CN102329727A - Preparing device of cell sheet for cold and hot regulation of temperature sensitive gel by utilizing semiconductor refrigerating sheets - Google Patents
Preparing device of cell sheet for cold and hot regulation of temperature sensitive gel by utilizing semiconductor refrigerating sheets Download PDFInfo
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- CN102329727A CN102329727A CN201110286443A CN201110286443A CN102329727A CN 102329727 A CN102329727 A CN 102329727A CN 201110286443 A CN201110286443 A CN 201110286443A CN 201110286443 A CN201110286443 A CN 201110286443A CN 102329727 A CN102329727 A CN 102329727A
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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
Technical field
The present invention relates to a kind of cell sheet preparation facilities, particularly utilize the cell sheet preparation facilities of semiconductor chilling plate the cold and hot adjusting of thermo-sensitive gel.
Background technology
Cell sheet engineering makes culturing cell separate from cultivation surface through Method of Tissue Engineering and forms the complete sheet structure that contains extracellular matrix, be a new technology of gathering in the crops seed cell and seed cell being shifted in the organizational project.Cell sheet engineering contains ionic channel, growth factor receptors and is connected important cells surface proteins such as albumen; Can avoid the spinoff that use brought, in reparations such as skin, cornea, periodontium, eyeball surface, cardiac muscle and lung gas leakage, be used widely because of carriers such as supports.
In early days, there is the scholar that cell evenly is inoculated in the glass culture dish, treats that all colonies connect when in blocks basically, with mechanical means it is peeled off from petridish and obtain cell sheet (Zhao Xiongfei; Zhang Shijun, Hu Jianian, Lu Shuzhen, Wang Xu; Li Ao, the human epidermal cell sheet homotransplantation of cultivation is in burn wound, PLA's medical journal, 1989; 14 (2): 82-85), mechanical means is damaging cells sometimes, the operational requirement SC.Mainly prepare cell sheet engineering (Li Haijia, Wang Wanpeng, Shi Hongcan in recent years through methods such as temperature sensitive petridish, collagen gel, magnetic force organizational project, shaggy particle unimolecular layer and polyelectrolytes; The cell sheet engineering construction process, Chinese reconstruction surgical magazine, 2011; 25 (6): 741-744); Wherein temperature sensitive petridish method obtains the cell sheet through cooling, and simple and reliable method has obtained using widely.Utilizing temperature sensitive petridish to make up cell sheet engineering generally is with temperature-sensitive hydrogel covalency grafting to ordinary polystyrene tissue culture ware surface, utilizes it can experience the variation of ambient temperature and the characteristic that sol-gel transition or reversible volume change takes place prepares the cell sheet.The material that can be used for preparing temperature-sensitive hydrogel comprises poly N-isopropyl acrylamide (poly-N-isopropylacrylamide; PNIPAAm), methylvinylether (MVE), vinylformic acid-2-N-morpholinyl ethyl ester (MPEMA), N; N '-dimethylin ethyl ester (DMAEMA), methylacrylic acid (MAAC), Prist (Poloxamer), ROHM (PAA), SEPIGEL 305 synthetic high polymers such as (PAAm); And Mierocrystalline cellulose and natural polymers such as verivate, chitosan and verivate thereof thereof, wherein the poly N-isopropyl acrylamide temperature-sensitive hydrogel is particularly noticeable.The poly N-isopropyl acrylamide intramolecularly has a certain proportion of hydrophobic sec.-propyl (CH (CH
3)
2-) and hydrophilic carboxamido-group (NHCO-), and hydrophilic/hydrophobic balance temperature influence, hydrophobic relatively in the time of 37 ℃; Be beneficial to cell adhesion, stretching, extension and propagation, drop to lower critical solution temperature (Low critical solution temperature, the LTCST of polymkeric substance when temperature; 32 ℃) time; Hydrophilic, expansion that polymer surfaces becomes, and form a hydration layer between the culturing cell, the cell nature is separated with petridish.Utilize temperature sensitive petridish to make up cell sheet engineering and need not enzyme and handle, kept the complete of some important cells surface proteins, mild condition, yield is high, and can make the cell that desorption is got off on the gel surface keep higher activity.Yet; The realization and the control that prepare temperature change in the process at the cell sheet are still a difficult problem, realize that through reducing indoor environment required time is longer mostly at present; Also be not easy accurately to control the temperature of temperature-sensitive hydrogel in the temperature sensitive petridish, cause the damage of low temperature pair cell easily.
