Gelatin micro-nano motor substrate with micro-channel structure as well as preparation method and application of gelatin micro-nano motor substrate

Abstract

The invention provides a gelatin micro-nano motor substrate with a micro-channel structure and a preparation method and application of the gelatin micro-nano motor substrate, and belongs to the technical field of biological medicine. The surface of the gelatin micro-nano motor substrate is provided with a micro-channel, and the gelatin micro-nano motor substrate is prepared from the following raw materials: gelatin, glutamine transaminase and a phosphate buffer solution. The gelatin micro-nano motor substrate can be used for culturing animal cells or human cells, and can fix the cells and induce the morphological growth of the cells. Through verification, when myocardial cells of a mouse grow on the gelatin micro-nano motor substrate with the micro-channel structure, the cells are fixed in the micro-channel, and the cells grow into tissues with muscle fiber morphology after being cultured for 8 days.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a gelatin micro-nano motor substrate with a microfluidic structure, and a preparation method and application thereof. Background Art

[0002] With the development of medical robots, clinical medicine has continuously improved its requirements for operational accuracy, and drug delivery systems have gradually developed from passive diffusion to targeted transport. Micro-nano motors have attracted extensive attention from researchers in the direction of targeted drug transport due to their responsive drive and load capacity. At present, the materials used to prepare micro-nano motors are mainly inorganic metals, MOFs, enzymes, etc., and then the micro-nano motors are given the ability to move through different response mechanisms. Cells, as the basic structural and functional units of organisms, have certain movement capabilities and do not require additional signal stimulation. Therefore, how to combine cells with micro-nano motors has attracted the attention of researchers. However, cells have poor adhesion to materials such as gelatin and cannot grow on the wall, which limits the preparation and further application of cell-driven micro-nano motors. Therefore, the development of micro-nano motor substrates that can fix cells and induce cell morphological growth has become the key to the development of cell-driven micro-nano motors. Summary of the invention

[0003] The purpose of the present invention is to provide a gelatin micro-nano motor substrate with a microfluidic structure and a preparation method and application thereof. The gelatin micro-motor substrate with a microfluidic structure of the present invention can fix cells and induce cell morphological growth.

[0004] The present invention provides a gelatin micro-nano motor substrate, the surface of which has a microfluidic channel; the gelatin micro-nano motor substrate comprises the following raw materials for preparation: gelatin, transglutaminase and phosphate buffer; the ratio of the mass of the gelatin, the mass of the transglutaminase and the volume of the phosphate buffer solution is 15-25 mg: 10-20 mg: 1.5-2.5 mL.

[0005] Preferably, the gelatin micro-nanomotor substrate is solidified from a mixed solution of gelatin, transglutaminase and phosphate buffer.

[0006] Preferably, the microchannels are arranged in parallel with equal spacing; the microchannels are rectangular microchannels; the width of each microchannel is 25 to 30 μm; the depth of each microchannel is 4 to 6 μm; and the spacing between adjacent microchannels is 4 to 6 μm.

[0007] The present invention also provides a method for preparing the gelatin micro-nano motor substrate described in the above scheme, comprising the following steps:

[0008] The gelatin solution and the glutamine transaminase solution are mixed to obtain solution C; the microfluidic mold is placed horizontally, and a containment-type template is placed on the upper surface of the microfluidic mold; the solution C is added into the containment-type template, solidified, and demolded to obtain a gelatin micro-nano motor substrate; the solvent of the gelatin solution and the glutamine transaminase solution is phosphate buffer; the microfluidic mold is a substrate with grooves on the upper surface.

[0009] Preferably, the grooves are arranged at equal intervals and in parallel; the spacing between the bottom of the groove and the lower surface of the substrate is 1 mm; the groove is a rectangular groove; the depth of the groove is 4 to 6 μm; the width of the groove is 25 to 30 μm; and the width of the gap between two adjacent grooves is 4 to 6 μm.

[0010] Preferably, the volume ratio of the gelatin solution to the glutamine transaminase solution is 1:(0.8-1.2).

[0011] Preferably, the curing time is 24 hours; after adding the solution C into the enclosure-type template, it also includes covering another microfluidic mold on top of the solution C and applying pressure on the top of the other microfluidic mold; the groove of the other microfluidic mold faces downward; the pressure is 4 to 5N.

