3D Bioprinting Ink for Cultured Meat and Method for Preparing a Cell Culture Scaffold

Abstract

The present invention provides a 3D bioprinting ink and a method for preparing a cell culture scaffold. Among them, the 3D bioprinting ink uses nano-starch as a functional factor, greatly enhancing the 3D printing properties of the hydrogel under low-temperature conditions and significantly promoting the adhesion and proliferation of fish myoblasts. The cell culture scaffold printed based on this ink can effectively promote the adhesion and proliferation of myoblasts compared with ordinary gelatin scaffolds. The nano-starch, gelatin, and sodium alginate of the present invention are all food-grade raw materials and have great application prospects in the field of cultured meat.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a preparation method of an edible nano-starch enhanced bioink for cultured meat. Background Art

[0002] Cultured meat is "grown" from animal myoblasts, adipocytes or endothelial cells cultured in vitro in an artificial scaffold and culture medium, and is considered to be the most promising strategy to solve the future dilemma of meat production. In recent years, many researchers have focused on constructing structured cultured meat through forming methods such as electrospinning, microcarrier manufacturing, 3D bioprinting, etc. to mimic the texture and taste of native meat. The first two methods are usually used to inoculate cells on a preformed scaffold, but it is difficult to achieve three-dimensional (3D) growth of cells in the hierarchical structure to simulate native meat. 3D bioprinting, also known as 3D cell printing, combines the advantages of simulation design and precise control of the spatial structure and biological benefits of cultured cells, and is widely used in fields such as regenerative medicine and cultured meat. However, it is a great challenge for bioinks to maintain the high activity of seeded cells while maintaining good printability and mechanical strength, especially when used as an edible scaffold material.

[0003] Therefore, when printing cells in 3D, some nano-scale active particles are often added to enhance cell activity. Manganese dioxide (MnO2) nanoparticles, TiO2 metal nanoparticles, etc. help to improve the activity of osteoblasts and have certain potential in bone repair and bone induction. However, the use of nanoparticles often increases the viscosity of bioinks, increases the critical extrusion pressure of the nozzle, and reduces printability. In addition, previous studies on nanoparticles have mostly focused on the field of tissue engineering, and most of them are inedible and cannot be applied in the field of cultured meat. In the food field, starch and cellulose are usually used as the main components for 3D printing plant-based foods, and their addition often leads to an increase in the viscosity of the system and an increase in the critical extrusion pressure of the nozzle. Compared with mammalian cells, the growth temperature of fish myoblasts is relatively low, usually below 27°C. And the decrease in printing temperature will cause great changes in the properties of bioinks. All in all, for cultured meat, there is currently a lack of a highly applicable bioink to support the low-temperature (<25°C) 3D printing and growth of fish myoblasts. Summary of the Invention

[0004] To solve this problem, we propose a new type of bioink; a certain proportion of functional factors are added to the 3D printing substrate material. The functional factors are evenly distributed in the substrate material and form a cell culture scaffold after being printed together with cells. On the one hand, these functional factors have a nano size, which can improve the adhesion of cells, and the special carbon chain structure of the functional factors can effectively promote cell proliferation; on the other hand, the nano-sized functional factors can shear the substrate material more effectively, enabling the extrusion and printing of the substrate material under mild conditions.

[0005] The present invention adopts the following technical solution: a 3D bioprinting ink, comprising a substrate material and nano starch; the mass ratio of the substrate material to the nano starch is 10:1 to 2:1; the size of the nano starch is 10 nm to 500 nm; the substrate material can form a gel under the action of an initiator.

[0006] Further, the substrate material is gelatin and sodium alginate; the initiator is a CaCl2 solution.

[0007] Further, the nano starch is prepared by the following method:

[0008] Dissolve 5 g of commercially available food-grade corn starch (including but not limited to CAS No.: 9005-25-8, CAS: 9037-22-3) in 100 mL of NaOH solution (0.01 g / mL) to obtain a starch suspension; pour the starch suspension into 100 mL of ethanol for antisolvent precipitation, and then centrifuge the liquid phase system containing the self-assembled nano starch precipitate at 5000×g for 5 min. After removing the supernatant, the nano starch precipitate is obtained, and it is washed 3 times with anhydrous ethanol and deionized water respectively; finally, the nano starch is freeze-dried at -60°C for 48 h for dehydration and ground through a 200-mesh sieve.

