CN110004116A - A kind of method preparing three dimensional biological construct, three dimensional biological construct and application thereof - Google Patents
A kind of method preparing three dimensional biological construct, three dimensional biological construct and application thereof Download PDFInfo
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- CN110004116A CN110004116A CN201910214252.7A CN201910214252A CN110004116A CN 110004116 A CN110004116 A CN 110004116A CN 201910214252 A CN201910214252 A CN 201910214252A CN 110004116 A CN110004116 A CN 110004116A
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- tumor cells
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- sodium alginate
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
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Abstract
The present embodiments relate to a kind of methods for preparing three dimensional biological construct, three dimensional biological construct and application thereof, and the method includes the step of using bio-ink to carry out biological 3D printing and cross-linking steps；Wherein, the bio-ink is made of the material including the cell culture fluid including biomaterial and containing tumour cell；The biomaterial includes gelatin solution and sodium alginate soln；The tumour cell is primary tumor cell.The preparation method can be quickly obtained three dimensional biological construct, high-efficient, time-consuming short, can preferably keep the biological characteristics and biological function of tumour cell in three dimensional biological construct.
Description
Technical Field
The invention relates to the technical field of biological 3D printing, in particular to a method for preparing a three-dimensional biological construct, the three-dimensional biological construct and application thereof.
Background
In the fields of medical research, drug research, such as drug discovery, drug screening, drug efficacy and/or toxicity screening, investigation/mechanistic toxicology, target identification/identification, drug relocation studies, and pharmacokinetic and pharmacodynamic testing, conventional 2D (two-dimensional) cell, tissue culture techniques, i.e., planar culture of cells, are still commonly applied, and cells can only extend along a plane during the culture process.
However, in living tissue, cells exist in 3D (three-dimensional) microenvironments with intricate cell-cell and cell-matrix interactions and complex transport kinetics of nutrients and cells, and the 2D culture environment does not adequately represent this environment. Not only do 2D cultures differ in cell morphology, but the cultured cells gradually lose their biological properties and biological functions in vivo. Studies have shown that 2D culture methods yield cultures with drug resistance different from that of tumor cells in 3D microenvironments in vivo for tumor cells, and 2D cultures often fail to reliably predict drug efficacy and toxicity in vivo. Therefore, the in vitro tumor model research before clinical treatment is particularly important, the effectiveness of the tumor model is related to success or failure of related medical research and drug research, and the traditional 2D culture technology cannot meet practical requirements.
3D cultures are more similar to tissues in vivo in terms of cellular communication, biochemical and physicochemical gradient formation, and development of extracellular matrix, and thus, in addition to the above-mentioned drug-related research fields, 3D cell culture techniques are receiving increasing attention in research fields such as regenerative medicine, pathology models, cell/tissue chips, and the like. As one of the 3D cell culture techniques, the biological 3D printing technique is a technique of printing a three-dimensional biological product by using a computer-aided design, taking a digital model file as a basis, and taking a biological material as a "biological ink", and particularly, it can take a material having biological activity, such as a cell, as a constituent of the "biological ink" to obtain a three-dimensional biological construct in vitro in a three-dimensional printing manner, and the prepared biological construct has a certain biological function, can provide conditions for further growth of the cell/tissue, and has an excellent application prospect in the fields of medical research and pharmaceutical research, for example, can be used as an in vitro tumor model.
At present, in vitro tumor model studies before clinical practice, studies of constructing a three-dimensional biological construct as a tumor model by using tumor cells as a biological material and using a biological 3D printing technology are far from insufficient, and for example, the preparation speed of the tumor model is increased, and biological characteristics and biological functions of the tumor cells in the tumor model are better maintained, so that further improvement is needed; the printability of the tumor model and the survival rate of the tumor cells in the tumor model are also to be improved.
Disclosure of Invention
In order to solve at least one of the above technical problems, the present invention provides a method for preparing a three-dimensional biological construct, a three-dimensional biological construct and use of the three-dimensional biological construct in drug screening. Other technical problems that can be solved by the embodiments of the present invention will be set forth in the description that follows.
According to a first aspect of the invention, embodiments of the invention provide a method of preparing a three-dimensional biological construct, the method comprising a step of bio-3D printing using a bio-ink and a cross-linking step; wherein the biological ink is made of materials including biological materials and cell culture solution containing tumor cells; the biological material comprises a gelatin solution and a sodium alginate solution; the tumor cells are primary tumor cells.
Further, the primary tumor cells are extracted from fresh tumor tissue by the extraction step.
Further, after the step of extracting the primary tumor cells from the fresh tumor tissue, performing 2D culture or 3D culture on the primary tumor cells, and mixing a cell culture solution containing the primary tumor cells subjected to the 2D culture step or the 3D culture step with the biological material; or,
after the step of extracting the primary tumor cells from the fresh tumor tissue, directly mixing a cell culture solution containing the primary tumor cells with the biological material.
Further, the step of extracting the primary tumor cells comprises the following steps:
s111: collecting fresh tumor tissue;
s112: digestion treatment;
s113: terminating digestion, collecting tumor cells, and resuspending the cells; or,
the extraction step of the primary tumor cells comprises the following steps:
s121: collecting fresh tumor tissue;
s122: performing shaking digestion treatment by using the first digestion solution;
s123: digesting with a second digestive juice;
s124: digestion was terminated.
Further, the first and second digestive juices are different; preferably, the first digestion solution is an EDTA-free trypsin digestion solution, and the second digestion solution is a digestion solution containing collagenase and dnase.
Further, the biological material comprises a gelatin solution and a sodium alginate solution; preferably, the biological material consists of a gelatin solution and a sodium alginate solution; in the biological ink, the mass-volume ratio of gelatin is 0.05-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises a gelatin solution, a sodium alginate solution and a fibrinogen solution; preferably, the biological material consists of a gelatin solution, a sodium alginate solution and a fibrinogen solution; in the biological ink, the mass-volume ratio of gelatin is 0.05-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, and the mass-volume ratio of fibrinogen is 1-5
mg/mL, tumor cell density 1X 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and matrigel; preferably, the biomaterial consists of a gelatin solution, a sodium alginate solution and matrigel; in the biological ink, the mass-volume ratio of gelatin is 0.0375-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, the volume fraction of matrigel is 5-30%, and the density of tumor cells is 1 multiplied by 106-5×106one/mL.
