Extraction method and application of urogenic stem cell exosome

An extraction method and exosome technology, 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 material extraction, and simple preparation process

Active Publication Date: 2018-10-30
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 a...

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  • Extraction method and application of urogenic stem cell exosome
  • Extraction method and application of urogenic stem cell exosome
  • Extraction method and application of urogenic stem cell exosome

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Experimental program
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Embodiment 1

[0056] Isolation and culture of urine-derived stem cells:

[0057] (1) Collection of urine-derived stem cells

[0058] After obtaining the informed consent of the donor, a 50mL sterile centrifuge tube containing 1% Antibiotic-Antimycotic was used to collect clean midsection urine (30-50mL) of healthy adults.

[0059] (2) Isolation of urine-derived 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 0.2 mL of liquid at the bottom of the tube), resuspend the pellet with 3 mL of primary culture medium, and then evenly divide it into 3 wells of a 12-well plate. All operation steps are carried out at room temperature. 37°C, 5% CO 2 Cultivate under conditions. The primary culture medium is DMEM / F-12 medium containing 10% fetal bovine serum, 100U / mL penicilli...

Embodiment 2

[0064] Extraction and identification of urinary stem cell exosomes:

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

Embodiment 3

[0067] The role of urine-derived stem cell exosomes in preventing and treating osteoporosis:

[0068] (1) Mouse model:

[0069] 16-month-old SPF C57BL / 6 male mice were selected as the model of senile osteoporosis.

[0070] Two-month-old SPF-grade C57BL / 6 female mice were selected to construct a postmenopausal osteoporosis model by bilateral ovariectomy (OVX), and a sham operation (Sham) group was established, in which only a little adipose tissue around the ovary was cut off.

[0071] Three-month-old female mice were selected for tail suspension (TS) to reduce the weight of their hind limbs, and a disuse osteoporosis model was constructed. The control group moved freely. The experimental mice were purchased from Hunan Slack Jingda Animal Experiment Co., Ltd.

[0072] (2) Group experiment:

[0073] Senile osteoporosis models: divided into urine-derived stem cell exosomes group, USC-Exos group, and equal volume solvent (PBS) control group (Control group). Inject 100 μg USC-Exos (dissolve...

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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, and in particular to a method for extracting urine-derived stem cell exosomes and its preparation for the prevention and treatment of bone The application of porosity in medicine, food or health products. Background technique [0002] Osteoporosis is a metabolic bone disease characterized by decreased bone mass, bone microstructure destruction 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 them, primary osteoporosis is divided into postmenopausal, senile and idiopathic osteoporosis, and secondary osteoporosis Mostly caused by diseases or other factors, including disuse osteoporosis. According to statistics from the International Osteoporosis Foundati...

Claims

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Application Information

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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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