Stem cell bioactive composition and preparation method and application thereof

A stem cell biology and composition technology, applied in biochemical equipment and methods, cell culture active agents, non-embryonic pluripotent stem cells, etc. The effect of promoting growth and maintaining undifferentiated state, avoiding safety hazards, and high yield

Active Publication Date: 2016-12-07
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the stem cell bioactive substances currently used in practice have the following deficiencies: 1. Extract directly from tissues such as placenta, resulting in low purity of the obtained stem cell bioactive substances; 3. Stem cells are rich in biologically active substances, which can affect the proliferation and differentiation of stem cells. etc. are very important, but these substances are not secreted outside the cells, so they are often missed during the extraction process; 4. A single embryonic stem cell or adult stem cell is used to culture and collect the supernatant, which does not take into account the complex growth environment of cells in vivo and Interactions between different types of cells, resulting in low yields of extracted bioactive substances

Method used

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  • Stem cell bioactive composition and preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0021] Embodiment 1, separation and purification of adipose stromal cells

[0022] After informed consent of the patients, adipose tissue was obtained by liposuction. The age of the patient is between 37-54 years old, and the body mass index (kg / m 2 ) between 28-35, in good health except obesity, without diabetes or other complications. After liposuction, put the adipose tissue in normal saline and send it to the laboratory within 2 hours after the operation, wash with 2 times the volume of Krebs-Ringer Bicarbonate buffer 3-4 times to remove blood stains, and then use 1 times the volume of type Ⅰ collagenase (1g / L of KRB, 1% BSA) was digested at 37°C for 60min with intermittent stirring. After digestion, centrifuge at 300 g for 5 min to remove floating adipocytes. Inoculate the obtained stromal cells into cell culture flasks at 37°C, 5% CO 2 The cells were cultured in an incubator at an initial density of 3.5×10 3 piece / cm 2 , the cells grow to about 80% after 4-5 days o...

Embodiment 2

[0023] Embodiment 2, separation and purification of placental mesenchymal stem cells

[0024] Take the placental tissue of full-term normal delivery or caesarean section, strip off the maternal side decidua, cut small pieces of fetal decidua side placental tissue, wash with PBS, and remove blood. Cut the placental tissue into pieces, add 0.1% type IV collagenase, digest at 37°C for 30 minutes, pass through a 100-mesh sieve, collect the cell suspension, add the prepared medium (containing DMEM, 10% FBS, 5ng / mL bFGF, 100U / mL penicillin, 100μg / mL streptomycin, 25mmol / L glutamine), adjust the culture cell density to 1×10 5 cells / mL, placed at 37°C, 5% CO 2 cultured in an incubator, and the medium was changed every 3-4 days. Only a small amount of adherent cells were obtained in the cells obtained after the fetal decidua side placental tissue was digested. After 2 weeks, flat monolayer cells gradually formed. The cells were passaged and frozen according to conventional methods. S...

Embodiment 3

[0025] Example 3, Establishment of a co-culture system of adipose stromal cells and mesenchymal stem cells

[0026] A non-contact co-culture of adipose stromal cells and mesenchymal stem cells was carried out using a Transwell hanging culture chamber with a PET membrane combined with a 6-well plate. Cells in the logarithmic growth phase were taken, digested with 0.05% / 0.25% trypsin to make a single cell suspension, counted, adjusted the cell density and inoculated, and the medium was MSC serum-free medium. In the experimental group, adipose stromal cells were inoculated in the lower chamber of the Transwell culture chamber, and then the upper chamber of the Transwell suspension culture chamber was placed in the hole, and MSCs were inoculated in the Transwell suspension culture chamber, so that the culture medium in the upper and lower chambers could communicate with each other, thereby A co-culture system of adipose stromal cells and mesenchymal stem cells was established; in ...

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Abstract

The invention discloses a stem cell bioactive composition and a preparation method and application thereof. The stem cell bioactive composition comprises 1.69 to 22.24 pg/mL of EGF (epidermal growth factor), 2443.17 to 4459.43 pg/mL of bFGF (basic fibroblast growth factor), 1.44 to 134.36 pg/mL of beta-NGF (Beta-nerve growth factor), 1.55 to 355.14 pg/mL of VEGF (vascular endothelial growth factor), 11.87 to 159.70 pg/mL of PDGF-DD (platelet-derived growth factor), 135.79 to 688.13 pg/mL of KGF (keratinocyte growth factor), and 1176.99 to 3825.79 pg/mL of TGF-Beta1 (transforming growth factor beta-1); the stem cell bioactive composition has high purity, high yield and high bioactivity.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to bioactive substances, and more specifically, the present invention relates to a stem cell bioactive composition and its preparation method and application. Background technique [0002] Stem cells are a type of cells with self-renewal, high proliferation and multi-directional differentiation potential. They are the origin cells of the body and the "universal cells" that form various tissues and organs of the human body. According to their developmental stage, they can be divided into embryonic stem cells and adult stem cells. Among them, adult stem cells (ASC) are widely distributed, have strong proliferation ability, have multi-directional differentiation potential, and have no ethical or religious issues. They can be derived from various tissues such as bone marrow, fat, skin, muscle, and cornea, including mesenchyme. Stem cells (Mesenchymal stem cells, MSCs), hematopoieti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P1/00C12N5/074A61K8/98A61Q19/08A61Q19/00A23L33/18
CPCA61K8/981A61Q19/00A61Q19/08C12N5/0607C12N2501/11C12N2501/115C12N2501/117C12N2501/135C12N2501/15C12N2501/165C12N2501/195C12P1/00
Inventor 裴雪涛南雪单紫筠贾茜媛吕洋李静姚海雷岳文
Owner ACADEMY OF MILITARY MEDICAL SCI
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