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Preparation method of human skin stem cell factor nano-liposome-exosome complex

A nano-liposome and skin stem cell technology, applied in the field of cell biology, can solve problems such as difficult to reach target tissue, cause immune response, weak binding force of target tissue cells, etc., and achieve good stability

Inactive Publication Date: 2020-03-06
JINAN PANSHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Phospholipids are the components of the cell membrane. Therefore, nano-liposomes have good biocompatibility when injected into the body; but because nano-liposomes are foreign substances, they are easily phagocytized and cleared by mononuclear macrophages after entering the body. It is difficult to reach the target tissue, and the binding force with the target tissue cells is weak, and the amount of deposition in the target tissue is small, which may cause an immune response

Method used

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  • Preparation method of human skin stem cell factor nano-liposome-exosome complex
  • Preparation method of human skin stem cell factor nano-liposome-exosome complex
  • Preparation method of human skin stem cell factor nano-liposome-exosome complex

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Embodiment 1

[0049] The preparation of the nanoliposome of embodiment 1 coating cytokines

[0050] 1. Materials and steps

[0051] 1. Isolation of skin cells

[0052] Disinfection treatment of human skin tissue: Use a scalpel or scissors to remove the subcutaneous tissue (remove as clean as possible), wash once in 70% ethanol, and transfer the tissue to a PBS culture dish containing 2× double antibody for 3 min / time* 2 times;

[0053] The tissue was cut into 2mm*2mm, and digested with an appropriate amount of 0.1-5mg / ml Dispase in a 4°C refrigerator overnight; the next morning, the epidermis and dermis were separated with tweezers.

[0054] 2. Obtain primary skin epidermal stem cells

[0055] Add 10ml of 0.05% trypsin to the collected epidermis and digest it in a water bath at 37°C until a large number of cells fall off. Repeatedly pipette with DMEM medium containing 10% FBS for more than 20 times to obtain a single cell suspension, filter with a 100 micron filter, 1000rpm Centrifuge f...

Embodiment 2

[0075] Example 2 Obtaining of exosomes

[0076] 1. Materials and methods

[0077] (1) Preparation of exosome-free cell culture medium:

[0078] Centrifuge at 100,000g in an ultra-high-speed centrifuge at 4°C for 8 hours, collect the supernatant, and remove the exosomes carried by the medium itself.

[0079] (2) Collection of skin cell supernatant

[0080] Select P5 with good growth status to represent skin stem cells and dermal fibroblasts P5, and culture them with medium containing exosomes. When the fusion degree reaches 70%-80%, discard the supernatant of the old medium and add physiological saline to wash Twice, replace without culture medium and cultivate for 48h. Collect the medium supernatant, place it in a centrifuge tube, and put it at 4°C for later use.

[0081] (3) Extraction of exosomes

[0082] ①Put the collected cell culture supernatant into a centrifuge at 300g, centrifuge at 4°C for 8-10min, and save the supernatant A into an ultracentrifuge tube.

[0083...

Embodiment 3

[0092] Example 3 Preparation of Human Skin Stem Cell Cytokine Nanoliposome-Exosome Complex

[0093] 1. Steps and methods

[0094] (1) Take the exosome stock solution and mix it with liposomes. After mixing, 3000g, centrifuge at 4°C and discard the supernatant to retain the precipitate.

[0095] (2) Add pre-cooled cell culture medium to the precipitate obtained above, shake in an ice bath at 80 rpm for 20 minutes, then place in a water bath at 37° C. for 30 minutes, and shake at 80 rpm.

[0096] (3) Freeze the solution obtained above for 5 to 10 minutes at ultra-low temperature, thaw it by ultrasonication at room temperature for 30 to 60 minutes, and repeat the above freezing and thawing steps several times to obtain exosome-liposome complexes, liposome-exosome complexes body membrane fusion image 3 shown.

[0097] 2. Results analysis

[0098] Depend on Figure 4 It can be seen that the nanoliposome-exosome complex is in the shape of spherical particles, and the particle ...

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Abstract

The invention discloses a preparation method of a human skin stem cell factor nano-liposome-exosome complex. According to the method, the exosome and the liposome are compounded, and through the synergistic effect between the exosome and the nano-liposome coated with the cytokine, the obtained compound system has good biocompatibility and immune escape performance, is free of cytotoxicity, not prone to being degraded by macrophages, good in stability and capable of continuously playing a role in vivo.

Description

technical field [0001] The invention relates to a method for preparing a human skin stem cell factor nanoliposome-exosome complex, belonging to the field of cell biology. Background technique [0002] In today's world, due to changes in the environment, people's skin is facing various problems, such as skin damage caused by continuous exposure to ultraviolet rays; as the age increases, the skin begins to appear fine lines, wrinkles, pigmentation, and shrinkage due to atrophy of the dermis and epidermis. These skin problems all affect people's lives. [0003] Exosome is a kind of membranous vesicle released into the extracellular matrix after the multivesicular body (multivesicular body, MVB) in the cell fuses with the cell membrane under pathological or normal conditions. It is 30-150mm long and contains biologically active substances such as protein, mRNA, miRNA and DNA fragments. Cellular exosomes participate in many important physiological or pathological processes of t...

Claims

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Application Information

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IPC IPC(8): A61K35/36A61K9/127A61P17/00A61P17/02A61P17/10A61P37/08A61K8/99A61K8/14A61Q19/00A61Q19/02A61Q19/08C12N5/071
CPCA61K8/14A61K8/99A61K9/1271A61K35/36A61P17/00A61P17/02A61P17/10A61P37/08A61Q19/00A61Q19/005A61Q19/02A61Q19/08C12N5/0625C12N2509/00A61K2300/00
Inventor 张甜甜邢志青张平王杰李霄王红丽
Owner JINAN PANSHENG BIOTECH
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