CN114306735B - 3D printing biological ink and preparation and application thereof - Google Patents
3D printing biological ink and preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of 3D biological printing, and particularly relates to 3D printing biological ink and preparation and application thereof. The biological ink comprises a sodium alginate solution, a hyaluronic acid-protein-chitosan system and a cell membrane wrapping system. The invention improves the toughness of the biological ink, constructs the hyaluronic acid-protein-chitosan matrix, and adds fibronectin, laminin and elastin, which can not only exist in the biological ink stably, but also promote the growth of cells in vitro. The application of the cell membrane wrapping technology greatly improves the utilization rate of the protein. The cell membrane wrapped laminin, the elastin and the sphingosine phosphate are added into the biological ink, so that the action protein is specifically combined with the printing cells, the degradation rate of the protein is reduced, long-term protein nutrition support is provided for in vitro culture of a printing structure, and the integrity of a printing result can be maintained for a long time.
Description
The technical field is as follows:
the invention belongs to the technical field of 3D biological printing, and particularly relates to 3D printing biological ink and preparation and application thereof.
Background art:
3D printing (3DP), a type of rapid prototyping technology, also known as additive manufacturing, is a technology that builds objects by layer-by-layer printing using bondable materials such as powdered metals or plastics based on digital model files. The technology has applications in jewelry, footwear, industrial design, construction, engineering and construction (AEC), automotive, aerospace, dental and medical industries, education, geographic information systems, civil engineering, firearms, and other fields. Nowadays, 3D printing is one of the representative leading technologies, and its application value has been recognized by many persons in the industry. In the medical field, 3D printing has gradually penetrated into a number of subdivided application scenarios such as surgical model rehearsal, rehabilitation medical device manufacturing, and the like. Under the promotion of the 3D printing front-edge technology, the service mode of the traditional medical industry is accelerating to change, and the intelligent, efficient and specialized medical service mode is accelerating to be developed.
Bio-ink is an ink that can be used in 3D printers, and is a technology for manufacturing living or non-living biological products using living cells, extracellular matrix, biological factors, and other biological materials as raw materials.
At present, most of biological inks have good mechanical property and biocompatibility, but facing in-vitro 3D cell model culture, how to construct a complex extracellular matrix becomes one of the problems to be solved urgently, and at present, many researchers add some cell growth factors into the biological inks, but the stability of the cell growth factors is poor, and the cell growth factors cannot exist in the biological inks for a long time, so that the culture of many in-vitro 3D cell models is unsatisfactory.
The invention content is as follows:
in order to solve the technical problems, the invention improves the toughness of the biological ink, constructs the hyaluronic acid-protein-chitosan matrix, and adds fibronectin, laminin and elastin, the proteins can stably exist in the biological ink and can promote the growth of in vitro cells, the hyaluronic acid-protein-chitosan matrix can delay the degradation of the proteins, and a more suitable matrix environment is created for the cells cultured in vitro for a long time.
The invention provides one of the technical schemes, which is a preparation method of 3D printing biological ink, and the preparation method specifically comprises the following steps:
step1, preparing a sodium alginate solution;
step2, preparing a hyaluronic acid-protein-chitosan system: dissolving hyaluronic acid, chitosan, collagen I and fibronectin in deionized water;
step3, preparing a cell membrane wrapping system: using cell membrane of target cell, dissolving and mixing laminin, elastin and sphingosine phosphate, and wrapping;
and Step4, mixing the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane wrapping system to obtain the biological ink.
