Cell-loaded pore-forming hydrogel bio-ink as well as preparation method and application thereof

Abstract

The invention discloses cell-loaded pore-forming hydrogel bio-ink as well as a preparation method and application thereof. The preparation method of the cell-loaded pore-forming hydrogel bio-ink comprises the following steps: preparing filiform or multi-arm sacrificial hydrogel; preparing a matrix hydrogel precursor solution; wherein living cells are loaded in the sacrificial hydrogel and / or the matrix hydrogel precursor solution; the sacrificial hydrogel and the matrix hydrogel precursor solution are mixed, and the cell-loaded pore-forming hydrogel bio-ink is obtained. According to the invention, hydrogel in a special form, such as filamentous or multi-arm-shaped hydrogel, is taken as a sacrificial template, after the sacrificial hydrogel is removed, a bionic blood vessel lumen form with a pore structure having better connectivity can be obtained, and cells can be loaded in the filamentous sacrificial hydrogel, so that in-situ release of cells in a three-dimensional pore network is realized, and the in-situ release of cells in the three-dimensional pore network is realized. Secondary planting is not needed, and the cell planting efficiency and uniformity are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a cell-carrying porogenic hydrogel bio-ink and a preparation method and application thereof. Background Art

[0002] Hydrogel is a network system formed by cross-linking of hydrophilic polymers dispersed in an aqueous phase. Hydrogel can simulate the three-dimensional microphysiological environment of the extracellular matrix in the body and is the most widely used three-dimensional cell culture matrix material. It can be applied to large-scale expansion of (stem) cells in vitro, construction of in vitro tissue / organ models, and repair of in vivo tissue / organs. Hydrogel is also the most important type of ink material for biological three-dimensional printing, which is used to load cells and other material molecules, and provide mechanical support and a three-dimensional culture environment for cells, so as to achieve direct three-dimensional printing of complex tissue / organ models containing living cells.

[0003] However, the penetration of oxygen and nutrients in three-dimensional matrix materials has a certain distance limit, resulting in the distance that cells in three-dimensional culture can receive sufficient nutrition is generally within 100-200 microns. Internal cells often have problems such as reduced activity and necrosis due to insufficient nutrition. How to create a three-dimensional hydrogel environment with higher material exchange efficiency for cells is an important issue.

[0004] Many existing methods for preparing porous hydrogels use salt particles, porogens, etc. as sacrificial templates to create pores in hydrogels. However, many porogens themselves or their removal process will introduce factors such as high salt, acidity and alkali, and high temperature, which will affect the in situ display and loading of living cells. Some studies have used biocompatible hydrogel particles as sacrificial templates to create pores in another hydrogel, which can meet the needs of encapsulating living cells inside the latter without affecting their activity. However, the pores created by the above-mentioned sacrificial template method are mostly spherical or other granular shapes with strong convergence. Pores of this shape have poor connectivity with each other and low material exchange efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide a cell-loaded porous hydrogel bio-ink and a preparation method and application thereof. The method uses a hydrogel in a special form such as a filamentous or multi-arm hydrogel as a sacrificial template. After removing the sacrificial hydrogel, a bionic vascular lumen morphology with a pore structure having better connectivity can be prepared. Cells can be loaded inside the filamentous sacrificial hydrogel, thereby achieving in situ release of cells in a three-dimensional porous network without the need for secondary implantation, thereby improving the efficiency and uniformity of cell implantation.

[0006] In a first aspect, the present invention provides a method for preparing a cell-carrying porous hydrogel bio-ink, comprising:

[0007] Preparation of filamentous or multi-arm sacrificial hydrogels;

[0008] preparing a matrix hydrogel precursor solution;

[0009] Wherein, the sacrificial hydrogel and / or the matrix hydrogel precursor solution are loaded with living cells;

[0010] The sacrificial hydrogel and the matrix hydrogel precursor solution are mixed to obtain the cell-carrying porous hydrogel bio-ink.

[0011] In the above-mentioned method for preparing the cell-laden porous hydrogel bio-ink, the diameter of the filamentous sacrificial hydrogel or the diameter of the filamentous side wall of the multi-arm sacrificial hydrogel is 10 to 1000 microns.

