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US-20260124341-A1 - FISH LIVER DECELLULARIZED EXTRACELLULAR MATRIX BASED MICROFLUIDIC 3D PRINTING HYDROGEL, AND PREPARATION METHOD AND APPLICATION THEREOF

US20260124341A1US 20260124341 A1US20260124341 A1US 20260124341A1US-20260124341-A1

Abstract

A fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel for liver regeneration, and a preparation method thereof are provided. A fish liver decellularized extracellular matrix (dECM) is combined with gelatin methacryloyl (GelMA), and loaded with hepatic spheroids derived from induced pluripotent stem cells (iPSC-hep) for liver regeneration. The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel of the present disclosure has excellent biocompatibility and retains intact endogenous growth factors, maintains the biological activity of cells, ensures effective cell encapsulation, and is conducive to robust functional expression of iPSC-hep. After being transplanted in vivo, the hydrogel significantly improves the survival rate and liver function of mice with acute liver failure, and promotes liver regeneration and repair.

Inventors

  • Jinglin WANG

Assignees

  • NANJING DRUM TOWER HOSPITAL

Dates

Publication Date
20260507
Application Date
20241223
Priority Date
20241104

Claims (9)

  1. 1 . A fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel, wherein a fish liver decellularized extracellular matrix is combined with gelatin methacryloyl as a scaffold, hepatic spheroids derived from human induced pluripotent stem cells are used as a cell source, and a microfluidic 3D printing technology is used to prepare a mixed material hydrogel scaffold.
  2. 2 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 1 , wherein preparation steps are as follows: 1) preparing the hepatic spheroids derived from the human induced pluripotent stem cells; 2) preparing the fish liver decellularized extracellular matrix; and 3) designing a 3D scaffold model; fully mixing the gelatin methacryloyl, the fish liver decellularized extracellular matrix prepared in the step 2), the hepatic spheroids derived from the human induced pluripotent stem cells prepared in the step 1), and a photoinitiator in a proportion in water to form a biological link; and solidifying the biological link under an ultra violet (UV) irradiation into the scaffold with a fixed shape.
  3. 3 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 2 , wherein the step 2) comprises steps as follows: a, extracting a liver and intact blood vessels from a fresh fish; b, continuously perfusing the liver with a surfactant solution at a room temperature for a decellularization to obtain a decellularized material; c, washing the decellularized material obtained from the step b with a phosphate buffered saline to obtain the fish liver decellularized extracellular matrix; and d, dissolving the fish liver decellularized extracellular matrix obtained from the step c in an acetic acid aqueous solution containing pepsin.
  4. 4 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 2 , wherein the photoinitiator is 2-hydroxy-2-methyl-1phenyl-1acetone.
  5. 5 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 3 , wherein a surfactant in the surfactant solution is sodium dodecyl sulfonate or sodium deoxycholate.
  6. 6 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 3 , wherein a concentration of the surfactant solution used in the step b is 1%.
  7. 7 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 3 , wherein a continuous perfusion rate for the liver is 10 mL/min, and a perfusion time is 3 h in the step b.
  8. 8 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 3 , wherein in the acetic acid aqueous solution containing the pepsin in the step d, a concentration of the pepsin is 1%, and a concentration of acetic acid is 100 mM.
  9. 9 . The fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel according to claim 2 , wherein a mass ratio of the fish liver decellularized extracellular matrix to the gelatin methacryloyl to the photoinitiator in a reaction system is (1.5-3):7.5:1.

Description

CROSS REFERENCE TO THE RELATED APPLICATIONS This application is based upon and claims priority to Chinese Patent Application No. 2024115549091, filed on Nov. 4, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD The present disclosure belongs to the field of biomedical materials, and specifically relates to a fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel for liver regeneration and a preparation method thereof. BACKGROUND Liver transplantation is an efficient method for treating end-stage liver diseases, but the scarcity of donor livers limits the clinical application of liver transplantation. Tissue engineering is considered as a promising alternative to donor livers due to an ability to construct functional hepatic tissues. Hydrogels are an advanced material for shaping various structures required for liver transplantation, and have good biocompatibility and cell proliferation ability for hepatic spheroids derived from induced pluripotent stem cells (iPSC-heps). However, hydrogels usually exhibit limited biological activity, and are significantly different from natural hepatic tissues in composition. In addition, most hydrogels require complex manufacturing processes, causing potential biosafety problems and reducing therapeutic effects. Therefore, the demand for innovative hydrogels that have inherent biological activity and provide sufficient support for liver function has not been met. As a natural material, decellularized fish liver has excellent biocompatibility and retains intact endogenous growth factors, including collagen and glycosaminoglycans. Compared with terrestrial animal liver, fish liver resources from aquatic products are abundant and cost-effective. In addition, microfluidics technology is recognized for precise control of fluid dynamics, and is an advanced form used for additive manufacturing in drug delivery, cell culture, biosensors, and the like. 3D printing allows personalized design of implantable hydrogels to meet specific clinical needs. With these advantages, the combination of microfluidics and 3D printing technology makes it possible to produce customizable hydrogels for liver regeneration. Therefore, it is very desirable to combine a fish liver decellularized hydrogel as a biological chain with a microfluidic assisted 3D printing technology to develop a new scaffold that can effectively promote liver repair. SUMMARY Objective of the disclosure: In order to solve the above technical problems, the present disclosure proves a simple microfluidic 3D printing technology to prepare a fish liver decellularized extracellular matrix based hydrogel for liver regeneration, which is used for liver repair by orthotopic transplantation. The hydrogel is prepared by a method using the microfluidic 3D printing technology, which is simple, versatile and convenient for large-scale production. The technical solution: A fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel, wherein a fish liver decellularized extracellular matrix is combined with gelatin methacryloyl as a scaffold, hepatic spheroids derived from human induced pluripotent stem cells are used as a cell source, and a microfluidic 3D printing technology is used to prepare a mixed material hydrogel scaffold. The preparation steps of a fish liver decellularized extracellular matrix based microfluidic 3D printing hydrogel are as follows: 1) preparing hepatic spheroids derived from human induced pluripotent stem cells;2) preparing a fish liver decellularized extracellular matrix; and3) designing a 3D scaffold model; fully mixing gelatin methacryloyl, the fish liver decellularized extracellular matrix prepared in step 2), the hepatic spheroids derived from human induced pluripotent stem cells prepared in step 1), and a photoinitiator to form a biological link; and solidifying the biological link under ultra violet (UV) irradiation into a scaffold with a fixed shape. The specific steps of step 2) are as follows: a. extracting the liver and intact blood vessels from a fresh fish;b. continuously perfusing the liver with a surfactant solution at room temperature for decellularization;c. washing the material obtained from step b with phosphate buffered saline; andd. dissolving the fish liver decellularized extracellular matrix obtained from step c in an acetic acid aqueous solution containing pepsin. Preferably, wherein the photoinitiator is 2-hydroxy-2-methyl-1phenyl-1acetone. Preferably, wherein the surfactant is sodium dodecyl sulfonate or sodium deoxycholate. Preferably, wherein the concentration of the surfactant solution used in step b is 1%(g/100 mL, i.e., 1 g of a surfactant dissolved in 100 mL of water), the continuous perfusion rate for the fish liver is 10 mL/min and the perfusion time is 3 h. Preferably, wherein the mass concentration of pepsin in the acetic acid aqueous solution of pepsin in step d is 1% (g/100 mL, i.e.,