CN109337436A - A kind of bio-ink and its preparation method and application - Google Patents
A kind of bio-ink and its preparation method and application Download PDFInfo
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- CN109337436A CN109337436A CN201811249690.9A CN201811249690A CN109337436A CN 109337436 A CN109337436 A CN 109337436A CN 201811249690 A CN201811249690 A CN 201811249690A CN 109337436 A CN109337436 A CN 109337436A
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/04—Printing inks based on proteins
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/03—Printing inks characterised by features other than the chemical nature of the binder
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
Abstract
The present invention provides a kind of bio-inks and its preparation method and application, belong to bio-ink preparation technical field.Bio-ink provided by the invention; the nanometer hectorite for being 0.01~5wt% including weight percentage; the methacrylation gelatin of 5~40wt%; the photoinitiator of 0.01~0.5wt% and the cell culture medium of 55~90wt%, the photoinitiator are 2- hydroxyl -4- (2- hydroxy ethoxy) -2- methyl phenyl ketone.Bio-ink good biocompatibility provided by the invention, cell survival rate is high, and print performance is good, the application suitable for cell 3D printing field.
Description
Technical field
The present invention relates to bio-ink preparation technical field more particularly to a kind of bio-ink and preparation method thereof and answer
With.
Background technique
Bio-ink is the ink that can be used for cell 3D printer, has excellent biocompatibility and bioactivity.It can
To replace extracellular matrix, three-dimensional space and transmission nutriment are provided to living cells in print procedure, is a kind of special
Biomedical material.Being suitable for the biomaterial of 3D printing at present mainly includes synthetic material, the degradable absorption polyester of thermoplasticity
Plastics (PLA, PLGA, PCL, PEG and its copolymer) and some natural macromolecular materials (alginate, fibrin, glue
Original, gelatin, chitosan, hyaluronic acid etc.).Above-mentioned synthetic material mostly uses greatly organic solvent or carries out 3D under the high temperature conditions
Printing, the supporting structure printed is stablized, but cannot print with mixing with cells.Natural macromolecular material can be used for cell 3D and beat
Print, but biocompatibility is poor, cell survival rate is lower, and the hydrogel stability formed after printing is poor, limits biology
It is printed upon the application of organizational project and regenerative medicine field.
No matter independent existing 3D printing biomaterial is or is used in combination, and is all difficult to take into account cell compatibility, bioactivity
And mechanical property, lead to existing bio-ink there are biocompatibilities poor, cell survival rate is lower, squeeze out print performance compared with
Difference can only be physical crosslinking or be chemically crosslinked, and form the problems such as hydrogel is unstable.
Summary of the invention
In consideration of it, the purpose of the present invention is to provide a kind of bio-inks and its preparation method and application.The present invention provides
Bio-ink good biocompatibility, cell survival rate is high, and print performance is good, answering suitable for cell 3D printing field
With.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of bio-ink, the component including following weight percentage:
The photoinitiator is 2- hydroxyl -4- (2- hydroxy ethoxy) -2- methyl phenyl ketone.
Preferably, the partial size of the nanometer hectorite is 20~200nm.
Preferably, the methacrylation degree of the methacrylation gelatin is 70~100%.
The present invention also provides the preparation methods of the bio-ink described in above-mentioned technical proposal, comprising the following steps:
Methacrylation gelatin and cell culture medium are mixed, mixed solution is obtained；
Nanometer hectorite, photoinitiator are sequentially added into the mixed solution, obtain bio-ink.
The present invention also provides application of the bio-ink described in above-mentioned technical proposal in cell 3D printing field.
Preferably, the application, comprising the following steps:
After cell pellets are mixed with bio-ink, it is packed into 3D printer feed bin, under ultraviolet light, squeezes out printing.
Preferably, the movement speed of the spray head of the 3D printer is 100~400mm/min.
Preferably, the extruded velocity of the bio-ink is 120~420mm/min.