Furthermore, the biological material is a gelatin solution and a sodium alginate solution, the mass-volume ratio of gelatin in the biological ink is 0.0625g/mL, the mass-volume ratio of sodium alginate is 0.01g/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and fibrinogen solution, wherein in the biological ink, the mass-volume ratio of gelatin is 0.05g/mL, the mass-volume ratio of sodium alginate is 0.01g/mL, the mass-volume ratio of fibrinogen is 0.03g/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and matrigel, wherein in the biological ink, the mass volume ratio of the sodium alginate is 0.01g/mL, the volume fraction of the matrigel is 30%, the mass volume ratio of the gelatin is 0.05g/mL, and the density of tumor cells is 1 multiplied by 106-5×106one/mL.
Further, the primary tumor cells include, but are not limited to, liver cancer primary tumor cells, cholangiocarcinoma primary tumor cells, and kidney cancer primary tumor cells.
Further, the cross-linking step is performed after the bio 3D printing step, or, alternatively, during the bio 3D printing process.
According to a second aspect of the invention, embodiments of the invention provide a three-dimensional biological construct prepared using one of the methods described above.
According to a third aspect of the present invention, the embodiments of the present invention provide a use of the three-dimensional biological construct as described above for drug discovery, or for drug screening, or for detecting the inhibition rate of a drug on the growth of tumor cells in the three-dimensional biological construct, or for evaluating the effect of a chemical agent or a physical stimulus on tumor cells in the three-dimensional biological construct.
The embodiment of the invention has the following beneficial effects: the method for preparing the three-dimensional biological construct provided by the embodiment of the invention can quickly obtain the three-dimensional biological construct, has high efficiency and short time consumption, can better keep the biological characteristics and biological functions of tumor cells in the three-dimensional biological construct, and is more suitable for medical research and drug research. Additional advantages and technical effects of embodiments of the present invention will be set forth in the description that follows.
Drawings
FIG. 1a is a photograph of a three-dimensional biological construct according to example 1 of the present invention.
FIG. 1b is a photograph of another three-dimensional biological construct according to example 1 of the present invention.
FIG. 2 is a photomicrograph on a scale of 100. mu.m, taken after 1 day, 3 days, 5 days, and 7 days of the 3D culture of cholangiocarcinoma primary tumor cells in example 1 of the present invention.
FIG. 3 is a photomicrograph on a scale of 100. mu.m, taken after 1 day, 3 days, 5 days, and 7 days of the 2D culture of cholangiocarcinoma primary tumor cells in example 1 of the present invention.
FIG. 4 is a confocal laser micrograph with a scale of 100 μm obtained by performing a death detection at the time of 0 culture and 7 days after culture in the 3D culture step of bile duct cancer primary tumor cells in example 1 of the present invention.
FIG. 5a is a photomicrograph on a scale of 100 μm showing cytoskeleton staining after 7 days of culture in the 2D culture step of bile duct cancer primary tumor cells in example 1 of the present invention.
FIG. 5b is a photomicrograph on a 100 μm scale showing cytoskeleton staining after 7 days of culture in the 3D culture step of bile duct cancer primary tumor cells in example 1 of the present invention.
FIG. 6 shows the relative gene expression levels of the stem cell markers CD133/EpCAM and matrix metalloproteinase MMP2/MMP9 for tumor cells in the 3D culture method and the 2D culture method in example 1 of the present invention.
FIG. 7 is a photomicrograph of hepatoma primary tumor cells cultured for 7 days in the biological 3D-printed three-dimensional biostructure, with a scale of 100 μm, according to example 4 of the present invention.
FIG. 8 is a photomicrograph, at 100 μm scale, of renal cancer primary tumor cells after 7 days of culture in biological 3D-printed three-dimensional biological constructs in example 5 of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will appreciate that the present invention is not limited to the drawings and the following examples.
Illustratively, some of the reagents, instruments and 3D printing parameters used in the examples of the invention and comparative examples are as follows:
sodium alginate: sigma, A0682.
Gelatin: sigma, G1890.
Poly-L-lysine (Poly-L-lysine solution): sigma, P8920.
Matrigel (matrigel): BD Biosciences, 354234.
Fibrinogen (fibronectin): sigma, F1141.
Calcein (Calcein-AM): sigma, 17783.
Propidium iodide (Propidium iodide): sigma, P4170.
Physiological saline: 0.9g/100mL aqueous sodium chloride (NaCl).
Cell counting instrument: countstar Autocytometer, product model IC 1000.
Biological 3D printing device: shangpo Boyuan (Beijing) Biotechnology Inc., product type SUNPALPHA-CPT 1.
Biological 3D printing parameters: extrusion speed of 5mm/s, scanning speed of 35mm/s, needle specification of 25G, layer thickness of 0.25mm, sleeve temperature of 25 deg.C, and molding chamber temperature of 4 deg.C
Those skilled in the art will appreciate that the above reagents, instruments and 3D printing parameters are merely exemplary embodiments set forth to fully disclose the invention, and are not intended to limit the invention.
According to a first aspect of the invention, the invention proposes a method for preparing a three-dimensional biological construct comprising the following steps:
the step of bio-3D printing a three-dimensional biological construct is performed using a bio-ink made of materials including a cell culture fluid (e.g., a cell suspension) containing tumor cells and a biological material.
The method for preparing the three-dimensional biological construct is described in detail below.
Step 101: preparation of Bio-ink (i.e., biological 3D printing composition)
A cell culture fluid (e.g., a cell suspension) containing tumor cells is mixed with a biological material to obtain a bio-ink.
In one embodiment, the tumor cell is a primary tumor cell, i.e., a P0 generation tumor cell that has not been subcultured.
Further, the primary tumor cells are extracted from fresh tumor tissue of a tumor patient by an extraction step.
