Cell-laden porogenic hydrogel bio-ink, preparation method and application thereof

Abstract

The application discloses a cell-loaded porous hydrogel biomaterial and a preparation method and application thereof. The preparation method of the cell-loaded porous hydrogel biomaterial comprises the following steps: preparing a filamentous or multi-arm sacrificial hydrogel; preparing a matrix hydrogel precursor solution; wherein, the sacrificial hydrogel and / or the matrix hydrogel precursor solution are loaded with living cells; and mixing the sacrificial hydrogel and the matrix hydrogel precursor solution to obtain the cell-loaded porous hydrogel biomaterial. The special-shaped hydrogel such as the filamentous or multi-arm hydrogel is used as a sacrificial template, and after the sacrificial hydrogel is removed, a biomimetic blood vessel lumen form with better connectivity of the pore structure can be obtained, and cells can be loaded in the filamentous sacrificial hydrogel, so that the cells can be released in situ in the three-dimensional pore network, and secondary planting is not needed, and the efficiency and uniformity of cell planting are improved.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and more particularly to a cell-loaded pore-forming hydrogel bio-ink, its preparation method, and its application. Background Technology

[0002] Hydrogels are network systems formed by the cross-linking of hydrophilic polymers dispersed in an aqueous phase. Hydrogels can simulate the three-dimensional microphysiological environment of the extracellular matrix in vivo, and are one of the most widely used three-dimensional cell culture substrates. They can be applied to the large-scale expansion of (stem) cells in vitro, the construction of in vitro tissue / organ models, and the repair of tissues / organs in vivo. Hydrogels are also one of the most important ink materials for bioprinting, used to load cells and other molecules, providing mechanical support and a three-dimensional culture environment for cells, enabling the direct three-dimensional printing of complex tissue / organ models containing living cells.

[0003] However, the penetration of oxygen and nutrients into three-dimensional matrix materials is limited by distance. This means that cells in a three-dimensional culture state can generally receive sufficient nutrients within a distance of 100-200 micrometers. Internal cells often experience reduced activity and necrosis due to insufficient nutrition. Therefore, creating a three-dimensional hydrogel environment with higher material exchange efficiency for cells is an important issue.

[0004] Existing methods for preparing porous hydrogels often employ salt particles or porogens as sacrificial templates to create pores within the hydrogel. However, many porogens themselves, or their removal processes, introduce factors such as high salt, acid / alkali, and high temperatures, affecting the in-situ display and loading of live cells. Some studies have used biocompatible hydrogel particles as sacrificial templates to create pores within another type of hydrogel, allowing live cells to be encapsulated within the latter without affecting their activity. However, the pores created by these sacrificial template methods are mostly spherical or other highly converging granular shapes. These pore shapes have poor interconnectivity and low material exchange efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a cell-loaded porous hydrogel bio-ink, its preparation method, and its application. This method uses hydrogels with special morphologies such as filamentous or multi-armed hydrogels as sacrificial templates. After removing the sacrificial hydrogel, a biomimetic vascular lumen morphology with better connectivity can be prepared. Cells can be loaded inside the filamentous sacrificial hydrogel, thereby realizing the in-situ release of cells within a three-dimensional porous network without the need for secondary seeding, thus improving the efficiency and uniformity of cell seeding.

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

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

[0008] Preparation of matrix hydrogel precursor solution;

[0009] The sacrificial hydrogel and / or the matrix hydrogel precursor solution contain live cells.

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

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

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

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

[0014] In the above-mentioned method for preparing cell-borne pore-forming 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 cell-borne pore-forming hydrogel bio-ink, the sacrificial hydrogel is gelatin hydrogel or sodium alginate hydrogel.

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

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

[0018] In the above-mentioned method for preparing cell-loaded pore-forming hydrogel bio-ink, the method for preparing sacrificial hydrogels loaded with living cells includes:

[0019] The single-cell suspension of the live cells is mixed with the precursor solution of the sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution.

