture flask

Abstract

The utility model relates to cell culture device technical field discloses a kind of temperature-sensitive adherent cell culture bottles.The culture bottle includes bottle body, bottle cap and built-in temperature-sensitive porous carrier, built-in temperature-sensitive porous carrier is core innovative structure, plane porous structure body is made of temperature-sensitive polymer, covers entire culture bottle bottom, by the stacking of countless microslides into sponge-like carrier, can be replaced and installed by extraction mode, flatly paste in bottle body inner bottom culture surface.The utility model realizes stem cell enzyme-free mild subculture by the temperature response characteristic of temperature-sensitive material, protects cell membrane protein and function, improves cell survival rate and stem cell stemness maintenance ability;Built-in temperature-sensitive porous carrier provides huge specific surface area, improves culture efficiency and mass transfer efficiency, and easy to operate, strong compatibility, controllable cost, can be widely applied to adherent cell large-scale culture and subculture, with very high practical value and popularization prospect.

Description

Technical Field

[0001] This application relates to the field of cell culture device technology, and in particular to a temperature-sensitive adherent cell culture flask. Background Technology

[0002] Currently, the passage of adherent cells mainly relies on digestive enzymes such as trypsin. The basic principle of this method is to use digestive enzymes to cleave the connective proteins between the cells and the culture surface, causing the cells to detach and thus achieving passage. However, this method has the following significant drawbacks: First, the risk of enzyme stimulation is unavoidable. Digestive enzymes, while cleaving connective proteins, inevitably damage other functional proteins and receptors on the cell membrane. These proteins and receptors are crucial for cellular physiological functions such as signal transduction, substance transport, proliferation, and differentiation; damage directly affects cell viability and functional integrity. For stem cells, such damage may lead to loss of stemness, rendering them unable to maintain self-renewal and multi-lineage differentiation capabilities, severely impacting subsequent research and applications.

[0003] Secondly, the enzyme digestion process is difficult to control precisely, and there is a risk of over-digestion. Excessive digestion time or too high enzyme concentration can exacerbate cell damage, even leading to cell lysis and death; while insufficient digestion results in incomplete cell detachment, affecting passage efficiency. Operators need extensive experience to control digestion conditions, which not only increases the operational difficulty but also reduces the reproducibility and stability of experiments.

[0004] Furthermore, the risk of enzyme residue is significant. After digestion, some digestive enzymes remain in the culture system, and these residual enzymes can continue to damage the cells. To remove enzyme residues, multiple centrifugation and washing operations are required. This process is not only cumbersome and time-consuming, but the mechanical force generated during centrifugation can also damage the cells again, further reducing cell viability.

[0005] Furthermore, existing culture equipment is not cost-effective. Both conventional cell culture flasks and existing temperature-sensitive culture dishes are disposable consumables, requiring disposal after each use. For large-scale cell culture, long-term use results in high costs, hindering efforts to reduce experimental and production costs. Simultaneously, existing temperature-sensitive culture dishes are mostly disposable plates with limited surface area, failing to meet the demands of large-scale cell culture and proving difficult to integrate into standard culture procedures. This makes the transition from small-scale laboratory culture to large-scale industrial production challenging.

[0006] In summary, existing adherent cell culture and passage technologies suffer from problems such as enzyme damage, complex operation, high cost, and low culture efficiency. There is an urgent need in this field for a culture device that can be integrated into standard culture procedures, can efficiently culture cells, and can achieve gentle enzyme-free passage in order to solve the above-mentioned technical problems. Utility Model Content

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies, such as cell damage caused by reliance on digestive enzymes for adherent cell passage, cumbersome operation, high cost, low culture efficiency, and poor compatibility of existing temperature-sensitive culture devices. This invention provides a temperature-sensitive adherent cell culture flask that increases the culture surface area, facilitates gentle, enzyme-free passage, and offers better cost-effectiveness. Through its unique structural design, this culture flask achieves gentle, enzyme-free passage, protects cell viability and function, improves culture efficiency, simplifies the operation process, reduces costs, and meets the needs of large-scale culture and passage of adherent cells (especially stem cells).