[0012] In the above-mentioned method for preparing the cell-carrying porous hydrogel bio-ink, the filamentous sacrificial hydrogel is one or more filamentous hydrogels with an aspect ratio of (2 to 1000):1;

[0013] The number of side arms of the multi-arm sacrificial hydrogel is 3 to 16, and the aspect ratio of each side arm is (1 to 50):1.

[0014] In the above-mentioned method for preparing the cell-carrying porous hydrogel bio-ink, the volume ratio of the sacrificial hydrogel to the matrix hydrogel precursor solution is 1:(1-9).

[0015] In the above-mentioned method for preparing the cell-carrying porous hydrogel bio-ink, the sacrificial hydrogel is a gelatin hydrogel or a sodium alginate hydrogel;

[0016] The matrix hydrogel is selected from a photocrosslinked hydrogel, a temperature or pH responsive natural hydrogel or agarose; preferably, the photocrosslinked hydrogel is methacrylated gelatin, methacrylated hyaluronic acid, methacrylated heparin, acrylated multi-arm polyethylene glycol, methacrylated dextran or methacrylated chondroitin sulfate.

[0017] Furthermore, the matrix hydrogel is methacrylated gelatin, and the working concentration of its precursor solution is 0.025-0.3 g / mL.

[0018] In the above-mentioned method for preparing the cell-loaded porous hydrogel bio-ink, the method for preparing the sacrificial hydrogel loaded with living cells comprises:

[0019] Mixing the single cell suspension of the living cells with the precursor solution of the sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution;

[0020] cross-linking the cell-containing sacrificial hydrogel precursor solution to obtain a sacrificial hydrogel loaded with living cells;

[0021] The method of preparing a precursor solution of a matrix hydrogel loaded with living cells comprises:

[0022] The single cell suspension of the living cells is mixed with the precursor solution of the matrix hydrogel to obtain a precursor solution of the matrix hydrogel loaded with living cells.

[0023] In a second aspect, the present invention provides a cell-laden porous hydrogel bio-ink obtained by any of the preparation methods described above.

[0024] In a third aspect, the present invention provides the use of the cell-laden porous hydrogel bio-ink in preparing tissue and / or organ models.

[0025] In the above application, the method for preparing the tissue and / or organ model comprises:

[0026] Performing biological three-dimensional printing on the cell-carrying porous hydrogel bio-ink, and cross-linking and curing the matrix hydrogel precursor solution;

[0027] The sacrificial hydrogel cross-distributed in the matrix hydrogel is removed to form interconnected luminal pores, thereby obtaining a tissue and / or organ model having a cell-laden porous hydrogel structure.

[0028] The present invention has the following beneficial effects:

[0029] (1) The method of the present invention uses a special form of hydrogel sacrificial template to achieve another pore structure with better connectivity and biomimetic lumen morphology in the hydrogel, providing a new ink form for biological three-dimensional printing.

[0030] (2) In the method of the present invention, living cells can be pre-loaded in a sacrificial hydrogel or a matrix hydrogel precursor solution, or in both at the same time; wherein the cells in the matrix phase present a typical three-dimensional culture state. The cells in the sacrificial phase are released as the sacrificial component dissolves, and grow in the constructed lumen pores, thereby achieving in situ cell planting and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the preparation principle of the cell-laden porous hydrogel bio-ink of the present invention.

[0032] Figure 2 This is a schematic diagram of the structure of the coaxial microfluidic device in Example 1 of the present invention. The markings are as follows: 1-outer needle tube; 2-inner needle tube; 3-silicone tube; P1-sodium alginate solution; P2-calcium chloride solution.

[0033] Figure 3The microfilamentous hydrogels obtained by fixing the flow rate of the calcium chloride solution at 2 ml / min, changing the flow rate of the sodium alginate solution to 0.1, 0.2, and 0.5 ml / min, and directly squeezing the sodium alginate solution into the calcium chloride solution in Example 1 of the present invention are recorded as 2 ml / min-0.1 ml / min, 2 ml / min-0.2 ml / min, 2 ml / min-0.5 ml / min, and Non-coaxial, respectively. From left to right are the first to fourth groups of micrographs at different magnifications and the corresponding diameter bar graphs. The average diameters of the first to fourth groups are approximately 50 microns, 100 microns, 150 microns, and 200 microns, respectively.

