Construction method and application of centimeter-level bioartificial liver

Abstract

The invention relates to a construction method of a centimeter-level bioartificial liver. The construction method comprises the following steps: respectively preparing a norbornene modified gelatin solution, a tetrazine modified gelatin solution and a norbornene modified RGDS solution; the method comprises the following steps: irrigating primary parenchymal hepatic cells from the liver of a mouse, performing in-vitro amplification culture by adopting a commercially available hepatic cell culture medium, collecting the cells, and resuspending by using the culture medium to obtain a primary parenchymal hepatic cell suspension; uniformly mixing the norbornene modified gelatin solution, the norbornene modified RGDS solution and the tetrazine modified gelatin solution, adding the primary parenchymal hepatic cell suspension, and continuously mixing; transferring the mixed solution into a cell culture plate, putting the cell culture plate into an incubator, and adding a cell culture medium for in-vitro culture after norbornene and tetrazine are fully reacted and solidified to form, so as to obtain the centimeter-level bioartificial liver; the obtained centimeter-level bioartificial liver is transplanted into the mesentery of the mouse, the liver function of the 90% liver cut mouse can be effectively compensated, and the survival rate of the 90% liver cut mouse is increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering, biomaterials, cell biology, tissue engineering, etc., and particularly relates to a method for constructing a centimeter-scale bioartificial liver. BACKGROUND

[0002] So far, there are still few reports on the use of cells to manufacture macro-size liver tissue simulators, which can be divided into two types: with and without scaffolds. Among them, the cell sheet as a high cell density microstructure without a scaffold can reproduce the continuous cell arrangement in liver tissue; however, the single-layer cell sheet structure is fragile and difficult to operate, and it is difficult to construct a three-dimensional structure of a bioartificial liver. Traditional liver tissue engineering includes seeding cells into a pre-made porous 3D scaffold, such as a decellularized extracellular matrix (dECM), a collagen-coated polyglycolic acid scaffold, and a modified hyaluronic acid (HA) tubular object. However, it is very difficult to fix cells in a pre-made ECM analog scaffold, and the cell density is usually low and the spatial distribution is uneven. Although biological 3D printing can produce large-size bioartificial livers, the demand for printing equipment increases the manufacturing cost. At the same time, the printable scaffold biomaterials are limited, and the most commonly used are photocurable biomaterials, such as methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), methacrylated dextran (DexMA), polyethylene glycol diacrylate (PEGDA) / thiolated polymer, etc. Although photocurable materials can be quickly formed, a photoinitiator is required, and the free radicals generated during the curing process can cause irreversible damage to cells. Therefore, there is an urgent need for a new curing method that meets the requirements of good biocompatibility, no catalyst, rapid reaction under physiological conditions, no by-products or non-toxic by-products. SUMMARY

[0003] In view of the technical problems existing in the prior art, the present application designs a method for constructing a centimeter-scale bioartificial liver and its application. By introducing primary hepatocytes into the matrix material forming process, a macro-size, transplantable bioartificial liver is manufactured from the bottom up.

[0004] Specifically, the present application introduces a biological orthogonal chemical strategy into gelatin-based materials, which can realize the construction of a macro-size, physiological cell density bioartificial liver, and is expected to open up a new biofabrication technology.

[0005] Biological orthogonal chemistry has high selectivity, can occur in biological systems, and does not interfere with endogenous biochemical processes. Biological orthogonal chemistry has broad application prospects in the field of biomedicine, and introducing a biological orthogonal chemical strategy into biomaterials is expected to form a series of tissue engineering materials with clinical application prospects.

[0006] Gelatin is a partially hydrolyzed product of collagen, easy to obtain, low cost, has been widely used in the field of biological medicine, such as hemostatic sponge, pharmaceutical excipients, plasma expander, tissue engineering scaffold materials, etc. Compared with collagen, gelatin has good water solubility, low immunogenicity, and retains cell adhesion sites (RGD and GFOGER) and matrix metalloproteinase (MMPs) sensitive amino acid sequences. Therefore, gelatin can be recognized and adhered by cells, and can be degraded by MMPs enzymes secreted by cells

[0007] In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] The first technical object of the present application is to provide a construction method of a centimeter-level bioartificial liver, comprising the following steps:

[0009] (1) Prepare norbornene-modified gelatin solution, tetrazine-modified gelatin solution and norbornene-modified RGDS solution respectively;

[0010] (2) Perfusion of primary hepatocytes from mouse liver, using commercially available hepatocyte culture medium for in vitro expansion culture, replacing the culture medium every two days, collecting the cells and resuspending them with the culture medium to obtain a primary hepatocyte suspension;

[0011] (3) Mix the norbornene-modified gelatin solution, tetrazine-modified gelatin solution and norbornene-modified RGDS solution uniformly, then add the primary hepatocyte suspension and continue mixing;

[0012] (4) Transfer the mixed solution in step (3) to a cell culture plate (48-well plate or 24-well plate) and place it in a cell culture incubator, wait for norbornene and tetrazine to fully react and solidify, then add cell culture medium for in vitro culture, to obtain a centimeter-level bioartificial liver.