[0012] The present invention also provides the use of the gelatin micro-nano motor substrate described in the above scheme or the gelatin micro-nano motor substrate prepared by the preparation method in animal cell or human cell culture.

[0013] The present invention also provides a method for culturing animal cells or human cells, comprising the following steps:

[0014] Animal cells or human cells are inoculated on the sterilized gelatin micro-nano motor substrate described in the above scheme or the gelatin micro-nano motor substrate prepared by the preparation method for culture.

[0015] Preferably, the animal cell or human cell includes a cardiomyocyte; and the animal includes a mouse.

[0016] The present invention provides a gelatin micro-nano motor substrate, the surface of which has a microfluidic channel; the gelatin micro-nano motor substrate comprises the following raw materials for preparation: gelatin, transglutaminase (TG) and phosphate buffered saline (PBS). The gelatin micro-nano motor substrate of the present invention can be used to culture animal cells or human cells, fix cells and induce cell morphological growth. It has been verified that when mouse cardiomyocytes are grown on the gelatin micro-nano motor substrate with a microfluidic channel structure of the present invention, the cells are fixed in the microfluidic channel and grow into tissues with muscle fiber morphology after 8 days of culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 Schematic diagram of the microfluidic mold structure;

[0019] Figure 2 The figure is a comparison diagram of the cell culture effects of Application Example 1 and Comparative Example 1, wherein the left figure is a microfluidic gelatin substrate cell culture effect diagram; the right figure is a planar gelatin substrate cell culture effect diagram;

[0020] Figure 3 It is a comparison chart of cell growth between Application Example 1 and Comparative Example 1; wherein A to D are the effect charts of cells growing on the microfluidic gelatin substrate for 2, 4, 6, and 8 days respectively; A' to D' are the effect charts of cells growing on the culture flask for 2, 4, 6, and 8 days respectively;

[0021] Figure 4 This is a diagram of the growth status of cells on the gelatin prepared in Example 2, wherein A to D are the states of cells growing for 2, 4, 6, and 8 days, respectively;

[0022] Figure 5 This is a diagram showing the growth status of cells on the solid G prepared in Comparative Example 2, wherein A to C represent the status of cells growing for 2, 4, and 6 days, respectively. DETAILED DESCRIPTION

[0023] The present invention provides a gelatin micro-nano motor substrate, the surface of which has a microfluidic channel; the gelatin micro-nano motor substrate comprises the following raw materials for preparation: gelatin, transglutaminase and phosphate buffer; the ratio of the mass of the gelatin, the mass of the transglutaminase and the volume of the phosphate buffer solution is 15-25 mg: 10-20 mg: 1.5-2.5 mL.

[0024] In the present invention, microchannels are distributed in parallel and at equal intervals on the surface of the gelatin micro-nano motor substrate.

[0025] In one embodiment of the present invention, the width of each microchannel is 25-30 μm; the depth of each microchannel is 4-6 μm; and the spacing between adjacent microchannels is 4-6 μm.

[0026] In the specific implementation of the present invention, both the upper and lower surfaces of the gelatin micro-nano motor substrate have microfluidic channel structures.

[0027] In the present invention, the gelatin, transglutaminase and phosphate buffer are from conventional commercial sources. In the specific implementation of the present invention, the gelatin (Fish Gelatin) G7041 was purchased from Sigma-Aldrich; the transglutaminase (TG) A3 type was purchased from Shandong Yuantai Bioengineering Co., Ltd.; and the phosphate buffer (1X PBS) was purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0028] In the specific implementation process of the present invention, the role of the transglutaminase is to cross-link and solidify with gelatin; the gelatin micro-nano motor substrate is obtained by solidifying a mixed solution of gelatin, transglutaminase and phosphate buffer.

[0029] In the specific implementation of the present invention, the phosphate buffer is 1× phosphate buffer.

[0030] In one embodiment of the present invention, the ratio of the mass of gelatin, the mass of transglutaminase and the volume of phosphate buffer solution is 20 mg:16 mg:2 mL.

[0031] The gelatin micro-nano motor substrate of the present invention is a motor substrate that can fix cells and induce cell morphological growth at a micro-nano scale.