[0034] Figure 4 These are fluorescent photographs of microfilamentous hydrogels with different aspect ratios obtained by cutting using blades arranged at different intervals in Example 1 of the present invention, from left to right: uncut, cut length 1 mm, cut length 2.5 mm and cut length 4 mm, with a diameter of 140 microns.

[0035] Figure 5 This is a fluorescent photograph of the porous hydrogel structure obtained after dissolving the porogenic bio-ink using EDTA / sodium citrate solution in Example 1 of the present invention.

[0036] Figure 6 This is a fluorescent photograph of the grid structure obtained by 3D printing the porous hydrogel bio-ink using needles of different thicknesses in Example 2 of the present invention.

[0037] Figure 7 It is the culture effect of cells loaded in the sacrificial phase and the matrix phase in the porogenic bio-ink in Example 2 of the present invention. NO L929 means that only human umbilical vein blood endothelial cells HUVEC are loaded in the microfilamentous hydrogel A, and L929 means that human umbilical vein blood endothelial cells HUVEC are loaded in the microfilamentous hydrogel A, and the fibroblast cell line L929 is loaded in the matrix phase. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0039] As described in the background art, the pores created by the existing porous hydrogels are mostly spherical or other granular shapes with strong convergence. The pores of this shape have poor interconnectivity and low material exchange efficiency. In order to solve the above technical problems, the present invention provides a method for preparing a cell-carrying porous hydrogel bio-ink, comprising: preparing a filamentous or multi-armed sacrificial hydrogel, wherein the multi-armed sacrificial hydrogel has a structure with multiple filamentous side arms extending from the center to the surroundings; preparing a matrix hydrogel precursor solution; wherein the sacrificial hydrogel and / or the matrix hydrogel precursor solution are loaded with living cells; mixing the sacrificial hydrogel and the matrix hydrogel precursor solution to obtain the cell-carrying porous hydrogel bio-ink.

[0040] The inventive concept of the present invention is as follows Figure 1 As shown, a filamentous or multi-armed hydrogel is used as a sacrificial template, and a filamentous or multi-armed sacrificial hydrogel A cross-linked gel is cross-distributed in the precursor solution of the matrix hydrogel B. After removing the sacrificial hydrogel, interconnected tubular pores are formed in the matrix hydrogel B cross-linked gel. The pore structure is a biomimetic vascular lumen morphology with better connectivity. Living cells can be pre-loaded in the sacrificial hydrogel or the matrix hydrogel precursor solution alone, or loaded in both at the same time. The cells in the matrix phase present a typical three-dimensional culture state. The cells in the sacrificial phase are released as the sacrificial components are dissolved and grow in the constructed tubular pores, which can realize the in situ planting and culture of cells. It should be noted that in the present invention, the sacrificial hydrogel can be filamentous or multi-armed. When a cell-carrying porous hydrogel bio-ink is prepared using a multi-armed sacrificial hydrogel, the tissue and / or organ model prepared using the bio-ink has interconnected tubular pores similar to the filamentous hydrogel structure. Based on the same inventive concept, in the specific embodiments of the present invention, only the silky hydrogel is taken as an example for detailed description, but this does not constitute any limitation to the present invention.