[0013] Optionally, the mass concentration of the norbornene-modified gelatin solution is 10wt%-15wt%, the mass concentration of the tetrazine-modified gelatin solution is 10wt%-15wt%, and the mass concentration of the norbornene-modified RGDS solution is 4wt%-8wt%.

[0014] Optionally, the cell density of the primary hepatocyte suspension is 0.9-11.4x10 6 per mL.

[0015] Optionally, in step 3), the adding amount of the norbornene-modified gelatin solution, the norbornene-modified RGDS solution, the tetrazine-modified gelatin solution and the primary hepatocytes suspension can be adjusted according to actual needs, preferably, when the volume of the norbornene-modified gelatin solution is 1 equivalent, the volume of the norbornene-modified RGDS solution is 0-0.17 equivalent, the volume of the tetrazine-modified gelatin solution is 1.11-3.62 equivalent, and the volume of the primary hepatocytes suspension is 1-1.5 equivalent.

[0016] Optionally, in step 4), the mixed solution in step 3) is transferred to a 48-well plate or a 24-well plate, so that each well contains 1-60×10 5 primary hepatocytes.

[0017] Further, in step 4), the incubator culture conditions are as follows: the culture time is 1-7 days, and the culture temperature is 35-40°C, preferably 37°C.

[0018] A second technical purpose of the present application is to provide an application of the centimeter-level bioartificial liver constructed by the method in biomedicine.

[0019] Specifically, the application of the centimeter-level bioartificial liver constructed by the present application in the treatment of acute severe liver failure, adjuvant therapy before liver transplantation, prevention and treatment of liver failure after massive liver resection, treatment of drug-induced liver injury, and adjuvant therapy for chronic liver disease.

[0020] It should be noted that the centimeter-level bioartificial liver of the present application is composed of tetrazine-modified gelatin, norbornene-modified gelatin, norbornene-modified RGDS and primary hepatocytes, and its size can be obtained by changing the cell culture well plate, wherein the size obtained by culture in a 48-well plate is 1.0×0.3 cm and the size obtained by culture in a 24-well plate is 1.5×0.3 cm, and the density of hepatocytes is as high as 5×10 7 cells / cm 3 , and the internal diameter is 180.5±24.5 μm. The bioartificial liver cultured by the 48-well plate can be transplanted into the mesentery of a mouse to effectively compensate for the liver function of 90% of the liver-cut mice and improve the survival rate of 90% of the liver-cut mice, and has a wide range of application scenarios in the field of bioartificial organs.

[0021] Compared with the prior art, the present application provides a construction method and application of a centimeter-level bioartificial liver, which has the following beneficial effects:

[0022] (1) The bioartificial liver constructed by the present application reaches the centimeter scale, has a cell density close to that of human liver (5×10 7 cells / cm 3), the internal aperture is 180.5±24.5 mu m, which guarantees the nutrient delivery and the cell activity in the center of the bioartificial liver.

[0023] (2) The bioartificial liver constructed in the application has good biological safety, and the main material is gelatin, which can be slowly and completely degraded in the body, and does not cause heterogenic reaction and fiber capsule formation.

[0024] (3) The bioartificial liver constructed in the application can form liver organoids in the process of in-vitro culture, the size of the liver organoids is 118±22 mu m, and the liver function is more mature.

[0025] (4) The bioartificial liver constructed in the application has a treatment function, and can effectively compensate the liver function of 90% of the mice with liver resection, and increase the survival rate of the 90% of the mice with liver resection from 0% to 45%. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0027] Figure 1 It is a construction process schematic diagram of the centimeter-level bioartificial liver of the application;

[0028] Figure 2 It is the live and dead conditions of the liver parenchymal cells in the bioartificial liver prepared in Example 1 of the application during in-vitro culture;

[0029] Figure 3 It is the uptake and release evaluation of ICG by the bioartificial liver prepared in Example 2 of the application;

[0030] Figure 4 It is the microscope photos of the liver organoids formed by the liver parenchymal cells in the bioartificial liver prepared in Example 3 of the application during in-vitro culture and the quantification results thereof;

[0031] Figure 5 It is the optical photo of the bioartificial liver prepared in Example 4 of the application;

[0032] Figure 6 It is the cross-sectional SEM photo and aperture quantification diagram of the bioartificial liver prepared in Example 4 of the application;

[0033] Figure 7 It is the gene expression of the bioartificial liver prepared in Example 4 of the application during in-vitro culture for 1 day and 3 days;

[0034] Figure 8 In vivo transplantation treatment effect of the bioartificial liver prepared in Example 4 of the present application;

[0035] Figure 9 HE section staining and HNF4α / DAPI fluorescence staining of the bioartificial liver prepared in Example 4 of the present application 12 days after in vivo transplantation;

[0036] Figure 10 Microscope photograph of the bioartificial liver prepared in Example 5 of the present application;

[0037] Figure 11 Section staining photograph of the bioartificial liver prepared in Example 5 of the present application;

[0038] Figure 12 In vivo degradation of the bioartificial liver prepared in Example 5 of the present application;

[0039] Figure 13 Optical photograph of the bioartificial liver prepared in Example 5 of the present application 12 days after in vivo transplantation and section fluorescence staining photograph thereof. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0041] Herein, the special term "embodiment" as explained in any embodiment does not necessarily mean that it is superior or better than other embodiments. In the performance index test of the embodiments of the present application, unless otherwise specified, the conventional test method in the art is adopted. It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the disclosure of the present application.

