A biomimetic decellularized matrix hydrogel and its application

Abstract

This invention discloses a biomimetic decellularized matrix hydrogel and its applications. It relates to the field of biomaterials technology. It comprises the following components: decellularized liver matrix, gelatin, and sodium alginate, and its applications are provided. The therapeutic artificial liver provided by this invention can be mass-produced in vitro using 3D bioprinting technology, exhibiting mature liver functional phenotypes such as ICG uptake / release and drug metabolism in vitro. It was transplanted into a type I tyrosinemia model of liver failure (Fah). ‑ / ‑ It can alleviate liver damage in mice and has the potential for artificial liver transplantation.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically to a biomimetic decellularized matrix hydrogel and its applications. Background Technology

[0002] The liver plays an irreplaceable role in maintaining many physiological functions, such as ICG uptake / release, drug metabolism, and the synthesis of various hormones. Normally, the liver has a strong regenerative capacity sufficient to cope with most liver injuries. However, diseases such as acute liver failure and cirrhosis can cause irreversible damage to the liver, even endangering the patient's life, necessitating liver transplantation. Currently, the limited number of organ donors is far from meeting the medical needs of liver transplant patients. Therefore, artificial livers fabricated using methods such as 3D bioprinting have received widespread attention from researchers in recent years.

[0003] 3D bioprinting technology can precisely control the spatial distribution of cells and biomaterials, which has a significant advantage for the reconstruction of large organs such as the liver. Currently, several studies have overcome the limitations of 2D culture by arranging commercially available gels (GelMA, matrigel, etc.) containing hepatocytes in specific spatial configurations to create artificial livers with some phenotypic functions, mimicking to some extent the mechanical microenvironment of hepatocyte growth in vivo.

[0004] However, artificial livers are insufficient to provide adequate and mature liver function to alleviate liver failure. Therefore, there is an urgent need for an artificial liver that can highly simulate the physiological environment of a real liver to address the shortage of liver transplants.

[0005] Therefore, how to provide a biomimetic decellularized matrix hydrogel and its applications, and overcome the above-mentioned technical shortcomings, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a biomimetic decellularized matrix hydrogel and its application. The biomimetic decellularized matrix hydrogel has good 3D printing performance, and can realize the large-scale construction of therapeutic artificial livers through bio-3D printing.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A biomimetic decellularized matrix hydrogel comprises the following components: liver decellularized matrix, gelatin, and sodium alginate.

[0009] Preferred concentration of decellularized liver matrix: 5%–15%;

[0010] The mass concentration of gelatin is 4-8%;

[0011] The mass concentration of sodium alginate is 0.5%–1.2%.

[0012] The present invention also provides the application of the above-mentioned biomimetic decellularized matrix hydrogel in the preparation of biological consumables.

[0013] Preferred: Biological consumables: therapeutic artificial liver.

[0014] This invention also provides a method for preparing the above-mentioned biomimetic decellularized matrix hydrogel, comprising the following steps:

[0015] 1) The liver tissue was perfused and then washed with PBS;

[0016] 2) The washed liver tissue was incubated in PBS containing sodium dodecyl sulfate and Triton X-100 to obtain decellularized liver tissue;

[0017] 3) The decellularized liver tissue was freeze-dried to obtain freeze-dried powder, which was then dissolved in PBS containing acetic acid and pepsin and shaken to obtain decellularized liver matrix. Finally, the pH was adjusted to neutral to obtain decellularized liver matrix hydrogel.

[0018] 4) Dissolve gelatin powder and sodium alginate powder in liver decellularized matrix hydrogel, sterilize, and obtain biomimetic decellularized matrix hydrogel.

[0019] Preferred: Step 1) The liver tissue is mouse liver tissue;

[0020] Step 2) The mass concentration of sodium dodecyl sulfate is 1%; the volume percentage of Triton X-100 is 1%; the incubation time is 72 hours.

[0021] Step 3) Freeze-drying conditions: -45℃, 24h; acetic acid concentration: 3%; pepsin concentration: 1mg / ml; shaking conditions: 37℃, 72h; pH adjustment using NaOH.

[0022] This invention also provides a method for preparing a therapeutic artificial liver based on the above-mentioned biomimetic decellularized matrix hydrogel, comprising the following steps:

[0023] A: After mixing the biomimetic decellularized matrix hydrogel with primary hepatocytes and refrigerating it, a biomimetic decellularized hydrogel containing hepatocytes was obtained.

[0024] B: Select the extrusion nozzle and adjust the print nozzle temperature and print platform temperature;

[0025] C: A biomimetic decellularized hydrogel containing hepatocytes was printed in a sterile culture dish to obtain a liver print;

[0026] D: The printed liver is rapidly cross-linked and then cultured using hepatocyte culture medium to obtain a therapeutic artificial liver.

