CN117504004A - Bionic acellular matrix hydrogel and application thereof - Google Patents
Bionic acellular matrix hydrogel and application thereof Download PDFInfo
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- CN117504004A CN117504004A CN202311592046.2A CN202311592046A CN117504004A CN 117504004 A CN117504004 A CN 117504004A CN 202311592046 A CN202311592046 A CN 202311592046A CN 117504004 A CN117504004 A CN 117504004A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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Abstract
The invention discloses a bionic acellular matrix hydrogel and application thereof. Relates to the technical field of biological materials. Comprises the following components: liver decellularized matrix, gelatin and sodium alginate, and uses thereof are provided. The therapeutic artificial liver provided by the invention can be produced in large scale in vitro by a 3D biological printing technology, and shows mature liver function phenotypes such as ICG uptake/release, drug metabolism and the like in vitro. It was transplanted into type i tyrosinemia model liver failure (Fah ‑/‑ ) Can relieve liver injury of mice in vivo, and has therapeutic potential for artificial liver transplantation.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a bionic acellular matrix hydrogel and application thereof.
Background
The liver plays an irreplaceable role in the maintenance of multiple physiological functions, such as ICG uptake/release, drug metabolism, and synthesis of various hormones. In general, the liver has extremely strong regeneration function enough to cope with most liver injuries, but acute liver failure, liver cirrhosis and other diseases can bring irreversible damage to the liver, even endanger the life of a patient, and liver transplantation treatment is needed for the patient. The current limited organ donors are far from meeting the medical needs of liver transplant patients. Therefore, artificial livers prepared by means of 3D bioprinting and the like have received extensive attention from researchers in recent years.
The 3D bioprinting technique can precisely control the spatial distribution of cells and biological materials, which has great advantages for reconstruction of large organs such as liver. Several studies have now formed artificial livers with partial phenotypic functions by means of a specific spatial arrangement of commercial gels containing hepatocytes (GelMA, matrigel, etc.), which overcome the limitations of 2D culture and to some extent mimic the mechanically powered microenvironment of hepatocyte growth in vivo.
However, it is not sufficient for artificial liver to possess sufficiently mature liver function to alleviate liver failure. Therefore, there is a need for an artificial liver that can highly mimic the physiological environment of a real liver to solve the problem of liver transplantation shortage.
Therefore, how to provide a bionic acellular matrix hydrogel and application thereof, and to overcome the above technical shortcomings are the problems that a person skilled in the art needs to solve.
Disclosure of Invention
In view of the above, the present invention provides a bionic acellular matrix hydrogel and application thereof. The bionic acellular matrix hydrogel has good 3D printing performance, and can realize large-scale construction of the therapeutic artificial liver through biological 3D printing.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a biomimetic decellularized matrix hydrogel comprising the following components: liver decellularized matrix, gelatin and sodium alginate.
Preferably: the mass concentration of the hepatic decellularized matrix is 5% -15%;
the mass concentration of the gelatin is 4-8%;
the mass concentration of the sodium alginate is 0.5-1.2%.
The invention also provides application of the bionic acellular matrix hydrogel in preparing biological consumables.
Preferably: biological consumables: therapeutic artificial liver.
The invention also provides a preparation method of the bionic acellular matrix hydrogel, which comprises the following steps:
1) Washing liver tissues with PBS after perfusion;
2) Incubating the cleaned liver tissue in PBS containing sodium dodecyl sulfate and Triton X-100 to obtain decellularized liver tissue;
3) Freeze-drying decellularized liver tissue to obtain freeze-dried powder, dissolving in PBS containing acetic acid and pepsin, performing shock treatment to obtain liver decellularized matrix, and finally adjusting pH to neutrality to obtain liver decellularized matrix hydrogel;
4) Dissolving gelatin powder and sodium alginate powder in the hepatic acellular matrix hydrogel, and sterilizing to obtain the bionic acellular matrix hydrogel.
Preferably: step 1) liver tissue is mouse liver tissue;
the mass concentration of the sodium dodecyl sulfate in the step 2) is 1%; the volume ratio of Triton X-100 is 1%; incubation time was 72h;
step 3) conditions of lyophilization treatment: -45 ℃,24h; the mass concentration of acetic acid is 3%; the concentration of pepsin is 1mg/ml; conditions of vibration treatment: 37 ℃ for 72h; naOH was used for pH adjustment.
The invention also provides a preparation method of the therapeutic artificial liver based on the bionic acellular matrix hydrogel, which comprises the following steps:
a: mixing the bionic acellular matrix hydrogel with primary hepatocytes, and refrigerating to obtain bionic acellular hydrogel containing hepatocytes;
b: selecting an extrusion nozzle, and adjusting the temperature of the printing nozzle and the temperature of a printing platform;
c: printing bionic decellularized hydrogel containing liver cells in a sterile culture dish to obtain liver print;
d: and rapidly crosslinking the printed liver print, and then culturing the liver print by using a liver cell culture medium to obtain the therapeutic artificial liver.
