CN113481163B - Composite material and preparation method thereof, tumor model and preparation method thereof - Google Patents

Composite material and preparation method thereof, tumor model and preparation method thereof Download PDF

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CN113481163B
CN113481163B CN202110939275.1A CN202110939275A CN113481163B CN 113481163 B CN113481163 B CN 113481163B CN 202110939275 A CN202110939275 A CN 202110939275A CN 113481163 B CN113481163 B CN 113481163B
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composite material
transparent substrate
laser
mixed solution
dimensional
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CN113481163A (en
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马名泽
邹晓敏
许改霞
杨成彬
孔湉湉
王晓梅
张楠楠
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Shenzhen University
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

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Abstract

The invention relates to a composite material and a preparation method thereof, a tumor model and a preparation method thereof. The preparation method of the composite material comprises the following steps: dissolving protein and a photosensitizer in water to prepare a mixed solution, wherein the concentration of the protein in the mixed solution is 20-90 mg/mL, the concentration of the photosensitizer is 0.2-0.4 w/v%, and the protein is at least one of albumin, fibronectin and laminin; and adding the mixed solution onto a transparent substrate, and curing the mixed solution by adopting a laser 3D printing technology to form a three-dimensional protein body to prepare the composite material, wherein the wavelength of laser is 750-850 nm, and the energy of the laser is 48-58 mW. The composite material prepared by the preparation method of the composite material can be used for three-dimensional cell culture.

Description

Composite material and preparation method thereof, tumor model and preparation method thereof
Technical Field
The invention relates to the technical field of biological materials, in particular to a composite material and a preparation method thereof, a tumor model and a preparation method thereof.
Background
The occurrence of tumors is a complex process and is still largely unknown, whereas in vitro culture of tumor cells plays a critical role in the study of the complex pathological mechanisms of tumors. The culture of cells in vitro is mainly divided into two-dimensional culture and three-dimensional culture. In a two-dimensional culture system, cells generally grow in a single-layer adherence manner, and lack dynamic interactions between cells and between ECM under a real in-vivo microenvironment. The three-dimensional cell culture is to construct an in-vitro model by utilizing a three-dimensional bracket, so as to aim at simulating the real in-vivo growth environment through the three-dimensional bracket. Compared with the traditional two-dimensional culture methods such as a culture dish, a culture bottle and the like, the three-dimensional bracket is adopted to culture the tumor cells, so that the physiological complexity of in-vivo tissues can be simulated more truly in vitro, and the accuracy of the preclinical antitumor drug curative effect evaluation is improved.
At present, scaffolds in three-dimensional culture can be classified into natural scaffolds and synthetic scaffolds according to different sources of scaffold materials. Common natural scaffold materials include collagen, fibrin, chitin, hyaluronic acid, silk, gelatin, etc.; common materials for scaffold synthesis are Polycaprolactone (PCL), poly (lactic-co-glycolic acid), polyethylene glycol (poly (ethylene glycol), PEG), etc. However, the current three-dimensional scaffold simulates a microenvironment with a certain gap from the real environment in the body, and cannot simulate invasion of tumor cells.
Disclosure of Invention
Based on this, it is necessary to provide a composite material in which the tumor cells cultured have invasive behaviors.
A method of preparing a composite material comprising the steps of:
dissolving protein and a photosensitizer in water to prepare a mixed solution, wherein the concentration of the protein in the mixed solution is 20-90 mg/mL, the concentration of the photosensitizer is 0.2-0.4 w/v%, and the protein is at least one of albumin, fibronectin and laminin; and
And after the mixed solution is added onto a transparent substrate, the mixed solution is solidified by adopting a laser 3D printing technology to form a three-dimensional protein body on the transparent substrate, and the composite material is prepared, wherein the wavelength of laser is 750-850 nm, and the energy of the laser is 48-58 mW.
According to the preparation method of the composite material, the three-dimensional protein body is formed by adopting the laser solidified protein on the transparent base material, so that the composite material can better simulate the real environment in the body when being applied to the three-dimensional culture of tumor cells, and the invasion behavior of the tumor cells can be easily observed.