[0008] This application provides a temperature-sensitive adherent cell culture flask, including a flask body, a flask cap, and a built-in temperature-sensitive porous carrier; the flask body is made of transparent polymer and has a standard threaded opening; the flask cap matches the threaded opening of the flask body and has a vent hole and a sealing cap; the built-in temperature-sensitive porous carrier is a planar porous structure that covers the entire bottom surface of the culture flask, is flat against the bottom culture surface of the flask body, and can be replaced and installed by extraction.

[0009] Furthermore, the transparent polymer of the bottle is polyethylene terephthalate or polystyrene, and the shape is a square T175 cell culture flask.

[0010] Furthermore, the built-in temperature-sensitive porous carrier is made of temperature-sensitive polymer and is a sponge-like carrier composed of countless stacked microcarrier sheets. It has an interconnected pore structure, including macroscopic pores and microscopic surfaces. The macroscopic pore size is 100-500μm, and the microscopic surface has temperature-sensitive adhesion properties.

[0011] Furthermore, the thermosensitive polymer is polyisopropylacrylamide.

[0012] Furthermore, the built-in temperature-sensitive porous carrier was prepared by freeze-drying, with an average pore size of 150 μm and a porosity greater than 90%.

[0013] Furthermore, the bottle cap has a breathable membrane inside the vent hole for gas exchange and to prevent contamination.

[0014] Furthermore, the built-in temperature-sensitive porous carrier is a replaceable module, and the bottle body can be reused or used as a disposable consumable.

[0015] Compared with the prior art, the temperature-sensitive adherent cell culture flask of this invention has the following significant advantages: (1) Enzyme-free and gentle passage, protecting cell viability and function: Cell detachment is achieved through the temperature-responsive properties of thermosensitive materials, eliminating the need for digestive enzymes such as trypsin. This fundamentally avoids damage to cell membrane proteins and receptors by enzymes, effectively protecting cell viability and physiological function. For stem cells, this can significantly improve cell viability while maintaining stem cell stemness, ensuring the self-renewal and multi-lineage differentiation capabilities of stem cells, and providing a high-quality cell source for subsequent research and applications.

[0016] (2) Ultra-high culture efficiency and increased cell yield per bottle: The built-in temperature-sensitive porous carrier has a sponge-like porous structure, providing a huge specific surface area far exceeding that of traditional two-dimensional bottle walls. Traditional cell culture bottles can only utilize the two-dimensional surface of the bottle wall for cell culture, while the porous carrier of this invention provides a three-dimensional growth space for cells, which significantly increases the cell yield per bottle, meets the needs of large-scale cell culture, and improves experimental and production efficiency.

[0017] (3) Simple operation, strong compatibility, and easy conversion: The culture flask of this utility model is basically the same as the standard cell culture flask in terms of structure and usage. It can be used directly as a standard culture flask without changing the existing cell culture process and operating habits, which reduces the learning cost and operation difficulty for operators. At the same time, the culture flask is perfectly compatible with existing culture equipment (such as incubators, microscopes, centrifuges, etc.), which makes it easy to realize the conversion from small-scale laboratory culture to large-scale industrial production, and has wide applicability.

[0018] (4) Controllable cost and reduced cost of large-scale use: The core built-in temperature-sensitive porous carrier is designed as a replaceable module, and the bottle can be reused. Only the carrier needs to be replaced periodically, which avoids the waste caused by the overall disposal of traditional disposable culture bottles and significantly reduces the cost of long-term large-scale use. At the same time, the entire culture bottle can also be designed as a disposable low-cost consumable according to actual needs, flexibly meeting the needs of different users and further improving cost-effectiveness.

[0019] (5) Excellent mass transfer efficiency, supporting high-density cell culture: The interconnected pore structure of the built-in temperature-sensitive porous carrier (3) not only increases the specific surface area but also optimizes the mass transfer efficiency. Oxygen and nutrients can be quickly and evenly transported to each cell inside the carrier through the pores, while waste products generated by cell metabolism can also be discharged in time, effectively avoiding the problems of insufficient nutrient supply and waste accumulation that are prone to occur in traditional high-density culture, providing a good microenvironment for high-density and high-quality cell growth, and further improving culture efficiency and cell quality.