Further, dissolving sodium alginate powder in deionized water in Step1 to prepare a sodium alginate solution with the concentration of 16-18% (m/v);
further, sterilizing with sodium alginate solution in Step1 with high pressure steam, and storing at 4 deg.C;
further, the weight ratio of hyaluronic acid, chitosan, collagen I and fibronectin in Step2 is 3.8-4.2: 2.2-2.6: 0.8-1.2: 0.8-1.2;
further, the ratio of the total mass of the hyaluronic acid, the chitosan, the collagen I and the fibronectin to the water in Step2 is 180-220: 4.8-5.2 (mg: ml);
further, Step2 is carried out at 4 deg.C to obtain hyaluronic acid-protein-chitosan system;
further, in Step3, firstly, extracting the cell membrane of the target cell by using a membrane protein cell component extraction kit;
further, the cell membrane used in Step3 can also be purchased commercially;
further, Step3 shows that the target cell is a target cell for printing, for example, if the target cell for printing is an HSF cell, the cell membrane of the HSF cell is extracted;
further, the weight ratio of the cell membrane, laminin, elastin and sphingosine phosphate of the cell in Step3 is 4.5-5: 1-1.2: 1-1.2: 1-1.2;
further, the ratio of the total mass of the cell membrane, laminin, elastin and sphingosine phosphate of the cell in Step3 to water is 38-45: 1.8-2.2 (mg: ml);
further, a cell membrane-encapsulated system was prepared by a liposome extruder in Step 3: mixing the cell membrane of the target cell with laminin, elastin, sphingosine phosphate and water, loading the mixture into a liposome extruder, and collecting the final liposome solution through the liposome extruders with different particle sizes;
further, Step3, loading 0.8 μm, 0.4 μm and 0.2 μm PC membranes into the liposome extruder, and removing impurities in the cell membrane system with gradient;
further, the volume ratio of the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane wrapping system in Step4 is 4.8-5: 4.8-5: 1.8-2.2;
further, the volume ratio of the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane wrapping system in Step4 is 5:5: 2;
further, after mixing a sodium alginate solution, a hyaluronic acid-protein-chitosan system and a cell membrane wrapping system, regulating the pH value of the system to 7.2-7.4 by adopting a HEPES buffer solution;
further, 4.7-5.2% CaCl is prepared 2 (m/v) solution as a coupling agent for bio-ink.
The second technical scheme provided by the invention is the biological ink prepared by the method in the first technical scheme.
The third technical scheme provided by the invention is the application of the bio-ink in the second technical scheme, in particular to the application in 3D printing, especially the application in skin tissue printing;
further, the method for printing the skin tissue by using the biological ink comprises the following steps:
culturing skin cells for 1-3 generations, mixing with biological ink, printing with a biological printer, controlling the thickness of the dermis and epidermis layers of the skin cells according to subsequent requirements, for example, printing 8 layers on the dermis layer and 4 layers on the epidermis layer, and culturing the printed tissue. The characteristics of the biological ink and the control of the culture conditions can lead the printed skin tissues to show a differentiation trend and keratinization of epidermal layers, can be used for the subsequent research of medicines and cosmetics, breaks through the inherent thinking of using animal models in the past, and lays a foundation for a large number of experimental researches.
Has the advantages that:
1. the porous structure of the hyaluronic acid-protein-chitosan matrix is beneficial to the slow release of protein, greatly delays the degradation speed of the protein and creates favorable conditions for the long-term culture of 3D cells.
2. The preparation process of the biological ink is improved, so that the biological ink has stronger toughness, the printing structure can be maintained for 8-12 weeks, and the biological ink on the market can be generally maintained for 1-6 weeks, thereby laying a foundation for subsequent researches on cosmetic toxicity detection tests, skin wound repair and the like.
3. The application of the cell membrane wrapping technology greatly improves the utilization rate of the protein. The cell membrane wrapped laminin, the elastin and the sphingosine phosphate are added into the biological ink, so that the action protein is specifically combined with the printing cells, the degradation rate of the protein is reduced, long-term protein nutrition support is provided for in vitro culture of the printing structure, and the integrity of the printing structure can be maintained for a long time.
4. The sphingosine phosphate is particularly added, the sphingosine phosphate is combined with cell membranes, so that the utilization rate of the sphingosine phosphate can be greatly improved, an important supporting effect on a printing structure is achieved, and the death of normal cells is inhibited, so that the method provides good help for long-term in-vitro culture of printing tissues.
5. The biological ink is safe and nontoxic in material.
Description of the drawings:
FIG. 1 is a flow chart of the preparation of bio-ink according to the present invention.
FIG. 2 shows a finished product of cell printing by using the bio-ink of the present invention.
FIG. 3 HE staining patterns of cells printed using the bio-ink of the present invention.
FIG. 4 is a graph of viable and dead cell staining of cells printed using the bio-ink of the present invention.
The specific implementation mode is as follows:
the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The materials, reagents and the like used are commercially available unless otherwise specified.
Example 1A 3D printing bio-ink and a method for preparing the same
The preparation process is shown in figure 1.
Dissolving 0.85g of sodium alginate powder in 5ml of deionized water, performing high-pressure steam sterilization after complete dissolution, and storing at 4 ℃.