[0042] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the present application belongs; as the test methods and technical means not specially noted in the present application all refer to the experimental methods and technical means generally used by those skilled in the art.

[0043] In order to better illustrate the content of the present application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In the embodiments, some methods, means, instruments, devices and the like which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0044] The technical features disclosed in the embodiments of the present application can be combined arbitrarily without conflict, and the resulting technical solutions belong to the disclosure of the present application.

[0045] The application discloses a construction method of a centimeter-level bioartificial liver.

[0046] In order to better understand the present application, the following examples are further specifically described, but it should not be understood as limiting the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the above disclosure are also regarded as falling within the protection scope of the present application.

[0047] The construction method of the centimeter-level bioartificial liver of the present application is as shown in Figure 1 , and specifically as follows:

[0048] Step (1): Prepare norbornene-modified gelatin solution (10wt%-15wt%), tetrazine-modified gelatin solution (10wt%-15wt%) and norbornene-modified RGDS solution (4wt%-8wt%) respectively;

[0049] Preparation of norbornene-modified gelatin solution: Gelatin is dissolved in PBS buffer solution in a 40℃ water bath with stirring, and gelatin solution is obtained by sufficient stirring and dissolution; norbornene dianhydride (Nb-COOH) powder is weighed and slowly added to the gelatin solution under stirring, so that the molar ratio is Nb-COOH:NH2=5, and the reaction is carried out under stirring for 9 hours, 24 hours and 48 hours respectively; the reaction liquid is dialyzed and freeze-dried to obtain norbornene-modified gelatin with different grafting rates, which are named Gel-N low , Gel-N med and Gel-N high respectively; weigh the norbornene-modified gelatin, expose it to ultraviolet light irradiation in a clean bench for 30 minutes, and then dissolve it in sterile PBS solution in a 40℃ water bath with stirring, thereby obtaining norbornene-modified gelatin solution (10wt%-15wt%);

[0050] Preparation of tetrazine-modified gelatin solution: Gelatin is dissolved in PBS buffer solution in a 40℃ water bath with stirring, and gelatin solution is obtained by sufficient stirring and dissolution; methyl tetrazine active ester (Tz-NHS) is dissolved in DMSO solution, and after complete dissolution, it is added dropwise to the gelatin solution, so that the molar ratio of Tz-NHS and amino group is NHS:NH2=1, and the reaction is carried out under stirring for 24 hours; the reaction liquid is dialyzed and freeze-dried to obtain tetrazine-modified gelatin (Gel-T); weigh the tetrazine-modified gelatin Gel-T, expose it to ultraviolet light irradiation in a clean bench for 30 minutes, and then dissolve it in sterile PBS solution in a 40℃ water bath with stirring, thereby obtaining tetrazine-modified gelatin solution (10wt%-15wt%);

[0051] Preparation of norbornene-modified RGDS solution: under the condition of stirring at room temperature, 216.6 mg of linear RGDS tetrapeptide (0.5 mmol) was dissolved in 30 mL of PBS buffer solution to obtain an RGD solution; 1 g of NHS-PEG-Nb was weighed and dissolved in 8 mL of PBS buffer solution, and then added to the RGD solution under stirring to make the molar ratio of NHS:RGDS = 1.0; the reaction was carried out at room temperature for 2 hours under stirring, and then the reaction solution was placed in a dialysis bag for dialysis for 2 days, and then freeze-dried to obtain norbornene-modified RGD (RGD-N); the RGD-N was weighed and dissolved in sterile PBS solution, and then filtered and sterilized by using a 0.2 μm sterile filter to obtain a norbornene-modified RGDS solution (4 wt%-8 wt%).

[0052] Step (2): Collection of primary hepatocytes

[0053] Regarding the extraction, culture and collection of primary hepatocytes in the present application: male C57BL / 6 mice were anesthetized by intraperitoneal injection of tribromoethanol, the skin was alcohol-disinfected, the abdominal cavity was opened, and the portal vein and inferior vena cava were exposed; a small incision was made at the distal end of the portal vein, and a retention needle was inserted after no blood flowed out; a small incision was made in the inferior vena cava as the outlet for perfusion fluid; physiological saline (about 30 ml) was perfused into the liver until it turned gray; 0.05% collagenase IV was continuously perfused (the inferior vena cava was ligated before perfusion, and the liver was kept for an hour after swelling, the inferior vena cava was opened to make the collagenase flow out, and this was repeated for 4-5 times, for about 10 min); the liver lobe was cut, the liver was cut into a milky state, filtered, and centrifuged with 40% percoll separation solution for 5 minutes to obtain mouse primary hepatocytes, which were cultured, and the fresh commercial hepatocyte culture medium was replaced every two days; after the primary hepatocytes grew, trypsin was used for digestion, centrifugation, PBS washing, and the cells were resuspended with commercial hepatocyte culture medium to obtain a primary hepatocyte suspension with a cell density of 0.9-11.4 x 10 6 According to the same method, mTmG positive primary hepatocytes were extracted, cultured and collected from male 6-8 week old mT / mG reporter mice.