In one embodiment, the albumin is selected from at least one of bovine serum albumin, human recombinant serum albumin, sheep serum albumin, and rabbit serum albumin;
and/or the photosensitizer is tiger red sodium salt or methylene blue;
and/or the transparent substrate is made of glass or plastic.
In one embodiment, in the step of solidifying the mixed solution by using a laser 3D printing technology to form a three-dimensional protein body on the transparent substrate, part of the mixed solution is scanned layer by layer through the transparent substrate by using a laser to locally solidify the mixed solution to form a plurality of spaced three-dimensional protein bodies on the transparent substrate.
In one embodiment, the spacing between two adjacent three-dimensional protein bodies is 2 μm to 5 μm.
In one embodiment, the three-dimensional protein body is cylindrical, and the diameter of the three-dimensional protein body is 1-5 μm; the height of the three-dimensional protein body is 2-10 mu m;
or the three-dimensional protein body is in a quadrangular prism shape, and the length of the three-dimensional protein body is 1-5 mu m; the width of the three-dimensional protein body is 1-5 mu m, and the height of the three-dimensional protein body is 2-10 mu m.
In one embodiment, the operation of forming the three-dimensional protein body is performed under an oxygen environment, wherein the oxygen partial pressure of the oxygen environment is 25kPa to 30kPa, and the volume concentration of oxygen in the oxygen environment is 20 to 80 percent;
and/or adopt laser to penetrate the transparentScanning the mixed solution layer by layer on a substrate to form the three-dimensional protein body on the transparent substrate, wherein the scanning sections of all layers are mutually parallel, and the area of the scanning section is 471.435 mu m 2 ～8256.5μm 2 The distance between adjacent scanning sections is 0.2-0.6 μm.
In one embodiment, the transparent substrate is sheet-like;
or the transparent substrate is in a groove shape, and the three-dimensional protein body is positioned on the inner bottom of the transparent substrate.
A composite material is prepared by the preparation method of the composite material.
A method for preparing a tumor model, comprising the steps of:
tumor cells were inoculated into the above composite material and cultured to prepare a tumor model.
A tumor model, prepared by the method for preparing a tumor model, wherein tumor cells in the tumor model have invasive behaviors.
Drawings
FIG. 1 is a schematic illustration of a method of preparing a composite material according to an embodiment;
FIG. 2 is a confocal three view of three-dimensional protein bodies formed on the composite material of example 1;
FIG. 3 is a confocal orthogonal view of three-dimensional protein bodies formed on the composite material of example 1;
FIG. 4 is a scanning electron microscope image of a three-dimensional protein body formed on the composite material of example 1;
FIG. 5 is a scanning electron microscope image of a single three-dimensional protein body formed on the composite material of example 1;
FIG. 6 shows the growth of tumor cells cultured on the composite material of example 11 under an optical microscope;
FIG. 7 shows the growth of tumor cells cultured on the composite material of example 12 under an optical microscope;
FIG. 8 shows the growth of tumor cells cultured on the composites of example 11 and example 12 under scanning electron microscopy;
FIG. 9 shows the fluorescent staining of tumor cells cultured on the composite material of example 1.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the invention, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The term "three-dimensional protein body" refers to a protein material having a three-dimensional structure, and the shape of the three-dimensional protein body may be arbitrary, such as a cylinder, a rectangular parallelepiped, or other irregular shape. The term "culture vessel" refers to a vessel in which cells are cultured, e.g., a cell culture plate, a cell culture dish, a cell culture flask, a cell culture tube, etc.
Unless defined otherwise, 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 invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In FIG. 1, "Ti: sapphire laser" refers to a titanium Sapphire laser; "two photon laser" refers to a two-photon laser; "dichromatic mirror" refers to a Dichroic mirror; "Side view" refers to a Side view; "Top view" refers to a Top view; "RB" is an abbreviation for Rose Bengal and refers to tiger red sodium salt.
Referring to fig. 1, an embodiment of the present application provides a method for preparing a composite material, which includes step S110 and step S120. Specifically:
step S110: the protein and the photosensitizer are dissolved in water to prepare a mixed solution.