[0020] The cell-containing sacrificial hydrogel precursor solution is crosslinked to obtain a sacrificial hydrogel loaded with living cells;

[0021] Methods for preparing precursor solutions of matrix hydrogels loaded with living cells include:

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

[0023] Secondly, the present invention provides a cell-loaded pore-forming hydrogel bio-ink obtained by any of the preparation methods described above.

[0024] Thirdly, the present invention provides the application of the cell-loaded pore-forming hydrogel bio-ink in the preparation of tissue and / or organ models.

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

[0026] The cell-loaded pore-forming hydrogel bio-ink was used for bio-3D printing, and the matrix hydrogel precursor solution was cross-linked and cured.

[0027] Sacrificial hydrogels that are cross-distributed in the matrix hydrogel are removed to form interconnected luminal pores, resulting in tissue and / or organ models with cell-loaded porous hydrogel structures.

[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 realize a pore structure with better internal connectivity and biomimetic tubular shape in another hydrogel, providing a new ink form for bio-3D printing.

[0030] (2) In the method of the present invention, live cells can be pre-loaded individually in a sacrificial hydrogel or a matrix hydrogel precursor solution, or simultaneously in both; wherein the cells in the matrix phase exhibit a typical three-dimensional culture state. The cells in the sacrificial phase are released as the sacrificial components dissolve and grow in the constructed lumen pores, enabling in-situ seeding and culture of cells. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the preparation principle of the cell-loaded pore-forming hydrogel bio-ink of the present invention.

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

[0033] Figure 3In Example 1 of this invention, the flow rate of the calcium chloride solution was fixed at 2 ml / min, and the flow rate of the sodium alginate solution was changed to 0.1, 0.2, and 0.5 ml / min. The microfilamentous hydrogels obtained by directly extruding the sodium alginate solution into the calcium chloride solution were denoted 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, the first to fourth groups are micrographs and corresponding diameter bar graphs at different magnifications. The average diameters of the first to fourth groups are approximately 50 μm, 100 μm, 150 μm, and 200 μm, respectively.

[0034] Figure 4 The images show fluorescence images of microfilamentous hydrogels with different aspect ratios obtained by cutting with blades of different spacing in Example 1 of the present invention. From left to right, they are uncut, cut with a length of 1 mm, cut with a length of 2.5 mm, and cut with a length of 4 mm, with a diameter of 140 micrometers.

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

[0036] Figure 6 The image shows a fluorescent photograph of the mesh structure obtained by 3D printing porous hydrogel bio-ink using needles of different diameters in Example 2 of this invention.

[0037] Figure 7 To illustrate the cell culture effect in the sacrificial and matrix phases of the porous bio-ink in Example 2 of this invention, NO L929 indicates that human umbilical vein blood endothelial cells (HUVECs) are loaded only in microfilamentous hydrogel A, and L929 indicates that human umbilical vein blood endothelial cells (HUVECs) are loaded in microfilamentous hydrogel A, and fibroblast cell line L929 is loaded in the matrix phase. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] As described in the background section, existing porous hydrogels mostly create pores in spherical or other highly converging granular shapes. These pore shapes have poor interconnectivity and low material exchange efficiency. To address the aforementioned technical problems, this invention provides a method for preparing a cell-loaded 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 outwards; preparing a matrix hydrogel precursor solution; wherein the sacrificial hydrogel and / or the matrix hydrogel precursor solution are loaded with live cells; and mixing the sacrificial hydrogel and the matrix hydrogel precursor solution to obtain the cell-loaded porous hydrogel bio-ink.