[0009] The present invention also provides a method for preparing a cell culture scaffold using the above ink, comprising the following steps:

[0010] (1) Disperse the nano starch in a culture medium to form a nano particle suspension; add the substrate material, mix evenly, and then add cells to form a printing solution;

[0011] (2) After 3D printing, use an initiator to initiate crosslinking to form a cell culture scaffold.

[0012] Further, in step (2), the extrusion pressure of 3D printing is below 0.2 MPa.

[0013] Further, in step (2), the printing temperature of 3D printing is below 25°C.

[0014] Further, in the step 1, the substrate material is a mixture of gelatin and sodium alginate. In the printing solution, the concentration of gelatin is 0.05 - 0.1 g / mL; the concentration range of sodium alginate is 0.01 - 0.05 g / mL; the dissolution temperature range of gelatin and sodium alginate in the nanoparticle suspension is 40°C - 70°C, and the dissolution time is more than 2 h. The initiator in step 2 is CaCl2, with a concentration of 1%, and the crosslinking time range is 1 min - 1 h.

[0015] Further, in the printing solution of step 1, the cell concentration is 5×10 6 cells / mL.

[0016] The beneficial effects of the present invention are as follows: The present invention uses nano starch as a functional factor to form a bioink, which is used to print with cells to form a cell culture scaffold. Under low temperature conditions, it has good 3D printability. Compared with ordinary gelatin scaffolds, it can effectively promote the adhesion and proliferation of myoblasts. Moreover, the nano starch, gelatin, and sodium alginate used in this method are all food-grade raw materials, and it has great application prospects in the field of cultured meat.

[0017] (1) Using the bioink prepared by this method, it has excellent biocompatibility with fish myoblasts. After culturing for 5 days, the cell survival rate increases by 20.8%, and the adhesion rate increases by 36.1%, greatly improving the growth effect of cells in the three-dimensional scaffold.

[0018] (2) Using the bioink prepared by this method, the addition of nano starch improves the printing properties of the gelatin-based bioink, enabling fish cells to be printed and extruded at 25°C with an extrusion pressure less than 0.2 MPa, greatly expanding the cell applicability range of 3D printing.

[0019] (3) Using the bioink prepared by this method, cell-cultured meat products with a higher cell density, a higher myotube content, and a water content similar to natural meat can be obtained. Description of the Drawings

[0020] Figure 1 is the 3D printing extrusion effect in Comparative Example 1;

[0021] Figure 2 is the cell adhesion situation after culturing for 5 days in Comparative Example 1. The round ones are non-adherent cells, and the spindle-shaped ones are adherent cells;

[0022] Figure 3 is the cell proliferation situation in the hydrogel after culturing for 10 days in Comparative Example 1;

[0023] Figure 4 is the nanoparticle size distribution of nano starch in Example 1;

[0024] Figure 5 is the 3D printing extrusion effect in Example 1;

[0025] Figure 6 It is the 3D printed scaffold in Example 1;

[0026] Figure 7 It is the cell adhesion situation after 5 days of culture in Example 1. The round ones are non-adherent cells, and the fusiform ones are adherent cells;

[0027] Figure 8 It is the cell proliferation situation in the hydrogel after 10 days of culture in Example 1;

[0028] Figure 9 It is the particle size distribution of nano-starch in Example 2. Specific implementation mode

[0029] Comparative example 1

[0030] In this example, nano-starch is not added, serving as a control group for other examples.

[0031] Preparation of bioink: Take 1 g of gelatin and 0.1 g of sodium alginate, dissolve them in 10 mL of water, and heat at 50 °C for 3 h; after cooling to 27 °C, mix with cells until the cell concentration is 5×10 6 / mL, and incubate at 27 °C for standby. The gel point of this formulated bioink measured by a rheometer is 27.3 °C, and it can barely be printed at a low temperature (25 °C). The extrusion pressure of the nozzle is 0.2 MPa, and the microfilaments printed by 3D are not continuous, and the extrusion property is not good ( Figure 1 ).