[0020] The above description is merely an overview of the technical solutions of the embodiments of this application. In order to better understand the technical means of the embodiments of this application and to implement them in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the embodiments of this application more obvious and understandable, specific implementation methods of this application are described below. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a structural diagram of the temperature-sensitive adherent cell culture flask of this utility model.

[0023] Figure 2 This is a diagram showing the internal structure of the built-in temperature-sensitive porous carrier of this utility model.

[0024] Explanation of reference numerals in the attached figures: 1. Bottle body; 2. Bottle cap; 3. Temperature-sensitive porous carrier; 4. Pore path of temperature-sensitive carrier. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims and drawings of this application are intended to cover non-exclusive inclusion.

[0027] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of the phrase "embodiment" in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0028] The directional terms appearing in the following description refer to the directions shown in the figures and are not intended to limit the specific structure of this application. For example, in the description of this application, terms such as "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the figures. They are used only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0029] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, "connection" or "joining" in mechanical structures can refer to a physical connection, such as a fixed connection, for example, a connection fixed by fasteners, such as a connection fixed by screws, bolts, or other fasteners; a physical connection can also be a detachable connection, such as a snap-fit ​​or interlocking connection; a physical connection can also be an integral connection, such as a connection formed by welding, bonding, or integral molding. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0030] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0031] This utility model provides, for example Figure 1 , Figure 2 The temperature-sensitive adherent cell culture flask shown includes a flask body 1, a flask cap 2, and an internal temperature-sensitive porous carrier 3. The structure and connection relationship of each part are as follows: A temperature-sensitive adherent cell culture flask includes a flask body 1, a flask cap 2 with a breathable membrane, and a planar porous temperature-sensitive carrier 3 that covers the entire bottom surface of the culture flask, with the temperature-sensitive carrier pores 4 evenly distributed inside the carrier.

[0032] The bottle body 1 is made of polyethylene terephthalate (PET) and is a square T175 cell culture flask. The bottle body has a standard threaded opening, good transparency and biocompatibility, which facilitates observation of cell growth status and does not affect cell activity.

[0033] The cap 2 matches the standard threaded opening of the bottle body 1, using a screw-type sealing connection. The vent hole on the cap 2 is equipped with a polytetrafluoroethylene breathable membrane with a pore size of 0.22μm, which can ensure the smooth exchange of oxygen and carbon dioxide, and effectively prevent external microorganisms from entering the bottle, ensuring the sterility of the culture environment; the sealing cap is made of silicone, which is soft and has good sealing performance, and can close the vent hole when needed.

[0034] The temperature-sensitive porous carrier 3 is prepared from polyisopropylacrylamide (PIPAAm) hydrogel by freeze-drying. The specific preparation steps are as follows: First, PIPAAm monomer is dissolved in deionized water, and an appropriate amount of crosslinking agent (such as N,N'-methylenebisacrylamide) and initiator (such as ammonium persulfate) are added. After stirring evenly, a prepolymer solution is formed. The prepolymer solution is poured into a mold matching the bottom size of the bottle 1 and refrigerated at 4°C to allow for preliminary polymerization. Subsequently, the prepolymerized gel is placed in a freeze dryer and freeze-dried at -50°C and a vacuum degree less than 10 Pa for 24 hours to remove moisture from the gel, forming a temperature-sensitive porous carrier 3 with an interconnected porous structure. The average pore size of this carrier is 150 μm, and the porosity is greater than 90%. It adheres flat to the bottom culture surface of the bottle 1 and can be removed and replaced by extraction from the bottle opening.