The primary tumor cells can be extracted from solid tumor tissues and can also be extracted from non-solid tumor tissues, including but not limited to fresh tumor tissues of patients with liver cancer, fresh tumor tissues of patients with bile duct cancer and fresh tumor tissues of patients with kidney cancer; such "fresh tumor tissue", for example, includes, but is not limited to: tumor tissue collected from the body of a patient immediately or tumor tissue in a state where desired tumor cells are maintained in a biologically active and characteristic state after storage for several hours, several tens of hours or several days after collection from the body of a patient.
In one embodiment, after the step of extracting the primary tumor cells from fresh tumor tissue of the tumor patient, the primary tumor cells are 2D cultured, and a cell suspension containing the primary tumor cells after the 2D culturing step is mixed with the biological material.
In another embodiment, after the step of extracting the primary tumor cells from the fresh tumor tissue of the tumor patient, the primary tumor cells are subjected to 3D culture, and a cell suspension containing the primary tumor cells after the 3D culture step is mixed with the biological material, wherein the 3D culture can be a 3D culture method in the prior art (which is well known to those skilled in the art and needs no further description) or a 3D culture method disclosed in the present specification.
In another preferred embodiment, the cell suspension containing primary tumor cells is mixed with the biological material directly after the extraction step of primary tumor cells from fresh tumor tissue of a tumor patient.
It is noted that the term "directly" refers to the primary tumor cells not being subjected to a 2D culture (i.e., planar culture) step, and does not mean that no other manipulations may be included after the extraction step.
The inventors of the present invention found that, after extracting primary tumor cells, the preferred embodiment, which directly mixes the cell culture solution containing primary tumor cells obtained by the extraction step with a biological material to obtain a bio-ink without a 2D culture step, has excellent technical effects. There is a prejudice among those skilled in the art that since the fresh tumor tissue of the patient is difficult to obtain, resulting in a very short supply of tumor cells derived from the fresh tumor tissue of the patient, especially for early-stage tumor patients, where the tumor tissue is small and the sample size is severely insufficient, those skilled in the art are strongly inclined to perform 2D culture on the tumor cells to proliferate the tumor cells, and then perform other experimental operations/attempts after obtaining enough tumor cells, thereby avoiding experimental failures due to the problem of cell size. The inventors of the present invention have realized the above-mentioned prejudice in their research and have further found that the culturing speed of tumor cells is not reduced but greatly increased by the 2D culturing step of discarding primary tumor cells, and in particular, this embodiment can achieve at least the following technical effects: the inventor finds that the growth speed of the tumor cells in the three-dimensional biological construct obtained by the biological 3D printing step is higher than that of the 2D culture step, and the actual culture time of the tumor cells is greatly shortened after the 2D culture step is abandoned; and moreover, the loss of biological characteristics and functions of the tumor cells in the in-vitro 2D culture process is avoided.
In another embodiment, the tumor cell is derived from a tumor cell obtained by 3D culturing of a primary tumor cell in the context of a biological 3D printed three-dimensional biological construct, or from a tumor cell obtained by 3D subculturing of a primary tumor cell in the context of a biological 3D printed three-dimensional biological construct.
In one embodiment, the step of extracting the primary tumor cells comprises the steps of:
s111: collecting fresh tumor tissue
S112: digestion treatment;
s113: digestion was stopped, tumor cells were collected and cells were resuspended.
Specifically, the extraction step of the primary tumor cells comprises the following steps:
s1111: collecting fresh tumor tissue, cleaning, and cutting into pieces;
s1112: digesting for 2-4 hours at 37 ℃ by using a digestion solution containing 0.125mg/mL collagenase and 0.1mg/mL deoxyribonuclease (DNase I);
s1113: digestion was stopped, tumor cells were collected and cells were resuspended.
After a preset number of tissues in the visual field are decomposed into single cells, adding cell culture solution with serum to stop digestion, centrifugally collecting tumor cells, discarding supernatant, and resuspending the cells by using the cell culture solution containing the serum to obtain cell suspension.
In another preferred embodiment, the step of extracting said primary tumor cells comprises the steps of:
s121: collecting fresh tumor tissue;
s122: performing shaking digestion treatment by using the first digestion solution;
s123: digesting with a second digestive juice; preferably, the second digestive juice is different from the first digestive juice;
s124: terminating digestion;
s125: fibroblasts were removed.
Specifically, the extraction step of the primary tumor cells comprises the following steps:
s1211: collecting fresh tumor tissue, cleaning, and cutting into pieces;
s1212: adopting 0.05% trypsin (trypsin, without EDTA) digestive juice, and performing shake digestion treatment on the tumor tissue at 4 deg.C, for example, for 6-18 hr, preferably 12-16 hr.
S1213: the digestion is carried out at 37 ℃ for 0.2 to 3 hours, preferably 0.5 to 1 hour, using a digestion solution containing 0.125mg/mL collagenase and 0.1mg/mL DNase (I).
S1214: after a preset number of tissues in the visual field are decomposed into single cells, adding cell culture solution with serum to stop digestion, centrifugally collecting tumor cells, discarding supernatant, and resuspending the cells by using the cell culture solution containing the serum to obtain cell suspension.
S1215: carrying out differential adherent treatment on the cell suspension at 37 ℃ to remove fibroblasts; collecting supernatant, centrifuging again, and resuspending to obtain cell suspension containing tumor cells.
This preferred embodiment adds a shaking digestion step, resulting in a substantial increase in cell anchorage rate, which in one embodiment is approximately 50% higher, with a significant increase in the number of cells extracted.
In one embodiment, the biological material comprises a gelatin solution and a sodium alginate solution, wherein in the biological ink, the mass-to-volume ratio of gelatin is 0.05-0.1g/mL, the mass-to-volume ratio of sodium alginate is 0.01-0.02g/mL, and the density of tumor cells is 1 x 106-5×106Per mL; more preferably, in the bio-ink, the mass-to-volume ratio of the gelatin is 0.0625g/mL, the mass-to-volume ratio (w/v) of the sodium alginate is 0.01g/mL, and the density of the tumor cells is 1 × 106-5×106one/mL. The biological material may comprise only gelatinThe glue solution and the sodium alginate solution, or other auxiliary biological materials.
