CN102329727B - Preparing device of cell sheet for cold and hot regulation of temperature sensitive gel by utilizing semiconductor refrigerating sheets - Google Patents

Abstract

The invention discloses a preparing device of a cell sheet for the cold and hot regulation of a temperature sensitive gel by utilizing semiconductor refrigerating sheets, which comprises a fixing frame (1). The semiconductor refrigerating sheets (2) are arranged on the fixing frame; the semiconductor refrigerating sheets (2) are coated with heat-conducting oil and are in close connection with a culture utensil (3); the semiconductor refrigerating sheets (2) are connected in parallel or in series and are connected with a controller (6) connected with a power supply through a conducting wire; a temperature sensitive gel (4) coats the bottom of the inner wall of the culture utensil (3); a temperature sensor (5) is arranged at the bottom of the outer wall of the culture utensil; and the temperature of the temperature sensitive gel (4) is regulated through the semiconductor refrigerating sheets (2) in the culture utensil, so as to achieve the preparation of a tissue engineering cell sheet. The device has a simple and compact structure and a quick temperature change speed, and further can be monitored in a timely way.

Description

Cell sheet preparation device using semiconductor refrigeration sheet to adjust temperature-sensitive gel cold and heat

technical field

The invention relates to a cell sheet preparation device, in particular to a cell sheet preparation device which utilizes a semiconductor refrigerating sheet to regulate temperature-sensitive gel cold and heat.

Background technique

Tissue engineered cell sheets separate cultured cells from the culture surface to form a complete sheet structure containing extracellular matrix through tissue engineering methods, which is a new technology for harvesting and transferring seed cells in tissue engineering. Tissue engineered cell sheets contain important cell surface proteins such as ion channels, growth factor receptors and connexins, which can avoid the side effects caused by the use of carriers such as scaffolds. It is widely used in the repair of air leakage and so on.

In the early days, some scholars evenly inoculated cells in glass culture dishes, and when all the colonies were basically connected into sheets, they were peeled off from the culture dishes by mechanical methods to obtain cell sheets (Zhao Xiongfei, Zhang Shijun, Hu Jianian, Lu Shuzhen, Wang Xu, Li et al. Ao, Homografting of cultured human epidermal cell sheets on burn wounds, PLA Medical Journal, 1989, 14(2): 82-85), mechanical methods sometimes damage cells, and special care is required for operation. In recent years, tissue-engineered cell sheets have been prepared mainly by methods such as temperature-sensitive culture dishes, collagen gels, magnetic tissue engineering, granular monolayers with rough surfaces, and polyelectrolytes (Li Haijia, Wang Wanpeng, Shi Hongcan, Tissue Engineering Cell Sheet Construction Methods, China Repair Journal of Reconstructive Surgery, 2011, 25(6): 741-744), in which the temperature-sensitive culture dish method is used to obtain cell sheets by cooling, which is simple and reliable, and has been widely used. The use of temperature-sensitive culture dishes to construct tissue engineering cell sheets is generally to covalently graft temperature-sensitive hydrogels onto the surface of ordinary polystyrene tissue culture dishes, and use them to sense changes in the external environment temperature to undergo sol-gel transition or reversibility. Characterization of volume transitions for preparation of cell sheets. Materials that can be used to prepare thermosensitive hydrogels include poly-N-isopropylacrylamide (poly-N-isopropylacrylamide, PNIPAAm), methyl vinyl ether (MVE), acrylate-2-N-morpholino ethyl ester (MPEMA), N, N'-dimethylaminoethyl ester (DMAEMA), methacrylic acid (MAAC), poloxamer (Poloxamer), polyacrylic acid (PAA), polyacrylamide (PAAm) and other synthetic polymers materials, as well as natural polymers such as cellulose and its derivatives, chitosan and its derivatives, among which poly-N-isopropylacrylamide temperature-sensitive hydrogels are of particular interest. Poly N-isopropylacrylamide has a certain proportion of hydrophobic isopropyl group (-CH(CH ₃ ) ₂ -) and hydrophilic amide group (-NHCO-), and the hydrophilic/hydrophobic balance is affected by Influenced by temperature, it is relatively hydrophobic at 37°C, which is conducive to cell adhesion, extension and proliferation. When the temperature drops to the low critical solution temperature (Low critical solution temperature, LTCST, 32°C) of the polymer, the polymer surface becomes hydrophilic , expand, and form a hydration layer with the cultured cells, so that the cells are naturally separated from the culture dish. The use of temperature-sensitive culture dishes to construct tissue engineering cell sheets does not require enzyme treatment, maintains the integrity of some important cell surface proteins, mild conditions, high yield, and can maintain high activity of cells desorbed from the surface of the gel. However, the realization and control of temperature changes in the process of cell sheet preparation is still a difficult problem. At present, most of them are realized by reducing the indoor environment, which takes a long time, and it is not easy to accurately control the temperature-sensitive hydrogel in the temperature-sensitive culture dish. low temperature, it is easy to cause damage to cells by low temperature.