[0032] The present invention also provides a method for preparing the gelatin micro-nano motor substrate described in the above scheme, comprising the following steps:

[0033] Mixing the gelatin solution and the transglutaminase solution to obtain solution C;

[0034] Placing the microfluidic channel mold horizontally, and placing a barrier-type template on the upper surface of the microfluidic channel mold;

[0035] Adding the solution C into the enclosure-type template, curing, and demoulding to obtain a gelatin micro-nano motor substrate;

[0036] The solvent of the gelatin solution and the glutamine transaminase solution is phosphate buffer; the microfluidic channel mold is a substrate with grooves on the upper surface.

[0037] The present invention dissolves gelatin in a phosphate buffer to obtain a gelatin solution (solution A).

[0038] In the specific implementation process of the present invention, the ratio of the mass of the gelatin to the volume of the phosphate buffer solution used to dissolve the gelatin is 20-25 mg:1 mL; the dissolution temperature of the gelatin in the phosphate buffer solution is 65°C.

[0039] The present invention dissolves transglutaminase in phosphate buffer to obtain a transglutaminase solution (solution B).

[0040] In the specific implementation of the present invention, the ratio of the mass of the transglutaminase to the volume of the phosphate buffer solution is 16 mg:1 mL; and the dissolution temperature of the transglutaminase in the phosphate buffer solution is 37°C.

[0041] In the specific implementation process of the present invention, the volume ratio of the phosphate buffer used to dissolve gelatin and the phosphate buffer used to dissolve glutamine transaminase is 1: (0.8-1.2) 。

[0042] In one embodiment of the present invention, the volume ratio of the phosphate buffer used to dissolve gelatin and the phosphate buffer used to dissolve transglutaminase is 1:1.

[0043] In the specific implementation process of the present invention, the dissolving or mixing is accompanied by magnetic stirring; the rotation speed of the magnetic stirring is 500 rpm.

[0044] After obtaining the gelatin solution and the glutamine transaminase solution, the present invention mixes the gelatin solution and the glutamine transaminase solution to obtain solution C.

[0045] In the specific implementation of the present invention, the volume ratio of the gelatin solution to the glutamine transaminase solution is 1:(0.8-1.2).

[0046] In one embodiment of the present invention, the volume ratio of the gelatin solution to the transglutaminase solution is 1:1.

[0047] The present invention places the microfluidic channel mold horizontally, and places a barrier-type template on the upper surface of the microfluidic channel mold; the microfluidic channel mold is a substrate with grooves on the upper surface.

[0048] In the specific implementation of the present invention, the grooves are arranged in parallel with equal spacing; the spacing between the bottom of the groove and the lower surface of the substrate is 1 mm; the groove is a rectangular groove; the depth of the groove is 4 to 6 μm; the width of the groove is 25 to 30 μm; the width of the gap between two adjacent grooves is 4 to 6 μm; the structural schematic diagram of the microfluidic mold is as shown in Figure 1 As shown; the overall material of the microfluidic mold is PDMS.

[0049] After obtaining solution C, the present invention adds the solution C into the enclosure-type template, solidifies and demoulds the template to obtain a gelatin micro-nano motor substrate.

[0050] After adding the solution C into the enclosure-type template, the present invention further includes covering another microfluidic channel mold on top of the solution C and applying pressure on the top of the other microfluidic channel mold; the groove of the other microfluidic channel mold faces downward.

[0051] In the specific implementation of the present invention, there is no special restriction on the planar shape of the gelatin micro-nano motor substrate. The present invention selects the shape of the enclosure type template according to the planar shape of the customized gelatin micro-nano motor substrate; the enclosure type template is used to shape the gelatin micro-nano motor substrate. In the specific implementation of the present invention, the enclosure type template is hollowed out from a material that does not react with gelatin. In one embodiment of the present invention, the enclosure type template is obtained by relative pasting of a general laboratory label tape; the length of the general laboratory label tape is 1.5 cm.

[0052] In a specific implementation of the present invention, applying pressure on another microfluidic channel mold includes placing a 500 g weight on another microfluidic channel mold to apply pressure.

[0053] In the specific implementation process of the present invention, the curing time is 24 hours; the curing temperature is 20-30° C., preferably 25-26° C.; the curing environment humidity is 30%-40% to ensure the gel forming effect.

[0054] Before the curing, the magnitude of the pressure applied to the upper part of the mold in the present invention is 4-5 N, preferably 4.9 N. In the specific implementation process of the present invention, a 500 g weight is placed on the upper part of the mold to apply pressure.