Preferably, the wavelength of the ultraviolet light is 400~315nm.
Preferably, the intensity of illumination of the ultraviolet light is 500~900mw/cm2。
The present invention provides a kind of bio-ink, the component including following weight percentage:
The photoinitiator is 2- hydroxyl -4- (2- hydroxy ethoxy) -2- methyl phenyl ketone.
The present invention, for main solid phase material, is main with cell culture medium with nanometer hectorite and methacrylation gelatin
Liquid phase material.Nanometer hectorite improves the thixotropy of mixed solution in the application, makes it after cryogenic freezing, has good
Print performance is squeezed out, hydrogel is quickly formed.Photoinitiator makes methacrylation gelatin under ultraviolet light, quickly completes
Chemical crosslinking forms stable hydrogel, improves the biocompatibility of bio-ink, and then improve the survival rate of cell, deposits
Motility rate is up to 85% or more, it can be achieved that application of the bio-ink in cell 3D printing field.
Specific embodiment
The present invention provides a kind of bio-ink, the component including following weight percentage:
The photoinitiator is 2- hydroxyl -4- (2- hydroxy ethoxy) -2- methyl phenyl ketone.
Bio-ink provided by the invention includes the nanometer hectorite of 0.01~5wt%, preferably 1~4wt%, more preferably
For 3wt%.In the present invention, the partial size of the nanometer hectorite is preferably 20~200nm, and more preferably 50~150nm is optimal
It is selected as 100nm.The present invention does not have special restriction to the source of the nanometer hectorite, and use is well known to those skilled in the art
Commercial product.In the present invention, methacrylation gelatin and cell culture medium mixed liquor is added in the nanometer hectorite
In after, can expand rapidly, formed include a large number of water molecules thixotropy gel, make it after cryogenic freezing, still have good
Extrusion print performance.
Bio-ink provided by the invention includes the methacrylation gelatin of 5~40wt%, preferably 10~30wt%,
More preferably 20wt%.In the present invention, the methacrylation degree of the methacrylation gelatin is 70~100%, more
Preferably 80~90%, most preferably 95%.In the present invention, the methacrylation gelatin physical state is sponge or cotton
Cotton-shaped or powdered, partial size is preferably 200~2000 μm, and more preferably 500~800 μm.The present invention is to the methacryl
The source for changing gelatin does not have special restriction, is made using preparation method well known to those skilled in the art or commercial product is equal
It can.In the present invention, methacrylation gelatin bioactivity with higher and Photocrosslinkable hydrogel character not only may be used
To improve survival rate of the cell in bio-ink, hydrogel that can be stable with forming properties.
Bio-ink provided by the invention includes the photoinitiator of 0.01~0.5wt%, preferably 0.1~0.4wt%, more
Preferably 0.2~0.3wt%, most preferably 0.25wt%.In the present invention, the photoinitiator is 2- hydroxyl -4- (2- hydroxyl second
Oxygroup) -2- methyl phenyl ketone.The present invention does not have special restriction to the source of the photoinitiator, using those skilled in the art
The known preparation method of member is made or commercial product.In the present invention, the addition of the photoinitiator makes methacrylation
Gelatin quickly completes chemical crosslinking and forms stable hydrogel, improve the survival rate of cell under ultraviolet light.
Bio-ink provided by the invention includes the cell culture medium of 55~90wt%, preferably 60~80wt%, more excellent
It is selected as 65~75wt%, most preferably 70wt%.In the present invention, the cell culture medium constituent preferably include 1000~
The glucose of 5000mg/L, the inorganic salts of 5000~7500mg/L, the nutriments such as amino acid of 800~1200mg/L.This hair
The bright source to the cell culture medium constituent does not have special restriction, using preparation side well known to those skilled in the art
Method is made or commercial product.In the present invention, the addition of the cell culture medium can be to cells with nutrient and promoting
The basic substance of germiparity proliferation, improves the survival rate of cell.