Contents of the invention

The technical problem to be solved by the present invention is to provide a simple and fast cell sheet preparation device, which can not only quickly realize the temperature change of the thermosensitive hydrogel, but also monitor the temperature of the culture vessel in a timely manner.

In order to solve the above-mentioned technical problems, the cell sheet preparation device of the present invention that utilizes a semiconductor refrigerating sheet to regulate temperature-sensitive gel cold and heat includes: a fixed frame, on which a semiconductor refrigerating sheet is arranged, and the semiconductor refrigerating sheet is coated with heat-conducting oil and tightly connected to the culture vessel , the semiconductor cooling chip is connected to the controller connected with the power supply through wires, a layer of temperature-sensitive gel is covered on the bottom of the inner wall of the culture vessel, and a temperature sensor is arranged on the bottom of the outer wall. The culture vessel can be a culture dish, a culture bottle or a culture plate (6-well, 12-well, 24-well, 48-well, 96-well, etc.) of various sizes, etc.

The lower critical solution temperature of the thermosensitive gel is lower than 37°C, poly N-isopropylacrylamide, methyl vinyl ether, 2-N-morpholinoethyl acrylate, N,N'-dimethyl Aminoethyl ester, methacrylic acid, poloxamer, polyacrylic acid, polyacrylamide and other synthetic high polymers, and natural polymers such as cellulose and its derivatives, chitosan and its derivatives, preferably poly N- Isopropylacrylamide can be obtained by monomer polymerization crosslinking, polymer crosslinking or graft copolymerization of carriers, and can also be made into films, microspheres, blocks, latex and other shapes. Graft copolymerization, phase transition polymerization, low temperature treatment, introduction of inorganic nanoparticles, and use of pore-forming agents can be used to increase the response rate of hydrogels, and the formation of interpenetrating network structures and physical cross-linking can also be used to increase the water retention rate. The response rate and mechanical properties of the gel, etc. The porous structure can also be introduced into the hydrogel by phase separation method, emulsion template method, porogen method, polymer interpenetrating network method, etc. to improve the response rate of poly-N-isopropylacrylamide hydrogel. Chitosan was introduced on the basis of poly-N-isopropylacrylamide to improve its biocompatibility while retaining the thermosensitivity of PNIPAAm. Hydrophilic monomers such as acrylic acid and acrylamide can be introduced into the gel skeleton structure to increase its lower critical solution temperature.

The semiconductor refrigeration chip is the key to realize the temperature change regulation of cell adhesion/desorption. Its principle is to use the Peltier effect of semiconductor materials and use special semiconductor material thermopiles for cooling, which can directly convert electrical energy into heat energy, and can realize cooling and heating at the same time. , Changing the positive and negative levels of the power supply can easily switch between cooling and heating. The semiconductor refrigeration chips are connected in parallel or in series, and are arranged in a fixed frame at uniform and symmetrical intervals. The number is 1-100 pieces, and the size of each piece is 0.5mm×0.5mm×0.5mm to 10mm×10mm×10mm. A small groove is engraved on the semiconductor refrigerating sheet to install a temperature sensor, or 1-200 temperature sensors are installed in the gap between the semiconductor refrigerating sheets; each temperature sensor is connected to the temperature input end of the controller. The controller is a temperature-controllable controller, and is provided with a display screen and a temperature alarm for displaying transient temperature.

The culture vessel is pressed in the fixed frame by a spring and a pressure plate, and is in close contact with the semiconductive refrigeration sheet. The upper edge of the culture vessel is 1mm-40mm higher than the upper edge of the fixed frame, so as to facilitate the removal of the culture vessel. 3-12 pairs of springs and pressure plates are symmetrically distributed on the upper edge of the fixed frame.