[0035] Bottle 1: Made of a standard transparent polymer, preferably polyethylene terephthalate (PET) or polystyrene (PS). These materials are chosen because they have good biocompatibility, do not have toxic effects on cell growth, and possess excellent transparency, allowing operators to easily observe cell growth under a microscope during culture. Bottle 1 is preferably a square T175 cell culture flask, conforming to standard cell culture flask specifications and facilitating use in existing culture equipment (such as incubators, centrifuges, etc.) without requiring additional equipment replacement. The bottle body has a standard threaded connection for a tight fit with the cap 2, ensuring a sealed culture environment.

[0036] Bottle Cap 2: Equipped with a vent and a sealing cap, it matches the threaded opening of bottle body 1 to achieve a screw-type sealing connection. The vent contains a permeable membrane with excellent gas permeability, allowing oxygen from the incubator to enter the bottle while simultaneously expelling carbon dioxide produced by cell metabolism, ensuring the necessary gaseous environment for cell growth. Simultaneously, the permeable membrane acts as a barrier, preventing external bacteria, fungi, and other contaminants from entering the bottle and avoiding cell culture contamination. The sealing cap can be placed over the vent when it is necessary to seal the culture bottle (such as during transfer or special culture operations), further ensuring the airtightness and sterility of the internal environment.

[0037] Built-in temperature-sensitive porous carrier 3: This is the core innovative structure of this invention, made of a temperature-sensitive polymer, preferably polyisopropylacrylamide (PIPAAm). PIPAAm is a typical temperature-sensitive polymer with unique temperature response characteristics: at 37℃ (normal human body temperature, which is also the conventional temperature for cell culture), the surface of PIPAAm is hydrophobic, facilitating cell attachment and growth; when the temperature drops below the critical temperature (such as 25℃ or room temperature), the surface of PIPAAm undergoes a hydrophilic transformation and swells, resulting in a weakening of the interaction force between the cell and the carrier surface, causing the cell to detach automatically, thereby achieving enzyme-free passage.

[0038] The built-in temperature-sensitive porous carrier 3 is a planar porous structure, a sponge-like carrier composed of numerous stacked microcarrier sheets. It can be replaced and installed by extraction, and is flat against the bottom culture surface of the bottle. This carrier has a huge specific surface area and an interconnected pore structure, with a macroscopic pore size of 100-500μm, preferably 150μm. This pore size allows cells and culture medium to permeate freely, ensuring that cells can enter the carrier for growth, while facilitating the flow of culture medium within the carrier to provide cells with sufficient nutrients and oxygen. Its microscopic surface (the material itself) provides temperature-sensitive adhesion properties, ensuring that cells can stably adhere and grow at suitable temperatures.

[0039] The built-in temperature-sensitive porous carrier 3 was prepared by freeze-drying. This preparation method allows for precise control of the pore size and porosity of the carrier. The resulting carrier has a uniform porous structure with an average pore size of 150 μm and a porosity greater than 90%. The high porosity not only further increases the specific surface area of ​​the carrier but also facilitates the transport of oxygen and nutrients within the carrier and the removal of cellular metabolic waste, providing a favorable microenvironment for high-density cell growth.

[0040] The method of using the temperature-sensitive adherent cell culture flask in this embodiment is as follows: Preparation: Sterilize the bottle body 1, bottle cap 2 and new temperature-sensitive porous carrier 3 by high-pressure steam (121℃, 0.1MPa, 20 minutes). After sterilization, remove them and place them in a clean bench to cool to room temperature for later use.

[0041] Cell seeding: In a clean bench, keep bottle 1 horizontal, unscrew bottle cap 2, and seed the prepared stem cell suspension (concentration 1×10⁻⁶). 5 Inject 15 mL of culture medium (cells / mL) into the culture flask from the neck, ensuring the medium completely submerges and fills the entire temperature-sensitive porous carrier. Gently shake the culture flask to distribute the cell suspension evenly within the carrier.

[0042] Culture process: Screw the cap 2 onto the culture flask inoculated with cells, ensuring the vent is unobstructed, and place it in a constant temperature incubator at 37℃ and 5% CO2 for static culture. During the culture process, observe the cell growth status daily using a microscope: After 12 hours of culture, cells begin to adhere to the inner and outer surfaces of the temperature-sensitive porous carrier 3; after 24 hours of culture, cells are firmly attached and begin to proliferate; after 72 hours of culture, cells form a dense cell layer inside the carrier, at which point passage can be performed.