[0032] Place the printed structure in 1% CaCl2 solution for crosslinking for 10 min to form a shape, and then transfer it to high-glucose DMEM medium for culture.

[0033] After 5 days of culture, the cell viability was detected by live-dead staining, and the cell viability was 73.5%, and 40.8% of them ( Figure 2 ) of the cells changed from round to spindle-shaped, proving successful adhesion.

[0034] After culturing the 3D bioprinted cell scaffold containing fish myoblasts in the growth medium for 10 d, the growth of PSCs was observed by Calcein-AM fluorescence staining ( Figure 3 ). After 10 d of proliferation, PSCs partially filled the nano-starch hydrogel scaffold, and its growth and proliferation were poor.

[0035] Comparative example 2

[0036] In this example, 100 nm polystyrene nanospheres are used in the printing solution.

[0037] Manufacture of nanoparticle suspension: 0.1 g of polystyrene nanospheres with a diameter of 100 nm was added to 10 mL of water, and the homogenizer was used to homogenize at a shear speed of 10000 rpm / min for 1 min to prepare a 0.01 g / mL nanoparticle suspension. 1 g of gelatin and 0.1 g of sodium alginate were dissolved in 10 mL of the nanoparticle suspension with a concentration of 0.01 g / mL, and heated at 50 °C for 3 h; after cooling to 27 °C, it was mixed with cells until the cell concentration was 5×10 6 / mL, and incubated at 27 °C for standby. The gel point of this formulated bioink was measured to be 27.8 °C by a rheometer, which did not support printing under low temperature conditions (25 °C), and could not be extruded when the nozzle extrusion pressure was 0.2 MPa.

[0038] Comparative Example 3

[0039] 0.1 g of ordinary corn starch (CAS No.: 9005-25-8) was added to 10 mL of water, and the homogenizer was used to homogenize at a shear speed of 10000 rpm / min for 1 min to make it stir evenly. 1 g of gelatin and 0.1 g of sodium alginate were dissolved in 10 mL of the starch particle suspension with a concentration of 0.01 g / mL, and heated at 40 °C for 3 h; after cooling to 27 °C, it was mixed with cells until the cell concentration was 5×10 6 / mL, and incubated at 27 °C for standby. The gel point of this formulated bioink was measured to be 28.1 °C by a rheometer, which did not support printing under low temperature conditions (25 °C), and could not be extruded when the nozzle extrusion pressure was 0.2 MPa.

[0040] Example 1

[0041] Manufacture of nano starch: 5 g of corn starch (CAS No.: 9005-25-8) was dissolved in 100 mL of NaOH solution (1% w / v) to obtain a starch suspension; the starch suspension was poured into 100 mL of ethanol for antisolvent precipitation, and then the liquid phase system containing the self-assembled nano starch precipitate was centrifuged at 5000×g for 5 min. After removing the supernatant, the nano starch precipitate was obtained, and washed 3 times with absolute ethanol and deionized water respectively; finally, the nano starch was freeze-dried at -60 °C for 48 h to dehydrate and ground through a 200-mesh sieve to obtain nano starch, and its particle size range was between 100 and 500 nm ( Figure 4 ). 0.1 g of nano starch was added to 10 mL of water, and the homogenizer was used to homogenize at a shear speed of 10000 rpm / min for 1 min to prepare a 0.01 g / mL nanoparticle suspension.

[0042] Preparation of bioink: 1 g of gelatin and 0.1 g of sodium alginate were dissolved in 10 mL of the nanoparticle suspension with a concentration of 0.01 g / mL, and heated at 50 °C for 3 h; after cooling to 27 °C, it was mixed with cells until the cell concentration was 5×10 6 / mL, incubate it at 27 °C for later use. The gel point of this formulated bioink was determined to be 25.6 °C by a rheometer, enabling printing at a low temperature (25 °C). The extrusion pressure of the nozzle was 0.1 MPa, and the microfilaments printed by 3D printing were relatively smooth ( Figure 5 ), and its printed scaffold is as Figure 6 shown.

[0043] Place the printed structure in a 1 wt% CaCl2 solution for crosslinking for 10 min to form a shape, and then transfer it to high-glucose DMEM medium for culture.