[0054] Step (3): In vitro construction of a centimeter-level bioartificial liver

[0055] The norbornene-modified gelatin solution, the norbornene-modified RGD solution and the tetrazine-modified gelatin solution were mixed uniformly according to a certain volume ratio, and were repeatedly extruded and mixed for 5 minutes by using a syringe, and then were placed in a cell culture box for 10-60 minutes to make the tetrazine and norbornene partially react. Then the above mixture and the primary hepatocyte suspension were mixed uniformly according to a certain volume ratio, and were repeatedly extruded and mixed for 5 minutes by using a syringe.

[0056] Step (4): in vitro culture of centimeter-scale bioartificial liver

[0057] The mixed solution in step (3) is transferred into a cell culture plate (48-well plate or 24-well plate) using a syringe, so that each well contains 1-60 x 10 5 primary hepatocytes, and is placed in a culture box for 30-120 minutes to allow the complete reaction of the tetrazine and norbornene, after which culture medium is added and in vitro culture is continued for 1-7 days, and the culture medium is discarded to obtain a centimeter-scale bioartificial liver.

[0058] The following are construction examples 1-5 of the centimeter-scale bioartificial liver of the present application:

[0059] Example 1

[0060] Step (1): Gel-N low , Gel-N med , Gel-N high and Gel-T

[0061] The norbornene-modified gelatin (Gel-N low , Gel-N med or Gel-N high ) and tetrazine-modified gelatin (Gel-T) of different modification degrees are irradiated under a UV lamp for 30 minutes, and then dissolved in sterile PBS solution in a 37°C water bath, with a concentration of 10 wt%.

[0062] Step (2): collection of primary hepatocytes

[0063] The primary hepatocytes in two-dimensional culture are trypsinized, centrifuged, washed with PBS, and resuspended in culture medium, with a cell concentration of 0.9-1.2 x 10 6 cells / mL.

[0064] Step (3): in vitro construction of bioartificial liver

[0065] Gel-N low and Gel-T are mixed in a volume ratio of 1:1.11, Gel-N med and Gel-T are mixed in a volume ratio of 1:1.76, and Gel-N high and Gel-T are mixed in a volume ratio of 1:2.56, and are repeatedly extruded and mixed for 5 minutes using a syringe, and then are placed in a cell culture box for 60 minutes to allow the partial reaction of the tetrazine and norbornene. After that, the primary hepatocyte suspension is mixed uniformly with the above-mentioned mixed solution, and the cell suspension and Gel-N low , Gel-N med or Gel-N highat a volume ratio of 1:1, and continue to extrude the mixture repeatedly with a syringe for 5 minutes.

[0066] Step (4) in vitro culture of bioartificial liver

[0067] The mixed solution in step (3) is transferred to a 48-well plate so that each 48-well contains 200 μL of the mixed solution, 1×10 5 primary hepatocytes, and the final concentration of gelatin is 5 wt%, and is placed in an incubator for 60 minutes to allow the tetrazine and norbornene to fully react, after which 200 μL of culture medium is added, and the in vitro culture is continued for 1 day or 7 days to obtain a bioartificial liver, which is named Gel-T / N low , Gel-T / N med , and Gel-T / N high , respectively, according to the crosslinking density of the bioorthogonal click gelatin.

[0068] For comparison, methacrylated gelatin (GelMA) with different modification degrees is used as a control group to construct photo-initiated solidified gelatin-based bioartificial livers with different crosslinking densities.

[0069] First, methacrylated gelatin with different modification degrees is synthesized: 1 g of gelatin (2.506 mmol of free NH2) is dissolved in 10 mL of PBS buffer at 40°C. Under magnetic stirring at 240 rpm, 0.8 mL of methacrylic anhydride (MA) is added to the dissolved gelatin solution at a flow rate of 0.2 ml / min, and the reaction is carried out at 40°C for 0.5 h, 1 h, and 2 h, respectively. 10 mL of preheated PBS is added to the reaction solution, and the reaction is terminated by magnetic stirring at 40°C for 30 min. The reaction solution is placed in a dialysis bag (molecular weight cut-off value of 8-14 kDa) and stirred in deionized water at 40°C for 24 hours to remove salt and unreacted MA. The solution is freeze-dried to obtain methacrylated gelatin (GelMA low , GelMA med , and GelMA high ) with different modification degrees.

[0070] GelMA low , GelMA med , and GelMA high are respectively irradiated with ultraviolet light for 30 minutes, and then dissolved in sterile PBS solution at a concentration of 10 wt% in a 37°C water bath. Sterile filtered photo-initiator LAP is added to the above three solutions to a concentration of 0.134 wt%.

[0071] 100 μL of GelMA low , GelMA med , and GelMAhigh The solution was mixed with 100 μL of primary hepatocyte suspension by repeated pipetting for 2 minutes, transferred into a 48-well plate, and immediately cured by light exposure for 1 minute (405 nm, intensity 10 mW cm -2 Each 48-well plate contained 250 μL of mixed solution and 1×10 5 Then, 200 μL of culture medium was added and cultured in vitro to obtain bioartificial livers. According to the cross-linking density of photoinduced curing gelatin, they were named GelMA. low , GelMA med and GelMA high .