In summary, the cell sheet preparation device of the present invention heats or cools the temperature-sensitive hydrogel through the semiconductor refrigeration chip, and utilizes the temperature-sensitive hydrogel to sense the change of the external environment temperature and undergo sol-gel transition or Properties of reversible volume transitions for preparation of tissue engineered cell sheets. The semiconductor refrigeration chip does not need any refrigerant and can work continuously. There is no pollution source, no rotating parts, no turning effect, and no sliding parts. It is a solid piece. There is no vibration, noise, long life and easy installation. Moreover, the thermal inertia of the semiconductor refrigeration sheet is very small, and the cooling and heating time is very fast. The temperature sensor installed in the culture vessel can timely monitor the temperature of the thermosensitive hydrogel. Compared with the existing tissue engineering cell sheet preparation device, the temperature change speed and timely monitoring have been significantly improved, and the damage to cells by temperature can be reduced, which is also one of the keys proposed by the present invention. The cell sheet preparation device provided by the present invention can select different temperature-sensitive hydrogel materials and the arrangement of semiconductor refrigeration sheets according to the requirements of cell sheet culture. The preparation device realized is simple and reliable, and is expected to be used in the preparation of more tissue engineering cell sheets. Play an important role. The temperature-sensitive polymer material selected is not limited to poly-N-isopropylacrylamide, as long as the low critical solution temperature of the selected polymer material meets the cultivation requirements. For the sake of brevity, the present invention is mainly described by taking poly-N-isopropylacrylamide as an example, and other temperature-sensitive hydrogels can adopt the same principle.

Description of drawings

Fig. 1 is a schematic structural view of a cell sheet preparation device that utilizes a semiconductor refrigeration chip to regulate temperature-sensitive gel cold and heat provided by the present invention;

Fig. 2 is a top view of the structure of the cell sheet preparation device utilizing the semiconductor refrigeration sheet to regulate the temperature-sensitive gel cold and heat provided by the present invention;

Fig. 3 is a schematic structural view of another embodiment of the cell sheet preparation device provided by the present invention that uses a semiconductor refrigeration chip to regulate the temperature-sensitive gel cold and heat;

Fig. 4 is a schematic structural view of the third embodiment of the cell sheet preparation device provided by the present invention that utilizes the semiconductor refrigeration sheet to regulate the temperature-sensitive gel cold and heat;

Fig. 5 is a schematic structural view of a fourth embodiment of a cell sheet preparation device provided by the present invention that uses a semiconductor refrigeration chip to regulate the temperature-sensitive gel.

Among them: 1. Fixed frame; 2. Semiconductor refrigeration sheet; 3. Culture vessel; 4. Temperature sensitive gel; 5. Temperature sensor; 6. Controller; 7. Spring; 8. Press plate; 9. Hole; 10. Heat sink; 11. Wire; 12. Wire.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Fig. 1 is a schematic structural view of a cell sheet preparation device provided by the present invention that uses a semiconductor refrigeration chip to regulate the temperature and temperature of a thermosensitive gel, and its structural top view is shown in Fig. 2 . 3, 4 and 5 are structural schematic diagrams of another three embodiments of the present invention. As can be seen from Fig. 1 and Fig. 2, the cell sheet preparation device utilizing semiconductor refrigerating sheet to regulate temperature-sensitive gel cold and heat of the present invention comprises: a fixed frame (1), is provided with semiconductor refrigerating sheet (2) on it, and semiconductor refrigerating sheet (2) is coated with heat-conducting oil and tightly connected with the culture vessel (3), the semiconductor refrigeration chips (2) are connected in parallel or in series, and connected to the controller (6) with power supply through wires, and the inner wall of the culture vessel (3) is covered with a layer A temperature-sensitive gel (4), a temperature sensor (5) is arranged at the bottom of the outer wall. Described culture vessel (3) can be culture dish, culture bottle or culture plate (6 holes, 12 holes, 24 holes, 48 holes, 96 holes etc.) etc. For the adjustment of the vessel (3), the fixed frame (1) and the culture vessel (3) are closely combined. The culture vessel (3) is pressed in the fixed frame (1) by the spring (7) and the pressure plate (8), and is in close contact with the semiconductor refrigeration sheet (2). The upper edge of the culture vessel (3) is 1mm-40mm higher than the upper edge of the fixed frame (1), so as to facilitate the removal of the culture vessel (3). 3-12 pairs of springs (7) and pressing plates (8) are symmetrically distributed on the upper edge of the fixed frame.