In the mixed solution, the concentration of the protein is 20 mg/mL-90 mg/mL, the concentration of the photosensitizer is 0.2 w/v-0.4 w/v%, and the protein is at least one of albumin, fibronectin and laminin.
Optionally, the albumin is selected from at least one of bovine serum albumin, human recombinant serum albumin, sheep serum albumin and rabbit serum albumin. It will be appreciated that the type of albumin is not limited to the above, but may be other albumin.
Specifically, a photosensitizer is a Photoinitiator (photo initiator) which is a substance capable of absorbing radiant energy and generating photochemical change through excitation to generate active intermediates (free radicals or cations) with polymerization initiating capability. Photoinitiators are key components of the photocurable materials and affect the speed of photocuring. Optionally, the photosensitizer is tiger red sodium salt or methylene blue. It will be appreciated that the type of photosensitizer is not limited to the above, but may be other materials that can cause protein light to cure.
In some embodiments, the concentration of protein in the mixture is 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, or 90mg/mL. The concentration of the photosensitizer is 0.2w/v%, 0.25w/v%, 0.3w/v%, 0.35w/v% or 0.4w/v%. Further, the concentration of the protein is 20 mg/mL-50 mg/mL; the concentration of the photosensitizer is 0.2w/v% -0.3 w/v%. Further, the concentration of the protein is 20 mg/mL-35 mg/mL; the concentration of the photosensitizer is 0.25w/v% -0.3 w/v%. W/v% means a mass-to-volume ratio. For example, a final concentration of the photosensitizer in the mixed solution of 0.4w/v% indicates a final concentration of the photosensitizer in the mixed solution of 4g/L.
Step S120: and (3) adding the mixed solution onto a transparent substrate, and curing the mixed solution by adopting a laser 3D printing technology to form a three-dimensional protein body on the transparent substrate to prepare the composite material.
Specifically, a laser is used to scan the mixed solution layer by layer through the transparent substrate to form a three-dimensional protein body on the transparent substrate. The mixed solution contains protein and photosensitizer, and when laser passes through the transparent base material and scans the mixed solution layer by layer, the mixed solution irradiated by the laser is gradually solidified on the surface of the transparent base material, so that a three-dimensional protein body is formed. Layer-by-layer scanning refers to scanning from one direction to the other, for example, laser is passed through a transparent substrate and starts scanning from the side of the mixed solution close to the transparent substrate, then the mixed solution moves upwards for a certain distance, the next layer of liquid level is continuously scanned, and the mixed solution gradually solidifies to form a three-dimensional protein body.
Specifically, the wavelength of the laser is 750-850 nm, and the energy of the laser is 48-58 mW. In some embodiments, the laser has a wavelength of 750nm, 790nm, 800nm, 820nm, or 850nm. The energy of the laser is 48mW, 50mW, 53mW, 55mW or 58mW. Further, the wavelength of the laser is 800-850 nm, and the energy of the laser is 50-55 mW.
Specifically, the transparent substrate provides support for the subsequently formed three-dimensional protein body, and the laser passes through the transparent substrate and acts on the mixed solution to form the three-dimensional protein body. Alternatively, the material of the transparent substrate is selected from glass or plastic. Specifically, the glass is tempered glass. The plastic is polystyrene or polycarbonate. It will be appreciated that the type of glass and the type of plastic are not limited to the above, but may be other transparent materials that can be used in cell culture.
In some embodiments, the transparent substrate is sheet-like. At this time, the step of forming the three-dimensional protein body includes: after the mixed solution is applied to the transparent substrate, the mixed solution is scanned layer by layer through the transparent substrate by using a laser to cure the mixed solution to form a three-dimensional protein body on the transparent substrate. In use, after the composite material is placed in a culture container, the culture to be cultured is added to the composite material for culture. Of course, in some embodiments, the above-described composite material is fixed to the bottom of the culture vessel. By fixing the composite material on the inner bottom of the culture container, the composite material is not easy to move in the culture container due to the action of the culture solution, thereby being beneficial to the growth of cells.