Preparing 5 percent (m/v) hyaluronic acid aqueous solution and 3 percent (m/v) chitosan aqueous solution at 4 ℃, wherein 2ml of hyaluronic acid aqueous solution and 2ml of chitosan aqueous solution are prepared according to the volume ratio of 1: 1, 500. mu.L of 50mg/ml collagen I and 500. mu.L of 50mg/ml fibronectin were added and stored at 4 ℃.
③ using a membrane protein cell component extraction kit (Shanghai Biyuntian biotechnology Co., Ltd. P0033) to respectively extract cell membranes of HSF and HaCaT cells, adjusting the cell number to obtain 50mg/ml cell membranes, mixing 500 uL of 10mg/ml laminin, 500 uL of 10mg/ml elastin and 500 uL of 10mg/ml sphingosine phosphate with 500 uL of HSF cell membranes and 500 uL of HaCaT cell membranes, standing at 4 ℃ for 15min to prepare two cell membrane encapsulation systems.
Loading 0.8 μm PC membrane (Whatman) into liposome extruder (Muger machine), loading the mixture into liposome extruder, repeatedly pushing and pulling push rod, repeating for 5 times, and pouring the product into clean test tube;
the final liposome solution was collected by passing the mixture in the test tube of the previous step sequentially through 0.4 μm, 0.2 μm PC membranes using the same procedure, at which time the lower cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HSF cells and the upper cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HaCaT cells were obtained, respectively.
Mixing the hyaluronic acid-protein-chitosan system with the sodium alginate solution and the lower layer cell membrane system according to the volume ratio of 5:5:2 to prepare the lower layer cell bio-ink;
mixing a hyaluronic acid-protein-chitosan system, a sodium alginate solution and an upper cell membrane system according to a volume ratio of 5:5:2 to prepare upper cell bio-ink;
the two bio-inks prepared at this time are different only in the kind of cell membrane, so that the preparation is more beneficial to the specific binding of the protein and the printed cells.
Adding HEPES buffer solution to regulate pH value to 7.2-7.4.
Sixthly, CaCl with the concentration of 5.0 percent is arranged 2 The solution is used as a coupling agent of the bio-ink.
EXAMPLE 23D cell printed Final product
1. Selecting Human Skin Fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) which grow vigorously after subculture for 24-48h, digesting the human skin fibroblasts and the human immortalized keratinocytes (HaCaT) for 5min by Trypsin-EDTA, and re-suspending the HaCaT cells by the upper cell bio-ink prepared in the example 1 to ensure that the concentration of the HaCaT cells in the bio-ink is 1 multiplied by 10 6 cells/ml, HSF cells were resuspended in the lower cell bio-ink prepared in example 1 to a HSF cell concentration of 5.5X 10 in the bio-ink 7 cells/ml (note that gentle blowing is needed to avoid generating bubbles and affecting printing), and the cells/ml are respectively loaded into a low-temperature charging barrel to prepare for printing. Meanwhile, other biological inks are adopted to prepare a control group, which comprises the following specific steps:
experimental groups: the bio-ink prepared in example 1 was used.
Control group 1: adopting the step three of removing the cell membrane wrapping system in the embodiment 1 and preparing the biological ink which is the same as the biological ink prepared in the embodiment 1;
control group 2: removing the components of the cell membrane in the step III in the embodiment 1, keeping the other components unchanged, and preparing the biological ink in the same way as the biological ink in the embodiment 1;
control group 3: removing sphingosine phosphate in the cell membrane wrapping system by adopting the step III of the embodiment 1, and preparing the biological ink with the concentration and the addition amount of the other components being the same as those of the biological ink prepared in the embodiment 1;
2. the cartridge mixed with cells prepared in step1 is loaded into a low-temperature shower head and loaded into a printer (
WS), with leading-in software of Bio3DPrintSKIN. stl model, set up to print the orbit and be "bow" shape, the lower floor prints the cell and is HSF, and the number of piles is 8 layers, and the upper strata prints the cell and is 4 layers for the HaCaT number of piles, places the culture dish at print platform, sets up printing pressure 60-80kPa, and printing speed is 5mm/S, and printing needle head diameter is 0.22mm, begins to print. After printing, 2 drops of CaCl with the concentration of 5 percent are dripped 2 And (3) crosslinking and fixing for 10min at 4 ℃, and washing for 2-3 times by using precooled PBS (phosphate buffer solution) so as to obtain a printed finished product. DMEM medium (10% fetal bovine serum, 1% cyan-chain double antibody) was added and the mixture was transferred to 37 ℃ with 5% CO 2 Culturing in a cell culture box.