In another embodiment, the biomaterial comprises a gelatin solution, a sodium alginate solution and a fibrinogen solution, wherein the mass to volume ratio of gelatin is 0.05-0.1g/mL, the mass to volume ratio of sodium alginate is 0.01-0.02g/mL, the mass to volume ratio of fibrinogen is 1-5mg/mL, and the density of tumor cells is 1 × 106-5×106Per mL; more preferably, in the biological ink, the mass-to-volume ratio of the gelatin is 0.05g/mL, the mass-to-volume ratio of the sodium alginate is 0.01g/mL, the mass-to-volume ratio of the fibrinogen is 0.03g/mL, and the density of the tumor cells is 1 × 106-5×106one/mL. The biomaterial may include only gelatin solution, sodium alginate solution and fibrinogen solution, or may further include other auxiliary biomaterials.
In another embodiment, in the bio-ink, the mass-to-volume ratio of gelatin is 0.0375-0.1g/mL, the mass-to-volume ratio of sodium alginate is 0.01-0.02g/mL, the volume fraction of matrigel is 5-30%, and the density of tumor cells is 1 × 106-5×106Per mL; more preferably, the biological material is gelatin solution, sodium alginate solution and matrigel, the mass volume ratio of sodium alginate in the biological ink is 0.01g/mL, the volume fraction of matrigel is 30%, the mass volume ratio of gelatin is 0.05g/mL, and the density of tumor cells is 1 × 106-5×106one/mL. The biomaterial may include only gelatin solution, sodium alginate solution and matrigel, or other auxiliary biomaterials.
The printability of the three-dimensional biological construct, the survival rate and the proliferation speed of tumor cells in the three-dimensional biological construct and the formation of the pore structure of the three-dimensional biological construct (the tumor cells in the three-dimensional biological construct are provided with nutrition through the pore structure) are closely related to the composition of the bio-ink.
Step 102, biological 3D printing and crosslinking
And (3) performing biological 3D printing by using a biological 3D printing device and taking the biological ink obtained in the step (101) as a raw material according to a computer model and a printing path preset based on nutrition and oxygen supply requirements required by the growth of the tumor cells, and crosslinking sodium alginate to obtain the three-dimensional biological construct containing the tumor cells.
The three-dimensional biological construct is printed layer by layer and comprises a plurality of layers, for example 3-10 layers.
In one embodiment, the cross-linking agent is provided to cross-link the sodium alginate during the printing process, for example, in layer-by-layer printing, the cross-linking agent is sprayed onto the layer to cross-link the layer-by-layer each time one layer is printed.
In another embodiment, a cross-linking agent is provided to cross-link the sodium alginate after the printing process is complete.
The crosslinking agent is, for example, an aqueous calcium chloride solution. Crosslinking agents capable of crosslinking sodium alginate are likewise known to the person skilled in the art and are not described in further detail.
The biological 3D printing technology can manufacture a personalized in-vitro three-dimensional biological function structural body or a coding biological model by using a three-dimensional printing technical means according to requirements of bionic morphology, biological structure or biological function, cell specific microenvironment and the like on biological units (cells/proteins/DNA and the like) and biological materials. The biological 3D printing step is carried out to enable the production of three-dimensional biological constructs having a predetermined pattern (e.g. any predetermined shape, structure), which may be in sheet-like structures, or hollow three-dimensional structures, or solid three-dimensional structures, or other three-dimensional structures, or any combination of various structures. Biological 3D printing methods are known to those skilled in the art and need not be described in further detail.
The three-dimensional biological construct obtained through the steps can be used as a tumor model, or a cell culture solution is applied to the three-dimensional biological construct obtained through the steps for culture, so that the tumor model meeting the preset conditions can be obtained. The method for culturing tumor cells in the three-dimensional biological construct belongs to the common technical means in the field and is not described in detail.
The tumor cells in the three-dimensional biological construct obtained by the step can be subjected to 3D subculture, wherein the 3D subculture step comprises the following steps:
s311, when the diameter of a tumor sphere in the three-dimensional biological construct reaches a preset size, cracking the three-dimensional biological construct;
s312, collecting the tumor balls, and digesting the tumor balls;
s313, terminating digestion to obtain tumor cells;
s314, mixing a cell culture solution (e.g., cell suspension) containing the tumor cells obtained in step S313 with the above-mentioned biological material of this example to prepare a bio-ink;
and S315, performing biological 3D printing on the three-dimensional biological construct by using the biological ink obtained in the step S314.
Thus, tumor cells cultured in 3D or subculture in 3D in the context of three-dimensional biological constructs may also be a source of tumor cells for 3D printing of organisms of the invention.
According to a second aspect of the invention, embodiments of the invention provide a three-dimensional biological construct prepared using the above-described method.
According to a third aspect of the present invention, the embodiments of the present invention provide a use of the above three-dimensional biological construct for drug discovery, or drug screening, or detecting the inhibition rate of a drug on the growth of tumor cells in the three-dimensional biological construct, or evaluating the effect of a chemical agent or physical stimulus on tumor cells in the three-dimensional biological construct.
The present invention is further illustrated by the following preferred embodiments, and it will be understood by those skilled in the art that the present invention may be embodied in various forms and should not be construed as limited by the following embodiments. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Example 1 three-dimensional biological construct for cholangiocarcinoma
The embodiment provides a method for 3D printing of a three-dimensional biological construct of cholangiocarcinoma, which comprises the following steps:
extraction of bile duct cancer Primary tumor cells
Culture solution: contains 15% (volume ratio) FBS, 1% (volume ratio) streptomycin mixed liquor (double antibody) and 1% (volume ratio) glutamine (glutamine), and the balance being DMEM.