[0043] Enzyme-free passage: Remove the culture flask from the incubator and place it at room temperature (approximately 25°C). Gently shake the flask at a frequency of 10-15 times per minute for 5 minutes. During this time, the PIPAAm material in the temperature-sensitive porous carrier 3 undergoes a hydrophilic transformation and swells due to the decrease in temperature, and the cells automatically detach from the carrier surface, forming a cell suspension. In a clean bench, unscrew the cap 2 and slowly aspirate the cell suspension from the flask using a pipette, transferring it to a centrifuge tube for subsequent passage and seeding operations.

[0044] Carrier replacement and bottle reuse: If the temperature-sensitive porous carrier 3 has been used multiple times, and there are many residual cells on the surface or the performance has deteriorated, after passage, open the bottle cap 2, use sterile forceps to reach into the bottle mouth, grasp the edge of the carrier, and pull it out to replace it with a new sterilized carrier. After cleaning (rinsing 3 times with deionized water, then soaking in 75% ethanol for 30 minutes, and finally rinsing clean with deionized water) and re-sterilizing, the bottle 1 can be reused up to 10 times or more.

[0045] The temperature-sensitive adherent cell culture flask of this embodiment effectively protects the activity and stemness of stem cells through a gentle, enzyme-free passage method. After testing, the cell viability after passage reached over 95%, which is significantly higher than that of traditional enzyme digestion passage methods (cell viability is high), demonstrating good practicality and promotional value.

[0046] The above-described embodiments are merely illustrative of the technical solutions of this application and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and this efficiency is approximately 80%. Meanwhile, because the temperature-sensitive porous carrier 3 provides a huge specific surface area, the cell yield per flask reaches 3-5 times that of traditional T175 culture flasks, significantly improving culture efficiency. Furthermore, this culture flask is easy to operate, requires no changes to the existing culture process, and the flask can be reused, significantly reducing the cost of large-scale cell culture. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A temperature-sensitive adherent cell culture flask, characterized in that: It includes a bottle body (1), a bottle cap (2), and a built-in temperature-sensitive porous carrier (3); the bottle body (1) is made of transparent polymer and has a standard threaded opening; the bottle cap (2) matches the threaded opening of the bottle body (1) and has a vent hole and a sealing cap; the built-in temperature-sensitive porous carrier (3) is a planar porous structure that covers the entire bottom surface of the culture bottle and is flat against the bottom culture surface inside the bottle, and can be replaced and installed by extraction.

2. The temperature-sensitive adherent cell culture flask according to claim 1, characterized in that: The transparent polymer of the bottle body (1) is polyethylene terephthalate or polystyrene, and the shape is a square T175 cell culture bottle.

3. The temperature-sensitive adherent cell culture flask according to claim 1, characterized in that: The built-in temperature-sensitive porous carrier (3) is made of temperature-sensitive polymer and is a sponge-like carrier composed of countless micro-carrier sheets stacked together. It has an interconnected pore structure, including macro-pores and micro-surfaces. The macro-pore size is 100-500μm, and the micro-surface has temperature-sensitive adhesion properties.

4. The temperature-sensitive adherent cell culture flask according to claim 3, characterized in that: The temperature-sensitive polymer is polyisopropylacrylamide.

5. The temperature-sensitive adherent cell culture flask according to claim 3, characterized in that: The built-in temperature-sensitive porous carrier (3) is prepared by freeze-drying, with an average pore size of 150 μm and a porosity greater than 90%.

6. The temperature-sensitive adherent cell culture flask according to claim 1, characterized in that: The bottle cap (2) has a breathable membrane inside the vent hole for gas exchange and to prevent contamination.

7. The temperature-sensitive adherent cell culture flask according to claim 1, characterized in that: The built-in temperature-sensitive porous carrier (3) is a replaceable module, and the bottle (1) can be reused or used as a disposable consumable.

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