[0040] The inventive concept of this invention is as follows: Figure 1 As shown, using filamentous or multi-armed hydrogels as sacrificial templates, the filamentous or multi-armed sacrificial hydrogel A cross-linked gel is cross-distributed in the precursor solution of matrix hydrogel B. After removing the sacrificial hydrogel, interconnected luminous pores are formed in the cross-linked gel of matrix hydrogel B. The constructed pore structure resembles a biomimetic blood vessel lumen with better connectivity. Live cells can be pre-loaded individually in the sacrificial hydrogel or matrix hydrogel precursor solution, or simultaneously in both. Cells in the matrix phase exhibit a typical three-dimensional culture state, while cells in the sacrificial phase are released as the sacrificial component dissolves and grow in the constructed luminous pores, enabling in-situ seeding and culture of cells. It should be noted that in this invention, the sacrificial hydrogel can be filamentous or multi-armed. When cell-loaded pore-forming hydrogel bio-ink is prepared using multi-armed sacrificial hydrogels, the tissue and / or organ models prepared with this bio-ink have interconnected luminous pores similar to the structure of filamentous hydrogels. Based on the same inventive concept, in the specific embodiments of the present invention, only filamentous hydrogels are used as an example for detailed description, but this does not constitute any limitation on the present invention.

[0041] To improve the connectivity of the pore structure and enable in situ seeding and culture of cells, preferably, the diameter of the filamentous sacrificial hydrogel or the diameter of the filamentous sidewalls of the multi-arm sacrificial hydrogel is 40–500 micrometers, including but not limited to 50–200 micrometers, 50–150 micrometers, 50–100 micrometers, 100–200 micrometers, 100–150 micrometers, 150–200 micrometers, etc. Taking filamentous sacrificial hydrogels as an example, in specific embodiments of the present invention, the diameter of the sacrificial hydrogel is 50 micrometers, 100 micrometers, 150 micrometers, or 200 micrometers. Preferably, the filamentous sacrificial hydrogel is one or more filamentous hydrogels with an aspect ratio of (2-1000):1, such as an aspect ratio of 50:1. In specific embodiments of the present invention, the filamentous sacrificial hydrogel is several filamentous hydrogels with a diameter of 50 micrometers and an aspect ratio of 50:1. Similarly, preferably, the number of side arms of the multi-armed sacrificial hydrogel is 3-8 (including any value and intermediate values, such as 3, 4, 5, 6, 7, 8), and the aspect ratio of each side arm is (2-10):1. The connectivity of the pores is related to the volume ratio of the sacrificial hydrogel and the length of a single sacrificial filamentous hydrogel. Generally, the larger the volume ratio and the longer the length of the sacrificial hydrogel, the greater the probability of the sacrificial hydrogels contacting each other, and the better the connectivity of the pores. Similarly, for sacrificial multi-armed hydrogels, the higher the proportion of the sacrificial phase, the longer the length / aspect ratio of the side arms, and the greater the number of side arms within a certain range, the greater the possibility of contact between the sacrificial phases, and the better the connectivity of the pores formed. Here, the aspect ratio is the ratio of the length to the diameter of the microfilaments.

