CN111197022A - Voxel printing biological ink and preparation method thereof - Google Patents
Voxel printing biological ink and preparation method thereof Download PDFInfo
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Abstract
A voxel printing biological ink and a preparation method thereof are provided, the method comprises the following steps: premixing two ultra-long chain biocompatible molecules with an extracellular matrix material respectively to obtain two premixed components, and injecting the two premixed components into microdroplets to generate mixture microdroplets; the droplets are made to flow in the micro-fluidic pipeline, two ultra-long chain biocompatible molecules serving as cross-linking agent molecules and the extracellular matrix material and two ultra-long chain biocompatible molecules are fully mixed and wound under the circulation flow in the droplets, chain nodes are formed by interweaving, and the extracellular matrix material is locked in the wound ultra-long chain molecule chains to form an integral network. The voxel printing biological ink obtained by the invention has good biocompatibility, high designability, simple application and no harm to occluded cells.
Description
Technical Field
The invention relates to biotechnology, in particular to voxel printing biological ink and a preparation method thereof.
Background
Extracellular matrix (ECM) is a network structure composed of macromolecules such as proteins and polysaccharides secreted and distributed outside cells, and the main components are various materials such as proteins (e.g., collagen, elastin, etc.), non-collagenous glycoproteins, and proteoglycans (proteoglycan). ECM plays a supporting, protecting and nourishing role for tissue cells, and is also bound to cells through cell surface specific receptors, and is closely related to basic vital activities such as proliferation, differentiation, metabolism, recognition, adhesion and migration of cells. Bio-inks based on extracts (components) of the mammalian natural extracellular matrix, such as collagen or matrigel, are important foundations for stem cell transplantation, 3D organ printing and in vitro pathology model fabrication. The biological ink has the potential of remarkably improving the treatment efficiency of stem cells by being used as an implantation carrier to assist stem cell transplantation; both 3D organ printing and pathological organ model fabrication rely on biomaterials to provide structural retention and support stem cell survival. However, native ECM has the disadvantages of low mechanical strength and slow gelation, which limits its application as a voxel printing ink (e.g., gel cell soft spheres). The introduction of chemical crosslinking (including thermal crosslinking and photoreactive crosslinking, etc.) can promote gelation and enhance mechanical properties, but the crosslinking agent or crosslinking conditions are generally detrimental to the cells, while the rate of harmless thiol-ene reaction is low.
Disclosure of Invention
The invention mainly aims to overcome the technical defects and provide the voxel printing biological ink and the preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of voxel printing bio-ink comprises the following steps:
premixing a first ultra-long chain biocompatible molecule with an extracellular matrix material to obtain a first premixed component;
premixing the second ultra-long chain biocompatible molecule with an extracellular matrix material to obtain a second premixed component;
injecting the first premixed component and the second premixed component into droplets, resulting in mixture droplets;
and (2) driving the micro-droplets to flow in a micro-fluidic pipeline by using a micro-fluidic technology, fully mixing and winding the first and second ultra-long chain biocompatible molecules serving as cross-linking agent molecules with the extracellular matrix material and the first and second ultra-long chain biocompatible molecules under the circulation flow in the micro-droplets, interweaving to form chain nodes, and locking the extracellular matrix material in the wound ultra-long chain molecule chains to form an integral network.
Further:
injecting the first premixed component and the second premixed component separately into the droplets, or mixing the first premixed component and the second premixed component prior to injecting the droplets.
The first ultra-long chain biocompatible molecule is a DNA single chain, a macromolecular chain or a polypeptide chain, and the second ultra-long chain biocompatible molecule is a complementary chain of the DNA single chain, a homogeneous/heterogeneous macromolecular chain or a polypeptide chain which can generate chain reaction or acting force with the macromolecular chain or the polypeptide chain.
The chain nodes are formed by one or more of hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, and physical entanglement.
The extracellular matrix material includes a protein fiber material including collagen and matrigel.
The ultra-long chain biocompatible molecules are crosslinked and solidified with extracellular matrix materials to generate soft spheres, or a plurality of soft spheres are fused into a strip glue after flowing and extruding.
A voxel printing biological ink comprises a first super-long chain biocompatible molecule, a second super-long chain biocompatible molecule and an extracellular matrix material, wherein the first super-long chain biocompatible molecule, the second super-long chain biocompatible molecule and the extracellular matrix material are fully mixed and wound as cross-linking agent molecules, the first super-long chain biocompatible molecule and the second super-long chain biocompatible molecule are interwoven to form chain nodes, and the extracellular matrix material is locked in the wound chain of the super-long chain molecules to form an integral network.