The specific extraction steps are as follows:
a. preparing 4 culture dishes, adding HBSS buffer solution (without calcium ions, magnesium ions and phenol red) into the culture dishes 1-3, adding 5mL of trypsin (trypsin, EDTA-free) with the mass percent of 0.05% into the culture dish 4, taking fresh bile duct tumor tissue of a patient with bile duct cancer into the culture dish 1, cleaning bloodstains, transferring the fresh bile duct tumor tissue into the culture dish 2, removing necrotic tissues, transferring the fresh bile duct tumor tissue into the culture dish 3 for cleaning, transferring the fresh bile duct tumor tissue into the culture dish 4 after cleaning, and shearing the fresh bile duct tumor tissue into 0.5-1cm3Tissue mass of size.
b. 5mL of trypsin (trypsin, EDTA-free) at 0.05% by mass was added, the flask was sealed with a sealing film, and the dish 4 was fixed on a shaker and shaken overnight (for example, 12 to 16 hours) at 4 ℃ at a rotation speed of 60 rmp.
c. The tissue fragments were collected by a pipette, transferred to a new petri dish, and a new digestion solution (containing 0.125mg/mL of collagenase and 0.1mg/mL of DNase I) was added to the new petri dish and digested at 37 ℃ for 0.5 to 1 hour.
d. After 90% of tissues in the visual field are decomposed into single cells, adding cell culture solution with serum to stop digestion, centrifuging for 3min at 1000rmp to collect bile duct cancer tumor cells, discarding supernatant, and resuspending the cells with 2mL of cell culture solution containing serum.
e. Adding the heavy suspension into a T175 culture flask containing cell culture solution containing serum, and differentially adhering to the wall in a carbon dioxide incubator for 1 hour at 37 ℃ to remove fibroblasts; collecting supernatant, centrifuging for 5min at 1200rmp, re-suspending to obtain cell culture solution containing bile duct cancer primary tumor cells, and counting cells for later use.
The extraction procedure of this example increased the agitation digestion procedure and increased the cell attachment rate from about 30% to about 45% as compared to the extraction procedure without agitation digestion (specifically: digestion at 37 ℃ for 2-4 hours using a digestion solution containing 0.125mg/mL collagenase and 0.1mg/mL DNase I).
Preparation of Bio-ink (i.e. biological 3D printing composition)
According to the density of preset tumor cells in the biological ink, calculating, taking a proper volume of cell culture solution containing the bile duct cancer primary tumor cells obtained by the extraction step, centrifuging, adding the cell culture solution for heavy suspension, and obtaining 600 mu L of cell suspension containing the preset number of bile duct cancer primary tumor cells; mixing 600 mu L of cell suspension containing a preset amount of cholangiocarcinoma primary tumor cells, 750 mu L of 15g/100mL gelatin solution and 450 mu L of 4g/100mL sodium alginate solution to obtain the bio-ink, wherein the mass-volume ratio of gelatin in the bio-ink is 0.0625g/mL, the mass-volume ratio of sodium alginate is 0.01g/mL, and the density of the tumor cells is 1 multiplied by 106-5×106one/mL.
Wherein the gelatin solution is prepared by dissolving gelatin in normal saline, the sodium alginate solution is prepared by dissolving sodium alginate in normal saline, the gelatin solution and the sodium alginate solution are prepared in advance and stored at 4 ℃, and when the biological ink is prepared, the biological ink is preheated at 37 ℃ and then used. In another embodiment, the gelatin solution and sodium alginate solution may also be used directly in the preparation of bio-ink after preparation at 37 ℃.
Printing and crosslinking
Performing biological 3D printing by using the biological ink obtained in the step as a raw material by using a biological 3D printing device and using a culture dish coated by poly-L-lysine as a carrier according to the printing parameters based on a preset computer model and a preset printing path, and adding 200mM CaCl into the culture dish after printing2And (5) carrying out cross-linking on the aqueous solution for 3min to obtain the three-dimensional biological construct containing the bile duct cancer primary tumor cells. The three-dimensional biological construct can be used as a tumor model and used for medical research and drug research.
Referring to FIG. 1a, in this example, the three-dimensional biological construct is a 6-layer lattice structure with dimensions of 10mm by 2mm, and the hydrogel filaments are about 500 μm in diameter. Fig. 1b is another three-dimensional biological construct obtained by 3D bioprinting according to this example.
The poly-L-lysine coated culture dish is prepared, for example, according to the following steps: diluting poly-L-lysine with distilled water to concentration of 0.0125g/100mL, and adding the solution at 0.0625mL/cm2The supernatant was applied to a petri dish, allowed to stand at room temperature for 20min, and the supernatant was discarded, washed with distilled water.
The computer model is designed by solidworks, for example, based on nutrition and oxygen supply requirements required by tumor cell growth, and is subjected to layered transformation to generate a corresponding STL file, and the STL file is identified by a biological 3D printer to generate a printing path program.
Optionally, this example also includes a 3D culture step of cholangiocarcinoma primary tumor cells
After the crosslinking step, the liquid phase is discarded and added to the cell cultureLiquid, put in a container containing 5% CO2And culturing in an incubator at 37 ℃ to obtain the three-dimensional biological construct of the bile duct cancer under preset conditions, wherein the three-dimensional biological construct can be used as a tumor model and used for medical research and drug research.
Optionally, this example also includes a 3D subculture step, including the steps of:
a. when the diameter of the tumor ball in the three-dimensional biological construct reaches about 80 mu m, discarding the culture solution, and washing with PBS for 3 times;
b. digesting for 20min in an incubator by using a lysis solution at 37 ℃, blowing and beating for 5 times by using a 1mL gun head at an interval of 5min, wherein the lysis solution is a mixed solution containing 55mM sodium citrate, 0.05% pancreatin (containing EDTA) and 60% Tryle Express;
c. after the three-dimensional biological construct is completely dissolved, centrifuging at 1000rmp for 3min to collect tumor spheres, discarding the supernatant, washing with PBS, and centrifuging at 1000rmp for 3min to collect tumor spheres;
d. digesting the tumor balls into single cells by using a Stempro accumtase digestive juice in an incubator at 37 ℃, and adding a culture solution to stop digestion after digestion is finished;
e. centrifuging at 1000rmp for 3min to collect single cells, mixing the single cells with the biological material, and then performing the steps of biological 3D printing of the three-dimensional biological construct, crosslinking of sodium alginate and 3D culture.
Comparative example: 2D culture of bile duct cancer tumor cells
In this comparative example, a predetermined number of bile duct cancer primary tumor cells were inoculated into a T25 flask containing 6ml of the same cell culture solution as in example 1, and 2D culture was performed, and when the cells reached 90% or more confluence, subculture was performed.