[0072] The product in Example 1 is analyzed as follows with reference to the accompanying drawings:

[0073] Figure 2 The bioartificial liver prepared in Example 1 of the present invention was cultured in vitro for 1 day and 7 days to measure the viability of the hepatocytes. The cell viability staining kit Calcein-AM / PI was used to quantify the viability of the hepatocytes in the bioartificial liver. Figure 2 As shown in A, after one day of in vitro culture, the hepatocytes in the bioartificial liver prepared by bioorthogonal gelatin in the present invention have better cell viability than those in the photoinduced solidified gelatin ( Figure 2 BD). In addition, there was no significant difference in the viability of hepatocytes in bioartificial livers prepared with bioorthogonal gelatin of different crosslinking densities, indicating that bioorthogonal chemistry has excellent biocompatibility and is suitable for the preparation of bioartificial livers. On the 7th day of in vitro culture, hepatocyte aggregation was clearly observed in the bioorthogonal gelatin, accompanied by strong green fluorescence of Calcein-AM and sporadic red fluorescence of PI ( Figure 2 E). In contrast, only minimal cell aggregation and weak green fluorescence were observed in the photocured gelatin ( Figure 2 FH).

[0074] Figure 2 This indicates that the activity of hepatocytes in the bioartificial liver prepared by bioorthogonal gelatin is significantly higher than that in the bioartificial liver prepared by photoinduced curing gelatin.

[0075] Example 2

[0076] Step (1) Prepare Gel-N high and Gel-T ​​solution

[0077] Gel-N high and Gel-T ​​were irradiated under UV light for 30 minutes, and then placed in a 37° C. water bath and dissolved in sterile PBS solution, with a concentration of 10 wt %.

[0078] Step (2) collection of primary hepatocytes

[0079] The trypsin-digested primary hepatocytes in two-dimensional culture were centrifuged, washed with PBS, resuspended in culture medium, and the cell concentration was 1.9-2.2 x 10 6 million / mL.

[0080] Step (3) in vitro construction of bioartificial liver

[0081] Gel-N high and Gel-T were mixed uniformly at a volume ratio of 1:2.56, repeatedly extruded with a syringe for 5 minutes, and then placed in a cell culture incubator for 30 minutes to allow partial reaction of the tetrazine and norbornene. Then the primary hepatocyte suspension was mixed uniformly with the above mixture, and the volume ratio of the cell suspension to Gel-N high was 1:1, and the mixture was repeatedly extruded with a syringe for 5 minutes.

[0082] Step (4) in vitro culture of bioartificial liver

[0083] The mixed solution in step (3) was transferred to a 24-well plate, so that each 24-well contained 300 μL of the mixed solution and 3 x 10 5 million primary hepatocytes, and was placed in a culture incubator for 120 minutes to allow complete reaction of the tetrazine and norbornene. Then 300 μL of culture medium was added, and in vitro culture was continued for 3 days to obtain a bioartificial liver, Gel-T / N high .

[0084] The function of the bioartificial liver was evaluated by indocyanine green (ICG) uptake and release, as follows:

[0085] ICG stock solution (100 mg / ml) dissolved in DMSO was diluted with culture medium to a working solution of 1 mg / mL. 500 μL of 1 mg / ml ICG working solution was added to the bioartificial liver prepared in Example 2, and was incubated in a cell culture incubator for 24 h. After discarding the ICG-containing culture medium, the bioartificial liver was washed 3 times with PBS, and the ICG uptake was observed by taking photographs. The bioartificial liver was then placed in a cell culture incubator, and the ICG release was observed under a microscope after 6 h.

[0086] Figure 3 The ICG uptake and release evaluation of the bioartificial liver prepared in Example 2 of the present application belongs to the function evaluation of the bioartificial liver. As can be seen from the figure, the positive ICG uptake cells are mainly located in the liver organoids, indicating that the bioartificial liver has obtained mature liver function.

[0087] Example 3

[0088] Step (1) Preparation of Gel-N low , Gel-N med , Gel-N high and Gel-T

[0089] Different modification degree of norbornene modified gelatin (Gel-N low , Gel-N med or Gel-N high ) and tetrazine modified gelatin (Gel-T) were placed under UV light for 30 minutes, and then dissolved in sterile PBS solution in a 37°C water bath, with a concentration of 10wt%.

[0090] Step (2) Collection of primary hepatocytes

[0091] The trypsin-digested mTmG positive primary hepatocytes in two-dimensional culture were centrifuged, washed with PBS, and resuspended in culture medium, with a cell concentration of 1.9-2.2×10 6 / mL.

[0092] Step (3) In vitro construction of bioartificial liver

[0093] Gel-N low and Gel-T were mixed at a volume ratio of 1:1.11, Gel-N med and Gel-T were mixed at a volume ratio of 1:1.76, or Gel-N high and Gel-T were mixed at a volume ratio of 1:2.56, and then repeatedly extruded with a syringe for 5 minutes, and then placed in a cell incubator for 30 minutes to allow the tetrazine and norbornene to partially react. Then the primary hepatocyte suspension was mixed with the above mixture, and the volume ratio of the cell suspension to Gel-N low , Gel-N med or Gel-N high was 1:1, and the mixture was repeatedly extruded with a syringe for 5 minutes.