Preferably: step A primary hepatocytes are mouse primary hepatocytes, and the final concentration after mixing is 5×10 6 /mL；
Specification of extrusion nozzle in step B: 23G-27G; the temperature of the printing nozzle is 10-15 ℃; the temperature of the printing platform is adjusted to 5-10 ℃;
and C, printing: extrusion speed of 1-2 mm 3 /s；
Step D, crosslinking: placing the printed liver print body in CaCl with mass concentration of 3% 2 In solution; culturing: fresh hepatocyte medium was changed every two days and cultured for one week.
Compared with the prior art, the invention discloses a bionic acellular matrix hydrogel and application thereof, and the technical effects are achieved: the therapeutic artificial liver provided by the invention can be produced in large scale in vitro by a 3D biological printing technology, and shows mature liver function phenotypes such as ICG uptake/release, drug metabolism and the like in vitro. It was transplanted into type i tyrosinemia model liver failure (Fah -/- ) Can relieve liver injury of mice in vivo, and has therapeutic potential for artificial liver transplantation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing rheological tests of bionic acellular matrix hydrogels provided by the invention.
Fig. 2 is a schematic diagram of a preparation process of the bionic acellular matrix hydrogel provided by the invention.
Fig. 3 is a diagram showing the proliferation of hepatocytes in the therapeutic artificial liver according to the present invention.
Fig. 4 is a diagram showing liver function test patterns of the therapeutic artificial liver according to the present invention.
FIG. 5 is a diagram showing the detection of CYP1A2 enzyme activity provided by the invention, wherein 3-methylflange: 3-methylcholanthrene; DMSO: dimethyl sulfoxide.
Fig. 6 is a graph showing survival curves of mice with liver failure treated by the bionic artificial liver provided by the invention.
Fig. 7 is a diagram showing the weight change of mice with liver failure treated by the bionic artificial liver provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a bionic acellular matrix hydrogel and application thereof.
The raw materials involved in the examples are all commercially available or prepared by conventional methods and are not described in detail herein.
Example 1
A biomimetic decellularized matrix hydrogel comprising the following components: liver decellularized matrix (5% -15% w/v), gelatin (4-8% w/v) and sodium alginate (0.5-1.2% w/v).
Example 2
Preparation and culture of therapeutic artificial liver:
(1) Preparation of bionic acellular matrix hydrogel:
liver tissues (the mass is about 1.5-2.5 g) of the C57 mice with the age of 6-8 weeks are separated by operation, and PBS is used for removing blood stains on the surfaces after perfusion. The liver tissue was then incubated in 50ml PBS containing 1% (w/v) Sodium Dodecyl Sulfate (SDS) and 1% (v/v) Triton X-100 for 72 hours for decellularization to give a decellularized liver tissue. The decellularized liver tissue was subjected to lyophilization at-45℃for 24 hours, and 0.1g of the lyophilized powder was dissolved in 1ml of PBS containing 3% acetic acid (MerckMillipore, USA) and 1mg/ml pepsin (Sigma Aldrich, USA) and shaken at 37℃for 72 hours to obtain a liquid-like liver decellularized matrix. Finally, naOH is used for adjusting the pH value of the dissolved liver acellular matrix to be neutral, so as to obtain the liver acellular matrix hydrogel, gelatin powder (0.05 g) and sodium alginate powder (0.008 g) are respectively dissolved in the liver acellular matrix hydrogel according to the proportion of 5% and 0.8% (w/v), the liver acellular matrix hydrogel is sterilized and packaged, and then stored at 4 ℃ to obtain the bionic type acellular matrix hydrogel, the liquid is heated to 37 ℃ until the liquid is melted before use, and the rheological property of the liquid is measured, and is shown in figure 1.
(2) Biological 3D printing preparation treatment type artificial liver
After preparing the biomimetic decellularized matrix hydrogel according to step (1), it is mixed with the mouse primary hepatocytes obtained by perfusion (final concentration 5×10) 6 and/mL) is placed at 4 ℃ for refrigeration for 20 minutes, so as to obtain the bionic decellularized hydrogel containing liver cells, and the printing performances such as viscosity, shear strain and the like are measured by using a rheometer. The extrusion nozzle with the specification of 23G-27G is used, the temperature of the printing nozzle is adjusted to 10-15 ℃, and the temperature of the printing platform is adjusted to 5-10 ℃. Using 1-2 mm 3 Extrusion speed/s bionic decellularized hydrogel containing liver cells was printed in 6cm sterile petri dishes to obtain liver prints. The printed liver prints were placed in 3% (w/v) CaCl 2 The cells were rapidly cross-linked in solution and then cultured using hepatocyte medium (see document Conversion ofTerminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity for specific ingredients, infra), fresh hepatocyte medium was changed every two days, and after one week of culture, the therapeutic artificial liver was obtained, the flow chart of which is shown in fig. 2.