[0041] In order to improve the connectivity of the pore structure and realize in situ planting and culture of cells, preferably, the diameter of the filamentous sacrificial hydrogel or the diameter of the filamentous side wall of the multi-arm sacrificial hydrogel is 40 to 500 microns, including but not limited to 50 to 200 microns, 50 to 150 microns, 50 to 100 microns, 100 to 200 microns, 100 to 150 microns, 150 to 200 microns, etc. Taking the filamentous sacrificial hydrogel as an example, in a specific embodiment of the present invention, the diameter of the sacrificial hydrogel is 50 microns, 100 microns, 150 microns, and 200 microns; preferably, the filamentous sacrificial hydrogel is one or more filamentous hydrogels with an aspect ratio of (2 to 1000): 1, such as an aspect ratio of 50: 1; in a specific embodiment of the present invention, the filamentous sacrificial hydrogel is a plurality of filamentous hydrogels with a diameter of 50 microns and an aspect ratio of 50: 1; similarly, preferably, the number of side arms of the multi-armed sacrificial hydrogel is 3 to 8 (including any numerical value and intermediate value therein, such as 3, 4, 5, 6, 7, 8), and the aspect ratio of each side arm is (2 to 10): 1. The connectivity of the pores is related to the volume proportion of the sacrificial hydrogel and the length of a single sacrificial filamentous hydrogel. Generally, the larger the volume proportion of the sacrificial hydrogel and the longer the length, the greater the probability of the sacrificial hydrogels contacting each other, and the better the connectivity of the pores. Similarly, for sacrificial multi-arm hydrogels, the higher the proportion of the sacrificial phase, the longer the length of the side arm / the higher the aspect ratio, and the more side arms there are within a certain range, the greater the possibility of contact between the sacrificial phases, and the better the connectivity of the pores. The aspect ratio is the ratio of the length to the diameter of the microfilament.

[0042] Under normal circumstances, the volume of blood vessels in human body tissues generally accounts for 10%-50%. Considering the printability of the bio-ink in the present invention and the mechanical properties of the final manufactured structure, preferably, the volume ratio of the sacrificial hydrogel and the matrix hydrogel precursor solution is 1: (1-9), for example, 1: 4. Optionally, the sacrificial hydrogel can be a gelatin hydrogel or a sodium alginate hydrogel; wherein the filamentous sacrificial hydrogel can be prepared by a method of preparing straight filaments such as coaxial microfluidics, convergent microfluidics, wet spinning, and template method. Preferably, in order to facilitate the control of the aspect ratio of the sacrificial hydrogel, the preparation of the sacrificial filamentous hydrogel includes: using a coaxial microfluidic device, the coaxial microfluidic device includes a coaxial needle and a microfluidic channel, and the precursor solution and the cross-linking agent of the sacrificial hydrogel are respectively passed into the inner layer and the outer layer of the coaxial needle, and the two are contacted to form the filamentous hydrogel. The following example is used to explain the details: for example, the sacrificial hydrogel is a sodium alginate hydrogel, the precursor solution of the sacrificial hydrogel is a sodium alginate solution, preferably, the mass concentration of the sodium alginate solution is 0.5-5%, and the sacrificial hydrogel crosslinker is Ca 2+ Or Ba 2+, such as calcium chloride solution, preferably, the concentration of calcium chloride solution is 20 mg / ml, using its reversible ionic crosslinking properties, with Ca 2+ , Ba 2+ The divalent cations are used as cross-linking agents to cross-link the hydrogel to form a sacrificial gel; in step S1, the preparation of the sacrificial hydrogel includes: using a coaxial microfluidic device, the coaxial microfluidic device includes a coaxial needle and a microfluidic pipeline, respectively passing a sodium alginate solution and a cross-linking agent into the inner layer and the outer layer of the coaxial needle, and the two contact to form a filamentous hydrogel; using coaxial microfluidic technology, using a coaxial needle microfluidic device, controlling the size of the inner and outer needles, the length and size of the microfluidic hose, the flow rate and concentration of each phase fluid to obtain microfilamentous hydrogels of different diameters, and obtaining microfilaments with controllable aspect ratios through subsequent shearing and other methods. The coaxial microfluidic device is composed of a coaxial needle and a microfluidic pipeline. Among them, the coaxial needle is composed of an inner and outer layer of needle tubes, and the axes of the two layers of needle tubes coincide. In a specific embodiment of the present invention, the outer needle tube size is selected as 22G (inner diameter is 410 microns), and the inner needle tube size is selected as 32G (inner diameter is 110 microns) or 34G (inner diameter is 60 microns). A transparent silicone tube with an inner diameter of 510 microns and a length of 20 cm is connected to the end of the coaxial needle. In a specific embodiment of the present invention, the mass concentration of the sodium alginate solution is 1%, the cross-linking agent is a calcium chloride solution with a concentration of 20 mg / ml, the flow rate of the calcium chloride solution is fixed at 2 ml / min, and the flow rate of the sodium alginate solution is changed to 0.1, 0.2, and 0.5 ml / min, which can realize the preparation of microfilamentous hydrogels with diameters of about 50 microns, 100 microns, and 150 microns; the sodium alginate solution is directly squeezed into the calcium chloride solution with a 34G (inner diameter of 60 microns) inner needle tube, which can realize the preparation of 200 micron microfilamentous hydrogels; the microfilamentous hydrogel is cut by a row of knives with different blade spacings (such as 1mm, 2.5mm or 4mm spacings), and microfilamentous hydrogels with different aspect ratios can be obtained. Among them, the preparation of multi-arm sacrificial hydrogels can be prepared by template method or 3D printing, photopatterning and other methods. Optionally, the matrix hydrogel is selected from a photocrosslinked hydrogel, a temperature or pH responsive natural hydrogel or agarose; preferably, the photocrosslinked hydrogel is methacrylated gelatin, methacrylated hyaluronic acid, methacrylated heparin, acrylated multi-arm polyethylene glycol, methacrylated dextran or methacrylated chondroitin sulfate. For example, the matrix hydrogel is methacrylated gelatin, and the working concentration of the matrix hydrogel precursor solution is 0.025-0.3 g / mL, i.e., 2.5%-30% (w / v), preferably 5%-10% (w / v), such as 7.5% (w / v), wherein w / v refers to g / mL.

