Ion-specific high-cell density microcarrier, bioartificial liver system, preparation method, and application

Abstract

The present invention discloses ion-specific high-cell-density microcarriers, a bioartificial liver system, and preparation methods and applications. Protein particle microcarriers are produced by shearing between an aqueous phase and an oil phase, wherein the aqueous phase is composed of ions and a photocurable protein hydrogel, and the oil phase is composed of a high-viscosity oil. The protein particles are recrystallized using a quenching-annealing method. The polymer structure network produced during the recrystallization process is retained by freeze-drying to form a biomimetic niche pore structure, thereby obtaining ion-specific high-cell-density microcarriers. The present invention combines protein-based hydrogels with the Hofmeister effect through microfluidic technology to prepare in vitro biomimetic liver microcarriers with high liver cell density. Liver cells can proliferate in large numbers on the microcarriers and express normal metabolites. The microcarriers can be used to prepare bioartificial liver systems for the treatment of acute liver failure.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to an ion-specific high-cell-density microcarrier, a bioartificial liver system, and a preparation method and application thereof. Background Art

[0002] A variety of factors, including viral infection, alcohol abuse, obesity, and genetic susceptibility, can lead to liver injury and acute liver failure. Although liver transplantation remains a lifesaving treatment for end-stage liver disease, its availability is limited by organ shortages and availability. Emerging bioartificial liver (BAL) systems mimic liver function and maintain essential activities in vitro, offering a promising alternative to transplantation. However, achieving high-density hepatocyte culture systems in BAL is challenging, as simply increasing the surface area by culturing cells on spheroids does not actually increase cell density. Although many commercially available biomaterials can provide high-surface-area scaffolds for cell culture, most are inherently biotoxic due to complex synthesis processes. Hepatocytes are highly polarized and require specific environmental cues within a three-dimensional niche to perform functions such as glycogen storage, ICG absorption, and LDL uptake. Therefore, the design of new biomaterials that promote hepatocyte growth is imperative. Efficient integrated BAL systems are essential. Summary of the Invention

[0003] The purpose of the present invention is to provide an ion-specific high-cell density microcarrier, a bioartificial liver system, and a preparation method and application thereof, so as to simulate the growth environment of different cells in tissue niches and achieve cytokine secretion and functional reproduction.

[0004] To achieve the above purpose, the technical solution provided by the present invention is

[0005] A method for preparing ion-specific high-cell-density microcarriers comprises the following steps:

[0006] Step S1: using microfluidic technology to generate protein particle microcarriers through shearing and UV curing between the aqueous phase and the oil phase, wherein the aqueous phase is composed of a photoinitiator, ions and a photocurable protein hydrogel, and the oil phase is composed of a viscous oil;

[0007] Step S2: recrystallizing the protein particles using a quenching-annealing method;

[0008] Step S3: freeze-drying is performed to preserve the polymer structure network generated during the recrystallization process, thereby forming a bionic niche pore structure and obtaining ion-specific high-cell density microcarriers.

[0009] To optimize the above technical solutions, specific measures / limitations adopted also include:

[0010] The ion-specific high-cell density microcarriers are prepared by capillary microfluidics technology, with an aqueous phase as the inner phase and an oil phase as the outer phase. Droplets are sheared into circles at the end of the inner phase, and the sheared circular droplets are solidified by ultraviolet light in the oil phase to form protein particle microcarriers.

[0011] The ions used in the aqueous phase follow the Ostwald ripening process, including anions and cations, and their concentrations in the aqueous phase are 0.001M-0.1M respectively.

[0012] Furthermore, the anion is selected from at least one of chloride ion, bromide ion or iodide ion; and the cation is selected from at least one of potassium ion and sodium ion.

[0013] The light-curable protein hydrogel used in the aqueous phase is a tyrosine-containing protein hydrogel; the concentration of the light-curable protein hydrogel in the aqueous phase is 5%-15% w / w.

[0014] Furthermore, the light-curable protein hydrogel is selected from at least one of silk fibroin, collagen, and gelatin.

[0015] The oil phase is silicone oil or vegetable oil.

[0016] In the quenching-annealing method, the annealing time is 10 min-100 min, and the annealing temperature is -40°C to -5°C.

[0017] The present invention also protects the ion-specific high-cell-density microcarrier prepared by the method.

[0018] The present invention also protects the application of the ion-specific high-cell-density microcarrier in liver cells, and the liver cells grow in the bionic niche pore structure of the ion-specific high-cell-density microcarrier.