The present invention also provides the preparation methods of bio-ink described in above-mentioned technical proposal, comprising the following steps:
Methacrylation gelatin and cell culture medium are mixed, mixed solution is obtained；
Nanometer hectorite, photoinitiator are sequentially added into the mixed solution, obtain bio-ink.
The present invention mixes methacrylation gelatin and cell culture medium, obtains mixed solution.
In the present invention, the methacrylation gelatin and cell culture medium preferably mix in a brown bottle.The present invention
There is no special restriction to the hybrid mode of methacrylation gelatin and cell culture medium, it is bright to can satisfy methacrylation
The requirement that glue and cell culture medium are uniformly mixed, specifically, such as mechanical oscillation.Speed of the present invention to the mechanical oscillation
And time of vibration is not particularly limited, and can satisfy the uniformly mixed requirement of methacrylation gelatin and cell culture medium i.e.
Can, specifically, time of vibration is preferably 5~10min if vibration velocity is preferably 1000~5000 turns/min.
After obtaining mixed solution, nanometer hectorite, photoinitiator are sequentially added the mixed solution by the present invention, are given birth to
Object ink.
The present invention does not have special limit to the hybrid mode of the nanometer hectorite, photoinitiator and the mixed solution
It is fixed, it can satisfy the uniformly mixed requirement of a nanometer hectorite, photoinitiator and the mixed solution, specifically, as mechanical
Vibration.The present invention is not particularly limited the speed and time of vibration of the mechanical oscillation, can satisfy a nanometer hectorite, light draws
The requirement that hair agent and the mixed solution are uniformly mixed, specifically, if vibration velocity is preferably 1000~5000 turns/min,
Time of vibration is preferably 5~10min.
Obtain the bio-ink, the present invention preferably the bio-ink is placed in low temperature environment refrigerate it is spare.At this
In invention, the temperature of the deepfreeze is preferably 0~4 DEG C, and the deepfreeze environment is further in embodiments of the present invention
Preferably 4 DEG C.
The present invention also provides application of the bio-ink described in above-mentioned technical proposal in cell 3D printing field.
In the present invention, the application preferably includes following steps:
After cell pellets are mixed with bio-ink, it is packed into 3D printer feed bin, under ultraviolet light, squeezes out printing.
In the present invention, the ratio of the cell pellets (cell quantity) and bio-ink is preferably 104A/mL~107
A/mL.
In the present invention, the bio-ink preferably needs to be homogenized using preceding.Homogenizing side of the present invention to the bio-ink
Formula does not have special restriction, can satisfy the requirement of bio-ink homogenizing, specifically, such as mechanical oscillation.The present invention is to institute
The speed and time for stating mechanical oscillation do not have particular/special requirement, can satisfy the requirement of bio-ink homogenizing, specifically, as shaken
Dynamic speed is preferably 1000~5000 turns/min, and time of vibration is preferably 1~5min.
The present invention does not have special restriction to the hybrid mode of the cell pellets and bio-ink, can satisfy cell ball
The requirement that group is uniformly mixed with bio-ink, specifically, such as injecting mixed.In the present invention, the injecting mixed is preferred
To use the multiple injecting mixed of liquid-transfering gun, in embodiments of the present invention, the blowing number is preferably 10~20 times.
In the present invention, the movement speed of the spray head of the 3D printer is preferably 100~400mm/min, more preferably
200~300mm/min, most preferably 250mm/min.In the present invention, the extruded velocity of the bio-ink be preferably 120~
420mm/min, more preferably 150~400mm/min, most preferably 200~350mm/min.