[0043] According to the present invention, in order to achieve the loading of living cells, the method for preparing a sacrificial hydrogel loaded with living cells includes: mixing a single cell suspension of living cells with a precursor solution of a sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution; cross-linking the cell-containing sacrificial hydrogel precursor solution to obtain a sacrificial hydrogel loaded with living cells. For example, a coaxial microfluidic device is used, and the coaxial microfluidic device includes a coaxial needle and a microfluidic channel. The cell-containing sacrificial hydrogel precursor solution and a cross-linking agent are respectively introduced into the inner layer and the outer layer of the coaxial needle. After the two are in contact, a sacrificial hydrogel loaded with living cells is formed. In the present invention, before preparing the sacrificial hydrogel, the single cell suspension is mixed with a sodium alginate solution, and then the obtained single cell-containing sodium alginate solution is made into a filamentous sodium alginate hydrogel by a coaxial needle method. Since the manufacturing process has good biocompatibility, cell activity is maintained. It can be understood that for the present invention, other methods for preparing filamentous hydrogels such as wet spinning can achieve the preparation of sacrificial hydrogels loaded with living cells and maintain cell activity. The preparation of a precursor solution of a matrix hydrogel loaded with living cells includes: mixing a single cell suspension of living cells with a precursor solution of a matrix hydrogel to obtain a precursor solution of a matrix hydrogel loaded with living cells. Optionally, depending on the application scenario, the living cells can be vascular endothelial cells, muscle cells, cardiomyocytes, etc., corresponding to the vascularization of in vitro tissue models, muscle or myocardial tissue reconstruction, such as loading human umbilical vein endothelial cells (HUVEC) in a sacrificial hydrogel and loading a fibroblast cell line L929 in a matrix hydrogel, wherein the cells in the sacrificial phase are released as the sacrificial components are dissolved, grow in the constructed lumen pores, and the cells in the matrix phase present a typical three-dimensional culture state.

[0044] The present invention also provides a cell-carrying porous hydrogel bio-ink prepared by any of the preparation methods described above.

[0045] The present invention further provides the use of the cell-loaded porous hydrogel bio-ink in the preparation of tissue and / or organ models. Furthermore, the method for preparing a tissue and / or organ model comprises: performing biological three-dimensional printing on the cell-loaded porous hydrogel bio-ink, and cross-linking and curing the matrix hydrogel precursor solution; removing the sacrificial hydrogel cross-distributed in the matrix hydrogel to form interconnected tubular pores, and obtaining a tissue and / or organ model with a cell-loaded porous hydrogel structure. The structure of the biological three-dimensional printing can be a common structure in tissue and / or organ models, such as a multi-layer grid structure that can be 3D printed.