[0042] Under normal circumstances, the volume ratio of blood vessels in human tissues is generally between 10% and 50%. Considering the printability of the bio-ink and the mechanical properties of the final manufactured structure, the volume ratio of the sacrificial hydrogel to the matrix hydrogel precursor solution is preferably 1:(1-9), for example, 1:4. Optionally, the sacrificial hydrogel can be gelatin hydrogel or sodium alginate hydrogel; wherein, the filamentous sacrificial hydrogel can be prepared into straight filaments using coaxial microfluidic technology, convergent microfluidic technology, wet spinning, template method, etc. Preferably, to facilitate control of the aspect ratio of the sacrificial hydrogel, the preparation of the sacrificial filamentous hydrogel includes: using a coaxial microfluidic device, which includes a coaxial needle and a microfluidic channel, and introducing the sacrificial hydrogel precursor solution and a crosslinking agent into the inner and outer layers of the coaxial needle, respectively, to form the filamentous hydrogel upon contact. The following example provides a detailed explanation: For instance, the sacrificial hydrogel is a sodium alginate hydrogel, the precursor solution of the sacrificial hydrogel is a sodium alginate solution, preferably with a mass concentration of 0.5-5%, and the sacrificial hydrogel crosslinking agent is Ca. 2+ Or Ba 2+For example, calcium chloride solution, preferably with a concentration of 20 mg / ml, utilizes its reversible ionic cross-linking properties to use Ca... 2+ Ba 2+ Divalent cations are used as crosslinking agents to crosslink sacrificial hydrogels. In step S1, the preparation of the sacrificial hydrogel includes: using a coaxial microfluidic device, which includes a coaxial needle and microfluidic channels; introducing sodium alginate solution and a crosslinking agent into the inner and outer layers of the coaxial needle, respectively, forming filamentous hydrogels upon contact; using coaxial microfluidic technology, the dimensions of the inner and outer needles, the length and dimensions of the microfluidic tubing, and the flow rate and concentration of each phase fluid are controlled to obtain microfilamentous hydrogels of different diameters. Subsequent shearing or other methods are then used to obtain microfilaments with a controllable aspect ratio. The coaxial microfluidic device consists of a coaxial needle and microfluidic channels. The coaxial needle consists of two layers of needle tubes with their axes overlapping. In a specific embodiment of the present invention, the outer needle tube size is selected as 22G (inner diameter of 410 micrometers), and the inner needle tube size is selected as 32G (inner diameter of 110 micrometers) or 34G (inner diameter of 60 micrometers). A transparent silicone tube with an inner diameter of 510 micrometers and a length of 20 centimeters is attached to the end of the coaxial needle tip. In a specific embodiment of the present invention, the sodium alginate solution has a mass concentration of 1%, the crosslinking 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 achieve the preparation of microfilamentous hydrogels with diameters of approximately 50 μm, 100 μm, and 150 μm. Using a 34G (60 μm inner diameter) inner-layer needle to directly extrude the sodium alginate solution into the calcium chloride solution, microfilamentous hydrogels with diameters of 200 μm can be prepared. By using a array of blades with different blade spacings (e.g., 1 mm, 2.5 mm, or 4 mm spacing) to cut the microfilamentous hydrogels, microfilamentous hydrogels with different aspect ratios can be obtained. The preparation of multi-armed sacrificial hydrogels can be achieved using template methods, 3D printing, or photopatterning. Optionally, the matrix hydrogel is selected from photocrosslinked hydrogels, temperature- or pH-responsive natural hydrogels, or agarose; preferably, the photocrosslinked hydrogel is methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide heparin, propylene glycol multi-arm polyethylene glycol, methacrylamide dextran, or methacrylamide chondroitin sulfate. For example, the matrix hydrogel is methacrylamide 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), where w / v refers to g / mL.

[0043] According to the present invention, a method for preparing a sacrificial hydrogel loaded with live cells includes: mixing a single-cell suspension of live cells with a precursor solution of a sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution; and crosslinking the cell-containing sacrificial hydrogel precursor solution to obtain a sacrificial hydrogel loaded with live cells. For example, a coaxial microfluidic device is used, comprising a coaxial needle and a microfluidic channel. The cell-containing sacrificial hydrogel precursor solution and a crosslinking agent are respectively introduced into the inner and outer layers of the coaxial needle, and upon contact, a sacrificial hydrogel loaded with live cells is formed. In this invention, before preparing the sacrificial hydrogel, a single-cell suspension is mixed with a sodium alginate solution, and then the resulting sodium alginate solution containing single cells is used to prepare a filamentous sodium alginate hydrogel using a coaxial needle method. Because this manufacturing process has good biocompatibility, cell viability is maintained. It is understood that, for the purposes of this invention, other methods for preparing filamentous hydrogels, such as wet spinning, can also achieve the preparation of sacrificial hydrogels loaded with live cells and maintain cell viability. The preparation of the precursor solution for the matrix hydrogel loaded with live cells includes: mixing a single-cell suspension of live cells with the precursor solution of the matrix hydrogel to obtain the precursor solution for the matrix hydrogel loaded with live cells. Optionally, depending on the application scenario, the live cells can be vascular endothelial cells, muscle cells, cardiomyocytes, etc., corresponding to vascularization of in vitro tissue models, reconstruction of muscle or myocardial tissue, respectively. For example, human umbilical vein blood endothelial cells (HUVECs) are loaded into a sacrificial hydrogel, and the fibroblast cell line L929 is loaded into a matrix hydrogel. In this case, the cells in the sacrificial phase are released as the sacrificial component dissolves and grow in the constructed luminal pores. The cells in the matrix phase exhibit a typical three-dimensional culture state.

