Method for preparing xenogeneic acellular matrix and product thereof

Abstract

The invention relates to a method for preparing a xenogeneic acellular matrix. The method comprises the following steps: preparing a split-thickness skin from an animal skin; separating epidermis from dermis; crosslinking; decellularizing; freeze-drying; and inactivating viruses to finally obtain the acellular dermal matrix. The method provided by the invention has the advantages that the method for crosslinking by N, N-dicyclohexyl carbodiimide is short in time and high in efficiency; in addition, on the premise of crosslinking, the treatment progress of the following crosslinking agent residue is extremely simplified, so that the cost is saved; enzymes, SDS and alkalis are combined in the decellularizing process, the acellular matrix is under micro-control, and the acellular matrix is under macro-control by freeze drying, so that decellularization is thorough and cells are easy to grow on the matrix.

Description

technical field

[0001] The invention relates to the field of tissue engineering, in particular to a method for preparing heterogeneous acellular matrix. Background technique

[0002] The purpose of the preparation of acellular dermal matrix is ​​to remove as much as possible the highly immunogenic cell components in the skin tissue (epidermal cells, hair follicles, sebaceous glands, sweat glands, and vascular endothelial cells in the dermis, fibroblasts, etc.) Weaker extracellular matrix components and complete three-dimensional spatial structure of dermal tissue. This can not only avoid rejection and excessive immune inflammatory response after transplantation, thereby affecting the transplantation effect, but also provide a biological "template" or scaffold to guide tissue regeneration, so as to achieve the purpose of maximally repairing tissue defects.

[0003] At present, the preparation methods of acellular dermal matrix mainly include the following: 1) DispaseII-Trito...

Examples

Embodiment 1

[0048] 1.0 Preparation of split skin slices: use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.15% bromogeramine for 30 minutes, wash with 100 ml of PBS each time, and wash 3 times , the effect picture of HE staining of pig medium-thick skin obtained from the treatment is shown in figure 2 .

[0049] 2.0 Separation of epidermis and dermis: Use 100ml of 0.15% trypsin-PBS solution, stop at 7°C for 24 hours.

[0050] 3.0 Cross-linking: use 100ml ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L, then add N,N-dicyclohexyl to the solution Carbodiimide (DCC) and N-hydroxysuccinimide (NHS), according to the mass ratio, 1.8 grams of N,N-dicyclohexylcarbodiimide and 0.415 grams of NHS per gram of decellularized tissue, soaked for 4 Hour.

[0051] 4.0 Decellularization: Trea...

Embodiment 2

[0055] 1.0 Preparation of split skin slices: use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.1% bromogeramine for 30 minutes, wash with 100 ml PBS each time, and wash 3 times .

[0056] 2.0 Separation of epidermis and dermis: use 0.1% trypsin-PBS solution 100ml, stop at 4°C for 6 hours.

[0057] 3.0 Cross-linking: Use 100ml of ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L. Add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS) to the solution, and the mass ratio is 5.4 grams of N,N-dicyclohexylcarbodiimide per gram of decellularized tissue imine and 1.245 g NHS, soaked for 4 hours.

[0058] 4.0 Decellularization: Treat with 100ml of 2% sodium hydroxide solution for 24 hours, then treat with 0.1% trypsin-PBS solution at 4°C for 36 hours. Rinse with PBS...

Embodiment 3

[0062] 1.0 Preparation of split skin slices: Use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.15 bromogeramine for 30 minutes, wash each time with 100 ml of PBS, and wash 3 times.

[0063] 2.0 Separation of epidermis and dermis: use 0.5% trypsin-PBS solution 100ml, stop at 20°C for 4 hours.

[0064] 3.0 Cross-linking: Use 100ml of ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L. Add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS) to the solution, and the mass ratio is 3.6 grams of N,N-dicyclohexylcarbodiimide per gram of decellularized tissue imine and 0.930 g NHS, soaked for 4 hours.

[0065] 4.0 Decellularization: Treat with 100ml of 1.5% sodium hydroxide solution for 24 hours, then treat with 0.5% trypsin-PBS solution at 20°C for 20 hours. Rinse wit...

technical field

[0001] The invention relates to the field of tissue engineering, in particular to a method for preparing heterogeneous acellular matrix. Background technique

[0002] The purpose of the preparation of acellular dermal matrix is ​​to remove as much as possible the highly immunogenic cell components in the skin tissue (epidermal cells, hair follicles, sebaceous glands, sweat glands, and vascular endothelial cells in the dermis, fibroblasts, etc.) Weaker extracellular matrix components and complete three-dimensional spatial structure of dermal tissue. This can not only avoid rejection and excessive immune inflammatory response after transplantation, thereby affecting the transplantation effect, but also provide a biological "template" or scaffold to guide tissue regeneration, so as to achieve the purpose of maximally repairing tissue defects.

[0003] At present, the preparation methods of acellular dermal matrix mainly include the following: 1) DispaseII-Trito...

Examples

Embodiment 1

[0048] 1.0 Preparation of split skin slices: use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.15% bromogeramine for 30 minutes, wash with 100 ml of PBS each time, and wash 3 times , the effect picture of HE staining of pig medium-thick skin obtained from the treatment is shown in figure 2 .

[0049] 2.0 Separation of epidermis and dermis: Use 100ml of 0.15% trypsin-PBS solution, stop at 7°C for 24 hours.

[0050] 3.0 Cross-linking: use 100ml ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L, then add N,N-dicyclohexyl to the solution Carbodiimide (DCC) and N-hydroxysuccinimide (NHS), according to the mass ratio, 1.8 grams of N,N-dicyclohexylcarbodiimide and 0.415 grams of NHS per gram of decellularized tissue, soaked for 4 Hour.

[0051] 4.0 Decellularization: Trea...

Embodiment 2

[0055] 1.0 Preparation of split skin slices: use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.1% bromogeramine for 30 minutes, wash with 100 ml PBS each time, and wash 3 times .

[0056] 2.0 Separation of epidermis and dermis: use 0.1% trypsin-PBS solution 100ml, stop at 4°C for 6 hours.

[0057] 3.0 Cross-linking: Use 100ml of ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L. Add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS) to the solution, and the mass ratio is 5.4 grams of N,N-dicyclohexylcarbodiimide per gram of decellularized tissue imine and 1.245 g NHS, soaked for 4 hours.

[0058] 4.0 Decellularization: Treat with 100ml of 2% sodium hydroxide solution for 24 hours, then treat with 0.1% trypsin-PBS solution at 4°C for 36 hours. Rinse with PBS...

Embodiment 3

[0062] 1.0 Preparation of split skin slices: Use a skin harvester to make animal skin slices with a length of 6 cm, a width of 4 cm, and a thickness of 0.6 mm, wash with pure water, sterilize and degrease with 0.15 bromogeramine for 30 minutes, wash each time with 100 ml of PBS, and wash 3 times.

[0063] 2.0 Separation of epidermis and dermis: use 0.5% trypsin-PBS solution 100ml, stop at 20°C for 4 hours.

[0064] 3.0 Cross-linking: Use 100ml of ethanol solution (volume ratio of ethanol is 40%), add morpholineethanesulfonic acid (MES) monohydrate to make the concentration reach 0.06mol / L. Add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS) to the solution, and the mass ratio is 3.6 grams of N,N-dicyclohexylcarbodiimide per gram of decellularized tissue imine and 0.930 g NHS, soaked for 4 hours.

[0065] 4.0 Decellularization: Treat with 100ml of 1.5% sodium hydroxide solution for 24 hours, then treat with 0.5% trypsin-PBS solution at 20°C for 20 hours. Rinse wit...

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