The bile duct cancer tumor cells in the 3D culture method of the present example and the 2D culture method of the comparative example
In this embodiment, the culture condition of the cholangiocarcinoma tumor cells is known by observing the growth condition, survival condition, proliferation condition, morphology change and the like of the cholangiocarcinoma tumor cells in the three-dimensional biological construct over time.
(1) Growth conditions
Fig. 2 is a micrograph after 1 day, 3 days, 5 days, and 7 days of culture in the 3D culture step of cholangiocarcinoma primary tumor cells, and it can be observed that the tumor cells stably grow and form tumor spheres.
Fig. 3 is a photomicrograph of bile duct cancer primary tumor cells after 1 day, 3 days, 5 days, and 7 days of 2D culture, which shows that the cells grow in a lamellar manner on a plane, and that tumor spheres obtained by the 3D culture method cannot be formed, and the difference between the microenvironment of the tumor cells and the actual microenvironment of the tumor cells in vivo is large.
(2) Survival rate
Fig. 4 is a confocal laser micrograph of bile duct cancer primary tumor cells obtained by performing a death detection at 0 th and 7 days after the culture in the 3D culture step.
Dead and alive detection solution: contains 1 μ M Calcein-AM, 2 μ M propidium iodide, and the balance PBS buffer.
A detection step: the three-dimensional biological constructs are respectively taken and washed 3 times by PBS buffer solution, then 1mL of dead and alive detection solution is added, the mixture is kept stand for 15min in a dark place, and the pictures are observed by using a laser confocal microscope (LSCM, Nikon, Z2). For each sample, 3 fields were selected for statistical analysis.
In fig. 4, the upper image is a photograph of the test of the dead or alive at time 0, and the lower image is a photograph of the test of the dead or alive after 7 days of culture, from left to right: the photograph of the combination of dead cells and live cells, the photograph of dead cells staining, and the photograph of live cells staining, wherein the bright spots in the photographs indicate live cells. Therefore, according to the method provided by the embodiment of the invention, the survival rate of the bile duct cancer tumor cells reaches 90%.
(3) Proliferation assay
In the 3D culture step of the cholangiocarcinoma primary tumor cells, after 1 day, 3 days, 5 days and 7 days of culture, proliferation detection is respectively carried out by adopting proliferation detection kits (Cell counting kit-8, Dojindo, CCK-8), the number of cells shows a stable growth trend in the 7-day culture process, and after 7 days of culture, the number of tumor cells is more than 10 times of the number of initial cells.
In the 2D culture step of the cholangiocarcinoma primary tumor cells, after 1 day, 3 days, 5 days and 7 days of culture, proliferation detection is carried out by adopting a proliferation detection kit (Cell counting kit-8, Dojindo, CCK-8); during the 7-day culture process, the cell number shows a steady increase trend, and after 7 days of culture, the number of tumor cells is 5 times of the initial cell number.
It can be seen that the proliferation rate of tumor cells in the 3D culture method is much higher than that in the 2D culture.
(4) Morphology of
FIG. 5a is a micrograph of biliary duct cancer primary tumor cells after staining cytoskeleton after 7 days of culture in the 2D culture step.
FIG. 5b is a micrograph of biliary duct cancer primary tumor cells after 7 days of culture and cytoskeletal staining in the 3D culture step.
It can be seen that the difference of cell morphology of the tumor cells is significant in 2D and 3D environments, the cells are fusiform in the 2D environment, and the cells grow into a tightly connected tumor-like sphere structure in the 3D environment.
Evaluation of the biological Properties of the three-dimensional biological constructs
In the embodiment, the biological characteristics of the obtained three-dimensional biological construct for the cholangiocarcinoma are evaluated by detecting the levels of tumor cell markers, fibrosis detection indexes and the like
(1) Tumor cell markers
1.1 CD133/EpCAM and MMP2/MMP9
In the 3D culture and 2D culture steps of the cholangiocarcinoma primary tumor cells, after 7 days of culture, the gene expression levels of stem cell markers CD133/EpCAM and matrix metalloproteinase MMP2/MMP9 of the tumor cells are respectively detected.
The detection method comprises extracting RNA, reverse transcribing to cDNA, and detecting gene expression level (internal reference is GAPDH gene) by fluorescence quantitative PCR.
The results of the detection are shown in FIG. 6. It can be seen that the relative gene expression levels of the stem cell markers CD133/EpCAM and matrix metalloproteinase MMP2/MMP9 of the tumor cells in the 3D culture method are significantly increased compared to the 2D culture method, and the relative gene expression levels of CD133, EpCAM, MMP9, and MMP2 in the 3D culture method are 3.7 times, 9.7 times, 4.4 times, and 5.1 times, respectively, of the relative gene expression level of the 2D culture method, and show relatively high dryness and malignancy.
1.2 CA19-9 and CEA
In the steps of 3D culture and 2D culture of the bile duct cancer primary tumor cells, after 7 days of culture, culture supernatants are respectively taken, and the levels of tumor markers CA19-9 and CEA in the supernatants are detected.
Compared with the supernatant cultured in 2D, the levels of the tumor markers CA19-9 and CEA in the supernatant cultured in 3D are remarkably increased, the expression level of CA19-9 is 2.9 times that of 2D, and the expression level of CEA is 5.7 times that of 2D.
(2) Index of fibrosis detection
In the steps of 3D culture and 2D culture of the cholangiocarcinoma primary tumor cells, after 7 days of culture, culture supernatants are respectively taken to detect the levels of fibrosis detection indexes (expression amounts of type III procollagen, type IV collagen and laminin) in the supernatants, and the expression level of each detection index of the 3D culture method is obviously higher than that of each detection index of the 2D culture method.
Wherein the expression level of type III procollagen in 3D culture is 2.8 times that of 2D culture, the expression level of type IV collagen is 2.4 times that of 2D culture, and the expression level of laminin is 6.3 times that of 2D culture.
It can be seen that the microenvironment created by the 3D construct facilitates the growth of tumor cells and the accumulation of extracellular matrix components, making them more accessible to in vivo growth.