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

[0045] This invention further provides the application of the aforementioned cell-loaded porous hydrogel bio-ink in the preparation of tissue and / or organ models. Further, the method for preparing tissue and / or organ models includes: bio-3D printing the cell-loaded porous hydrogel bio-ink, and cross-linking and curing the matrix hydrogel precursor solution; removing sacrificial hydrogels cross-distributed in the matrix hydrogel to form interconnected luminous pores, thereby obtaining a tissue and / or organ model with a cell-loaded porous hydrogel structure. The bio-3D printed structure can be a common structure in tissue and / or organ models, such as a multi-layered mesh structure that can be 3D printed.

[0046] According to the present invention, a suitable crosslinking and curing method can be selected according to the type of matrix hydrogel, such as crosslinking under a UV curing lamp; in a specific embodiment of the present invention, the matrix hydrogel is methacrylamide gelatin, and the working concentration of its precursor solution (GelMA solution) is 7.5% (w / v), using 60mW / cm 2 Crosslinking was performed using a 405nm wavelength ultraviolet curing lamp.

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

[0048] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

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

[0051] according to Figure 1 The flowchart shown illustrates the preparation of porous hydrogel bio-inks, with the specific steps as follows:

[0052] (1) Preparation of microfilament hydrogels

[0053] like Figure 2 As shown, the coaxial microfluidic device consists of a coaxial needle and a microfluidic conduit. The coaxial needle comprises an outer needle tube 1 and an inner needle tube 2, with their axes overlapping. The outer needle tube 1 is sized 22G (inner diameter 410 micrometers), and the inner needle tube 2 is sized either 32G (inner diameter 110 micrometers) or 34G (inner diameter 60 micrometers). A transparent silicone tube 3 with an inner diameter of 510 micrometers and a length of 20 centimeters is attached to the end of the coaxial needle.

[0054] The specific method for preparing sacrificial microfilamentous hydrogel A is as follows: Sodium alginate solution (1% by mass) and calcium chloride solution P2 (20 mg / ml) are respectively introduced into the inner and outer layers of a coaxial needle using an injection pump at specific flow rates. Upon contact with the calcium chloride solution, the sodium alginate solution rapidly cross-links, forming a microfilamentous hydrogel. By adjusting the flow rates of the sodium alginate and calcium chloride solutions, microfilamentous hydrogels of different diameters can be prepared. 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, microfilamentous hydrogels with diameters of approximately 50 μm, 100 μm, and 150 μm can be prepared. The fourth group refers to directly squeezing the sodium alginate solution into the calcium chloride solution using a 34G needle to obtain a microfilamentous hydrogel with a diameter of 200 μm. See the micrograph below. Figure 3 After successfully preparing the microfilamentous hydrogels, the microfilamentous hydrogels were cut using a array of blades with different blade spacings (1 mm, 2.5 mm, and 4 mm) to obtain microfilamentous hydrogels with different aspect ratios. Microscopic images are shown below. Figure 4 .Depend on Figures 3-4 It can be seen that microfilamentous hydrogels with different diameters and aspect ratios can be cross-distributed to create interconnected porous structures.

