Umbilical cord mesenchymal stem cell preparation and preparation method and application thereof

A stem cell preparation and stem cell technology, applied in the field of preparations containing umbilical cord mesenchymal stem cells and their preparation, can solve the problems of insufficient utilization of umbilical cord mesenchymal stem cells, low yield of active ingredients, limited types and quantities, etc., and achieve effective factor Enrich, optimize product quality, and ensure the effect of cell state

Inactive Publication Date: 2019-09-24
湖南丰晖生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current methods for preparing stem cell preparations have low yields of active ingredients. For example, the invention patent with application number CN201810621158 uses the supernatant culture fluid of umbilical cord mesenchymal stem cells as the essence. However, it is well known that high-generation stem cells are being cultured In the process, not only beneficial cytokines will be produced, but also cell metabolic wastes will be produced, and the cytokines of this invention only contain IGF, bFGF and EGF, and the types of cytokines obtained by using the supernatant culture fluid of umbilical cord mesenchymal stem cells Both the quantity and quantity are limited, and the umbilical cord mesenchymal stem cells cannot be fully utilized, nor can it meet the actual needs of the industry.

Method used

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  • Umbilical cord mesenchymal stem cell preparation and preparation method and application thereof
  • Umbilical cord mesenchymal stem cell preparation and preparation method and application thereof
  • Umbilical cord mesenchymal stem cell preparation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0051] Example 3 MTT detects the effects of ordinary medium and special medium on the proliferation of human umbilical cord mesenchymal stem cells

[0052] After recovery and culture of human umbilical cord mesenchymal stem cells, 3000 cells / well were plated into 96-well plates, 2 groups were plated, each group had 5 duplicate wells, and 4 96-well plates were plated in total. The cells in the two groups were cultured with ordinary medium and special medium, and MTT was added every 24 hours, and the OD value in the wells at a wavelength of 570nm was detected with a microplate reader. The ordinary medium did not add the above-mentioned 20 kinds of amino acids and 8 kinds of vitamins. The test results are shown in Table 1 and figure 1 As shown, the transfer culture medium adopted in the present invention can obviously promote the proliferation of human umbilical cord mesenchymal stem cells and maintain the viability and better cell state of stem cells, and after 72 hours of cult...

Embodiment 4

[0055] Example 4 Immunofluorescence Detection of CD44 Expression in Human Umbilical Cord Mesenchymal Stem Cells

[0056] Human umbilical cord mesenchymal stem cells of P3, P10, and P11 generations were divided into 5×10 4 The density of cells / well was spread into a 24-well plate. After the cells adhered to the wall, the cells were fixed with paraformaldehyde, blocked with BSA, then coated with CD44 antibody and fluorescent secondary antibody, and finally stained with DAPI and observed under a fluorescent microscope. ,Photograph. Test results such as figure 2 As shown, under the same experimental and microscopic examination conditions, the P3-P11 generation umbilical cord mesenchymal stem cells can express CD44 molecules, but the CD44 expression level decreased significantly since the P11 generation, and the P3 generation was the best. It can be seen that the umbilical cord mesenchymal stem cells The selection of young passages is necessary for cell viability, maintenance of...

Embodiment 5

[0057] Example 5 Preparation of Umbilical Cord Mesenchymal Stem Cell Extract Liquid Powder

[0058]Select the umbilical cord mesenchymal stem cells of passage 3-10, and inoculate them into T75 culture flasks, add 10ml of special medium to the culture flask, collect the medium after 24 hours, store it at 4°C, and replace it with 10ml of fresh special medium. Then collect again at 24 hours until the cell fusion rate in the T75 culture flask reaches 90%, the cells are digested and collected.

[0059] The collected cells were suspended in PBS, lysed by low-temperature ultrasonication, and then centrifuged to remove cell debris, and the supernatant was collected, and the remaining supernatant contained cell lysates.

[0060] Mix all cell culture supernatants and cell lysate supernatants collected and stored at 4°C, add mannitol, trehalose, and dextran as freeze-drying protective agents, filter and sterilize at 0.22 μm, and add them to freeze-drying bottles,- After pre-freezing at ...

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Abstract

The invention discloses an umbilical cord mesenchymal stem cell preparation and a preparation method and application thereof. Through exploration and verification by a large quantity of experiments, the 3rd-10th-generation umbilical cord mesenchymal stem cells are selected as raw materials, and by a method of dispersing tissue by a mechanical method and then performing treatment with mixed enzymes containing collagenase I, collagenase II and trypsin for a short time for many times, the vitality of the separated umbilical cord mesenchymal stem cells is guaranteed. A culture medium special for the umbilical cord mesenchymal stem cell preparation, containing DMEM/F12 (without phenol red), 10% FBS, 20 kinds of amino acids and 8 kinds of vitamins, is adopted, mixed liquor of repeated cell culture supernatant and cell lysate supernatant is collected, more umbilical cord mesenchymal stem cell active protein and active factors are obtained, the preparation cost is reduced, and industrial requirements for a stem cell product preparation are met. The prepared umbilical cord mesenchymal stem cell preparation can promote proliferation of epidermic cells and skin fibroblast, is safe and is free from toxic and side effects, an allergy does not exist, and when the umbilical cord mesenchymal stem cell preparation is applied to feature-beautifying skin-care products, the effects are notable.

Description

technical field [0001] The invention relates to the technical field of skin care products, in particular to a preparation containing umbilical cord mesenchymal stem cells and its preparation method and application. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are derived from the mesoderm and ectoderm in the early development, a type of stem cells with self-renewal, proliferation, and multidirectional differentiation capabilities. It was discovered in the bone marrow in 1968 and can be induced to differentiate into Myocardium, nerve, cartilage, fat, tendon and other tissue cells. Mesenchymal stem cells can be isolated from fat, bone marrow, umbilical cord, placenta, amniotic membrane and other tissues, and have been widely used clinically in various fields such as nervous system, cardiovascular, orthopedics, diabetes, liver, skin, gastrointestinal tract, etc. . As the earliest discovered bone marrow mesenchymal stem cells, the researc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K8/99A61K8/34A61K8/44A61K8/49A61K8/64A61K8/67A61Q19/00C12N5/0775
CPCA61K8/345A61K8/44A61K8/442A61K8/447A61K8/4913A61K8/492A61K8/4946A61K8/64A61K8/671A61K8/673A61K8/676A61K8/678A61K8/99A61K2800/84A61Q19/00C12N5/0665C12N2501/105C12N2501/11C12N2501/115C12N2501/12C12N2501/135C12N2501/165C12N2509/00
Inventor 许澎黄巧胡清云
Owner 湖南丰晖生物科技有限公司
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