[0094] Step (4) In vitro culture of bioartificial liver

[0095] The mixed solution in step (3) was transferred to a 48-well plate, so that each 48-well contained 200 μL of mixed solution and 2×10 5 primary hepatocytes, and was placed in an incubator for 60 minutes to allow the tetrazine and norbornene to fully react, and then 200 μL of culture medium was added for further in vitro culture for different time to obtain a bioartificial liver, which was named Gel-T / N low , Gel-T / N med and Gel-T / N high according to the crosslinking density of the bioorthogonal click gelatin.

[0096] The bioartificial liver prepared in Example 3 was cultured in vitro for 1 day, 3 days, 5 days, and 7 days, and photographed under a FLUOCA microscope. The diameter of the cell clusters was quantified using FCSnap.

[0097] Figure 4 Microscopic photographs of liver organoids formed by hepatocytes and quantification of the number and size of liver organoids after 1, 3, 5, and 7 days of in vitro culture for the bioartificial liver prepared in Example 3 of the present invention.

[0098] like Figure 4 As shown in Figure 2, after 1 day of in vitro culture, the hepatocytes in the bioartificial liver based on bioorthogonal gelatin quickly aggregated into liver organoids with a size of approximately 60 μm. After 3 days of in vitro culture, the size of the liver organoids reached its maximum, with the Gel-T / N low The maximum size is 98.1μm and remains basically unchanged thereafter. low 、Gel-T / N med and Gel-T / N high There was no significant difference between the two groups, indicating that cross-linking density had no significant effect on liver organoid size. In contrast, liver organoids in the bioartificial liver prepared using photocured gelatin were significantly smaller. These results demonstrate that bioorthogonal gelatin has better biocompatibility than photocured gelatin, facilitates the aggregation and proliferation of hepatocytes, and promotes the rapid formation of liver organoids.

[0099] Example 4

[0100] Step (1) Prepare Gel-N high , Gel-T ​​and RGD-N solutions

[0101] Gel-N high and Gel-T ​​were irradiated under UV light for 30 minutes, and then placed in a 37° C. water bath and dissolved in sterile PBS solution, with a concentration of 15 wt %.

[0102] RGD-N was weighed and dissolved in a sterile PBS solution, and then sterilized by filtration using a 0.2 μm sterile filter to obtain a norbornene-modified RGDS solution with a concentration of 8 wt%.

[0103] Step (2) Collection of primary hepatocytes

[0104] The primary hepatocytes cultured in two-dimensional culture were digested with trypsin, centrifuged, washed with PBS, and resuspended in culture medium to a cell concentration of 1.13-1.14×10 7 pieces / mL.

[0105] Step (3) in vitro construction of bioartificial liver

[0106] Gel-N high , RGD-N and Gel-T were mixed in a volume ratio of 1:0.17:3.62, and repeatedly extruded with a syringe for 5 minutes, and then placed in a cell incubator for 10 minutes to allow the tetrazine and norbornene moieties to react partially, to obtain a Gel-N high , RGD-N and Gel-T mixture solution. Then the cell suspension was mixed uniformly with the above mixture solution, wherein the volume ratio of the cell suspension to Gel-N high was 1.5:1, and the mixing was continued for 5 minutes by repeatedly extruding with a syringe.

[0107] Step (4) in vitro culture of bioartificial liver

[0108] The mixed solution in step (3) was transferred to a 48-well plate, so that each 48-well contained 200 μL of the mixed solution, 6×10 6 primary hepatocytes, and the final concentration of gelatin was 12 wt%, and was placed in an incubator for 60 minutes to allow the tetrazine and norbornene to react completely, and then 200 μL of culture medium was added, and the in vitro culture was continued for 3 days to obtain a bioartificial liver Livergraft with a cell density of 3×10 7 / mL. Under the same preparation conditions, a group without cells was named Gel-T / N high .

[0109] Figure 5 is an optical photograph of the bioartificial liver prepared in Example 4 of the present application; it can be seen from the figure that the light transmittance of the bioartificial liver (Livergraft) is poorer than that of the bioorthogonal gelatin without cells (Gel-T / N high ).

[0110] Figure 6 is a cross-sectional SEM photograph and pore size quantification diagram of the bioartificial liver prepared in Example 4 of the present application; it can be seen from the figure that the bioartificial liver has a highly developed pore structure, with an average pore size of 180.5±24.5 μm, which is sufficient for the exchange of nutrients / metabolites, thereby ensuring the activity of the cells in the central region. The high-magnification cross-sectional scanning electron microscope image further confirms that the hepatocytes exist in the form of liver organoids rather than single cells.

[0111] Figure 7Gene expression of the bioartificial liver prepared in Example 4 of the present application cultured in vitro for 1 day and 3 days; the qPCR results in the figure evaluate the function of the bioartificial liver, after 1 day of in vitro culture, the expression level of liver function genes (Alb, Ttr and Ck18) of the bioartificial liver is increased compared with two-dimensional culture cells and Matrigel, indicating that the bioorthogonal gelatin provides a good microenvironment for hepatocyte maturation. At the same time, compared with two-dimensional culture and Matrigel, the expression level of enriched transcription factors (Hnf4a, Hnf1a and Foxa2) of the bioartificial liver is increased, and these transcription factors are essential for maintaining the liver phenotype. This result shows that the hepatocyte fate determination is enhanced in the environment provided by the bioorthogonal gelatin. After 3 days of in vitro culture, the expression levels of Alb, Ttr and Hnf4a are further increased compared with two-dimensional culture, indicating that the liver function of the bioartificial liver is further enhanced as the culture time is prolonged.