The lower critical solution temperature of the thermosensitive gel (4) should be lower than 37°C, poly-N-isopropylacrylamide, methyl vinyl ether, 2-N-morpholinoethyl acrylate, N,N '-Dimethylaminoethyl ester, methacrylic acid, poloxamer, polyacrylic acid, polyacrylamide and other synthetic polymers, as well as natural polymers such as cellulose and its derivatives, chitosan and its derivatives, Poly N-isopropylacrylamide is preferred, which can be obtained by monomer poly-crosslinking, polymer cross-linking or graft copolymerization of carriers. Graft copolymerization, phase transition polymerization, low temperature treatment, introduction of inorganic nanoparticles, and use of pore-forming agents can be used to increase the response rate of hydrogels, and the formation of interpenetrating network structures and physical cross-linking can also be used to increase the water retention rate. The response rate and mechanical properties of the gel, etc. The porous structure can also be introduced into the hydrogel by phase separation method, emulsion template method, porogen method, polymer interpenetrating network method, etc. to improve the response rate of poly-N-isopropylacrylamide hydrogel. Chitosan is introduced on the basis of poly-N-isopropylacrylamide to improve its biocompatibility while retaining the thermosensitivity of poly-N-isopropylacrylamide. Hydrophilic monomers such as acrylic acid and acrylamide can be introduced into the gel skeleton structure to increase its lower critical solution temperature.

The semiconductor cooling chips (2) are arranged in the fixed frame (1) evenly and symmetrically at intervals, the number of which is 1-100 pieces, and the size of each piece is 0.5mm×0.5mm×0.5mm to 10mm×10mm×10mm. The semi-conductor refrigerating sheet (2) can be arranged in a single layer or in multiple layers, and when the layers are arranged, heat conduction oil is coated between each layer to reduce contact thermal resistance. According to the size and bottom thickness of the culture vessel (3), the layout and quantity of the semiconductive refrigeration sheets (2) are selected to obtain sufficient refrigeration and heating capacity. When only one semiconductor cooling chip (2) is arranged, a small groove is engraved on the semiconductor cooling chip (2) to install a temperature sensor (5) (such as thermocouple or thermal resistance, etc.); when a plurality of semiconductor cooling chips ( 2), 1-200 temperature sensors (5) are installed in the gaps between the semiconductor refrigerating sheets (2). Each temperature sensor (5) is connected with the temperature input end of the controller (6). The controller (6) is a temperature-controllable controller, and is provided with a display screen for displaying transient temperature and a temperature alarm. A programmed chip is installed in the controller (6), and different forms of current output are generated according to the set temperature and the temperature measured by the temperature sensor (5), so as to realize different cooling or heating. When the temperature obtained by the temperature sensor (5) is too high or too low, the temperature alarm in the controller (6) will prompt.

Example 1

As can be seen from Figures 1 and 2, in this embodiment, the fixed frame (1) and the controller (6) are integrated, and all wires are integrated in the device, with a compact structure. When the bottom area of the culture vessel (3) is small and the required cooling capacity is not large, a hollow hole (9) is provided at the bottom of the fixed frame (1) to expose the bottom surface of the semiconductor cooling chip (2) to facilitate the use of air to dissipate heat.

Example 2

It can be seen from Fig. 3 that in this embodiment, the fixed frame (1) and the controller (6) are integrated, and all wires are integrated in the device, so the structure is compact. When the bottom area of the culture vessel (3) is large and the required cooling capacity is large, the bottom of the fixed frame (1) is processed into heat sinks (10) to increase the heat dissipation area.

Example 3

It can be seen from Fig. 4 that in this embodiment, the fixed frame (1) and the controller (6) are separated separately, and the semiconductor refrigerator (2) and the temperature sensor (5) communicate with the controller ( 6) connection, the cooling and heating of the semiconductor cooling chip (2) can be remotely controlled by the controller (6). The fixed frame (1) can not only adopt the square shape shown in Figure 1 and Figure 2, but also can be processed into a circle or other shapes when the culture vessel (3) is a circle or other shapes. When the bottom area of the culture vessel (3) is small and the required cooling capacity is not large, a hollow hole (9) is provided at the bottom of the fixed frame (1) to expose the bottom surface of the semiconductor cooling chip (2) to facilitate the use of air to dissipate heat.