In the present invention, the ultraviolet light time is preferably 30~90s, more preferably 50~70s, most preferably
60s, the wavelength of the ultraviolet light are preferably 400~315nm, more preferably 380~330nm, most preferably 350nm, the purple
The intensity of illumination of outer light is preferably 500~900mw/cm2, more preferably 800~860mw/cm2, most preferably 850mw/cm2。
Bio-ink provided by the invention and its preparation method and application is described in detail below with reference to embodiment,
But they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
After the methacrylation gelatin of 20wt% is put into brown bottle, the cell culture medium of 79wt% is added, with
After the speed mechanical vibration 5min of 1000 turns/min, the nanometer hectorite of 0.5wt% and the 2- hydroxyl -4- (2- of 0.5wt% is added
Hydroxy ethoxy) -2- methyl phenyl ketone, vibrates 5min with the speed mechanical of 3000 turns/min, obtains bio-ink.
According to 5x105A/mL mixes l cell with obtained bio-ink, is blown with liquid-transfering gun through 10 times
After mixing, cell biological ink is obtained.
Cell biological ink 3D printing condition: light source is ultraviolet light, wavelength 315nm, intensity 830mw/cm2, ultraviolet
Light application time is 30s, and 3D printer spray head movement speed is 200mm/min, and cell biological ink extruded velocity is 220mm/
min。
The hydrogel compression modulus 100kPa of obtained load cell, after the completion of printing, l cell survival rate
After reaching 92%, culture 7 days, cell survival rate reaches 96%.
Embodiment 2
After the methacrylation gelatin of 25wt% is put into brown bottle, the cell culture medium of 70wt% is added, with
After the speed mechanical vibration 10min of 2000 turns/min, the nanometer hectorite of 4.9wt% and the 2- hydroxyl -4- of 0.1wt% is added
(2- hydroxy ethoxy) -2- methyl phenyl ketone vibrates 10min with the speed mechanical of 3500 turns/min, obtains bio-ink.
According to 1x106A/mL mixes human vascular endothelial with obtained bio-ink, is blown with liquid-transfering gun through 12 times
After mixing, cell biological ink is obtained.
Cell biological ink 3D printing condition: light source is ultraviolet light, wavelength 400nm, intensity 870mw/cm2, ultraviolet
Light application time is 90s, and 3D printer spray head movement speed is 400mm/min, and cell biological ink extruded velocity is 420mm/
min。
The hydrogel compression modulus 130kPa of obtained load cell, after the completion of printing, human vascular endothelial survival rate
After reaching 86%, culture 7 days, cell survival rate reaches 92%.
Embodiment 3
After the methacrylation gelatin of 28wt% is put into brown bottle, the cell culture medium of 70wt% is added, with
After the speed mechanical vibration 8min of 2500 turns/min, the nanometer hectorite of 1.9wt% and the 2- hydroxyl -4- (2- of 0.1wt% is added
Hydroxy ethoxy) -2- methyl phenyl ketone, vibrates 8min with the speed mechanical of 4000 turns/min, obtains bio-ink.
According to 2x106A/mL mixes human bone marrow stroma stem cell with obtained bio-ink, is sprayed with liquid-transfering gun through 15 times
After blowing mixing, cell biological ink is obtained.
Cell biological ink 3D printing condition: light source is ultraviolet light, wavelength 380nm, intensity 840mw/cm2, ultraviolet
Light application time is 80s, and 3D printer spray head movement speed is 350mm/min, and cell biological ink extruded velocity is 250mm/
min。
The hydrogel compression modulus 160kPa of obtained load cell, after the completion of printing, human bone marrow stroma stem cell survival
After rate reaches 80%, culture 7 days, cell survival rate reaches 87%.
Embodiment 4
After the methacrylation gelatin of 30wt% is put into brown bottle, the cell culture medium of 68wt% is added, with
After the speed mechanical vibration 5min of 3500 turns/min, the nanometer hectorite of 1.7wt% and the 2- hydroxyl -4- (2- of 0.3wt% is added
Hydroxy ethoxy) -2- methyl phenyl ketone, vibrates 5min with the speed mechanical of 4500 turns/min, obtains bio-ink.
According to 1.5x106A/mL mixes rat osteoblast with obtained bio-ink, is blown with liquid-transfering gun through 12 times
After mixing, cell biological ink is obtained.