3. After culturing for 12 weeks in a cell culture box, the printed product is placed under an optical microscope for morphological observation, and the result is shown in fig. 2, and the structure (experimental group) printed by the biological ink in the embodiment 1 of the invention still keeps relatively complete compared with other control groups; the first, second and third control groups showed collapse in different degrees in 3 rd, 6 th and 5 th weeks after printing, respectively, with the first control group being the most serious.
(1) Compared with a control group, the experimental group still keeps complete structure, and the cell membrane wrapping technology can improve the toughness of the biological ink.
(2) Compared with the control group III and the control group I, the structure of the contrast group II is kept relatively complete, which shows that sphingosine phosphate plays an important role in controlling the structural integrity in the culture of 3D printing tissues, so that the printing structure is more stable (the damage of the contrast group II can be obviously seen to be less).
In conclusion, the cell membrane wrapping system, particularly the sphingosine phosphate component in the cell membrane wrapping system, can improve the toughness of the printing tissue, maintain the structural integrity, enable the printing tissue to be cultured for a long time and facilitate the subsequent histological research.
Example 33 finished HE staining of cell prints
1. The printed product obtained in step2 of example 2 (printed tissue composed of human dermal fibroblasts (HSF) at the lower layer and human immortalized keratinocytes (HaCaT) at the upper layer) was cultured in DMEM medium for 7 days in an immersion manner, and then replaced with Epi-life medium for gas-liquid critical culture, so that HaCaT was exposed and cultured, and HSF was immersed in the culture medium to promote the formation of stratum corneum. The printed tissue at 12 weeks after printing (i.e., 11 weeks in gas-liquid critical culture) was embedded in o.c.t.compound and fixed at-80 ℃ for 24 hours, and then taken out to prepare a section.
2. The temperature of the microtome (Leica CM1950) and the blade was lowered in advance, and when the temperature of the box was-18 ℃ and the temperature of the quick freezing table was-25 ℃, the microtome and the blade were cut into sections of 8 μm thickness.
3. Immediately after the slicing, the cells were fixed in AAF mixed fixative at 4 ℃ for 10 min.
4. And (3) after dyeing with hematoxylin for 3min, returning to blue with warm water, dropwise adding eosin dye, washing off the eosin dye for about 15s, sealing with neutral resin, and observing and taking a picture.
5. The results are shown in FIG. 3.
6. The structure printed by the biological ink shows the differentiation trend of the horny layer after being cultured for 12 weeks, while the structures printed by other biological inks are cracked at the 4 th week (a control group 1), the 6 th week (a control group 2) and the 5 th week (a control group 3), and the structures are cultured to the 12 th week (counted according to the time after printing) under the same condition and then dyed (according to the steps 1-4 of the embodiment 3), and the dyeing result shows that:
(1) the biological ink disclosed by the invention is more stable in structure, can adapt to long-term in-vitro tissue culture, and the cell printing structure obviously develops towards tissues and is already divided into a dermis layer and an epidermis layer, so that a good guarantee is provided for subsequent research.
(2) Compared with the control group, the experimental group still keeps the complete structure, and the control group has a separation fault of the dermis layer and the epidermis layer, which shows that the cell membrane component plays a key role in whether the printed cells can tend to normal tissue growth, and the cell membrane component promotes the printed cells to generate tissues.
(3) The control group I and the control group III both have the dissociation and the breakage of the structure, the breakage degree of the control group I is more serious, and the structure of the control group II is relatively complete, so that the sphingosine phosphate plays a supporting role on the structure of the whole printing tissue and the death ratio of cells is reduced.
(4) From the dyeing result, the growth state of the cells can be found, although the control group II has faults between the dermis layer and the epidermis layer, the growth state of the cells is good, and the epidermis layer has keratinization due to gas-liquid critical culture; the number of cells in the control group I and the control group III is obviously reduced, and some cells are deeply stained and fragmented, and the control group I is more serious, which is enough to indicate that the protein and the sphingosine phosphate (especially the sphingosine phosphate) in the cell membrane wrapping system can inhibit the apoptosis of normal cells.
In conclusion, the cell membrane coating system has a supporting effect on the structure of the printing tissue and promotes the differentiation of the printing cells, wherein the supporting effect and the apoptosis inhibiting effect of the sphingosine phosphate are obvious, and the cell membrane coating system is favorable for the printing cells to tend to the tissue differentiation, and is favorable for subsequent animal experiments, pathological tests, morphology and other researches.