Summary of the invention
The technical problem that the present invention will solve provides a kind of sheet of cell simply fast preparation facilities, temperature change that not only can the Rapid Realization temperature-sensitive hydrogel, and can timing monitoring incubator utensil temperature.
For solving the problems of the technologies described above; The semiconductor chilling plate that utilizes of the present invention comprises the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel: a fixed frame; Which is provided with semiconductor chilling plate, scribble thermal oil on the semiconductor chilling plate and closely be connected with the cultivation vessel, semiconductor chilling plate is communicated with the unit that is connected to power supply through lead; Be covered with one deck thermo-sensitive gel at the bottom of cultivating the vessel inwall, the outer wall base fabric is equipped with TP.Said cultivation vessel can be petridish, culturing bottle or the culture plate (6 holes, 12 holes, 24 holes, 48 holes, 96 holes etc.) etc. of multiple size.
The lower critical solution temperature of said thermo-sensitive gel is lower than 37 ℃; Can select poly N-isopropyl acrylamide, methylvinylether, vinylformic acid-2-N-morpholinyl ethyl ester, N; Synthetic high polymers such as N '-dimethylin ethyl ester, methylacrylic acid, Prist, ROHM, SEPIGEL 305; And Mierocrystalline cellulose and natural polymers such as verivate, chitosan and verivate thereof thereof; Preferred poly N-isopropyl acrylamide can obtain through the methods such as graft copolymerization that monomer gathers crosslinked, crosslinked polymer or carrier, can also process shapes such as film, microballoon, piece, latex.Graft copolymerization be can adopt, polymerization, subzero treatment, introducing inorganic nano-particle and utilize method such as pore former to improve the speed of response of hydrogel changed mutually, also can be through forming the speed of response and the mechanical property etc. that inierpeneirating network structure and methods such as utilizing physical crosslinking improve hydrogels.Also can in hydrogel, introduce vesicular structure, to improve the speed of response of poly N-isopropyl acrylamide hydrogel through phase separation method, emulsion template method, pore-creating agent method, polymer interpenetration network method etc.On the poly N-isopropyl acrylamide basis, introduce chitosan, when keeping the temperature sensitive property of PNIPAAm, improve its biocompatibility.Can hydrophilic monomers such as vinylformic acid, acrylic amide be introduced the gel skeleton structure and can improve its lower critical solution temperature.
Semiconductor chilling plate is the key that realizes temperature change regulating cell adhesion/desorption; Its principle is to utilize the Peltier effect of semiconductor material; Adopt the particular semiconductor material thermopile to freeze; Can directly convert electric energy into heat energy, can realize refrigeration and heating simultaneously, the positive and negative level that changes power supply can conveniently be switched refrigeration and heating.Said semiconductor chilling plate parallel connection or series connection are spaced symmetrically in fixed frame, and its quantity is 1-100 piece, and every piece is of a size of 0.5mm * 0.5mm * 0.5mm to 10mm * 10mm * 10mm.Be carved with sulculus on the said semiconductor chilling plate 1 TP is installed, perhaps 1-200 TP is equipped with in the gap between the semiconductor chilling plate; Each TP is connected with the temperature input terminus of unit.Said unit is the unit of Controllable Temperature, is provided with the image display and the temperature buzzer that show transient temperature.
Said cultivation vessel through spring with closely contact with semiconductor chilling plate in pressing plate is pressed on fixed frame.Cultivate the vessel upper limb and exceed fixed frame upper limb 1mm-40mm, be beneficial to convenient the taking-up and cultivate vessel.3-12 is symmetrically distributed on the fixed frame upper limb spring and pressing plate.