Use of three-dimensional biological constructs
The three-dimensional biological constructs obtained in this example have a variety of uses. For example, for drug discovery, the three-dimensional biological construct obtained through the above steps is exposed to a set of compounds, the effect of each compound on the three-dimensional biological construct determines the therapeutic effect of each compound on the relevant disease, and in combination with the toxicity and side effects of each compound, the compound having the best therapeutic effect and/or the lowest toxicity and side effects, or the compound having the best balance between the therapeutic effect and the side effects is determined as a lead compound for further drug development. Also for example, for drug screening, for detecting the rate of inhibition of tumor cell growth in a three-dimensional biological construct by a drug, for assessing the effect of a chemical agent (e.g., one or more compounds) or a physical stimulus (e.g., radiation or heat) on tumor cells in a three-dimensional biological construct.
In the following, this example illustrates the use of three-dimensional biological constructs in a specific embodiment.
(1) In the 3D culture step of the cholangiocarcinoma primary tumor cells, after 7 days of culture, the culture solution was changed to a drug-containing culture solution, and culture was continued for 48 hours.
Drug-containing culture solution:
culture broth containing 25, 50, 100 or 150 μ M Sorafenib (Sorafenib).
Culture broth containing 0.1, 0.2, 0.25 or 0.6mM cisplatin (cissplatin).
Culture broth containing 1.25, 2.5, 5, 6.5 or 10mM 5-fluorouracil (5-fluorouracil).
Each drug concentration was set for 3 replicates. Cell viability was then determined using cell counting kit-8 (Dojindo; CCK8) and following the instructions.
Inhibition of tumor cell growth in three-dimensional biological constructs by drugs:
| sorafenib concentration (μ M) | 25 | 50 | 100 | 150 | / |
| Inhibition rate | 19.8％ | 62.2％ | 63.2％ | 66.5％ | / |
| Cisplatin concentration (mM) | 0.1 | 0.2 | 0.25 | 0.6 | / |
| Inhibition rate | 15.9％ | 17.9％ | 52.9％ | 62.1％ | / |
| 5-Fluorouracil (mM) | 1.25 | 2.5 | 5 | 6.5 | 10 |
| Inhibition rate | 31.3％ | 36.8％ | 33.9％ | 31.3％ | 42.9％ |
(2) In the 2D culture step of the cholangiocarcinoma primary tumor cells, after 7 days of culture, the culture solution was changed to a drug-containing culture solution, and culture was continued for 48 hours.
Drug-containing culture solution:
culture broth containing 25, 50, 100 or 150 μ M Sorafenib (Sorafenib).
Culture broth containing 0.1, 0.2, 0.25 or 0.6mM cisplatin (cissplatin).
Culture broth containing 1.25, 2.5, 5, 6.5 or 10mM 5-fluorouracil (5-fluorouracil).
Each drug concentration was set for 3 replicates. Cell viability was then determined using cell counting kit-8 (Dojindo; CCK8) and following the instructions.
Inhibition of tumor cell growth in three-dimensional biological constructs by drugs:
| sorafenib concentration (μ M) | 25 | 50 | 100 | 150 | / |
| Inhibition rate | 97.1％ | 99.6％ | / | / | / |
| Cisplatin concentration (mM) | 0.1 | 0.2 | 0.25 | 0.6 | / |
| Inhibition rate | 98.3％ | 98.9％ | / | / | / |
| 5-Fluorouracil (mM) | 1.25 | 2.5 | 5 | 6.5 | 10 |
| Inhibition rate | 69.0 | 72.4％ | 76.9％ | 77.9％ | 78.4％ |
The reason for the difference in inhibition rates is the difference in the effectiveness of the three-dimensional biological constructs as tumor models and tumor models obtained by 2D culture of tumor cells. In the tumor model obtained by the 2D culture method, during detection, the medicine directly contacts cells, so that the obstruction of the microenvironment of the tumor cells is avoided, and the detection is not consistent with the real condition of the environment where the tumor cells in a patient body are located.
Example 2 three-dimensional biological construct for cholangiocarcinoma obtained with another biological ink
The present example is different from example 1 in that the composition of the bio-ink of the present example is:
400 μ L of a cell suspension containing a predetermined amount of cholangiocarcinoma primary tumor cells (the method for obtaining the same is well known to those skilled in the art, and can be referred to example 1), 500 μ L of a 20g/100mL gelatin solution, 500 μ L of a 4g/100mL sodium alginate solution, and 600 μ L of a 10mg/mL fibrinogen solution were mixed at 37 ℃ to obtain a bio-ink. In the biological ink, the mass-volume ratio of gelatin is 0.05g/mL, the mass-volume ratio of sodium alginate is 0.01g/mL, and the fiberThe mass-to-volume ratio of the proprotein is 0.03g/mL, and the density of the tumor cells is 1 multiplied by 106-5×106one/mL.
Wherein, the fibrinogen solution is prepared by dissolving fibrinogen in DMEM culture solution, and is prepared on site.
The rest steps are the same and are not described again.
Example 3 three-dimensional biological construct for cholangiocarcinoma obtained with another biological ink
The present example is different from example 1 in that the composition of the bio-ink of the present example is:
400 μ L of a cell suspension containing a predetermined amount of cholangiocarcinoma primary tumor cells (the method for obtaining the same is well known to those skilled in the art and can be referred to in example 1), 500 μ L of a 4g/100mL sodium alginate solution, 600 μ L of matrigel, and 500 μ L of a 20g/100mL gelatin solution were mixed at 37 ℃ to obtain a bio-ink. In the biological ink, the mass volume ratio of sodium alginate is 0.01g/mL, the volume fraction of matrigel is 30 percent, the mass volume ratio of gelatin is 0.05g/mL, and the density of tumor cells is 1 multiplied by 106-5×106one/mL.
The rest steps are the same and are not described again.
The bio-ink provided by the embodiment of the invention has higher printability, higher survival rate of tumor cells in the obtained three-dimensional biological construct, higher proliferation speed of the tumor cells, and good pore structure, is beneficial to culture of the tumor cells in the three-dimensional biological construct, and has good tumor forming capability.