[0055] The present invention also provides the use of the gelatin micro-nano motor substrate described in the above scheme or the gelatin micro-nano motor substrate prepared by the preparation method in animal cell or human cell culture.

[0056] The present invention also provides a method for culturing animal cells or human cells, comprising the following steps:

[0057] Animal cells or human cells are inoculated on the sterilized gelatin micro-nano motor substrate described in the above scheme or the gelatin micro-nano motor substrate prepared by the preparation method for culture.

[0058] In a specific implementation of the present invention, the animal cell or human cell includes a cardiomyocyte; and the animal includes a mouse.

[0059] In order to further illustrate the present invention, a gelatin micro-nano motor substrate with a microfluidic structure provided by the present invention, its preparation method and application are described in detail below in combination with the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] 1. Dissolve 20 mg of gelatin in 1 mL of PBS (1×) phosphate buffer at 65°C with magnetic stirring at a speed of 500 rpm to obtain solution A.

[0062] 2. Dissolve 16 mg of glutamine transaminase in 1 mL of PBS (1×) phosphate buffer at 37° C. with magnetic stirring at a speed of 500 rpm to obtain solution B.

[0063] 3. Mix equal volumes of solution A and solution B by magnetic stirring for 10 min at a magnetic stirring speed of 500 rpm to obtain solution C.

[0064] 4. According to the plane shape of the customized gelatin micro-nano motor substrate, two sections of about 1.5 cm long laboratory general label tape are pasted relative to each other and hollowed out, and the tape is placed on the microfluidic mold ( Figure 1 ) on the grooves for shaping the gelatin micro-nano motor substrate, and then 500 μL of solution C is dripped into the hollow template and covered with another microfluidic mold; the grooves of the other microfluidic mold face downward, and then a 500 g weight is placed on top of the mold to apply pressure, and it is cured at a relative humidity of 30% to 40% and room temperature for 24 hours, and demolding is performed to obtain a gelatin micro-nano motor substrate (transparent solid D) with a microfluidic structure.

[0065] Application Example 1

[0066] The transparent solid D prepared in Example 1 was immersed in an ethanol aqueous solution with an ethanol volume concentration of 75% for 1 hour, sterilized with ultraviolet light for 3 minutes, and then mouse cardiomyocytes were cultured for 8 days on the transparent solid D. During the culture process, the cell growth morphology was observed every 2 days.

[0067] Comparative Example 1

[0068] The solution C in Example 1 was added dropwise to a culture dish and solidified at room temperature for 24 hours to obtain a transparent solid E.

[0069] The transparent solid E was immersed in an ethanol aqueous solution with an ethanol volume concentration of 75% for 1 hour, sterilized by ultraviolet light for 3 minutes, and then the mouse cardiomyocytes were cultured for 8 days on the transparent solid E. During the culture process, the cell growth morphology was observed every 2 days.

[0070] The results of Application Example 1 and Comparative Example 1 are as follows Figure 2 , 3 As shown. Figure 2 , 3 It can be seen that when cells grow on the flat gelatin substrate of Comparative Example 1, the cells have poor adhesion and cannot effectively grow on the wall. When cells grow on the gelatin substrate with a microfluidic structure of Example 1, the cells are fixed in the microfluidic channel and grow into tissue with muscle fiber morphology.

[0071] Example 2

[0072] 1. Dissolve 25 mg of gelatin in 1 mL of PBS (1×) phosphate buffer at 65°C with magnetic stirring at a speed of 500 rpm to obtain solution A.

[0073] Steps 2 to 4 are the same as in Example 1.

[0074] The transparent solid D prepared in Example 2 was immersed in an ethanol aqueous solution with an ethanol volume concentration of 75% for 1 hour, sterilized with ultraviolet light for 3 minutes, and then mouse cardiomyocytes were cultured for 8 days on the transparent solid D. During the culture process, the cell growth morphology was observed every 2 days.

[0075] Comparative Example 2

[0076] 1. Dissolve 30 mg of gelatin in 1 mL of PBS (1×) phosphate buffer at 65°C with magnetic stirring at a speed of 500 rpm to obtain solution E.

[0077] 2. Solution E and solution B in Example 2 were mixed in equal volumes by magnetic stirring for 10 min at a magnetic stirring speed of 500 rpm to obtain solution F.

