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Method for preparing cardiac muscle tissue engineering decellularization collagen diaphragm

A tissue engineering and decellularization technology, which is applied in tissue regeneration, medical science, prosthesis, etc., can solve the problems that have not been reported yet, and achieve the effect of low price, avoiding damage, and convenient material collection

Inactive Publication Date: 2017-06-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, it has been reported that bovine aponeurotic sheets are used for the culture of nerve cells; collagen sheets derived from trotter tendon are used for the construction of cardiomyocyte-like tissue, especially the report of using human induced pluripotent stem cell-derived cardiomyocytes as seed cells is unknown.

Method used

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  • Method for preparing cardiac muscle tissue engineering decellularization collagen diaphragm
  • Method for preparing cardiac muscle tissue engineering decellularization collagen diaphragm
  • Method for preparing cardiac muscle tissue engineering decellularization collagen diaphragm

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Preparation of decellularized collagen membrane sheets for myocardial tissue engineering with a thickness of 3 μm.

[0024] Wash the trotters to remove salt and dirt, further wash to remove oil stains, dissect the trotters, take out the tendons of the trotters, remove the surface fascia, and wash them with normal saline. Obtain pig’s tendons with a length of 0.5 cm and a diameter of 0.5 cm; place the pig’s tendons in a low-temperature-resistant container, pass through liquid nitrogen, and quickly transfer them to a container containing 37°C normal saline after 1 minute, and rewarm in a 37°C constant temperature water bath 5min, repeated freezing and thawing 5 times. Put the hoof tendon after repeated freezing and thawing into a container containing 100 μg / mL DNase and 100 μg / mL RNase, place it on a constant temperature shaker at 37°C for 12 hours, wash it with PBS 5 times, 30 min each time. The trotter tendon was embedded with OTC frozen embedding agent, pre-frozen at ...

Embodiment 2

[0026] Preparation of decellularized collagen membrane sheet for myocardial tissue engineering with a thickness of 30 μm.

[0027] Wash the trotters to remove salt and dirt, further wash to remove oil stains, dissect the trotters, take out the tendons of the trotters, remove the surface fascia, and wash them with normal saline. Obtain pig’s tendons with a length of 2 cm and a diameter of 1 cm; place the pig’s tendons in a low-temperature-resistant container, pass through liquid nitrogen, and quickly transfer them to a container filled with 37°C normal saline after 1 minute, and rewarm in a 37°C constant temperature water bath for 5 minutes. Repeated freezing and thawing 5 times. Put the hoof tendon after repeated freezing and thawing into a container containing 100 μg / mL DNase and 100 μg / mL RNase, place it on a constant temperature shaker at 37°C for 12 hours, wash it with PBS 5 times, 30 min each time. The trotter tendon was embedded with OTC frozen embedding agent, pre-froz...

Embodiment 3

[0030] Cell culture of decellularized collagen membrane and myocardial tissue construction.

[0031] Using the decellularized collagen membrane prepared in Example 2, the configuration is as follows figure 1 shown. The decellularized collagen membrane was soaked in 75% ethanol, sterilized by ultraviolet light for 2 hours, soaked in PBS solution containing 10% double antibody overnight, and the surface was modified with matrigel, and the decellularized collagen membrane was inoculated with human induced pluripotent stem cell-derived myocardium Cells were seeded at a density of 3 × 10 6Cells / mL, 24 hours after the cells adhered to the wall, the medium was changed. After the cells were cultured for 3 days, the life and death of the cells and the beating of the myocardium were observed under a microscope every day; , see the result Figure 5-8 .

[0032] The collagen membrane can maintain the survival of cardiomyocytes, and the cardiomyocytes grow in the form of single cells a...

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Abstract

The invention provides a method for preparing a cardiac muscle tissue engineering decellularization collagen diaphragm. The method comprises the following step: selecting natural pork tendon tissue, and performing decellularization on the tissue by using treatment methods of repeated freezing and thawing and nuclease treatment, thereby obtaining decellularization pork tendon diaphragms of different thicknesses, wherein the thicknesses of the diaphragms are within 5-30[mu] m; the obtained diaphragms comprise collagen as a main component; and the diaphragms are of structures of fibers which are arranged in parallel, are similar to extracellular matrix components and structures of cardiac muscle tissue cells, can induce cardiac muscle cells to extend along the fiber direction, and thus can be used for culturing cardiac muscle cells and establishing heart-like tissue. The method has the advantages of being convenient in material obtaining, low in price, stable in preparation process, uniform in product morphology, and simple and gentle, and collagen diaphragms can be highly controlled, have complete structures, morphology, biological properties and inherent mechanical properties of materials, furthermore have good biocompatibility and biodegradability, and are very applicable to cardiac muscle tissue engineering construction and regenerative medicine.

Description

technical field [0001] The invention relates to the technical field of novel biomaterials and tissue engineering, in particular to a preparation method of decellularized collagen membrane for myocardial tissue engineering. Background technique [0002] Myocardial tissue engineering is an emerging discipline that combines cell biology and material science to study the relationship between normal myocardial function, pathological myocardial tissue and the development of biological substitutes for repairing, maintaining and improving myocardial tissue function. The emergence of the invention breaks the limitations of traditional organ transplantation and biomaterial transplantation, and repairs damaged myocardial tissue. The four elements of myocardial tissue engineering mainly include seed cells, biomaterials, integration of cells and biomaterials, and integration of implants and in vivo microenvironment. [0003] In addition to maintaining tissue shape and function, myocardi...

Claims

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Application Information

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IPC IPC(8): A61L27/24A61L27/36A61L27/58
CPCA61L27/24A61L27/3604A61L27/365A61L27/367A61L27/3675A61L27/3683A61L27/58A61L2430/02A61L2430/10A61L2430/20A61L2430/30A61L2430/40
Inventor 秦建华张晓庆王丽徐聪
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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