[0112] Figure 8 The treatment effect of the bioartificial liver prepared in Example 4 of the present application in vivo transplantation; the experimental procedure is as shown in Figure 8 A, for mice, 70% partial hepatectomy (PHx) is well tolerated, while 90% PHx is mostly a lethal surgery, and most of them die within 24 hours due to acute liver failure. Therefore, in wild-type mice receiving 90% PHx, surgery-induced acute liver failure was performed to verify the compensatory effect of the bioartificial liver on liver function. The centimeter-scale bioartificial liver was transplanted into the mesentery of the mouse on day 0 Figure 8 B), and the recipient mice received 90% PHx surgery on day 7, removing the left lobe, middle lobe and right lobe, while retaining the caudate lobe Figure 8 B). On the first day after PHx surgery, the mice were obviously inactive, hunched, and had low body temperature, so all mice were injected intraperitoneally with glucose every 4 hours to prevent dehydration, and were placed in a 37°C environment to prevent hypothermia. The untransplanted mice (only 90% PHx group) had a 100% mortality rate within 20 hours Figure 8 C). The survival rate of the bioartificial liver transplanted mice was significantly improved, 5 out of 12 mice were able to eat independently 24 hours after 90% PHx surgery, and behaved like normal mice after 48 hours. At the same time, their body weight began to recover 72 hours after 90% PHx surgery, after reaching a minimum Figure 8 D). 120 hours after 90% PHx treatment, the liver volume of the mice transplanted with the bioartificial liver was significantly increased after resection, and the morphology of the newly formed part was irregular Figure 8B). Meanwhile, the liver function indicators, including total protein (TP), albumin (ALB), glucuronic acid (GLB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), total bile acid (TBA), total bilirubin (T-Bil) and direct bilirubin (D-Bil), showed no difference from normal mice Figure 8 E). However, the serum TP, ALB and GLB levels of the 90% PHx group mice decreased sharply, while the ALT, AST, ALP, CGT, TBA, total bilirubin (T-Bil) and direct bilirubin (D-Bil) levels increased. The above results suggest that the transplantation of the bioartificial liver successfully rescued the acute liver failure mice.

[0113] Figure 9 HE staining and HNF4α / DAPI fluorescence staining of the bioartificial liver prepared in Example 4 after in vivo transplantation for 12 days. As can be seen from the figure, the liver parenchymal cells in the bioartificial liver after in vivo transplantation for 12 days still exist in the form of liver organoids rather than single cells. Hepatocyte nuclear factor 4α (HNF4α) and DAPI nuclear staining are co-located in the bioartificial liver section, indicating that the liver parenchymal cells in the bioartificial liver maintain the phenotype of the liver.

[0114] Example 5

[0115] Step (1) Preparation of Gel-N med , Gel-T and RGD-N solutions

[0116] Gel-N med and Gel-T were respectively irradiated under a UV lamp for 30 minutes, and then dissolved in sterile PBS solution in a 37°C water bath, with a concentration of 15wt%.

[0117] RGD-N was weighed and dissolved in sterile PBS solution, and then filtered with a sterile filter head of 0.2 μm to obtain a norbornene-modified RGDS solution with a concentration of 4wt%.

[0118] Step (2) Collection of primary liver parenchymal cells

[0119] The trypsin-digested mTmG positive primary liver parenchymal cells were centrifuged, washed with PBS, and resuspended in cell culture medium to make the cell concentration 1.13-1.14×10 7 per mL.

[0120] Step (3) In vitro construction of bioartificial liver

[0121] Gel-N med, RGD-N and Gel-T were mixed at a volume ratio of 1:0.17:3.62, mixed repeatedly for 5 minutes by using a syringe, and then placed in a cell culture box for 15 minutes to allow the partial reaction of the tetrazine and norbornene, to obtain Gel-N med , RGD-N and Gel-T were mixed. Then the cell suspension was mixed with the above mixture uniformly, wherein the volume ratio of the cell suspension to Gel-N high was 1.5:1, and the mixing was continued for 5 minutes by repeatedly using a syringe.

[0122] Step (4) in vitro culture of the bioartificial liver

[0123] The mixed solution in step (3) was transferred to a 48-well plate, so that each 48-well contained 200 μL of the mixed solution and 6×10 6 primary hepatocytes, and was placed in a culture box for 30 minutes to allow the complete reaction of the tetrazine and norbornene, and then 200 μL of culture medium was added, and the in vitro culture was continued for 3 days to obtain a bioartificial liver Livergraft with a cell density of 3×10 7 cells / mL.