[0078] 3. According to the plane shape of the customized gelatin micro-nano motor substrate, two sections of about 1.5 cm long laboratory general label tape are pasted relative to each other and hollowed out, and the tape is placed on the microfluidic mold ( Figure 1 ) on the groove for shaping the gelatin micro-nano motor substrate, and then 500 μL of solution F is dripped into the hollow template and covered with another microfluidic mold; the groove of the other microfluidic mold faces downward, and then a 500 g weight is placed on top of the mold to apply pressure, and it is cured at a relative humidity of 30% to 40% and room temperature for 24 hours, and demolding is performed to obtain a gelatin micro-nano motor substrate (transparent solid G) with a microfluidic structure.

[0079] The transparent solid G was immersed in an ethanol aqueous solution with an ethanol volume concentration of 75% for 1 hour, sterilized by ultraviolet light for 3 minutes, and then the mouse cardiomyocytes were cultured for 8 days on the transparent solid E. During the culture process, the cell growth morphology was observed every 2 days.

[0080] The results of Example 2 and Comparative Example 2 are as follows Figure 4 , 5 As shown. Figure 4 It can be seen that the culture effect of gelatin dosage within a certain range is similar to that of Example 1. When cells are cultured on solid G in Comparative Example 2, it is difficult for cells to adhere to gelatin, and the surface microstructure will be lost over time, resulting in cells still suspended and eventually lysed.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gelatin micro-nano motor substrate, characterized in that: The surface of the gelatin micro-nano motor substrate has a microfluidic channel; the gelatin micro-nano motor substrate includes the following preparation raw materials: gelatin, glutamine transaminase and phosphate buffer solution; the ratio of the mass of the gelatin, the mass of the glutamine transaminase and the volume of the phosphate buffer solution is 15-25 mg: 10-20 mg: 1.5-2.5 mL.

2. The gelatin micro-nano motor substrate according to claim 1, characterized in that: The gelatin micro-nano motor substrate is solidified from a mixed solution of gelatin, glutamine transaminase and phosphate buffer.

3. The gelatin micro-nano motor substrate according to claim 1, characterized in that: The microchannels are arranged in parallel at equal intervals; the microchannels are rectangular microchannels; the width of each microchannel is 25 to 30 μm; the depth of each microchannel is 4 to 6 μm; and the spacing between adjacent microchannels is 4 to 6 μm.

4. The method for preparing the gelatin micro-nano motor substrate according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mixing the gelatin solution and the transglutaminase solution to obtain solution C; Placing the microfluidic channel mold horizontally, and placing a barrier-type template on the upper surface of the microfluidic channel mold; Adding the solution C into the enclosure-type template, curing, and demoulding to obtain a gelatin micro-nano motor substrate; The solvent of the gelatin solution and the glutamine transaminase solution is phosphate buffer; the microfluidic channel mold is a substrate with grooves on the upper surface.

5. The preparation method according to claim 4, characterized in that: The grooves are arranged in parallel with equal spacing; the spacing between the bottom of the groove and the lower surface of the substrate is 1 mm; the groove is a rectangular groove; the depth of the groove is 4 to 6 μm; the width of the groove is 25 to 30 μm; the width of the gap between two adjacent grooves is 4 to 6 μm.

6. The preparation method according to claim 4, characterized in that: The volume ratio of the gelatin solution to the glutamine transaminase solution is 1:(0.8-1.2).

7. The preparation method according to claim 4 or 6, characterized in that: The curing time is 24 hours; after adding the solution C into the enclosure template, it also includes covering another microfluidic mold on the solution C and applying pressure on the top of the other microfluidic mold; the groove of the other microfluidic mold faces downward; the pressure is 4 to 5N.

8. Use of the gelatin micro-nano motor substrate described in any one of claims 1 to 3 or the gelatin micro-nano motor substrate prepared by the preparation method described in any one of claims 4 to 7 in animal cell or human cell culture.

9. A method for culturing animal cells or human cells, characterized in that: The following steps are involved: Animal cells or human cells are inoculated on the sterilized gelatin micro-nano motor substrate described in any one of claims 1 to 3 or the gelatin micro-nano motor substrate prepared by the preparation method described in any one of claims 4 to 7 for culture.

10. The method according to claim 9, characterized in that The animal cell or human cell includes a cardiomyocyte; and the animal includes a mouse.

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