In another embodiment, the transparent substrate is in the shape of a groove. Optionally, the transparent substrate is a culture vessel. Such as a petri dish, a culture plate, etc. The three-dimensional protein body can be directly used as a culture container after being prepared on a groove-shaped transparent substrate. In this case, the three-dimensional protein body is located on the inner bottom of the transparent substrate. The step of forming a three-dimensional protein body includes: after the mixed liquid is added into the transparent substrate, laser is adopted to pass through the bottom of the transparent substrate from the outside of the bottom of the transparent substrate and scan the mixed liquid in the transparent substrate layer by layer, so that a three-dimensional protein body is formed on the inner bottom of the transparent substrate. When in use, the culture to be cultivated and the culture solution are directly added into the composite material for cultivation.
In some embodiments, when the mixture is scanned layer by layer with a laser, the scan sections of the layers are parallel to each other, the area of the scan section being 471.435 μm 2 ～8256.5μm 2 The distance between adjacent scanning sections is 0.2-0.6 μm. Further, the area of the scanning section was 981.74 μm 2 ～3926.99μm 2 The distance between adjacent scanning sections is 0.2-1 μm. In some embodiments, the area of the scan cross-section is 981.74 μm 2 ～2000μm 2 The distance between adjacent scanning sections is 0.2-1 μm. Alternatively, each layer is circular in scanning cross-section. Of course, the scanning section of each layer is not limited to a circular shape, and may be any pattern such as a rectangle or a triangle.
In some embodiments, the number of three-dimensional protein bodies is a plurality, the plurality of three-dimensional protein bodies being spaced apart on the transparent substrate. At this time, the step of forming the three-dimensional protein body includes: after the mixed solution is added on the transparent substrate, part of the mixed solution is scanned layer by layer through the transparent substrate by laser so as to locally solidify the mixed solution to form a plurality of spaced three-dimensional protein bodies. Optionally, the laser is divided into a plurality of laser units, and the laser units scan part of the mixed liquid layer by layer simultaneously, so that the mixed liquid is partially solidified to form a plurality of spaced three-dimensional protein bodies simultaneously, and batch production is simply and conveniently realized.
Further, the distance between two adjacent three-dimensional protein bodies is 2-5 μm. In one specific example, the spacing between two adjacent three-dimensional protein bodies is 2 μm or 5 μm. It will be appreciated that the spacing between adjacent three-dimensional protein bodies may be adjusted by adjusting the distance between the multiple laser beams. The pitch herein refers to an average pitch. That is, in some embodiments, a plurality of three-dimensional protein bodies are arranged at equal intervals, and the distances between all two adjacent three-dimensional protein bodies are equal and are any number in the interval of 2 μm to 5 μm; in other embodiments, the plurality of three-dimensional protein bodies are non-equally spaced, and the average of the distances between all adjacent two three-dimensional protein bodies is in the interval 2 μm to 5 μm.
In some embodiments, the three-dimensional protein body is cylindrical, the diameter of the three-dimensional protein body is 1 μm to 6 μm, and the height of the three-dimensional protein body is 2 μm to 10 μm. In other embodiments, the three-dimensional protein body has a quadrangular prism shape, and the three-dimensional protein body has a length of 1 μm to 5 μm; the width of the three-dimensional protein body is 1-5 mu m, and the height of the three-dimensional protein body is 2-10 mu m. It will be appreciated that the shape and size of the three-dimensional protein body may be adjusted by adjusting the shape of the cross-section of the laser beam and the time the laser is applied to the mixed liquor.
In some embodiments, the operation of forming the three-dimensional protein body is performed in an oxygen environment having an oxygen partial pressure of 25kPa to 30kPa and a volume concentration of oxygen in the oxygen environment of 20% to 80%. Further, the oxygen partial pressure of the oxygen environment is 25 kPa-28 kPa, and the volume concentration of oxygen in the oxygen environment is 40% -60%.
The preparation method of the composite material is simple and convenient, raw materials are easy to obtain, and the composite material prepared according to the composite material comprises a transparent substrate and a three-dimensional protein body positioned on the transparent substrate, so that when the composite material is applied to three-dimensional culture of tumor cells, the real environment in the body can be better simulated, and invasion behaviors of the tumor cells can be easily observed.