[0124] A 10-week-old male C57BL6 mouse weighing about 20 g was intraperitoneally injected with 1.25% tri- bromoethanol at a dose of 0.2-0.25 mL / 10 g of body weight (i.e., 200-250 mg / kg). After the mouse was anesthetized, the abdominal cavity was depilated, and the abdominal cavity was opened with a surgical knife. A bioartificial liver prepared in advance, which had a size of 1.5×0.3 cm, a density of 5×10 7 cells / cm 3 , and an internal pore diameter of 180.5±24.5 μm, was embedded on the mouse mesentery, the abdominal cavity was sutured, and the mouse was allowed to wake up. Three days, 5 days, 14 days, and 21 days after the transplantation in the mesentery, the bioartificial liver Livergraft in the mouse was taken out for observation.

[0125] Figure 10 It is a microscope photograph of the bioartificial liver prepared in Example 5 of the present application; as can be seen from the photograph, the bioartificial liver Livergraft was prepared using mTmG positive hepatocytes, and the microscope photograph shows that although the cell density is high, the hepatocytes are uniformly distributed.

[0126] Figure 11 It is a photograph of the staining of a section of the bioartificial liver prepared in Example 5 of the present application; the DAPI nuclear staining and mTmG fluorescence of the section of the bioartificial liver further prove that hepatocytes can form liver organoids in the microenvironment provided by the bioorthogonal gelatin.

[0127] Figure 12In vivo degradation of the bioartificial liver prepared in Example 5 of the present application; the in vivo degradation of the bioartificial liver was evaluated by performing a mesenteric implantation in C57BL / 6 mice. After the mesenteric implantation, the mice behaved normally, without fights or bites. The bioartificial liver was removed after three days of implantation, and a strong red fluorescence was observed, indicating the survival of mTmG positive hepatocytes. Five and 14 days after implantation, the volume of the bioartificial liver was significantly reduced. After 21 days of implantation, the bioartificial liver was completely degraded, and no mTmG positive hepatocytes were observed in the entire abdominal cavity.

[0128] More importantly, no fibrous capsule formation was observed on the surface of the bioartificial liver after 14 days of implantation. It is well known that the formation of a fibrous capsule between the implant and the tissue due to a foreign body reaction is one of the key factors that affect the reliability and functionality of the implant in the body. In general, the fibrous capsule begins to form 3 days after implantation, and the fibrous capsule formation is visible after 14 days. The results show that the foreign body reaction of the bioartificial liver prepared based on the bioorthogonal gelatin is negligible.

[0129] Figure 13 Optical photograph of the bioartificial liver prepared in Example 5 of the present application after 12 days of in vivo implantation and fluorescence staining photograph of its section; as can be seen from the figure, after 12 days of implantation, the local pore size of the bioartificial liver increased to nearly 1 mm.

[0130] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a centimeter-scale bioartificial liver, characterized in that: The steps include: 1) Prepare dilute norborneol-modified gelatin solution, tetrazine-modified gelatin solution, and dilute norborneol-modified RGDS solution respectively; 2) Primary hepatocytes were perfused from mouse livers and cultured in vitro using commercially available hepatocyte culture medium. The culture medium was changed every two days, and the cells were harvested and resuspended in culture medium to obtain a primary hepatocyte suspension. 3) Mix the dilute norborneol-modified gelatin solution, the dilute norborneol-modified RGDS solution, and the tetrazine-modified gelatin solution, then add the primary hepatocyte suspension and continue mixing; 4) The mixed solution from step 3) is transferred to a cell culture plate and placed in an incubator to wait for the norbornene and tetrazine to fully react and solidify, and then cell culture medium is added for in vitro culture to obtain a centimeter-scale bioartificial liver.

2. The method for constructing a centimeter-scale bioartificial liver according to claim 1, characterized in that: The mass concentration of the norbornene dilute modified gelatin solution is 10wt%-15wt%, the mass concentration of the tetrazine modified gelatin solution is 10wt%-15wt%, and the mass concentration of the norbornene dilute modified RGDS solution is 4wt%-8wt%.

3. The method for constructing a centimeter-scale bioartificial liver according to claim 1, characterized in that: The cell density of the primary hepatocyte suspension is 0.9-11.4×10 6 pieces / mL.

4. The method for constructing a centimeter-scale bioartificial liver according to claim 1, characterized in that: In step 3), the amount of norbornene-modified gelatin solution, norbornene-modified RGDS solution, tetrazine-modified gelatin solution and primary hepatocyte suspension added is adjusted according to actual needs. When the volume of norbornene-modified gelatin solution is 1 equivalent, the volume of norbornene-modified RGDS solution is 0-0.17 equivalents, the volume of tetrazine-modified gelatin solution is 1.11-3.62 equivalents, and the volume of primary hepatocyte suspension is 1-1.5 equivalents.

5. The method for constructing a centimeter-scale bioartificial liver according to claim 1, characterized in that: In step 4), the mixed solution of step 3) was transferred to a 48-well plate or a 24-well plate so that each well contained 1-60×10 5 Primary hepatocytes.

6. The method for constructing a centimeter-scale bioartificial liver according to claim 1 or 5, characterized in that: In step 4), the incubator culture conditions are: the culture time is 1-7 days, and the culture temperature is 35°C-40°C.

7. Application of the centimeter-scale bioartificial liver constructed by the method of claim 1 in biomedicine.

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