[0027] Preferred: In step A, the primary hepatocytes are mouse primary hepatocytes, and the final concentration after mixing is 5 × 10⁻⁶. 6 / mL;

[0028] Step B: Extrusion nozzle specifications: 23G~27G; Printing nozzle temperature: 10~15℃; Printing platform temperature adjusted to 5~10℃;

[0029] Step C: Printing: Extrusion speed 1-2 mm 3 / s;

[0030] Step D: Crosslinking: Place the printed liver body in a 3% CaCl2 solution; Cultivation: Change the liver cell culture medium every two days and cultivate for one week.

[0031] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a biomimetic decellularized matrix hydrogel and its application, achieving the following technical effects: The therapeutic artificial liver provided by the present invention can be mass-produced in vitro using 3D bioprinting technology, and it exhibits mature liver functional phenotypes such as ICG uptake / release and drug metabolism in vitro. When transplanted into a type I tyrosinemia model of liver failure (Fah... - / - It can alleviate liver damage in mice and has the potential for artificial liver transplantation. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0033] Figure 1 The attached figure shows the rheological test diagram of the biomimetic decellularized matrix hydrogel provided by the present invention.

[0034] Figure 2 The attached figure is a schematic diagram of the preparation process of the biomimetic decellularized matrix hydrogel provided by the present invention.

[0035] Figure 3 The attached figure shows a graph of hepatocyte proliferation detection in the therapeutic artificial liver provided by the present invention.

[0036] Figure 4 The attached figure shows a liver function test diagram of the therapeutic artificial liver provided by the present invention.

[0037] Figure 5 The attached figure is a CYP1A2 enzyme activity detection diagram provided by the present invention, wherein 3-methylcholanthrene is 3-methylcholanthrene; DMSO is dimethyl sulfoxide.

[0038] Figure 6 The attached figure shows the survival curve of mice with liver failure treated with the biomimetic artificial liver provided by this invention.

[0039] Figure 7 The attached figure shows the weight change of mice with liver failure treated with the biomimetic artificial liver provided by this invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] This invention discloses a biomimetic decellularized matrix hydrogel and its applications.

[0042] The raw materials involved in the examples are all commercially available or prepared by conventional methods, and will not be described in detail here.

[0043] Example 1

[0044] A biomimetic decellularized matrix hydrogel comprises the following components: decellularized liver matrix (5%–15% w / v), gelatin (4–8% w / v), and sodium alginate (0.5–1.2% w / v).

[0045] Example 2

[0046] Preparation and culture of therapeutic artificial liver:

[0047] (1) Preparation of biomimetic decellularized matrix hydrogel:

[0048] Liver tissue (approximately 1.5–2.5 g) from 6–8 week old C57 mice was surgically separated and perfused. After perfusion, surface blood was removed using PBS. The liver tissue was then incubated for 72 hours in 50 ml PBS containing 1% (w / v) sodium dodecyl sulfate (SDS) and 1% (v / v) Triton X-100 for decellularization, yielding decellularized liver tissue. The decellularized liver tissue was lyophilized at -45°C for 24 hours. 0.1 g of the lyophilized powder was dissolved in 1 ml PBS containing 3% acetic acid (Merck Millipore, USA) and 1 mg / ml pepsin (Sigma Aldrich, USA), and shaken at 37°C for 72 hours to obtain a liquid decellularized liver matrix. Finally, the pH of the dissolved decellularized liver matrix was adjusted to neutral using NaOH to obtain a decellularized liver matrix hydrogel. Gelatin powder (0.05 g) and sodium alginate powder (0.008 g) were dissolved in the decellularized liver matrix hydrogel at ratios of 5% and 0.8% (w / v), respectively. After sterilization and aliquoting, the hydrogel was stored at 4°C to obtain a biomimetic decellularized matrix hydrogel. Before use, the liquid was heated to 37°C until melted, and its rheological properties were measured. (See attached data.) Figure 1 .

[0049] (2) Bio-3D printing to prepare therapeutic artificial livers

[0050] After preparing the biomimetic decellularized matrix hydrogel according to step (1), it is mixed with mouse primary hepatocytes obtained by perfusion method (final concentration 5×10⁻⁶). 6 A biomimetic decellularized hydrogel containing hepatocytes was obtained by refrigerating the hydrogel at 4°C for 20 minutes ( / mL). Its viscosity, shear strain, and other printing properties were measured using a rheometer. An extrusion nozzle with a specification of 23G-27G was used, and the nozzle temperature was adjusted to 10-15°C, and the platform temperature to 5-10°C. A 1-2mm extrusion nozzle was used. 3 At an extrusion speed of / s, a biomimetic decellularized hydrogel containing hepatocytes was printed into a 6cm sterile culture dish to obtain a liver print. The printed liver print was then placed in a 3% (w / v) CaCl2 solution to rapidly cross-link, and subsequently cultured in hepatocyte culture medium (specific components can be found in the literature "Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity," hereinafter the same). The medium was replaced with fresh hepatocyte culture medium every two days. After one week of culture, a therapeutic artificial liver was obtained. The procedure is as follows: Figure 2 .