An embodiment of the present application also provides a composite material, which is prepared by the preparation method of the composite material.
The composite material can be used as a bracket or a culture container in three-dimensional cell culture.
In addition, the application also provides application of the composite material in three-dimensional cell culture.
Specifically, cells were inoculated onto the above composite material and cultured. Alternatively, the cell is a tumor cell.
In addition, an embodiment of the present application further provides a preparation method of a tumor model, which includes the following steps: tumor cells are inoculated into the composite material for culture, and a tumor model is prepared. In an alternative specific example, the tumor cell is human breast cancer cell MCF-7.
In one embodiment, the transparent substrate of the composite material is sheet-shaped. At this time, the preparation method of the tumor model includes the following steps: and (3) placing the composite material in a culture container, and inoculating tumor cells to the composite material for culture to prepare a tumor model. Of course, during the culturing process, the culture vessel contains a medium for the growth of tumor cells, and the medium is replaced with a new medium if necessary.
In another embodiment, the transparent substrate of the composite material is in a groove shape. In this case, the composite material can be directly used as a culture vessel for culturing tumor cells. Specifically, the preparation method of the tumor model comprises the following steps: inoculating tumor cells to the composite material for culture to prepare a tumor model. It will be appreciated that during the culturing process, the composite material has medium for the growth of tumor cells, and that new medium is replaced if necessary.
In some embodiments, after seeding tumor cells onto the composite material, the incubation is performed for 1-3 days, and invasion of tumor cells is observed.
In addition, an embodiment of the application also provides a tumor model, which is prepared by the preparation method of the tumor model, and tumor cells in the tumor model have invasive behaviors.
The tumor model can be used for evaluating the efficacy of anti-tumor drugs.
In addition, an embodiment of the application also provides application of the tumor model in evaluating the efficacy of the anti-tumor drug. The tumor model can show the invasion behavior of the tumor, so that the effect of evaluating the invasion capacity of the anti-tumor drug on tumor cells by adopting the tumor model is better.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.
Example 1
The composite material of example 1 consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located within the confocal laser petri dish. Referring to table 1, the method for preparing the composite material of example 1 includes the following steps:
(1) Bovine Serum Albumin (BSA) and a photosensitizer (Rose Bengal, tiger red sodium salt) were thoroughly mixed to prepare a mixed solution. Wherein, in the mixed solution, the concentration of BSA is 40mg/mL, and the concentration of the photosensitizer is 0.4w/v%.
(2) Dripping 20 mu L of the mixed solution prepared in the step (1) into a confocal laser culture dish, and placing the mixed solution on a confocal microscope sample stage to prepare a three-dimensional protein body;
(3) Near infrared light with the wavelength of 800nm is set as excitation light for photochemical crosslinking, and the scanning mixed liquid is composed of two photons in an oxygen environment to excite the photosensitizer, so that the laser scanning mixed liquid generates photochemical crosslinking reaction, and a plurality of non-wrapped cylindrical three-dimensional protein bodies (figures 2-5) are prepared on the confocal laser culture dish. Wherein the diameter of the three-dimensional protein body is 2 mu m, the height is 10 mu m, and the interval between the adjacent three-dimensional protein bodies is 2 mu m; the laser wavelength energy is 49mW, the oxygen partial pressure of the oxygen environment is 40kPa, and the volume concentration of oxygen in the oxygen environment is 60%; when the mixed liquid is scanned layer by layer, the scanning sections of all layers are mutually parallel, and the area of the scanning section is 981.74 mu m 2 The spacing between adjacent scan sections is 0.5 μm.