[0046] According to the present invention, a suitable cross-linking curing method can be selected according to the selected matrix hydrogel type, such as cross-linking under a UV curing lamp; in a specific embodiment of the present invention, the matrix hydrogel is methacrylated gelatin, and the working concentration of its precursor solution (GelMA solution) is 7.5% (w / v), and the cross-linking curing method is 60mW / cm 2 The cross-linking is carried out by a strong 405nm wavelength UV curing lamp.

[0047] Similarly, a suitable sacrificial method can be selected according to the selected sacrificial hydrogel, such as soaking the hydrogel with a sodium alginate hydrogel de-crosslinking agent to selectively remove the sacrificial hydrogel. In a specific embodiment of the present invention, the sodium alginate hydrogel de-crosslinking agent comprises sodium citrate and / or ethylenediaminetetraacetic acid, such as 1% EDTA + 1.5% sodium citrate solution, and the crosslinking time can be adaptively adjusted according to the de-crosslinking situation, such as soaking for 30 minutes.

[0048] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0049] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0050] Example 1: Preparation of porous hydrogel bio-ink using microfilamentous hydrogel as sacrificial template

[0051] according to Figure 1 The flowchart shown in the figure is used to prepare the porous hydrogel bio-ink, and the specific steps are as follows:

[0052] (1) Preparation of microfilamentous hydrogel

[0053] like Figure 2 As shown, the coaxial microfluidic device is composed of a coaxial needle and a microfluidic pipeline. Among them, the coaxial needle is composed of an outer needle tube 1 and an inner needle tube 2, and the axes of the two needle tubes coincide. The size of the outer needle tube 1 is selected as 22G (inner diameter is 410 microns), and the size of the inner needle tube 2 is selected as 32G (inner diameter is 110 microns) or 34G (inner diameter is 60 microns). A transparent silicone tube 3 with an inner diameter of 510 microns is connected to the end of the coaxial needle, and the length is 20 cm.

[0054] The specific method for preparing sacrificial microfilamentous hydrogel A is as follows: sodium alginate solution (mass concentration is 1%) and calcium chloride solution P2 (concentration is 20 mg / ml) are introduced into the inner and outer layers of the coaxial needle respectively by using a syringe pump at a certain flow rate. The sodium alginate solution rapidly cross-links after contacting the calcium chloride solution to form a microfilamentous hydrogel. The preparation of microfilamentous hydrogels of different diameters can be achieved by adjusting the flow rates of the sodium alginate solution and the calcium chloride solution. Different flow rate combinations can prepare microfilamentous hydrogels of different diameters. For example, by fixing the flow rate of the calcium chloride solution at 2 ml / min and changing the flow rate of the sodium alginate solution to 0.1, 0.2, and 0.5 ml / min, the preparation of microfilamentous hydrogels with diameters of about 50 microns, 100 microns, and 150 microns can be achieved. The fourth group refers to directly squeezing the sodium alginate solution into the calcium chloride solution with a 34G needle to obtain a microfilamentous hydrogel with a diameter of 200 microns. See the micrograph. Figure 3 After the microfilamentous hydrogel was successfully prepared, the microfilamentous hydrogel was cut using a row of knives with different blade spacings (1mm, 2.5mm, and 4mm spacings) to obtain microfilamentous hydrogels with different aspect ratios. See the micrographs. Figure 4 .Depend on Figure 3-Figure 4 It can be seen that microfilamentous hydrogels with different diameters and aspect ratios can be cross-distributed to produce interconnected pore structures.