(3) Hepatocyte proliferation assay for therapeutic artificial liver
The therapeutic artificial livers cultured for 0,2,4 and 6 days were dissolved by using dissociation solutions containing sodium citrate (55 mM), EDTA (20 mM) and NaCl (150 mM), respectively, and the cell numbers in the prints were measured using a cell counter and the cell proliferation curves of hepatocytes in the prints were calculated, thus demonstrating that the above bionic decellularized matrix hydrogel has good biocompatibility and can provide good proliferation environments for hepatocytes (see FIG. 3).
(4) Liver function detection of therapeutic artificial liver
The therapeutic artificial liver was cultured in the hepatocyte medium for 48 hours, then the artificial liver was incubated with the hepatocyte medium containing ICG (1 mg/ml) at 37℃for 1 hour, washed three times with PBS, changed to PBS, and the uptake/release of ICG was observed under a microscope, and the results showed that the therapeutic artificial liver prepared using the biomimetic decellularized matrix hydrogel described above had good metabolic function (see FIG. 4).
3-methylcholanthrene was incubated with the therapeutic artificial liver at 37℃for 72 hours, and the control group incubated DMSO with the therapeutic artificial liver at 37℃for 72 hours. The CYP1A2 enzyme activity of two groups of artificial livers is detected by using a CYP1A2 quantitative kit, and the result shows that the therapeutic artificial livers prepared by using the bionic acellular matrix hydrogel can express the CYP1A2 enzyme and have good drug metabolism function (see figure 5).
(5) Transplanting the therapeutic artificial liver to a commercially available source of Fah -/- In mice (mesenteric part), the treatment of the Fah-deficient mice with NTBC was performed after 1 week, and the survival time and weight change of the mice in the control group and the experimental group were recorded, which indicated that the above-mentioned therapeutic artificial liver prepared by using the biomimetic acellular matrix hydrogel had a certain liver function in vivo, and the injury caused by liver injury was alleviated (see FIGS. 6 and 7).
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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 (8)
1. A biomimetic decellularized matrix hydrogel, comprising the following components: liver decellularized matrix, gelatin and sodium alginate.
2. The biomimetic decellularized matrix hydrogel of claim 1, wherein the mass concentration of the hepatic decellularized matrix is 5% -15%;
the mass concentration of the gelatin is 4-8%;
the mass concentration of the sodium alginate is 0.5-1.2%.
3. Use of a biomimetic decellularized matrix hydrogel according to claim 1 or 2 in the preparation of a biological consumable.
4. The use according to claim 3, wherein the biological consumable: therapeutic artificial liver.
5. A method for preparing the biomimetic acellular matrix hydrogel according to claim 1 or 2, comprising the steps of:
1) Washing liver tissues with PBS after perfusion;
2) Incubating the cleaned liver tissue in PBS containing sodium dodecyl sulfate and TritonX-100 to obtain decellularized liver tissue;
3) Freeze-drying decellularized liver tissue to obtain freeze-dried powder, dissolving in PBS containing acetic acid and pepsin, performing shock treatment to obtain liver decellularized matrix, and finally adjusting pH to neutrality to obtain liver decellularized matrix hydrogel;
4) Dissolving gelatin powder and sodium alginate powder in the hepatic acellular matrix hydrogel, and sterilizing to obtain the bionic acellular matrix hydrogel.
6. The method of preparing a biomimetic decellularized matrix hydrogel of claim 5, wherein the liver tissue of step 1) is mouse liver tissue;
the mass concentration of the sodium dodecyl sulfate in the step 2) is 1%; the volume ratio of Triton X-100 is 1%; the incubation time is 72 hours;
step 3) conditions of the lyophilization process: -45 ℃,24h; the mass concentration of the acetic acid is 3%; the concentration of the pepsin is 1mg/ml; the conditions of the oscillation treatment are as follows: 37 ℃ for 72h; the pH adjustment is performed by using NaOH.
7. A method for preparing a therapeutic artificial liver based on the biomimetic acellular matrix hydrogel according to claim 1 or 2, comprising the steps of:
a: mixing the bionic acellular matrix hydrogel with primary hepatocytes, and refrigerating to obtain bionic acellular hydrogel containing hepatocytes;
b: selecting an extrusion nozzle, and adjusting the temperature of the printing nozzle and the temperature of a printing platform;
c: printing bionic decellularized hydrogel containing liver cells in a sterile culture dish to obtain liver print;
d: and rapidly crosslinking the printed liver print, and then culturing the liver print by using a liver cell culture medium to obtain the therapeutic artificial liver.
8. The method of claim 7, wherein the primary hepatocytes of step a are murine primary hepatocytes, and the final concentration after mixing is 5 x 10 6 /mL；
Specification of the extrusion nozzle described in step B: 23G-27G; the temperature of the printing nozzle is 10-15 ℃; the temperature of the printing platform is adjusted to 5-10 ℃;
and C, printing: extrusion speed of 1-2 mm 3 /s；
Step D, crosslinking: placing the printed liver print body in CaCl with mass concentration of 3% 2 In solution; the culture: fresh hepatocyte medium was changed every two days and cultured for one week.
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