[0051] (3) Detection of hepatocyte proliferation in therapeutic artificial liver

[0052] Therapeutic artificial livers cultured for 0, 2, 4, and 6 days were dissolved using a dissociation solution containing sodium citrate (55 mM), EDTA (20 mM), and NaCl (150 mM). The number of cells in the printed body was measured using a cell counter, and the cell proliferation curve of hepatocytes in the printed body was calculated. The results indicate that the above-mentioned biomimetic decellularized matrix hydrogel has good biocompatibility and can provide a favorable environment for hepatocyte proliferation (see...). Figure 3 ).

[0053] (4) Liver function testing of therapeutic artificial liver

[0054] The therapeutic artificial liver was cultured in hepatocyte culture medium for 48 hours, and then co-incubated with hepatocyte culture medium containing ICG (1 mg / ml) at 37°C for 1 hour. It was washed three times with PBS, and the PBS was replaced afterward. ICG uptake / release was observed under a microscope. The results indicate that the therapeutic artificial liver prepared using the biomimetic decellularized matrix hydrogel has good metabolic function (see...). Figure 4 ).

[0055] 3-methylcholanthrene was co-incubated with a therapeutic artificial liver at 37°C for 72 hours, while the control group was co-incubated with DMSO at 37°C for 72 hours. The CYP1A2 enzyme activity of the two artificial livers was detected using a CYP1A2 quantitative kit. The results indicate that the therapeutic artificial liver prepared using a biomimetic decellularized matrix hydrogel can express the CYP1A2 enzyme and has good drug metabolism function (see...). Figure 5 ).

[0056] (5) Transplanting a therapeutic artificial liver into a commercially available Fah - / - In mice (mesenteric region), after one week, Fah-deficient mice were treated with NTBC depletion. Survival time and weight changes were recorded in both the control and experimental groups. The results indicate that the therapeutic artificial liver prepared using biomimetic decellularized matrix hydrogel possesses certain liver function in vivo and can alleviate liver damage (see...). Figure 6 , 7 ).

[0057] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a therapeutic artificial liver based on a biomimetic decellularized matrix hydrogel, wherein the biomimetic decellularized matrix hydrogel is characterized in that... It includes the following components: decellularized liver matrix, gelatin, and sodium alginate; The mass concentration of the decellularized liver matrix is ​​5% to 15%. The mass concentration of the gelatin is 4-8%; The mass concentration of sodium alginate is 0.5–1.2%; The preparation method of the biomimetic decellularized matrix hydrogel includes the following steps: 1) The liver tissue was perfused and then washed with PBS; 2) The washed liver tissue was incubated in PBS containing sodium dodecyl sulfate and Triton X-100 to obtain decellularized liver tissue; 3) The decellularized liver tissue was freeze-dried to obtain freeze-dried powder, which was then dissolved in PBS containing acetic acid and pepsin and shaken to obtain decellularized liver matrix. Finally, the pH was adjusted to neutral to obtain decellularized liver matrix hydrogel. 4) Dissolve gelatin powder and sodium alginate powder in liver decellularized matrix hydrogel, sterilize, and obtain biomimetic decellularized matrix hydrogel; The liver tissue mentioned in step 1) is mouse liver tissue; Step 2) The sodium dodecyl sulfate concentration is 1% by mass; the Triton X-100 volume percentage is 1%; the incubation time is 72 hours. Step 3) The freeze-drying conditions are: -45℃, 24h; the acetic acid concentration is 3%; the pepsin concentration is 1mg / ml; the shaking treatment conditions are: 37℃, 72h; the pH is adjusted using NaOH. The method for preparing a therapeutic artificial liver based on a biomimetic decellularized matrix hydrogel includes the following steps: A: After mixing the biomimetic decellularized matrix hydrogel with primary hepatocytes and refrigerating it, a biomimetic decellularized hydrogel containing hepatocytes was obtained. B: Select the extrusion nozzle and adjust the print nozzle temperature and print platform temperature; C: A biomimetic decellularized hydrogel containing hepatocytes was printed in a sterile culture dish to obtain a liver print; D: The printed liver is rapidly cross-linked and then cultured using hepatocyte culture medium to obtain a therapeutic artificial liver.

2. The method for preparing a therapeutic artificial liver based on a biomimetic decellularized matrix hydrogel as described in claim 1, characterized in that, The primary hepatocytes used in step A are mouse primary hepatocytes, and the final concentration after mixing is 5 × 10⁻⁶. 6 / mL; The specifications of the extrusion nozzle mentioned in step B are: 23G~27G; the printing nozzle temperature is 10~15℃; the printing platform temperature is adjusted to 5~10℃. Step C, printing: extrusion speed is 1-2 mm. 3 / s; The cross-linking step D involves placing the printed liver body in a 3% CaCl2 solution; the culture involves replacing the liver cell culture medium with fresh medium every two days and culturing for one week.

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