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Extraction method and application of urine-derived stem cell exosomes

A technology of stem cells and exosomes, applied in the field of stem cells, can solve problems not involved in the prevention and treatment of osteoporosis, and achieve the effects of strong self-renewal ability, safe and non-invasive materials, and simple preparation process

Active Publication Date: 2019-08-06
湖南鹤螈生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the patent discloses the extraction and separation method of urine-derived stem cells and is used for the study of diabetes, it does not involve the prevention and treatment of osteoporosis

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  • Extraction method and application of urine-derived stem cell exosomes
  • Extraction method and application of urine-derived stem cell exosomes
  • Extraction method and application of urine-derived stem cell exosomes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Isolation and culture of urinary stem cells:

[0057] (1) Collection of urinary stem cells

[0058] After obtaining the donor's informed consent, the clean midstream urine (30-50 mL) of healthy adults was collected in a 50 mL sterile centrifuge tube added with 1% antibacterial-antimycotic (Antibiotic-Antimycotic).

[0059] (2) Isolation of urinary stem cells

[0060] The collected urine was centrifuged at 400×g for 10 minutes to remove most of the supernatant (leave 1 mL of liquid at the bottom of the tube), add 10 mL of PBS to the centrifuge tube, and centrifuge at 200×g for 10 minutes to remove most of the supernatant ( Leave 0.2mL of liquid at the bottom of the tube), resuspend the pellet with 3mL of primary culture medium, and distribute it equally to 3 wells of a 12-well plate. All operating steps were performed at room temperature. 37°C, 5% CO 2 cultivated under conditions. The primary medium was DMEM / F-12 medium containing 10% fetal bovine serum, 100 U / mL pe...

Embodiment 2

[0064] Extraction and identification of urine-derived stem cell exosomes:

[0065] When the urinary stem cells reached 80% confluence, they were cultured for 48 h with fresh expansion medium without exosomes. Collect the cell culture supernatant, centrifuge at low speed to remove residual cells and cell debris in the supernatant, filter through a 0.22 μm sterile filter, transfer the filtered liquid to a 15 mL 10KD ultrafiltration centrifuge tube, centrifuge at 4000×g, 4°C for about After 20 minutes, the ultrafiltrate was concentrated to 1 mL. Add 14 mL of PBS to the ultrafiltrate, centrifuge at 4000×g for about 15 minutes at 4°C, and concentrate the ultrafiltrate to 1 mL. Transfer the ultrafiltrate to a new 1.5mL sterile eppendorf centrifuge tube, add Exoquick-TC at a ratio of 1:5 TM Exosome extraction reagent, incubate at 4°C for 12 hours, centrifuge at 1500×g, 4°C for 30 minutes, discard the supernatant, and resuspend the pellet with sterile PBS to obtain urine-derived ste...

Embodiment 3

[0067] The role of urinary stem cell exosomes in the prevention and treatment of osteoporosis:

[0068] (1) Mouse model:

[0069] A 16-month-old SPF grade C57BL / 6 male mouse was selected as a model of senile osteoporosis.

[0070] Two-month-old SPF-grade C57BL / 6 female mice were selected to establish a postmenopausal osteoporosis model by bilateral ovariectomy (OVX). At the same time, a sham operation (Sham) group was established, in which only a small amount of periovary adipose tissue was excised.

[0071] 3-month-old female mice were selected for tail suspension (TS) to reduce the load on their hind limbs to construct a disuse osteoporosis model, and the control group moved freely. Experimental mice were purchased from Hunan Slack Jingda Animal Experiment Co., Ltd.

[0072] (2) Group experiment:

[0073] Senile osteoporosis model: Divided into a urine-derived stem cell exosome group (USC-Exos group) and an equal volume solvent (PBS) control group (Control group). 100 μg...

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Abstract

The invention discloses an extraction method and the application of an urogenic stem cell exosome. The extraction method comprises the following specific steps: firstly, collecting urine, then carrying out first-time centrifugation, and adding PBS into a first residual liquid obtained after supernatant is removed; carrying out second-time centrifugation, resuspending the second-time residual liquid obtained after the supernatant is removed through a primary culture medium to obtain urogenic stem cell suspension solution; secondly, carrying out multiplication culture on the urogenic stem cellsand passing down to the second to the sixth generation for later use; thirdly, changing the culture medium obtained in the second step, then continuously culturing, collecting and culturing supernatant, centrifuging, filtering and carrying out centrifugal concentration; adding an exosome extraction reagent for extracting to obtain a resuspension solution of the urogenic stem cells. The urogenic stem cell exosome extracted by the invention is proved by animal experiments to have the advantages of good therapeutic effects on senile osteoporosis, postmenopausal osteoporosis and disuse osteoporosis.

Description

technical field [0001] The present invention relates to the technical field of stem cells, in particular, to a method for extracting urine-derived stem cell exosomes and its application, especially to a method for extracting urine-derived stem cell exosomes and its preparation for preventing and treating osteoporosis. The application in medicine, food or health product of porosis. Background technique [0002] Osteoporosis is a metabolic bone disease characterized by decreased bone mass, destruction of bone microarchitecture, and increased bone fragility. It often induces fractures and brings a heavy burden to individuals, families, and society. Osteoporosis is divided into two categories: primary and secondary, among which primary osteoporosis is further divided into postmenopausal, senile and idiopathic osteoporosis, and secondary osteoporosis Mostly caused by disease or other factors, including disuse osteoporosis. According to statistics from the International Osteopor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074A61K35/28A61P19/10
CPCA61K35/28A61P19/10C12N5/0684C12N2500/32C12N2500/84C12N2501/11C12N2501/115C12N2501/135
Inventor 谢辉陈春媛饶珊珊刘祎伟谭艺娟胡雄科罗明洁胡阴殷豪黄杰罗娟
Owner 湖南鹤螈生物科技有限公司
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