Cell biological ink 3D printing condition: light source is ultraviolet light, wavelength 350nm, intensity 860mw/cm2, ultraviolet
Light application time is 50s, and 3D printer spray head movement speed is 250mm/min, and cell biological ink extruded velocity is 300mm/
min。
The hydrogel compression modulus 180kPa of obtained load cell, after the completion of printing, rat osteoblast survival rate reaches
To after 87%, culture 7 days, cell survival rate reaches 93%.
Embodiment 5
After the methacrylation gelatin of 35wt% is put into brown bottle, the cell culture medium of 65wt% is added, with
After the speed mechanical vibration 10min of 4500 turns/min, the nanometer hectorite of 4.5wt% and the 2- hydroxyl -4- of 0.5wt% is added
(2- hydroxy ethoxy) -2- methyl phenyl ketone vibrates 10min with the speed mechanical of 5000 turns/min, obtains bio-ink.
According to 1x106A/mL mixes fibroblasts of adult human dermis with obtained bio-ink, is sprayed with liquid-transfering gun through 10 times
After blowing mixing, cell biological ink is obtained.
Cell biological ink 3D printing condition: light source is ultraviolet light, wavelength 330nm, intensity 850mw/cm2, ultraviolet
Light application time is 60s, and 3D printer spray head movement speed is 300mm/min, and cell biological ink extruded velocity is 400mm/
min。
The hydrogel compression modulus 180kPa of obtained load cell, after the completion of printing, fibroblasts of adult human dermis survival
After rate reaches 85%, culture 7 days, cell survival rate reaches 91%.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of bio-ink, the component including following weight percentage:
The photoinitiator is 2- hydroxyl -4- (2- hydroxy ethoxy) -2- methyl phenyl ketone.
2. bio-ink according to claim 1, which is characterized in that the partial size of the nanometer hectorite is 20~200nm.
3. bio-ink according to claim 1, which is characterized in that the methacryl of the methacrylation gelatin
Change degree is 70~100%.
4. the preparation method of the described in any item bio-inks of claims 1 to 3, comprising the following steps:
Methacrylation gelatin and cell culture medium are mixed, mixed solution is obtained；
Nanometer hectorite, photoinitiator are sequentially added into the mixed solution, obtain bio-ink.
5. application of the described in any item bio-inks of claims 1 to 3 in cell 3D printing field.
6. application according to claim 5, comprising the following steps:
After cell pellets are mixed with bio-ink, it is packed into 3D printer feed bin, under ultraviolet light, squeezes out printing.
7. application according to claim 6, which is characterized in that the movement speed of the spray head of the 3D printer be 100~
400mm/min。
8. application according to claim 6 or 7, which is characterized in that the extruded velocity of the bio-ink be 120~
420mm/min。
9. application according to claim 6, which is characterized in that the wavelength of the ultraviolet light is 400~315nm.
10. application according to claim 6 or 9, which is characterized in that the intensity of illumination of the ultraviolet light be 500~
900mw/cm2。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811249690.9A CN109337436A (en) | 2018-10-25 | 2018-10-25 | A kind of bio-ink and its preparation method and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109822889A (en) * | 2019-03-22 | 2019-05-31 | 河北雄安大洲智造科技有限公司 | A kind of multichannel 3D printing spray head and preparation method thereof, cell/bio-ink transportation system |
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2018
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| CN108367100A (en) * | 2015-12-02 | 2018-08-03 | 奥塔哥创新有限公司 | It is prepared by the photoactivation of hydrogel |
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| CN109822889A (en) * | 2019-03-22 | 2019-05-31 | 河北雄安大洲智造科技有限公司 | A kind of multichannel 3D printing spray head and preparation method thereof, cell/bio-ink transportation system |
| CN109822889B (en) * | 2019-03-22 | 2024-03-26 | 深圳大洲医学科技有限公司 | Multichannel 3D printing spray head, preparation method thereof and cell/biological ink conveying system |
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