Example 2
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that the concentration of protein in the preparation method of the composite material of this example was 20mg/mL. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 3
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located within the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the concentration of BSA was 90mg/mL. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 4
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located within the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the concentration of BSA was 50mg/mL. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 5
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that the concentration of the photosensitizer in the preparation method of the composite material of this example was 0.2w/v%. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 6
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that the concentration of the photosensitizer in the preparation method of the composite material of this example was 0.3w/v%. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 7
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the energy of the laser was 48mW. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 8
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the energy of the laser was 58mW. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 9
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the energy of the laser was 55mW. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 10
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the energy of the laser was 50mW. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 11
The composite of this example was substantially identical to the composite of example 1, except that the spacing between adjacent three-dimensional protein bodies of the composite of this example was 5 μm. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 12
The composite of this example was substantially identical to the composite of example 1, except that the spacing between adjacent three-dimensional protein bodies of the composite of this example was 3 μm. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Example 13
The composite material of this example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located in the confocal laser petri dish, and the preparation method of the composite material of this example was substantially the same as that of example 1, except that in the preparation method of the composite material of this example, the photosensitizer was methylene blue. The other raw materials and preparation parameters in the preparation method of the composite material of this embodiment are shown in table 1.
Comparative example 1
The composite of this comparative example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located within the confocal laser petri dish, and was prepared in a manner substantially identical to that of example 1, except that the concentration of BSA was 100mg/mL in the preparation of the composite of this comparative example. The other raw materials and preparation parameters in the preparation method of the composite material of the comparative example are shown in table 1.
Comparative example 2
The composite of this comparative example consisted of a confocal laser petri dish and a plurality of spaced three-dimensional protein bodies located within the confocal laser petri dish, and was prepared in a manner substantially identical to that of example 1, except that the concentration of photosensitizer in the preparation of the composite of this comparative example was 0.5w/v%. The other raw materials and preparation parameters in the preparation method of the composite material of the comparative example are shown in table 1.
Comparative example 3
The composite of this comparative example consisted of a laser confocal petri dish and a plurality of spaced three-dimensional protein bodies located within the same dish, and was prepared in substantially the same manner as in example 1, except that the energy of the laser was 45mW in the preparation of the composite of this comparative example. The other raw materials and preparation parameters in the preparation method of the composite material of the comparative example are shown in table 1.
TABLE 1
And (3) testing:
the composites of each example and each comparative example were each used to culture tumor cells (human breast cancer cells MCF-7) to evaluate the performance of each composite. Specifically:
(1) Culture conditions: tumor cells were cultured in low sugar Dulbecco's modified Eagle medium (DMEM, gibco) with 10% fetal bovine serum (FBS, gibco) and 2mM glutamine (Gibco). The cells were maintained at 37℃with 5% CO 2 Medium was changed every 2 days in a wet environment. After the cell density reached 80% of the T25 flask, it was digested with 0.05% trypsin EDTA (Gibco) and diluted to a density of 1X 10 5 A cell suspension of one/mL for culture on a three-dimensional protein body.
(2) Will be 5X 10 4 The cell suspension was cultured in three-dimensional protein bodies. During the culture:
the growth rate of the cells was monitored in real time using an optical microscope to compare the effect of the two-dimensional and three-dimensional environments on the growth rate of the cells and to record the morphology of the cells observed by a scanning electron microscope. The recording results of the tumor cells cultured in the composite material of example 11 under the optical microscope are shown in fig. 6 (three-dimensional culture area inside the frame in fig. 6, two-dimensional culture area outside the frame); the recorded results of tumor cells cultured in the composite material of example 12 under an optical microscope are shown in fig. 7 (three-dimensional culture area inside the frame of fig. 7, two-dimensional culture area outside the frame); the results of the scanning electron microscope for the third day of tumor cells cultured using the composites of example 11 and example 12 are shown in FIG. 8. In FIG. 8, A to D are the cases of two-dimensional monolayer culture of tumor cells, and E to L are the cases of culture of tumor cells in the case of the provision of a three-dimensional protein body; E-H, wherein the three-dimensional protein bodies with the diameters of 2 mu m and the intervals of 5 mu m form a protein array, and H is a partial enlarged view of G; in I-L, the three-dimensional protein bodies with the diameters of 2 μm and the intervals of 2 μm form a protein array, L is a partial enlarged view of K, and the scales in H and L are 3 μm.