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

[0056] Following the procedure in (1), microfilamentous hydrogel A was prepared by cutting with a 2.5 mm spacing blade using a flow rate of 2 ml / min for calcium chloride solution and 0.1 ml / min for sodium alginate solution. A 7.5% (w / v) GelMA solution was selected as the matrix hydrogel material. The precursor solutions of microfilamentous hydrogel A and matrix hydrogel B were mixed at a volume ratio of 1:4 to form a mixture solution, and a 60 mW / cm² solution was used. 2 Crosslinking was performed using a 405nm wavelength UV curing lamp. After crosslinking B, the entire structure was immersed in a 1% EDTA + 1.5% sodium citrate solution for 30 minutes, during which the sacrificial microfilament hydrogel A gradually decrosslinked and dissolved, ultimately yielding porous hydrogel B, as shown below. Figure 5 As shown.

[0057] Example 2: Preparation of cell-loaded pore-forming hydrogel bio-ink using microfilamentous hydrogels as sacrificial templates

[0058] Following the procedure in step (1) of Example 1, microfilamentous hydrogel A was prepared by cutting with a 2.5mm spacing blade using a flow rate of 2 ml / min for calcium chloride solution and 0.1 ml / min for sodium alginate solution. A 7.5% (w / v) GelMA solution was selected as the matrix hydrogel material. The precursor solutions of A and matrix hydrogel B were mixed at a volume ratio of 1:4 to form a composite solution, wherein both A and B could be optionally loaded with live cells. This composite solution was transferred to the sleeve of a 3D bioprinter, and a 3D structure was formed using a computer-aided design model, and printed as shown. Figure 6 The four-layer mesh structure is shown. After cross-linking it using a UV curing lamp, the entire structure was immersed in a 1% EDTA + 1.5% sodium citrate solution for 30 minutes. During this process, the sacrificial microfilament hydrogel A gradually decrosslinked and dissolved, ultimately yielding the 3D-printed porous hydrogel B. The printed structure was transferred to cell culture plates, the corresponding complete culture medium was added, and the plates were placed in a 37°C cell culture incubator, with the culture medium changed daily.

[0059] In this embodiment, live cells are loaded simultaneously in A and B. The specific steps for loading live cells into A and B are as follows:

[0060] A: Before preparing A using a coaxial needle, a digested single-cell suspension (human umbilical vein blood endothelial cells (HUVECs)) was cultured in a culture flask at 37°C with 5% carbon dioxide. When using the cells, the culture medium was aspirated, and 0.25% trypsin-EDTA solution was added for digestion for 3 minutes to detach the cells from the bottom of the flask and disperse them into single cells. Culture medium was then added to terminate the digestion. The number of cells obtained from digestion was determined by cell counting, and supplementation was calculated and performed. The culture medium (a single-cell suspension with a cell density of 1×10^7 cells / ml) was mixed with sodium alginate solution (2% by mass) at a 1:1 volume ratio to obtain a cell suspension with a concentration of 1% sodium alginate and a cell density of 5×10^6 cells / ml. This suspension was then processed into filamentous sodium alginate hydrogels using a coaxial needle method (cut with a flow rate of 2 ml / min of calcium chloride solution and 0.1 ml / min of sodium alginate solution using blades with a 2.5 mm spacing). Due to the good biocompatibility of this manufacturing process, cell viability was 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. The cell suspension was then directly mixed with the precursor solution of matrix hydrogel B (a 15% GelMA solution) at a volume ratio of 1:1 to obtain a 7.5% GelMA concentration and an L929 cell suspension containing a cell density of 1×10^6 cells / ml, thereby achieving cell loading in B.

[0062] In this embodiment, human umbilical vein blood endothelial cells (HUVECs) are loaded into microfilamentous hydrogel A. The HUVECs are released as the sacrificial component dissolves and grow in the constructed tubular pores. The matrix phase is either unloaded or loaded with the fibroblast cell line L929. Figure 7 In the image, No. L929 indicates unloaded, and L929 indicates loaded. Micrographs of cell-loaded porous hydrogels cultured for 1 day and 3 days using endothelial cell-specific medium (EGM-2, Lonza) are shown below. Figure 7 As shown. By Figure 7 It can be seen that the cells in the matrix phase exhibit 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 carrying live cells in the porous hydrogel of the present invention.