In sum; Cell sheet preparation facilities of the present invention heats temperature-sensitive hydrogel through semiconductor chilling plate or cools off, and utilizes temperature-sensitive hydrogel can experience the variation of ambient temperature and the characteristic that sol-gel transition or reversible volume change takes place prepares cell sheet engineering.Semiconductor chilling plate is without any need for refrigeration agent, but non-stop run does not have source of pollution not have turning unit, can not produce turning leverage, and not having slide unit is a kind of solid piece part, does not have vibrations, noise, life-span long during work, installs easily.And the semiconductor chilling plate thermal inertia is very little, and the cooling and warming time is very fast.Be installed in the temperature that the TP of cultivating vessel can the timing monitoring temperature-sensitive hydrogel.Compare with existing cell sheet engineering preparation facilities, on temperature change speed and timing monitoring, all be significantly improved, can reduce the damage of temperature pair cell, this also is one of key of proposing of the present invention.Cell sheet preparation facilities provided by the invention can be selected different temperature-sensitive hydrogel material and semiconductor chilling plate arrangement mode according to cell sheet culture requirement; The preparation facilities of being realized is simple and reliable, is expected in more cell sheet engineering preparations, play a significant role.Selected temperature-sensitive macromolecular material not only is confined to poly N-isopropyl acrylamide, as long as the lower critical solution temperature of selected macromolecular material meets the cultivation requirement.For for purpose of brevity, the present invention mainly is that example is set forth with the poly N-isopropyl acrylamide, and other temperature-sensitive hydrogels can adopt identical principle.
Description of drawings
Fig. 1 is the structural representation that utilizes semiconductor chilling plate to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention;
Fig. 2 is the structure vertical view that utilizes semiconductor chilling plate to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention;
Fig. 3 is the structural representation that utilizes semiconductor chilling plate to another embodiment of the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention;
Fig. 4 is the structural representation that utilizes semiconductor chilling plate to the 3rd embodiment of the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention;
Fig. 5 is the structural representation that utilizes semiconductor chilling plate to the 4th embodiment of the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention.
Wherein: 1, fixed frame; 2, semiconductor chilling plate; 3, cultivate vessel; 4, thermo-sensitive gel; 5, TP; 6, unit; 7, spring; 8, pressing plate; 9, hole; 10, radiator element; 11, lead; 12, lead.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Fig. 1 is the structural representation that utilizes semiconductor chilling plate to the cell sheet preparation facilities of the cold and hot adjusting of thermo-sensitive gel provided by the invention, and its structure vertical view is as shown in Figure 2.Fig. 3,4 and 5 is the present invention's structural representations of three embodiment in addition.Can know that by Fig. 1 and Fig. 2 the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel of the present invention comprises: a fixed frame (1); Which is provided with semiconductor chilling plate (2); Scribble thermal oil on the semiconductor chilling plate (2) and closely be connected with cultivation vessel (3), semiconductor chilling plate (2) parallel connection or series connection are communicated with the unit (6) that is connected to power supply through lead; Be covered with one deck thermo-sensitive gel (4) at the bottom of cultivating vessel (3) inwall, the outer wall base fabric is equipped with TP (5).Said cultivation vessel (3) can be petridish, culturing bottle or the culture plate (6 holes, 12 holes, 24 holes, 48 holes, 96 holes etc.) etc. of multiple size, and the physical dimension of fixed frame (1) is according to the adjustment of cultivating vessel (3), and fixed frame (1) and cultivation vessel (3) are combined closely.Said cultivation vessel (3) are pressed in the fixed frame (1) through spring (7) and pressing plate (8) and closely contact with semiconductor chilling plate (2).Cultivate vessel (3) upper limb and exceed fixed frame (1) upper limb 1mm-40mm, be beneficial to convenient the taking-up and cultivate vessel (3).3-12 is symmetrically distributed on the fixed frame upper limb spring (7) and pressing plate (8).