Examples 3-6 were followed for both the experimental and control groups of example 2.
EXAMPLE 43D Final Living and dead cell staining of cell printing
1. The printed product obtained in step2 of example 2 (printed tissue composed of human dermal fibroblasts (HSF) at the lower layer and human immortalized keratinocytes (HaCaT) at the upper layer) was cultured in DMEM medium for 7 days in an immersion manner, and then replaced with Epi-life medium for gas-liquid critical culture, so that HaCaT was exposed and cultured, and HSF was immersed in the culture medium to promote the formation of stratum corneum. The printed tissue at 12 weeks after printing was stained for viable and dead cells, and the staining reagents were purchased from Beyotime, inc, Calcein/PI kit for cell activity and cytotoxicity detection (Beyotime C2015).
The cells were washed 2-3 times with PBS, added with the appropriate volume of Calcein AM/PI and incubated at 37 ℃ for 30min in the absence of light. After the incubation, the staining effect was observed under a fluorescence microscope (Calcein AM: green fluorescence, Ex/Em: 494/517 nm; PI: red fluorescence, Ex/Em: 535/617 nm). Attention is paid to the whole process and the operation is carried out in a dark place.
2. The results are shown in FIG. 4. (Red fluorescence is dead cells, green fluorescence is live cells)
(1) The green fluorescence ratio of the experimental group is the highest compared with that of all the control groups, which indicates that the number of living cells in the experimental group is the largest, thereby confirming that the biological ink system of the experimental group can effectively inhibit the death of the cells.
(2) Compared with the experimental group, the control group II and the control group III both show two kinds of fluorescence. Compared with the control group, the experimental group has high occupation ratio of green fluorescence, which shows that the protein wrapped by the cell membrane can provide nutrient substances for long-term in-vitro culture of the printing cells. The experiment group is higher in occupation ratio of green fluorescence than the control group, and the edge of the printing structure at the lower right corner of the control group is unclear, which proves that the sphingosine phosphate plays an important role in supporting the printing structure.
(3) The control group (i) has almost no green fluorescence, indicating that the cells have very poor survival under the condition of no trophic protein and sphingosine phosphate.
In conclusion, the cell membrane encapsulation system can inhibit normal cell death.
Example 53D printed cell viability
The cell survival rate of the biological ink printed by the biological ink is compared with that of other biological inks.
1. The prepared product of step2 in example 2 (printing tissue consisting of Human Skin Fibroblasts (HSF) at the lower layer and human immortalized keratinocytes (HaCaT) at the upper layer) is soaked and cultured in a DMEM medium for 7 days, and then is replaced by an Epi-life medium for gas-liquid critical culture, so that HaCaT is exposed and cultured, and HSF is submerged and cultured to promote the formation of stratum corneum. Suspensions (100 μ L/well) of cells were seeded into 96-well plates for different culture times (1-13 weeks after printing), respectively;
2. add 10. mu.L of CCK8 solution to each well (care not to generate bubbles while protecting from light);
3. incubator (37 ℃ 5% CO) 2 ) After 2h incubation, the cells were removed and the absorbance at 450nm was measured.
4. The survival rate was converted according to the standard curve.
By comparison it can be seen that:
the survival rate of the printed cells of the experimental group and the control group is high in the first week, and the survival rate of the cells of the experimental group is higher than that of the control group from the second week, which shows that the biological ink of the experimental group provides the optimal growth environment for the cells. In conclusion, the cell membrane coating system can reduce the death rate of normal cells, supply nutrients to the cells for a long time and prolong the life cycle of the 3D printed cells.
Example 6 protein degradation Rate of Bio-ink
1. The finished product of step2 in example 2 (printed tissue consisting of human dermal fibroblasts (HSF) at the lower layer and human immortalized keratinocytes (HaCaT) at the upper layer) was cultured in DMEM medium for 7 days in a soaking manner, and then replaced with Epi-life medium for gas-liquid critical culture (12-well plate) to allow HaCaT to be cultured in an exposing manner and HSF to be cultured in a soaking manner, thereby promoting the formation of stratum corneum. And (3) cleaning the printed tissues with different culture times (1 st, 3 rd, 5 th, 7 th, 9 th and 12 th weeks after printing) by using PBS (phosphate buffer solution), discarding the PBS, adding 800 mu L of biuret reagent into each well (mlbio), fully immersing and uniformly mixing, standing for 15min, taking 150 mu L of biuret reagent into a 96-well plate, carrying out color comparison at 540nm, and determining the protein content in the printed structure.