Example 4

It can be seen from Fig. 5 that in this embodiment, the fixed frame (1) and the controller (6) are separated separately, and the semiconductor refrigerator (2) and the temperature sensor (5) communicate with the controller ( 6) connection, the cooling and heating of the semiconductor cooling chip (2) can be remotely controlled by the controller (6). The fixed frame (1) can not only adopt the square shape shown in Figure 1 and Figure 2, but also can be processed into a circle or other shapes when the culture vessel (3) is a circle or other shapes. When the bottom area of the culture vessel (3) is large and the required cooling capacity is large, the bottom of the fixed frame (1) is processed into heat sinks (10) to increase the heat dissipation area.

The cell sheet preparation device provided by the present invention heats or cools the temperature-sensitive hydrogel through a semiconductor refrigeration chip, and uses the temperature-sensitive hydrogel to sense changes in the temperature of the external environment and undergo sol-gel transition or reversible volume transition Characteristics Preparation of tissue engineered cell sheets. Compared with the current method of adjusting the temperature of the thermosensitive hydrogel by changing the indoor temperature, the present invention has the advantages of fast temperature change, timely temperature monitoring, compact structure and the like. It is precisely because of these comprehensive factors that the device provided by the present invention has a simple and reliable structure, and has great advantages over the previous preparation devices.

Claims (8)

1. one kind is utilized semiconductor chilling plate to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that, comprise: a fixed frame (1), which is provided with semiconductor chilling plate (2), scribbling thermal oil on the semiconductor chilling plate (2) closely is connected with culture vessel (3), in parallel or the series connection of semiconductor chilling plate (2), be communicated with the controller (6) that is connected to power supply by wire, be covered with one deck thermo-sensitive gel (4) at the bottom of culture vessel (3) inwall, the outer wall base fabric is equipped with temperature sensor (5).

2. the semiconductor chilling plate that utilizes according to claim 1 is to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that: described thermo-sensitive gel (4) lower critical solution temperature is lower than 37 ℃, material is poly N-isopropyl acrylamide, is that the graft copolymerization by poly-crosslinked, the crosslinked polymer of monomer or carrier obtains.

3. the semiconductor chilling plate that utilizes according to claim 1 is to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that: described semiconductor chilling plate (2) is spaced in the fixed frame (1) symmetrically, its quantity is 1-100 piece, and every piece is of a size of 0.5mm * 0.5mm * 0.5mm to 10mm * 10mm * 10mm.

According to claim 1 or the 3 described semiconductor chilling plates that utilize to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that: be carved with sulculus on the described semiconductor chilling plate (2) 1 temperature sensor (5) is installed, perhaps 1-200 temperature sensor (5) is equipped with in the gap between the semiconductor chilling plate (2); Each temperature sensor (5) is connected with the temperature input terminus of controller (6).

5. the semiconductor chilling plate that utilizes according to claim 1 is to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, and it is characterized in that: described controller (6) is the controlled controller of temperature, is provided with the display screen and the temperature buzzer that show transient temperature.

6. the semiconductor chilling plate that utilizes according to claim 1 is to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that: described culture vessel (3) is culture dish, culturing bottle or culture plate, be pressed in the fixed frame (1) and semiconductor chilling plate (2) close contact by spring (7) and pressing plate (8), culture vessel (3) upper limb exceeds fixed frame (1) upper limb 1mm-40mm.

7. the semiconductor chilling plate that utilizes according to claim 1 is characterized in that the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel: described fixed frame (1) and controller (6) are made into one, and all wires all are integrated in the device; Perhaps fixed frame (1) and controller (6) separately separately, semiconductor chilling plate (2) is connected 5 with temperature sensor) pass through respectively the first wire (11) and be connected wire (12) and be connected with controller (6).

8. the semiconductor chilling plate that utilizes according to claim 1 is to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel, it is characterized in that: the bottom surface of semiconductor chilling plate (2) is exposed in the hole (9) that described fixed frame (1) is provided with hollow out, perhaps is processed into radiator element (10) in fixed frame (1) bottom.

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