Further:
the first ultra-long chain biocompatible molecule is a DNA single chain, a macromolecular chain or a polypeptide chain, and the second ultra-long chain biocompatible molecule is a complementary chain of the DNA single chain, a homogeneous/heterogeneous macromolecular chain or a polypeptide chain which can generate chain reaction or acting force with the macromolecular chain or the polypeptide chain.
The chain nodes are formed by one or more of hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, and physical entanglement.
The extracellular matrix material comprises a protein fiber material comprising collagen and matrigel; the ultra-long chain biocompatible molecules are crosslinked and solidified with extracellular matrix materials to generate soft spheres, or a plurality of soft spheres are fused into a strip glue after flowing and extruding.
The invention has the following beneficial effects:
the invention provides a voxel printing biological ink and a preparation method thereof, wherein two ultra-long chain biocompatible molecules are used as a non-cytotoxic cross-linking agent and are injected into microdrops together with ECM, a microfluidic flow dynamics field is utilized to enable the two ultra-long chain biocompatible molecules and ECM to be fully mixed and wound and the two ultra-long chain biocompatible molecules to be interwoven to form chain nodes, and the ECM is locked in the wound ultra-long chain molecule chains to form an integral network, so that the cross-linking of natural ECM such as protein fiber materials and the like is effectively promoted, the voxel printing ink is obtained, and the dependence on toxic or complex chemical reaction conditions required for cells is avoided. The crosslinking strategy in the invention is different from the traditional crosslinking reagent or crosslinking condition with potential cytotoxicity such as chemistry (such as crosslinking by using chemical crosslinking molecules and generating heat or photoreaction) or physical crosslinking (such as ultraviolet ray or gamma ray irradiation, dehydration crosslinking and the like), and the like. The cross-linked native ECM and its derived materials of the invention may better reduce the native matrix microenvironment of stem cells and the like compared to synthetic hydrogels. The application of the invention can greatly expand the application of natural biological materials in regeneration and transformation medicine, and is expected to promote the development and application of the fields of stem cell transplantation, organoid manufacture, 3D organ printing and the like.
Drawings
Fig. 1 is a schematic diagram of an internal flow cycle of a droplet flowing in a microfluidic channel according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the embodiment of the present invention in which the ultra-long chain molecules are mixed and interlaced by internal circulation of microdroplets to form "locked" nodes of the immobilized fibrous scaffold material (e.g., collagen and matrigel).
FIG. 3(a) is a schematic diagram showing the mechanical properties of collagen of the ultra-long chain molecule interweaving node "locked" solidified collagen-based soft spheres according to the embodiment of the present invention; FIG. 3(b) is a partial SEM image of a soft sphere; FIG. 3(c) is a global view of a soft sphere.
Fig. 4 is a representation of a matrix gum base soft sphere with "locked" solidification of the interwoven nodes of ultralong chain molecules in an embodiment of the invention.
FIG. 5 is two schematic views showing how several collagen-based soft spheres "locked" and solidified by the interweaving nodes of very long chain molecules are fused into a long-strand glue after flowing and extruding.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for either a fixed or coupled or communicating function.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Fig. 1 is a schematic diagram of an internal flow cycle of a droplet flowing in a microfluidic channel according to an embodiment of the present invention. FIG. 2 is a schematic diagram of the formation of "locked" junctions of immobilized extracellular matrix materials (e.g., collagen and matrigel) by mixing and interlacing of ultralong-chain molecules through internal circulation of microdroplets in accordance with an embodiment of the present invention.
Referring to fig. 1 and 2, an embodiment of the present invention provides a method for preparing a voxel-printing bio-ink, including the following steps: premixing a first ultra-long chain biocompatible molecule 4 with an extracellular matrix material 6 to obtain a first premixed component; premixing a second ultra-long chain biocompatible molecule 5 with an extracellular matrix material 6 to obtain a second premixed component; wherein the first ultralong-chain biocompatible molecule 4 and the second ultralong-chain biocompatible molecule 5 are complementary cross-linker molecules; injecting the first premixed component and the second premixed component into microdroplets 1, resulting in mixture microdroplets; the droplets 1 are driven to flow in the microfluidic pipeline 2 by using a microfluidic flow dynamic field, and under the condition of circulating flow in the droplets 1 (see a circulating flow direction 3 in fig. 1), the first and second ultra-long chain biocompatible molecules 5 serving as cross-linking agent molecules and the extracellular matrix material 6, and the first and second ultra-long chain biocompatible molecules 4 and 5 are sufficiently mixed and wound to form chain nodes in an interweaving manner, so that the extracellular matrix material 6 is locked in the wound ultra-long chain molecule chains to form an integral network.
In some embodiments, the first and second premixed components may be injected into the microdroplets 1 separately to produce the mixture microdroplets 1. In other embodiments, the first premixed component and the second premixed component may be mixed prior to injection into the droplets 1.