[0019] The present invention also provides a bioartificial liver system, which integrates ion-specific high-cell-density microcarriers and hollow fiber tubes in a bioreactor. Plasma passes through the hollow fiber tubes, and the microcarriers containing hepatocytes filter impurities that permeate the plasma.

[0020] Furthermore, the ion-specific high cell density microcarriers and the hollow fiber tubes are arranged at intervals.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention utilizes the low sensitivity of protein-based biomaterials to immune rejection reactions, and with the help of microfluidics, various types of protein-based hydrogels can be customized into different forms by precisely controlling different fluids. In addition, the present invention utilizes the Hofmeister effect to conveniently and effectively control the recrystallization process of ice to generate microcarriers with an ecological niche structure. By combining protein-based hydrogels with the Hofmeister effect through microfluidics technology, in vitro bionic liver microcarriers with high hepatocyte density are prepared. Hepatocytes can proliferate in large numbers on the microcarriers and express normal metabolites, which can be used to prepare bioartificial liver systems for the treatment of acute liver failure.

[0023] The present invention provides a microcarrier with an ecological niche structure regulated by different ion types, and the size of the structure can be flexibly adjusted according to the ion type, annealing temperature, and time: the present invention produces a large number of silk fibroin particles based on a capillary microfluidic device, and can adjust the pore size after recrystallization within tens of microns by changing the type or concentration of ions in the aqueous solution in the initial stage. The in vitro culture of hepatocytes on the recrystallized microcarriers enhances cell proliferation activity, as well as metabolic and secretory functions.

[0024] The present invention uses pure protein-based hydrogel as raw material, and the raw materials include various easily available biological macromolecules such as collagen, gelatin, and silk fibroin. The cross-linking relies on the amino acid groups of the protein itself and does not introduce toxic substances.

[0025] In addition, the preparation process of the present invention is convenient and has good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of ion-specific high-cell density microcarriers for filtering blood metabolites.

[0027] Figure 2 : The photopolymerization process of silk fibroin-based hydrogel in Example 1.

[0028] Figure 3 : Light microscopy image of the silk fibroin microcarriers generated by microfluidics in Example 1.

[0029] Figure 4 : Schematic diagram of thin film preparation for observing the recrystallization effects of different ions in the embodiment.

[0030] Figure 5 : Relationship between ice crystal size and annealing time after recrystallization of different ions in the embodiment. DETAILED DESCRIPTION

[0031] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0033] The light-curable protein hydrogel used in the aqueous phase of the present invention is a protein hydrogel containing tyrosine, and the curing principle is based on the polymerization between tyrosines to form dityrosine.

[0034] The hollow fiber tube used in the examples is a commercial semi-permeable filter membrane.

[0035] Example 1 Preparation of ion-specific high cell density microcarriers

[0036] (1) Capillary microfluidic template: Microcarriers are produced using a microfluidic device composed of capillaries. The microcarriers are formed by shearing between the aqueous phase and the oil phase. The inner phase is composed of 5% w / w silk fibroin containing a photoinitiator (divalent ruthenium / sodium persulfate) and 10 mM sodium iodide ions. The outer phase uses silicone oil. A coaxial capillary with an inner diameter of 580 μm is used to make a microfluidic device. The droplets are sheared into circles at the tip of the inner phase to generate a large number of silk fibroin microcarriers in the oil phase, and then the excess oil droplets are removed by washing with ethanol.

[0037] (2) Recrystallization of silk fibroin microcarriers: The hydrogel was recrystallized using the quenching-annealing method. The silk fibroin microcarriers were placed in a solution containing sodium iodide ions. The solution was placed on dry ice to quickly form ice crystals. The solution was then annealed at -10°C for 60 min according to the experimental conditions.

[0038] (3) After further freeze-drying, the obtained microcarriers retain the polymer structure network left by ice crystal extrusion and retain a large number of biomimetic niche structures, thus obtaining ion-specific high cell density microcarriers.

[0039] Example 2 Preparation of hepatocyte-loaded microcarrier bionic liver system

[0040] (1) Capillary microfluidic template: Microcarriers are produced using a microfluidic device composed of capillaries. The microcarriers are formed by shearing between the aqueous phase and the oil phase. The inner phase is composed of 3% w / w recombinant human collagen containing a photoinitiator (divalent ruthenium / sodium persulfate) and 10 mM sodium iodide ions. The outer phase is made of corn oil using a coaxial capillary with an inner diameter of 580 μm to make a microfluidic device. The droplets are sheared into a circle at the tip of the inner phase to generate a large number of silk fibroin microcarriers in the oil phase, and then the excess oil droplets are removed by washing with ethanol.

