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Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof

A technology of conditioned medium and stem cells, which is applied in the field of tissue engineering and medical wound repair, can solve the problem that it is difficult to effectively exert the dual advantages of amnion secreted active factors and amnion natural matrix at the same time, so as to improve the quality of wound repair, promote wound healing, and avoid The effect of disease spread

Active Publication Date: 2015-11-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the above-mentioned methods of using amnion epithelial cells or acellular amnion matrix alone to improve wound healing are difficult to effectively utilize the dual advantages of amnion secretion active factors and amnion natural matrix at the same time.
[0005] So far, there has been no research report on the preparation of amnion into active amnion microparticles containing amnion epithelial cells and amnion stroma to construct active dermal substitutes

Method used

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  • Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof
  • Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof
  • Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1. Preparation of active amniotic membrane microparticles (LMAM) of the present invention

[0036] 1. Acquisition of amniotic membrane

[0037] With the approval of the Hospital Ethics Committee and the informed consent of the puerpera, fresh placental tissues from cesarean section women who were negative for hepatitis virus antibody, syphilis antibody, and HIV were selected, and the chorion was bluntly separated under sterile conditions, and the fibroblast layer and spongy layer were removed. After soaking and washing in phosphate buffered saline (PBS) containing 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin for 3×15 min, the amniotic membrane was cut into pieces of 8×6 cm in size.

[0038] 2. Preparation, cryopreservation, recovery and activity determination of active amnion microparticles

[0039] Prepare amniotic membrane particles of 300-600 μm in size from 8×6 cm amniotic membrane slices with a particle skin cutter, and add 3...

Embodiment 2

[0042] Example 2. Preparation of Active Amnion Microparticle Conditioned Medium

[0043] 1. Preparation of active amnion microparticles conditioned medium (conditioned medium, CM for short)

[0044] Take out the cryopreservation tube from liquid nitrogen and place it in a 37°C water bath to fully thaw for 1 minute. After repeated washing and centrifugation with PBS for 3 times, the supernatant is removed, and the ratio of 1ml of particles: 6ml of DMEM medium is added to a six-well plate. 5%CO 2 Continue to incubate in the incubator for 48 hours, collect the supernatant, centrifuge at 1000 rpm for ten minutes, filter the supernatant through a 0.2 μm filter, and store it in a -80°C refrigerator for later use.

[0045] 2. Transwell detects the effect of CM on chemotaxis and migration of epidermal cells, fibroblasts and endothelial cells

[0046] The transwell migration chamber (Corning, USA) with a pore size of 8 μm was used to detect the chemotaxis and migration ability of CM ...

Embodiment 3

[0048] Embodiment 3. Animal experiments

[0049] Take 15 db / db diabetic mice (provided by Shanghai Xipro-Bikay Experimental Animal Co., Ltd.), male or female, routinely anesthetized with 1% sodium pentobarbital intraperitoneally, and disinfected with alcohol. A circular full-thickness skin defect wound with a diameter of 1.2 cm was made on both sides of the back of each db / db diabetic mouse, and the wounds (30) of the db / db diabetic mice were randomly divided into LMAM groups (prepared in Example 1) Active amniotic membrane microparticles group), DMAM group (decellularized amniotic membrane microparticles group, see Ji SZ, Xiao SC, Luo PF, Huang GF, Wang GY, Zhu SH, et al. An epidermal stem cells niche microenvironment created by engineered human amniotic membrane.Biomaterials2011; 32(31):7801-11) and blank control group, 10 wounds in each group.

[0050] After LMAM and DMAM were routinely thawed and revived, the supernatant was removed after repeated washing and centrifugati...

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Abstract

The invention relates to the technical field of tissue engineering and medical wound repairing, in particular to an amnion innate stem cell carried frozen active amnion particle and a conditioned medium and application thereof. Discarded fresh amnions are prepared into particles and then frozen into liquid nitrogen by means of serum-free stem cell freezing liquid; thus, complete matrix components of amnion are maintained while amnion epithelial cell activity is maintained for a long time, and further, disease transmission is avoided effectively. The conditioned medium which is prepared and collected by means of the frozen active amnion particles can promote chemotaxis and migration of human epidermal cells, fibroblast and endothelial cells effectively. In terms of zoografting, wound healing can be improved by multiple ways of regulating inflammatory reaction, promoting vascularization, quickening epithelization and the like through the active amnion particles; further, the amnion matrix can be directly used as a dermis equivalent to induce dermal regeneration so as to improve wound healing quality remarkably. The amnion innate stem cell carried frozen active amnion particle and the conditioned medium thereof can provide simple but effective ways for wound repairing.

Description

technical field [0001] The invention relates to the technical fields of tissue engineering and medical wound repair, in particular to a cryoviable amnion microparticle loaded with amnion intrinsic stem cells, a conditioned medium and application thereof. Background technique [0002] The amniotic membrane is derived from the discarded placental tissue during childbirth. Its structure is similar to that of normal skin, including epithelial layer, basement membrane, compact layer, fibroblast layer and spongy layer, without blood vessels, nerves, and lymphoid tissues. Amniotic membrane has been used as an external covering for burns and corneal injury protection for more than 100 years. At present, there are commercialized decellularized amniotic membranes at home and abroad (Ruiji Bio-Amnion Jiangxi Ruiji Bioengineering Technology Co., Ltd. in China, and abroad). dHACM, MiMedx Group, Inc.), and is widely used in the protection of various acute and chronic wounds. [0003] Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073A01N1/02A61L27/38A61L27/36A61L27/60
Inventor 纪世召郑勇军肖仕初夏照帆李廷王光毅孙颖
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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