The lower critical solution temperature of said thermo-sensitive gel (4) should be lower than 37 ℃; Can select poly N-isopropyl acrylamide, methylvinylether, vinylformic acid-2-N-morpholinyl ethyl ester, N; Synthetic high polymers such as N '-dimethylin ethyl ester, methylacrylic acid, Prist, ROHM, SEPIGEL 305; And Mierocrystalline cellulose and natural polymers such as verivate, chitosan and verivate thereof thereof; Preferred poly N-isopropyl acrylamide can obtain through the methods such as graft copolymerization that monomer gathers crosslinked, crosslinked polymer or carrier.Graft copolymerization be can adopt, polymerization, subzero treatment, introducing inorganic nano-particle and utilize method such as pore former to improve the speed of response of hydrogel changed mutually, also can be through forming the speed of response and the mechanical property etc. that inierpeneirating network structure and methods such as utilizing physical crosslinking improve hydrogels.Also can in hydrogel, introduce vesicular structure, to improve the speed of response of poly N-isopropyl acrylamide hydrogel through phase separation method, emulsion template method, pore-creating agent method, polymer interpenetration network method etc.On the poly N-isopropyl acrylamide basis, introduce chitosan, when keeping the temperature sensitive property of poly N-isopropyl acrylamide, improve its biocompatibility.Can hydrophilic monomers such as vinylformic acid, acrylic amide be introduced the gel skeleton structure and can improve its lower critical solution temperature.
Said semiconductor chilling plate (2) is spaced symmetrically in fixed frame (1), and its quantity is 1-100 piece, and every piece is of a size of 0.5mm * 0.5mm * 0.5mm to 10mm * 10mm * 10mm.Semiconductor chilling plate (2) can be arranged also and can multilayer arrange by individual layer, scribbles thermal oil between every layer when multilayer is arranged, reduces thermal contact resistance.Decoration form and quantity according to size of cultivating vessel (3) and bottom thickness selection semiconductor chilling plate (2) obtain enough refrigeration and heating efficiency.When only arranging a block semiconductor cooling piece (2), on semiconductor chilling plate (2), be carved with sulculus 1 TP (5) (like thermoelectricity hot resistance etc. occasionally) is installed; When arranging polylith semiconductor chilling plate (2), 1-200 TP (5) is equipped with in the gap between semiconductor chilling plate (2).Each TP (5) is connected with the temperature input terminus of unit (6).Said unit (6) is the unit of Controllable Temperature, is provided with the image display and the temperature buzzer that show transient temperature.The chip of programmed is installed in unit (6), and the electric current output multi-form according to the temperature generation of temperature of setting and TP (5) measurement realizes different cooling or heating.The temperature that obtains when TP (5) is too high or cross when low and pointed out by the temperature buzzer of unit (6) lining.
Can be known that by Fig. 1 and 2 in the present embodiment, fixed frame (1) and unit (6) are made into one, all leads all are integrated in the device, compact construction.Vessel (3) floorage is less when cultivating, and when required refrigerating duty was little, the bottom surface of semiconductor chilling plate (2) was exposed in the hole (9) that is provided with hollow out in fixed frame (1) bottom, is beneficial to utilize the air heat radiation.
Can be known that by Fig. 3 in the present embodiment, fixed frame (1) and unit (6) are made into one, all leads all are integrated in the device, compact construction.Vessel (3) floorage is bigger cultivating, and when required refrigerating duty is big, is processed into radiator element (10) in fixed frame (1) bottom, increases area of dissipation.
Can know by Fig. 4; In the present embodiment; Separately, semiconductor refrigerating (2) is connected with unit (6) with lead (12) through lead (11) respectively with TP (5), can pass through the cooling and the heating of unit (6) teletype control semiconductor chilling plate (2) separately for fixed frame (1) and unit (6).Fixed frame (1) not only can adopt illustrated in figures 1 and 2 square, when cultivating vessel (3) for circular or other shapes, also can be processed into circular or other shapes.Vessel (3) floorage is less cultivating, and when required refrigerating duty was little, the bottom surface of semiconductor chilling plate (2) was exposed in the hole (9) that is provided with hollow out in fixed frame (1) bottom, is beneficial to utilize the air heat radiation.
Can know by Fig. 5; In the present embodiment; Separately, semiconductor refrigerating (2) is connected with unit (6) with lead (12) through lead (11) respectively with TP (5), can pass through the cooling and the heating of unit (6) teletype control semiconductor chilling plate (2) separately for fixed frame (1) and unit (6).Fixed frame (1) not only can adopt illustrated in figures 1 and 2 square, when cultivating vessel (3) for circular or other shapes, also can be processed into circular or other shapes.Vessel (3) floorage is bigger cultivating, and when required refrigerating duty is big, is processed into radiator element (10) in fixed frame (1) bottom, increases area of dissipation.