2. Standard and blank tubes were prepared for the assay and 540nm colorimetry as per the biuret reagent (mlbi) specifications. The standard and blank tubes need only be measured once.
3. The protein concentration in the sample was calculated and is shown in the table below.
By comparison it can be seen that:
(1) the protein content of the finished product printed by the biological ink is higher than that of all control groups, which shows that the protein in the biological ink is high in binding efficiency with printing cells, and the degradation amount of the protein in the biological ink is lowest after long-term culture.
(2) Compared with a control group, the experimental group has high protein content, which shows that the cell membrane wrapping component is beneficial to long-term maintenance of the protein in the printing structure, thereby providing sustainable nutrient supply for the printing cells.
In conclusion, the cell membrane encapsulation system creates opportunities for binding and long-term stabilization of proteins in the printed structure.
Example 7A 3D printing bio-ink
Dissolving 0.8g of sodium alginate powder in 4.8ml of deionized water, sterilizing by high-pressure steam after complete dissolution, and storing at 4 ℃.
Preparing 5 percent (m/v) hyaluronic acid aqueous solution and 3 percent (m/v) chitosan aqueous solution at 4 ℃, wherein 2ml of hyaluronic acid aqueous solution and 2ml of chitosan aqueous solution are prepared according to the volume ratio of 1: 1, 490. mu.L of 50mg/ml collagen I and 490. mu.L of 50mg/ml fibronectin were added and stored at 4 ℃.
③ using a membrane protein cell component extraction kit (Shanghai Biyuntian biotechnology Co., Ltd. P0033) to respectively extract cell membranes of HSF and HaCaT cells, adjusting the cell number to obtain 50mg/ml cell membranes, mixing 480 uL 10mg/ml laminin, 480 uL 10mg/ml elastin and 480 uL 10mg/ml sphingosine phosphate with 480 uLHSF cell membranes and 480 uLHaCaT cell membranes, and standing at 4 ℃ for 15min to prepare two cell membrane coating systems.
Loading 0.8 μm PC membrane (Whatman) into liposome extruder (Muger machine), loading the mixture into liposome extruder, repeatedly pushing and pulling push rod, reciprocating for 5 times, and pouring the product into clean test tube;
the final liposome solution was collected by passing the mixture in the test tube of the previous step sequentially through 0.4 μm, 0.2 μm PC membranes using the same procedure, at which time the lower cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HSF cells and the upper cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HaCaT cells were obtained, respectively.
Mixing the hyaluronic acid-protein-chitosan system with the sodium alginate solution and the lower layer cell membrane system according to the volume ratio of 4.8:4.98:1.92 to prepare the lower layer cell bio-ink;
mixing a hyaluronic acid-protein-chitosan system, a sodium alginate solution and an upper cell membrane system according to a volume ratio of 4.8:4.98:1.92 to prepare upper cell bio-ink;
the two bio-inks prepared at this time are different only in the kind of cell membrane, so that the preparation is more beneficial to the specific binding of the protein and the printed cells.
Adding HEPES buffer solution to regulate pH value to 7.2-7.4.
Sixthly, 4.7 percent of CaCl is arranged 2 The solution is used as a coupling agent of the bio-ink.
When the biological ink is used for printing finished products according to the method in the embodiment 2, the printing structure can be maintained for 12 weeks in the tissue culture process, and the biological ink can be used for subsequent cosmetic toxicity detection tests, skin wound repair and the like.
Example 8A 3D printing bio-ink
Dissolving 0.86g of sodium alginate powder in 4.9ml of deionized water, sterilizing by high-pressure steam after complete dissolution, and storing at 4 ℃.
Preparing 5 percent (m/v) hyaluronic acid aqueous solution and 3 percent (m/v) chitosan aqueous solution at 4 ℃, wherein 2ml of hyaluronic acid aqueous solution and 2ml of chitosan aqueous solution are prepared according to the volume ratio of 1: 1, adding 510. mu.L of 50mg/ml collagen I and 510. mu.L of 50mg/ml fibronectin, and storing at 4 ℃.