[0041] (2) Recrystallization of recombinant human collagen microcarriers: The hydrogel was recrystallized using the quenching-annealing method. The recombinant human collagen microcarriers were placed in a solution containing sodium iodide ions. The solution was placed on dry ice to quickly form ice crystals. The solution was then annealed at -6°C for 40 min according to the experimental conditions.

[0042] (3) After further freeze-drying, the obtained microcarriers retain the polymer structure network left by ice crystal extrusion and retain a large number of biomimetic niche structures, thus obtaining ion-specific high cell density microcarriers.

[0043] (4) Three-dimensional biomimetic microcarriers based on iodine ions, using mature hepatocytes induced by human pluripotent hepatocytes. These cells can cluster and proliferate in the microcarrier niche structure, express glycogen synthesis and secrete normal liver metabolic products. The proliferation method is to co-culture the hepatocytes with the ion-specific high cell density microcarriers to obtain microcarriers containing hepatocytes.

[0044] (5) The obtained hepatocyte-containing microcarriers and hollow fiber tubes are integrated (co-encapsulated) in a 6×12 cm cylindrical bioreactor to assemble a bioartificial liver system; plasma can pass normally through the hollow fiber tubes, and the hepatocyte-containing microcarriers can filter impurities permeated from the plasma for the treatment of acute liver failure; ion-specific high-cell density microcarriers and hollow fiber tubes can be arranged at intervals.

[0045] Example 3

[0046] The ice crystal size obtained after recrystallization using different ions and the required annealing time were studied (other operations were the same as in Example 1). The preparation process diagram is shown in FIG. Figure 4 As shown in the figure, the relationship between ice crystal size and annealing time is shown in the figure Figure 5 As shown,

[0047] The ability of three different ions (F - , Br - and I - ) regulated the formation of ice crystals in 2% silk fibroin solution; recrystallization mainly followed the Ostwald ripening process; ice crystals grew at the expense of smaller crystals, increasing the average crystal size and reducing the total number of crystals; the average size of ice crystals obtained from NaI solution was 105.6±6.5μm, while the ice crystal size of the NaF group was significantly reduced compared with that of the pure water group (78.6±7.1μm); after annealing for 60 minutes, the average recrystallization pore size of the NaBr group was similar to that of the pure water group; therefore, the average size of ice crystals can be adjusted by changing the type of anions in the silk fibroin solution.

[0048] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing ion-specific high cell density microcarriers, characterized in that: The following steps are involved: Step S1: using microfluidic technology to generate protein particle microcarriers through shearing and UV curing between the aqueous phase and the oil phase, wherein the aqueous phase is composed of a photoinitiator, ions and a photocurable protein hydrogel, and the oil phase is composed of a viscous oil; Step S2: recrystallizing the protein particles using a quenching-annealing method; Step S3: freeze-drying is performed to preserve the polymer structure network generated during the recrystallization process, thereby forming a bionic niche pore structure and obtaining ion-specific high-cell density microcarriers; The ions used in the aqueous phase follow the Ostwald ripening process and include anions and cations, with concentrations in the aqueous phase ranging from 0.001 M to 0.1 M; the anions are selected from at least one of chloride ions, bromide ions, or iodide ions; and the cations are selected from at least one of potassium ions and sodium ions. The light-curable protein hydrogel used in the aqueous phase is a tyrosine-containing protein hydrogel; the concentration of the light-curable protein hydrogel in the aqueous phase is 5%-15% w / w; the light-curable protein hydrogel is selected from at least one of silk fibroin, collagen, and gelatin; In the quenching-annealing method, the annealing time is 10 min-100 min, and the annealing temperature is -40°C to -5°C.

2. The method for preparing ion-specific high cell density microcarriers according to claim 1, characterized in that: The ion-specific high-cell density microcarriers are prepared by capillary microfluidics technology, with an aqueous phase as the inner phase and an oil phase as the outer phase. Droplets are sheared into circles at the end of the inner phase, and the sheared circular droplets are solidified by ultraviolet light in the oil phase to form protein particle microcarriers.

3. The method for preparing ion-specific high cell density microcarriers according to claim 1, characterized in that: The oil phase is silicone oil or vegetable oil.

4. The ion-specific high cell density microcarrier prepared by the method according to any one of claims 1 to 3.

5. A bioartificial liver system, characterized by: The ion-specific high-cell-density microcarriers and hollow fiber tubes described in claim 4 are integrated in a bioreactor, plasma passes through the hollow fiber tubes, and the microcarriers containing hepatocytes filter impurities permeated from the plasma.

6. The bioartificial liver system according to claim 5, characterized in that: Ion-specific high cell density microcarriers and hollow fiber tubes are arranged in an interspaced pattern.

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