In some embodiments, the first ultralong-chain biocompatible molecule 4 is a single DNA strand, a polymer strand, or a polypeptide chain, and the second ultralong-chain biocompatible molecule 5 is a complementary strand of the single DNA strand, a homo/hetero polymer strand, or a polypeptide chain that can produce an interchain reaction or force with the polymer strand or the polypeptide chain.
In some embodiments, the chain nodes 7 are formed by one or more of hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, physical entanglement.
The extracellular matrix material 6 comprises a protein fibrous material comprising collagen and matrigel.
As shown in fig. 3(c), fig. 4 and fig. 5, the ultra-long chain biocompatible molecule crosslinks and solidifies the extracellular matrix material 6 to form soft spheres, or further fuses a plurality of the soft spheres into a growing gelatin after flowing and extruding.
Referring to fig. 2 to 5, a bio-ink for voxel printing includes a first ultra-long chain biocompatible molecule 4, a second ultra-long chain biocompatible molecule 5 and an extracellular matrix material 6, wherein the first ultra-long chain biocompatible molecule 4, the second ultra-long chain biocompatible molecule 5 and the extracellular matrix material 6, and the first ultra-long chain biocompatible molecule 4 and the second ultra-long chain biocompatible molecule 5 are mixed and wound together as cross-linker molecules, and are interwoven to form chain nodes 7, so that the extracellular matrix material 6 is locked in the wound ultra-long chain molecule chains to form an integral network.
The first ultra-long chain biocompatible molecule 4 and the second ultra-long chain biocompatible molecule 5 are complementary cross-linking agent molecules, the first ultra-long chain biocompatible molecule 4 is a single DNA chain, a macromolecular chain or a polypeptide chain, and the second ultra-long chain biocompatible molecule 5 is a complementary chain of the single DNA chain, a homogeneous/heterogeneous macromolecular chain or a polypeptide chain which can generate an inter-chain reaction or acting force with the macromolecular chain or the polypeptide chain. The chain nodes 7 are formed by one or more of hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, and physical entanglement.
Wherein the extracellular matrix material 6 comprises a protein fiber material comprising collagen and matrigel; the ultra-long chain biocompatible molecules are crosslinked and solidified with extracellular matrix materials to generate soft spheres, or a plurality of soft spheres are fused into a strip glue after flowing and extruding.
In the embodiment of the invention, extracellular matrix extraction component materials such as collagen, matrigel and the like are used to obtain a fiber soft scaffold material, the fiber soft scaffold material is mixed with two ultra-long chain biocompatible molecules in micro-droplets, and the cross-linking is realized by micro-scale fluid flow regulation/drive. In the cross-linking agent, one kind of ultra-long chain biocompatible molecule (such as a DNA single chain (ultra-long single-chain ssDNA), a polymer chain, a polypeptide chain and the like) is premixed with the fiber material, intermolecular forces (such as electrostatic force, hydrogen bonding effect or covalent bonding effect and the like) exist between the cross-linking agent molecule and the fiber, and the other kind of ultra-long chain biocompatible molecule (such as a complementary chain of the DNA single chain, the same kind of polymer chain (or different kind of polypeptide chain) which can generate the interchain reaction or the acting force with the polymer chain (or polypeptide chain) and the like) is also premixed with the fiber material. The two pre-mixed components can be injected into the droplet-microfluidic to produce a mixture droplet, either separately or after mixing. As shown in fig. 1 and fig. 2, the micro-droplets are driven to flow in the micro-fluidic pipeline by using a micro-fluidic flow dynamic field, the mixed liquid promotes the molecules of the cross-linking agent to be fully mixed and wound with the fibers under the circulation flow in the micro-droplets, and the molecules of the complementary cross-linking agent are interwoven to form chain nodes to lock the fibers in the ultra-long chain winding to form an integral network. Junction formation may be hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, or physical entanglement.
Exemplary results of crosslinking collagen and matrigel by ultralong-chain biocompatible molecules to form soft structure spheres and elongated gel are shown in fig. 3(a) -5. FIG. 3(a) shows the collagen mechanical properties of the ultra-long chain molecule interweaving nodes "locked" into the solidified collagen-based soft spheres of the present invention; FIG. 3(b) is a partial SEM image of a soft sphere; FIG. 3(c) is a global view of a soft sphere. Fig. 4 shows an embodiment of the invention in which the interwoven nodes of ultralong chain molecules "lock" the solidified matrix gum-based softspheres. FIG. 5 shows two examples of how several collagen-based soft spheres "locked" and solidified by the interweaving nodes of very long chain molecules are fused into a long-strand glue after flowing and extruding.