Cell sheet preparation facilities provided by the invention heats temperature-sensitive hydrogel through semiconductor chilling plate or cools off, and utilizes temperature-sensitive hydrogel can experience the variation of ambient temperature and the characteristic that sol-gel transition or reversible volume change takes place prepares cell sheet engineering.Compare at present and regulate the temperature-sensitive hydrogel method of temperature through changing room temp, it is fast that the present invention has temperature change speed, but and the timing monitoring temperature, advantages such as compact construction.Just because of these composite factors, make that apparatus structure provided by the invention is simple, reliable, the preparation facilities of comparing in the past has very big advantage.
Claims (8)
1. a cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel is characterized in that, comprising: a fixed frame (1); Which is provided with semiconductor chilling plate (2); Scribble thermal oil on the semiconductor chilling plate (2) and closely be connected with cultivation vessel (3), semiconductor chilling plate (2) parallel connection or series connection are communicated with the unit (6) that is connected to power supply through lead; Be covered with one deck thermo-sensitive gel (4) at the bottom of cultivating vessel (3) inwall, the outer wall base fabric is equipped with TP (5).
2. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1; It is characterized in that: said thermo-sensitive gel (4) lower critical solution temperature is lower than 37 ℃; Material is N-NSC 11448, methylvinylether, vinylformic acid-2-N-morpholinyl ethyl ester, N; N '-dimethylin ethyl ester, methylacrylic acid, Prist, ROHM or SEPIGEL 305 synthetic high polymer; And Mierocrystalline cellulose and verivate or chitosan and verivate natural polymer thereof, the graft copolymerization that gathers crosslinked, crosslinked polymer or carrier through monomer obtains.
3. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1; It is characterized in that: said semiconductor chilling plate (2) is spaced symmetrically in fixed frame (1); Its quantity is 1-100 piece, and every piece is of a size of 0.5mm * 0.5mm * 0.5mm to 10mm * 10mm * 10mm.
4. according to claim 1 or the 3 described cell sheet preparation facilitiess that utilize semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel; It is characterized in that: be carved with sulculus on the said semiconductor chilling plate (2) 1 TP (5) is installed, perhaps 1-200 TP (5) is equipped with in the gap between the semiconductor chilling plate (2); Each TP (5) is connected with the temperature input terminus of unit (6).
5. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1, it is characterized in that: said unit (6) is the unit of Controllable Temperature, is provided with the image display and the temperature buzzer that show transient temperature.
6. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1; It is characterized in that: said cultivation vessel (3) are petridish, culturing bottle or culture plate; Be pressed on through spring (7) and pressing plate (8) and closely contact with semiconductor chilling plate (2) in the fixed frame (1), cultivation vessel (3) upper limb exceeds fixed frame (1) upper limb 1mm-40mm.
7. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1, it is characterized in that: said fixed frame (1) and unit (6) are made into one, and all leads all are integrated in the device; Perhaps separately, semiconductor refrigerating (2) is connected with unit (6) with lead (12) through lead (11) respectively with TP (5) separately for fixed frame (1) and unit (6).
8. the cell sheet preparation facilities that utilizes semiconductor chilling plate to the cold and hot adjusting of thermo-sensitive gel according to claim 1; It is characterized in that: the bottom surface of semiconductor chilling plate (2) is exposed in the hole (9) that said fixed frame (1) is provided with hollow out, perhaps is processed into radiator element (10) in fixed frame (1) bottom.
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| CN113036029B (en) * | 2021-03-03 | 2023-08-04 | 重庆大学 | High-performance low-cost hydrogel thermoelectric material |
| CN114350094A (en) * | 2021-12-08 | 2022-04-15 | 广东省科学院化工研究所 | Temperature-sensitive thermoelectric hydrogel and preparation method and application thereof |
| CN114350094B (en) * | 2021-12-08 | 2022-11-08 | 广东省科学院化工研究所 | Temperature-sensitive thermoelectric hydrogel and preparation method and application thereof |
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