Cell viability in two-dimensional and three-dimensional cultures were compared using Live/Dead stabilizing to verify that three-dimensional cultures did not decrease cell viability: on the first and third days of culture, no dead cells were found on the protein struts; on day 3 of culture, the fluorescent antibodies stained actin, integrin protein integrin αv and DAPI, and observed the state of actin filaments extending on the cell surface and integrin protein distribution on the protein struts under confocal microscopy. The results of example 1 are shown in FIG. 9. In FIG. 9, the first and third rows are pictures of the distribution of the three proteins integrin αv, actin and DAPI, respectively, of the cells on a two-dimensional plane. Wherein the first row is a picture of a single cell field of view and the third row is a picture of multiple cell fields of view; the second and fourth rows are pictures of the distribution of the three proteins integrin αv, actin and DAPI, respectively, of the cells on the three-dimensional microcolumn scaffold. Wherein the second row is a picture of a single cell field and the fourth row is a picture of multiple cell fields. In FIG. 9, the three-dimensional cultured cells had a sharper protein scaffold structure and also had more integrin secretion than the first and second rows, indicating a tendency for migration and invasion of tumor cells cultured with the three-dimensional protein scaffold. The same results are obtained for the comparison of the third and fourth rows.
As can be seen from fig. 6 and 7, when the interval between adjacent three-dimensional protein bodies is 5 μm, the two-dimensional culture growth rate of tumor cells is similar to the three-dimensional culture growth rate; at a spacing of 3 μm between adjacent three-dimensional protein bodies, the two-dimensional culture growth rate of tumor cells is faster than the three-dimensional culture growth rate.
As can be seen from fig. 8, in the three-dimensional culture performed on the composite materials of example 11 and example 12, the tumor cells may generate filaments adhered to the three-dimensional protein body (see fig. H and L), and the tumor cells have a tendency to invade, so that the invasion behavior of the tumor cells can be simulated.
As can be seen from FIG. 9, in the three-dimensional culture performed on the protein body of example 1, cytoskeletal actin of tumor cells was significantly changed, and migration-related protein integrin was secreted along the protein body, which was useful for studying stroma of tumor cell invasion.
The tumor cells cultured in comparative examples 1 to 3 had no invasive tendency and no invasive behavior.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims (5)

1. A method of preparing a composite material, comprising the steps of:
dissolving protein and a photosensitizer in water to prepare a mixed solution, wherein the concentration of the protein in the mixed solution is 40mg/mL, the concentration of the photosensitizer is 0.4w/v%, and the protein is bovine serum albumin; the photosensitizer is tiger red sodium salt; after the mixed solution is added to a transparent substrate, the mixed solution is solidified by adopting a laser 3D printing technology to form a three-dimensional protein body on the transparent substrate, so as to prepare a composite material;
wherein the wavelength of the laser is 800nm, and the energy of the laser is 49Mw;
in the step of solidifying the mixed solution by adopting a laser 3D printing technology to form a three-dimensional protein body on the transparent substrate, part of the mixed solution is scanned layer by adopting laser to penetrate through the transparent substrate so as to locally solidify the mixed solution to form a plurality of spaced three-dimensional protein bodies on the transparent substrate; the spacing between two adjacent three-dimensional protein bodies is 3 μm or 5 μm;
the operation of forming the three-dimensional protein body is performed under an oxygen environment, wherein the oxygen partial pressure of the oxygen environment is 40kPa, and the volume concentration of oxygen in the oxygen environment is 60%;
scanning the mixed solution layer by layer through the transparent substrate by adopting laser to form the three-dimensional protein body on the transparent substrate, wherein the scanning sections of all layers are mutually parallel, and the area of the scanning section is 981.74 mu m 2 The spacing between adjacent scan sections is 0.5 μm.
2. The method of producing a composite material according to claim 1, wherein the transparent substrate is sheet-like.
3. The method for preparing a composite material according to any one of claims 1 to 2, wherein the transparent substrate is in a groove shape, and the three-dimensional protein body is located on an inner bottom of the transparent substrate.
4. A composite material characterized by being produced by the method for producing a composite material according to any one of claims 1 to 3.
5. A method for preparing a tumor model, comprising the steps of:
tumor cells were inoculated into the composite material of claim 4 for culture to prepare a tumor model.
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