[0055] (2) Preparation of porous hydrogel bio-ink

[0056] According to the process in (1), the flow parameters of calcium chloride solution 2ml / min and sodium alginate solution 0.1ml / min were used, and the microfilamentous hydrogel A was cut with a row blade with a spacing of 2.5mm. The matrix hydrogel material was selected as 7.5% (w / v) GelMA solution. The precursor solutions of microfilamentous hydrogel A and matrix hydrogel B were mixed in a volume ratio of 1:4 to form a mixture solution. The mixture solution was heated at 60mW / cm 2 After B is cross-linked, the entire structure is immersed in 1% EDTA + 1.5% sodium citrate solution for 30 minutes, during which the sacrificial microfilamentous hydrogel A is gradually de-crosslinked and dissolved, and finally a porous hydrogel B is obtained, such as Figure 5 shown.

[0057] Example 2: Preparation of cell-carrying porous hydrogel bio-ink using microfilamentous hydrogel as sacrificial template

[0058] According to the operation in step (1) of Example 1, the flow parameters of calcium chloride solution 2 ml / min and sodium alginate solution 0.1 ml / min were used, and the microfilamentous hydrogel A was cut using a row blade with a spacing of 2.5 mm, and the matrix hydrogel material was selected as a 7.5% (w / v) GelMA solution. The precursor solution of A and the matrix hydrogel B were mixed in a volume ratio of 1:4 to form a composite solution, wherein both A and B can be loaded with living cells. The composite solution was transferred to the sleeve of a biological 3D printer, combined with a computer-aided design model, a 3D structure was formed, and printed as shown. Figure 6 The four-layer grid structure shown. After cross-linking with a UV curing lamp, the entire structure was immersed in a 1% EDTA + 1.5% sodium citrate solution for 30 minutes, during which the sacrificial microfilamentous hydrogel A gradually decrosslinked and dissolved, and finally a porous hydrogel B constructed by three-dimensional printing was obtained. The printed structure was transferred to a cell culture well plate, the corresponding complete culture medium was added, and it was placed in a 37°C cell culture incubator, and the culture medium was changed every day.

[0059] In this embodiment, living cells are loaded into A and B at the same time. The specific steps of loading living cells into A and B are as follows:

[0060] A: Before using the coaxial needle to prepare A, the digested single cell suspension (human umbilical vein blood endothelial cells (HUVEC)) was cultured in a culture bottle at 37 degrees Celsius and 5% carbon dioxide. When the cells were taken, the culture medium in the culture bottle was aspirated, and 0.25% trypsin-EDTA solution was added for digestion for 3 minutes to make the cells detach from the bottom of the culture bottle and disperse into single cells, and then the culture medium was added to terminate the digestion. The number of cells obtained by digestion was determined by cell counting, and the number of cells was calculated and supplemented. The culture medium was used to obtain a single cell suspension with a cell density of 1×10^7 cells / ml) and a sodium alginate solution (mass concentration of 2%) were mixed in a volume ratio of 1:1 to obtain a cell suspension with a concentration of 1% sodium alginate and a cell density of 5×10^6 cells / ml, which was then made into a filamentous sodium alginate hydrogel by a coaxial needle method (with a flow parameter of 2 ml / min for calcium chloride solution and 0.1 ml / min for sodium alginate solution, and cut with a row blade with a spacing of 2.5 mm). Since the manufacturing process has good biocompatibility, cell activity can be maintained;

[0061] B: Similarly, through the same digestion steps, a single cell suspension of the fibroblast cell line L929 with a cell density of 2×10^6 cells / ml was obtained, and the cell suspension was directly mixed with the precursor solution of the matrix hydrogel B (GelMA solution with a mass concentration of 15%) in a volume ratio of 1:1 to obtain a GelMA with a concentration of 7.5% and a L929 cell suspension with a cell density of 1×10^6 cells / ml to achieve cell loading in B.

[0062] In this embodiment, human umbilical vein endothelial cells (HUVEC) are loaded in the microfilamentous hydrogel A. The human umbilical vein endothelial cells (HUVEC) are released as the sacrificial component is dissolved and grow in the pores of the constructed lumen. The matrix phase is not loaded or loaded with fibroblast cell line L929 ( Figure 7 In the figure, No L929 means no loading, L929 means loading), micrographs of cell-loaded porous hydrogels cultured for 1 day and 3 days using endothelial cell-specific culture medium (EGM-2, Lonza) are shown in Figure 7 As shown. Figure 7 It can be seen that the cells in the matrix phase present a typical three-dimensional culture state, and the cells in the sacrificial phase are released as the sacrificial components dissolve and grow in the constructed tubular pores, further verifying the preliminary feasibility of the porous hydrogel carrying living cells of the present invention.