[0063] In summary, this invention uses hydrogels with special morphologies, such as filamentous or multi-armed hydrogels, as sacrificial templates to prepare cell-loaded porous hydrogel bio-inks. These bio-inks are then used for bio-3D printing, and the matrix hydrogel precursor solution is cross-linked and solidified. After removing the sacrificial hydrogel, tissue and / or organ models with a biomimetic vascular lumen morphology and better connectivity can be obtained. Cells can be loaded inside the filamentous sacrificial hydrogel, thereby achieving in-situ release of cells within a three-dimensional porous network without the need for secondary seeding, thus improving the efficiency and uniformity of cell seeding.

[0064] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing a cell-loaded pore-forming hydrogel bio-ink, characterized in that, include: Prepare filamentous or multi-armed sacrificial hydrogels, wherein the multi-armed sacrificial hydrogel has a structure in which multiple filamentous side arms extend from the center to the periphery; The diameter of the filamentous sacrificial hydrogel or the diameter of the filamentous sidewalls of the multi-arm sacrificial hydrogel is 10~1000 micrometers. The filamentous sacrificial hydrogel is one or more filamentous hydrogels with an aspect ratio of (2~1000):1; The multi-armed sacrificial hydrogel has 3 to 16 lateral arms, and the aspect ratio of each lateral arm is (1 to 50):

1. Preparation of matrix hydrogel precursor solution; The sacrificial hydrogel and / or the matrix hydrogel precursor solution contain live cells. The sacrificial hydrogel and the matrix hydrogel precursor solution are mixed to obtain the cell-loaded pore-forming hydrogel bio-ink; The volume ratio of the sacrificial hydrogel to the matrix hydrogel precursor solution is 1:(1~9). The filamentous or multi-armed sacrificial hydrogels are cross-distributed in the matrix hydrogel precursor solution. After the sacrificial hydrogels are removed, interconnected luminal pores are formed in the cross-linked gel of the matrix hydrogel. The resulting pore structure is a biomimetic vascular lumen morphology with better connectivity.

2. The method for preparing cell-loaded pore-forming hydrogel bio-ink according to claim 1, characterized in that: The sacrificial hydrogel is a gelatin hydrogel or a sodium alginate hydrogel. The matrix hydrogel is selected from photocrosslinked hydrogels, temperature or pH responsive natural hydrogels, or agarose.

3. The method for preparing cell-loaded pore-forming hydrogel bio-ink according to any one of claims 1-2, characterized in that: The matrix hydrogel is methacrylamide gelatin, and the working concentration of its precursor solution is 0.025~0.3 g / mL.

4. The method for preparing cell-loaded pore-forming hydrogel bio-ink according to any one of claims 1-2, characterized in that: Methods for preparing sacrificial hydrogels loaded with living cells include: The single-cell suspension of the live cells is mixed with the precursor solution of the sacrificial hydrogel to obtain a cell-containing sacrificial hydrogel precursor solution. The cell-containing sacrificial hydrogel precursor solution is crosslinked to obtain a sacrificial hydrogel loaded with living cells; Methods for preparing precursor solutions of matrix hydrogels loaded with living cells include: The single-cell suspension of the live cells is mixed with the precursor solution of the matrix hydrogel to obtain the precursor solution of the matrix hydrogel loaded with live cells.

5. The cell-loaded pore-forming hydrogel bio-ink obtained by the preparation method according to any one of claims 1-4.

6. The application of the cell-loaded pore-forming hydrogel bio-ink according to claim 5 in the preparation of tissue or organ models.

7. The application according to claim 6, characterized in that: The method for preparing the tissue or organ model includes: The cell-loaded pore-forming hydrogel bio-ink was used for bio-3D printing, and the matrix hydrogel precursor solution was cross-linked and cured. By removing sacrificial hydrogels that are cross-distributed in the matrix hydrogel to form interconnected luminal pores, tissue or organ models with cell-carrying porous hydrogel structures are obtained.

Images