	US 12,257,364 B2
	Preparation method and application of decellularized extracellular matrix tissue paper
Meifeng Zhu, Tianjin (CN); Wen Li, Tianjin (CN); Deling Kong, Tianjin (CN); and Zhen Zhang, Tianjin (CN)
Assigned to NANKAI UNIVERSITY, Tianjin (CN)
Filed by NANKAI UNIVERSITY, Tianjin (CN)
Filed on Mar. 15, 2023, as Appl. No. 18/122,009.
Claims priority of application No. 202210607526.0 (CN), filed on May 31, 2022.
Prior Publication US 2023/0405186 A1, Dec. 21, 2023
Int. Cl. A61L 27/36 (2006.01)

CPC A61L 27/3687 (2013.01) [A61L 27/3633 (2013.01); A61L 2430/40 (2013.01)]

7 Claims

1. A preparation method of decellularized extracellular matrix tissue paper, comprising the following steps:

(1) preparing decellularized extracellular matrix materials, comprising:

washing a tissue with water and then slicing the tissue;

disinfecting the washed sliced tissue with a peroxyacetic acid;

washing the disinfected tissue by adding sterilized water or normal saline; and

further washing the disinfected tissue with a buffer containing DNase and RNase so as to remove DNA and RNA;

(2) preparing tissue paper, comprising:

homogenizing and stirring the decellularized extracellular matrix materials from step (1) for a time ranging from 1 minute to 60 minutes to obtain a homogenate;

placing the obtained homogenate on a flat-bottom filter screen to filter out water from the obtained homogenate to obtain filtered homogenate, wherein the water passes through the flat-bottom filter screen, and air-drying the filtered homogenate on the flat-bottom filter screen to obtain air-dried homogenate, wherein placing the obtained homogenate on the flat-bottom filter screen and air-drying the filtered homogenate is performed in a range of from 1 hour to 120 hours; and

freeze-drying the air-dried homogenate at a temperature ranging from −196° C. to −20° C. for 4 hours to 48 hours, to obtain the tissue paper

(3) cross-linking the tissue paper, comprising:

cross-linking the tissue paper obtained in step (2) in a cross-linking solution, wherein the cross-linking solution in step (3) is an ethanol solution containing a cross-linking agent, the cross-linking agent comprising one or more of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimine, N-hydroxysuccinimide, glutaraldehyde, formaldehyde, and genipin, and wherein the cross-linking is conducted at a temperature of 4° C. to 27° C. for 4 hours to 12 hours; and

(4) freeze-drying the cross-linked tissue paper, comprising:

freeze-drying the cross-linked tissue paper obtained in step (3), to obtain the decellularized extracellular matrix tissue paper, wherein the freeze-drying temperature in step (4) ranges from −196° C. to −20° C. for 12 hours to 72 hours.