[0063] In summary, the present invention uses special forms of hydrogels such as filamentous or multi-arm hydrogels as sacrificial templates to prepare cell-laden porous hydrogel bio-ink, uses the bio-ink for biological three-dimensional printing, and cross-links and solidifies the matrix hydrogel precursor solution. After removing the sacrificial hydrogel, a tissue and / or organ model with a pore structure of a bionic vascular lumen with better connectivity can be obtained. Cells can be loaded inside the filamentous sacrificial hydrogel, thereby achieving in situ release of cells in the three-dimensional porous network without the need for secondary implantation, thereby improving the efficiency and uniformity of cell implantation.

[0064] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for preparing a cell-carrying porous hydrogel bio-ink, characterized in that: include: Preparing a filamentous or multi-arm sacrificial hydrogel, wherein the multi-arm sacrificial hydrogel has a structure in which a plurality of filamentous side arms extend from the center to the surrounding areas; preparing a matrix hydrogel precursor solution; Wherein, the sacrificial hydrogel and / or the matrix hydrogel precursor solution are loaded with living cells; The sacrificial hydrogel and the matrix hydrogel precursor solution are mixed to obtain the cell-carrying porous hydrogel bio-ink.

2. The method for preparing the cell-carrying porous hydrogel bio-ink according to claim 1, characterized in that: The diameter of the filamentous sacrificial hydrogel or the diameter of the filamentous sidewall of the multi-arm sacrificial hydrogel is 10 to 1000 microns.

3. The method for preparing the cell-carrying porous hydrogel bio-ink according to claim 1 or 2, characterized in that: The filamentous sacrificial hydrogel is one or more filamentous hydrogels with an aspect ratio of (2 to 1000):1; The number of side arms of the multi-arm sacrificial hydrogel is 3 to 16, and the aspect ratio of each side arm is (1 to 50):

1.

4. The method for preparing the cell-carrying porous hydrogel bio-ink according to any one of claims 1 to 3, characterized in that: The volume ratio of the sacrificial hydrogel to the matrix hydrogel precursor solution is 1:(1-9).

5. The method for preparing the cell-carrying porous hydrogel bio-ink according to any one of claims 1 to 4, characterized in that: The sacrificial hydrogel is a gelatin hydrogel or a sodium alginate hydrogel; The matrix hydrogel is selected from a photo-crosslinked hydrogel, a temperature or pH responsive natural hydrogel or agarose.

6. The method for preparing the cell-laden porous hydrogel bio-ink according to any one of claims 1 to 5, characterized in that: The matrix hydrogel is methacrylated gelatin, and the working concentration of its precursor solution is 0.025-0.3 g / mL.

7. The method for preparing the cell-laden porous hydrogel bio-ink according to any one of claims 1 to 6, characterized in that: The method of preparing a sacrificial hydrogel loaded with living cells comprises: Mixing the single cell suspension of the living cells with the precursor solution of the sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution; cross-linking the cell-containing sacrificial hydrogel precursor solution to obtain a sacrificial hydrogel loaded with living cells; The method of preparing a precursor solution of a matrix hydrogel loaded with living cells comprises: The single cell suspension of the living cells is mixed with the precursor solution of the matrix hydrogel to obtain a precursor solution of the matrix hydrogel loaded with living cells.

8. The cell-laden porous hydrogel bio-ink obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the cell-laden porous hydrogel bio-ink according to claim 8 in preparing tissue and / or organ models.

10. The use according to claim 9, characterized in that: The method for preparing the tissue and / or organ model comprises: Performing biological three-dimensional printing on the cell-carrying porous hydrogel bio-ink, and cross-linking and curing the matrix hydrogel precursor solution; The sacrificial hydrogel cross-distributed in the matrix hydrogel is removed to form interconnected luminal pores, thereby obtaining a tissue and / or organ model having a cell-laden porous hydrogel structure.

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