③ using a membrane protein cell component extraction kit (Shanghai Biyuntian biotechnology Co., Ltd. P0033) to respectively extract cell membranes of HSF and HaCaT cells, adjusting the cell number to obtain 50mg/ml cell membranes, mixing 520 uL of 10mg/ml laminin, 520 uL of 10mg/ml elastin and 520 uL of 10mg/ml sphingosine phosphate with 520 uLHSF cell membranes and 520 uLHaCaT cell membranes, standing at 4 ℃ for 15min, and preparing two cell membrane coating systems.
Loading 0.8 μm PC membrane (Whatman) into liposome extruder (Muger machine), loading the mixture into liposome extruder, repeatedly pushing and pulling push rod, reciprocating for 5 times, and pouring the product into clean test tube;
the final liposome solution was collected by passing the mixture in the test tube of the previous step sequentially through 0.4 μm, 0.2 μm PC membranes using the same procedure, at which time the lower cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HSF cells and the upper cell membrane system formed by cell membrane-encapsulated laminin, elastin and sphingosine phosphate of HaCaT cells were obtained, respectively.
Mixing the hyaluronic acid-protein-chitosan system with the sodium alginate solution and the lower layer cell membrane system according to the volume ratio of 4.9:5.02:2.08 to prepare the lower layer cell bio-ink;
mixing a hyaluronic acid-protein-chitosan system, a sodium alginate solution and an upper cell membrane system according to a volume ratio of 4.9:5.02:2.08 to prepare upper cell bio-ink;
the two bio-inks prepared at this time are different only in the kind of cell membrane, so that the preparation is more beneficial to the specific binding of the protein and the printed cells.
Adding HEPES buffer solution to regulate pH value to 7.2-7.4.
Sixthly, the CaCl with the concentration of 5.2 percent is arranged 2 The solution is used as a coupling agent of the bio-ink.
Finished product printing is carried out by using the biological ink according to the method in the embodiment 2, and a printing structure can be maintained for 12 weeks in the tissue culture process, so that the biological ink can be used for subsequent cosmetic toxicity detection tests and skin wound repair.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of 3D printing biological ink is characterized by comprising the following steps:
step1, preparing a sodium alginate solution;
step2, preparing a hyaluronic acid-protein-chitosan system: dissolving hyaluronic acid, chitosan, collagen I and fibronectin in deionized water;
step3, preparing a cell membrane wrapping system: using cell membrane of target cell, dissolving and mixing laminin, elastin and sphingosine phosphate, and wrapping;
step4, mixing the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane wrapping system to prepare the biological ink;
preparing a cell membrane wrapping system by a liposome extruder in Step 3: mixing the cell membrane of the target cell with laminin, elastin, sphingosine phosphate and water, loading into a liposome extruder, and collecting the final liposome solution through liposome extruders with different particle sizes.
2. The method for preparing 3D printing bio-ink according to claim 1, wherein sodium alginate powder is dissolved in deionized water in Step1 to prepare a sodium alginate solution with a concentration of 16% -18%.
3. The method for preparing a bio-ink for 3D printing according to claim 1, wherein the weight ratio of hyaluronic acid, chitosan, collagen I and fibronectin in Step2 is 3.8-4.2: 2.2-2.6: 0.8-1.2: 0.8-1.2;
the ratio of the total mass of the hyaluronic acid, the chitosan, the collagen I and the fibronectin to the water in Step2 is 180-220: 4.8-5.2.
4. The method of claim 1, wherein Step3 is performed by extracting the cell membrane of the target cell using a membrane protein cell fraction extraction kit, or by purchasing the cell membrane of the target cell commercially.
5. The method of claim 1, wherein the weight ratio of cell membrane, laminin, elastin and sphingosine phosphate in Step3 is 4.5-5: 1-1.2: 1-1.2: 1-1.2;
the ratio of the total mass of cell membranes, laminin, elastin and sphingosine phosphate to water in Step3 is 38-45: 1.8-2.2.
6. The method for preparing a bio-ink for 3D printing according to claim 1, wherein the volume ratio of the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane encapsulation system in Step4 is 4.8-5: 4.8-5: 1.8-2.2.
7. The method for preparing a 3D printing bio-ink according to claim 1, wherein after the sodium alginate solution, the hyaluronic acid-protein-chitosan system and the cell membrane encapsulation system are mixed, HEPES buffer solution is used to adjust the pH of the system to 7.2-7.4.
8. A bio-ink prepared by the method of any one of claims 1 to 7.
9. Use of the bio-ink of claim 8 in 3D printing.
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