In the embodiment of the invention, two ultra-long chain biocompatible molecules are used as a non-cytotoxic cross-linking agent to be injected into a microdrop together with ECM, and a microfluidic flow dynamics field is utilized to ensure that the two ultra-long chain biocompatible molecules are fully mixed and wound with the ECM and the two ultra-long chain biocompatible molecules are mutually interwoven to form chain nodes, and the ECM is locked in the wound ultra-long chain molecule chain to form an integral network, so that the cross-linking of natural ECM such as protein fiber materials and the like is effectively promoted, the voxel printing ink is obtained, and the condition of depending on toxic or complex chemical reaction to cells is avoided. The crosslinking strategy in the invention is different from the traditional crosslinking reagent or crosslinking condition with potential cytotoxicity such as chemistry (such as crosslinking by using chemical crosslinking molecules and generating heat or photoreaction) or physical crosslinking (such as ultraviolet ray or gamma ray irradiation, dehydration crosslinking and the like), and the like. The cross-linked native ECM and its derived materials of the invention may better reduce the native matrix microenvironment of stem cells and the like compared to synthetic hydrogels. The application of the invention can greatly expand the application of natural biological materials in regeneration and transformation medicine, and is expected to promote the development and application of the fields of stem cell transplantation, organoid manufacture, 3D organ printing and the like.
The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the claims.
Claims (10)
1. A preparation method of voxel printing biological ink is characterized by comprising the following steps:
premixing a first ultra-long chain biocompatible molecule with an extracellular matrix material to obtain a first premixed component;
premixing the second ultra-long chain biocompatible molecule with an extracellular matrix material to obtain a second premixed component;
injecting the first premixed component and the second premixed component into droplets, resulting in mixture droplets;
and (2) driving the micro-droplets to flow in a micro-fluidic pipeline by using a micro-fluidic technology, fully mixing and winding the first and second ultra-long chain biocompatible molecules serving as cross-linking agent molecules with the extracellular matrix material and the first and second ultra-long chain biocompatible molecules under the circulation flow in the micro-droplets, interweaving to form chain nodes, and locking the extracellular matrix material in the wound ultra-long chain molecule chains to form an integral network.
2. The method of making a voxel-printing bio-ink of claim 1, wherein the first pre-mixed component and the second pre-mixed component are separately injected into the droplets or the first pre-mixed component and the second pre-mixed component are mixed and then injected into the droplets.
3. The method of any one of claims 1-2, wherein the first ultralong-chain biocompatible molecule is a single DNA chain, a polymer chain, or a polypeptide chain, and the second ultralong-chain biocompatible molecule is a complementary chain of the single DNA chain, a homo/hetero polymer chain, or a polypeptide chain that can react or act with the polymer chain or the polypeptide chain.
4. The method of producing a voxel printing bio-ink according to any of claims 1 to 3, wherein the chain junctions are formed by one or more of hydrogen bond complementation, heterogeneous charge attraction, covalent bonding, physical entanglement.
5. The method of preparing a voxel printing bio-ink according to any of the claims 1 to 4, wherein the extracellular matrix material comprises a protein fiber material comprising collagen and matrigel.
6. The method for preparing a bio-ink for voxel printing according to any one of claims 1 to 5, wherein the ultra-long chain biocompatible molecule is cross-linked and solidified to form a soft sphere from an extracellular matrix material, or a plurality of the soft spheres are further fused into a long stripe glue after being flow extruded.
7. A voxel printing bio-ink is characterized by comprising a first super-long chain biocompatible molecule, a second super-long chain biocompatible molecule and an extracellular matrix material, wherein the first super-long chain biocompatible molecule, the second super-long chain biocompatible molecule and the extracellular matrix material and the first super-long chain biocompatible molecule and the second super-long chain biocompatible molecule which are used as cross-linking agent molecules are fully mixed and wound to form chain nodes in an interweaving mode, and the extracellular matrix material is locked in the wound super-long chain molecule chains to form an integral network.
8. The method of claim 7, wherein the first ultralong-chain biocompatible molecule is a single DNA chain, a polymer chain, or a polypeptide chain, and the second ultralong-chain biocompatible molecule is a complementary chain of the single DNA chain, a homo/hetero polymer chain or a polypeptide chain that can react or act with the polymer chain or the polypeptide chain.
9. The method of producing a voxel printing bio-ink according to any of claims 7 to 8, wherein the chain junctions are formed by one or more of hydrogen bond complementary action, heterogeneous charge attraction action, covalent bond action, physical entanglement action.
10. The method of preparing a voxel printing bio-ink according to any of the claims 7 to 9, wherein the extracellular matrix material comprises a protein fiber material comprising collagen and matrigel; the ultra-long chain biocompatible molecules are crosslinked and solidified with extracellular matrix materials to generate soft spheres, or a plurality of soft spheres are fused into a strip glue after flowing and extruding.
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Application publication date: 20200526 |