[0044] After culturing for 5 days, the cell viability was detected by live / dead staining ( Figure 7 ). Its cell viability was 91.2%, and 71.0% of the cells changed from round to spindle-shaped, proving successful adhesion and implantation.

[0045] After culturing the 3D bioprinted cell scaffold containing fish myoblasts in growth medium for 10 d, Calcein-AM fluorescence staining was used to observe the growth of PSCs ( Figure 8 ). After 10 d of proliferation, the PSCs basically filled the nano-starch hydrogel scaffold, and its growth and proliferation were good.

[0046] Example 2

[0047] Manufacture of nano-starch: Dissolve 5 g of corn starch (CAS: 9037-22-3) in 100 mL of NaOH solution (0.01 g / mL) to obtain a starch suspension; pour the starch suspension into 100 mL of ethanol for anti-solvent precipitation. Subsequently, centrifuge the liquid phase system containing the self-assembled nano-starch precipitate at 5000×g for 5 min, remove the supernatant to obtain the nano-starch precipitate, and wash it 3 times with absolute ethanol and deionized water respectively; finally, freeze-dry the nano-starch at -60 °C for 48 h to dehydrate it, and grind it through a 200-mesh sieve to obtain nano-starch, whose particle size range is between 100 and 500 nm ( Figure 9 ). Add 0.25 g of nano-starch to 10 mL of water, and homogenize it with a homogenizer at a shear speed of 10000 rpm / min for 1 min to prepare a 0.025 g / mL nano-particle suspension.

[0048] Preparation of bioink: Dissolve 0.5 g of gelatin in 10 mL of nano-particle suspension with a concentration of 0.025 g / mL, then add 0.5 g of sodium alginate, and heat it at 70 °C for 2 h; after cooling to 27 °C, mix it with cells until the cell concentration is 5×10 6 / mL, incubate it at 27 °C for later use. The gel point of this formulated bioink was determined to be 24.2 °C by a rheometer, enabling printing at a low temperature (25 °C). The extrusion pressure of the nozzle was 0.13 MPa, and the microfilaments printed by 3D printing were relatively smooth.

[0049] The printing structure was crosslinked in a 1 wt% CaCl2 solution for 1 h for molding, and then transferred to high-glucose DMEM medium for culture. After 5 days of culture, the cell viability was 89.6%, and 65.5% of the cells were successfully adhered and implanted. After culturing the 3D bioprinted cell scaffold containing fish myoblasts in the growth medium for 10 days, the growth and proliferation were good.

[0050] It can be seen that the addition of nano-starch can significantly improve the 3D printing properties of the bioink, greatly promote cell adhesion and proliferation, and a culture structure with a higher cell density can be obtained in the same time.

[0051] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed technical content to form equivalent embodiments with equivalent changes, but as long as the technical content of the present invention is not departed from, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a cell culture scaffold, characterized in that: Including the following steps: (1) Dispersing nano-starch in a culture medium to form a nano-particle suspension; Adding a substrate material, mixing evenly, and then adding cells to form a printing solution; the mass ratio of the substrate material to the nano-starch is 10:1 to 2:1; the size of the nano-starch is 10 nm to 500 nm; the substrate material can form a gel under the action of an initiator; the substrate material is gelatin and sodium alginate, and in the printing solution, the gelatin concentration is 0.05 to 0.1 g / mL; the sodium alginate concentration range is 0.01 to 0.05 g / mL; (2) After 3D printing, crosslinking is initiated by an initiator to form a cell culture scaffold; the extrusion pressure of 3D printing is below 0.2 MPa; the printing temperature of 3D printing is below 25 °C.

2. The method according to claim 1, wherein The initiator is a CaCl2 solution.

3. The method according to claim 1, characterized in that, The nano-starch is prepared by the following method: Dissolving 5 g of corn starch in 100 mL of NaOH solution (0.01 g / mL) to obtain a starch suspension; pouring the starch suspension into 100 mL of ethanol for anti-solvent precipitation, and then centrifuging the liquid phase system containing the self-assembled nano-starch precipitate at 5000×g for 5 min, removing the supernatant to obtain a nano-starch precipitate, and washing it 3 times with absolute ethanol and deionized water respectively; finally, the nano-starch is freeze-dried at -60 °C for 48 h for dehydration and ground through a 200-mesh sieve.

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