Example 4 three-dimensional biological constructs for liver cancer
Culture solution: contains 15% (volume ratio) of Fetal Bovine Serum (FBS), 1% (volume ratio) of streptomycin mixed liquor (double antibody), 1% (volume ratio) of glutamine (glutamine), 25ng/mL of Hepatocyte Growth Factor (HGF), 10mM HEPES, 1mM sodium pyruvate, 1mM NEAA and 0.01mg/mL of insulin, and the balance of DMEM.
The remaining features are the same as in example 1.
FIG. 7 is a photograph of the three-dimensional biological construct containing primary tumor cells of hepatocarcinoma obtained by biological 3D printing of this example.
This example also compares the following two embodiments:
(1) directly mixing cell suspension containing the liver cancer primary tumor cells with a biological material to prepare biological ink for biological 3D printing after the extraction step of the liver cancer primary tumor cells;
(2) after the extraction step of the primary tumor cells of the liver cancer, 2D culture is carried out on the primary tumor cells of the liver cancer.
Experimental results show that the growth time of primary liver cancer cells in a 2D culture dish to next passage is about 25 days approximately, and in a 3D model, the cell growth can be performed by passage culture only in 15-20 days. The present invention example abandons the 2D culturing step of tumor cells, and the proliferation rate of tumor cells is faster, which is further verified in this comparative experiment.
Example 5 three-dimensional biological constructs of renal carcinoma
Culture solution: contains 15% (volume ratio) of fetal bovine serum FBS, 1% (volume ratio) of streptomycin mixed liquor (double antibody), 1% (volume ratio) of glutamine (glutamine), 1mM of sodium pyruvate, 1mM of NEAA, 0.01mg/mL of insulin and 50ng/mLEGF, and the balance of DMEM.
The remaining features are the same as in example 1.
Fig. 8 is a photograph of a three-dimensional biological construct containing renal cancer primary tumor cells obtained by biological 3D printing of the present example.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (11)
1. A method of preparing a three-dimensional biological construct, comprising a step of bio-3D printing using bio-ink and a cross-linking step; wherein,
the biological ink is made of materials including biological materials and cell culture solution containing tumor cells;
the biological material comprises a gelatin solution and a sodium alginate solution;
the tumor cells are primary tumor cells.
2. The method of claim 1, wherein the primary tumor cells are extracted from fresh tumor tissue by the step of extracting.
3. The method for preparing a three-dimensional biostructure according to claim 2, wherein after the step of extracting the primary tumor cells from the fresh tumor tissue, the primary tumor cells are 2D-cultured or 3D-cultured, and a cell culture solution containing the primary tumor cells subjected to the 2D-culture step or 3D-culture step is mixed with the biomaterial; or,
after the step of extracting the primary tumor cells from the fresh tumor tissue, directly mixing a cell culture solution containing the primary tumor cells with the biological material.
4. The method for preparing a three-dimensional biological construct according to claim 2, wherein the step of extracting the primary tumor cells comprises the steps of:
s111: collecting fresh tumor tissue;
s112: digestion treatment;
s113: terminating digestion, collecting tumor cells, and resuspending the cells; or,
the extraction step of the primary tumor cells comprises the following steps:
s121: collecting fresh tumor tissue;
s122: performing shaking digestion treatment by using the first digestion solution;
s123: digesting with a second digestive juice;
s124: digestion was terminated.
5. The method of making a three-dimensional biological construct of claim 4, wherein the first digestive fluid is different from the second digestive fluid; preferably, the first digestion solution is an EDTA-free trypsin digestion solution, and the second digestion solution is a digestion solution containing collagenase and dnase.
6. The method of producing a three-dimensional biological construct of claim 1, wherein the biological material comprises a gelatin solution and a sodium alginate solution; preferably, the biological material consists of a gelatin solution and a sodium alginate solution; in the biological ink, the mass-volume ratio of gelatin is 0.05-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises a gelatin solution, a sodium alginate solution and a fibrinogen solution; preferably, the biological material consists of a gelatin solution, a sodium alginate solution and a fibrinogen solution; in the biological ink, the mass-volume ratio of gelatin is 0.05-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, the mass-volume ratio of fibrinogen is 1-5mg/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and matrigel; preferably, the biomaterial consists of a gelatin solution, a sodium alginate solution and matrigel; in the biological ink, the mass-volume ratio of gelatin is 0.0375-0.1g/mL, the mass-volume ratio of sodium alginate is 0.01-0.02g/mL, the volume fraction of matrigel is 5-30%, and the density of tumor cells is 1 multiplied by 106-5×106one/mL.
7. The method of claim 6, wherein the biological material is a gelatin solution and a sodium alginate solution, and the bio-ink comprises gelatin at a ratio of 0.0625g/mL, sodium alginate at a ratio of 0.01g/mL, and tumor cells at a density of 1 x 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and fibrinogen solution, wherein in the biological ink, the mass-volume ratio of gelatin is 0.05g/mL, the mass-volume ratio of sodium alginate is 0.01g/mL, the mass-volume ratio of fibrinogen is 0.03g/mL, and the density of tumor cells is 1 multiplied by 106-5×106Per mL; or,
the biological material comprises gelatin solution, sodium alginate solution and matrigel, wherein in the biological ink, the mass volume ratio of the sodium alginate is 0.01g/mL, the volume fraction of the matrigel is 30%, the mass volume ratio of the gelatin is 0.05g/mL, and the density of tumor cells is 1 multiplied by 106-5×106one/mL.
8. The method of preparing a three-dimensional biological construct according to claim 1, wherein the primary tumor cells include, but are not limited to, liver cancer primary tumor cells, cholangiocarcinoma primary tumor cells, kidney cancer primary tumor cells.
9. The method of making a three-dimensional biological construct of claim 1, wherein the cross-linking step is performed after the biological 3D printing step or during the biological 3D printing process.
10. A three-dimensional biological construct prepared by the method of any one of claims 1 to 9.
11. Use of the three-dimensional biological construct of claim 10 for drug discovery, or for drug screening, or for detecting the rate of inhibition of tumor cell growth in a three-dimensional biological construct by a drug, or for assessing the effect of a chemical agent or physical stimulus on tumor cells in a three-dimensional biological construct.
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