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Preparation method and application of mesenchymal stem cell cytokines

A technology of mesenchymal stem cells and cytokines, which is applied in the field of preparation of mesenchymal stem cell cytokines, can solve the problems that cytokine compositions cannot simulate cytokine types and proportions, adverse reactions, etc., and achieve simple preparation methods and inhibition of degradation , reduce the effect of rejection

Inactive Publication Date: 2017-03-22
四川华皓生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a preparation method and application of mesenchymal stem cell cytokines, which can effectively solve the problem that the artificially prepared cytokine composition in the prior art cannot simulate the cellular microenvironment. Types and proportions of cytokines, adverse reactions during use

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  • Preparation method and application of mesenchymal stem cell cytokines
  • Preparation method and application of mesenchymal stem cell cytokines
  • Preparation method and application of mesenchymal stem cell cytokines

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 In vitro expansion and identification of mesenchymal stem cells

[0028] After digesting the P5 generation placental mesenchymal stem cells with a confluence of 90% (tissue attachment method or enzyme digestion method), counting, and preparing a density of 1 × 10 6 cells / mL cell suspension, then filtered with a 100 μm cell mesh, and then 1 mL of the filtrate was taken, and 10 μL of fluorescently labeled cell surface marker antibodies were added to the filtrate, and incubated at 4 °C for 20 min in the dark. .

[0029] After incubation, wash once with 0.9% normal saline (4°C), centrifuge at 1200r / min for 5min, discard the supernatant, add 250μL 0.9% normal saline to the precipitate and mix well, then add 250μL 1% paraformaldehyde Fix, then adopt the flow cytometer of Beckman Company to carry out detection and analysis, the detection result sees figure 1 .

[0030] The antibodies used are CD11b-FITC, CD19-ECD, CD29-FITC, CD34-PE, CD44-FITC, CD45-PC7, CD73-PE, ...

Embodiment 2

[0032] Example 2 Preparation of clinical grade mesenchymal stem cell cytokines

[0033] Inoculate the mesenchymal stem cells into the culture vessel, add the medium, and then place it at 37°C, 5% CO 2 subculture under the following conditions; medium components: FBS with a volume concentration of 20% and GlutaMAX with a volume concentration of 2% TM -I, the rest is DMEM; after the confluence of P5 mesenchymal stem cells reaches 80-90%, adjust the oxygen concentration in the culture container to 1% (v / v), and then continue to culture the cells overnight;

[0034] Digest the overnight cells with 0.25% trypsin-EDTA solution for 2-3min, then centrifuge at 20°C and 2000r / min for 3min, remove the supernatant, wash the precipitate with 0.9% normal saline for 2-3 times and collect cells, and the cell viability was detected, and the cells were counted.

[0035] Adjust the cell concentration to 1 x 10 with 0.9% saline 8 cells / mL, then add 3 mg / mL EDTA and 10 mg / mL Vc, mix well, then ...

Embodiment 3

[0039] Example 3 Preservation Validity Experiment of Mesenchymal Stem Cell Cytokines

[0040] The prepared mesenchymal stem cell cytokines were stored at -80°C for 1 month, 3 months, 6 months, and 12 months. Freshly prepared mesenchymal stem cell cytokines were used as the control group, and ELISA kits were used to According to the instruction manual, various cytokines (EGF, FGF, IGF-1, HGF, PDGF-AA, VEGF) were detected, and the kits were purchased from R&D company. The experimental results are shown in image 3 .

[0041] Depend on image 3 It can be seen that, taking the cytokine content frozen for 3 months as a reference, the decrease of cytokines is not obvious within 6 months of frozen storage (P>0.05). When the frozen storage time increases to 12 months, the cytokine content There is a small amount of loss (P<0.01), so the validity period of the stem cell factor preparation is recommended to be 12 months.

[0042] The mesenchymal stem cell cytokine prepared by the inv...

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Abstract

The invention provides a preparation method for mesenchymal stem cell cytokines. The preparation method comprises the following steps: (1) extraction of mesenchymal stem cells; (2) culture; (3) continued culture under low-oxygen conditions; (4) digestion with a 0.25% pancreatin-EDTA solution; (5) centrifugation and cleaning with 0.9% normal saline; (6) adjustment of cell concentration with 0.9% normal saline and addition of EDTA with a concentration of 1 to 3 mg / mL and Vc with a concentration of 5 to 10 mg / mL; (7) freezing-thawing; and (8) centrifugation and filtering. The mesenchymal stem cell cytokines prepared in the invention can effectively overcome the problems that artificially prepared cytokine compositions in the prior art cannot simulate the variety and ratio of cytokines in a cell microenvironment and incur adverse reaction during usage.

Description

technical field [0001] The invention belongs to the technical field of mesenchymal stem cell cytokines, and in particular relates to a preparation method and application of mesenchymal stem cell cytokines. Background technique [0002] Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with self-renewal ability, which widely exist in a variety of adult tissues or perinatal tissues, such as bone marrow, fat, dental pulp, placenta, umbilical cord, etc. The clinical application of mesenchymal stem cells as pharmaceutical preparations mainly depends on their immune regulation ability and tissue regeneration ability, but these two abilities are mainly attributed to the powerful cytokine secretion ability of mesenchymal stem cells. Although mesenchymal stem cells have good multidirectional differentiation potential and can differentiate into cells from various tissue sources such as fat, cartilage, bone, and nerves under specific induction conditions, studies have ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12P21/02C07K14/475C07K1/34A61K35/28A61K38/19A61K8/64A61K8/98A61Q19/00
CPCC12N5/0668A61K8/64A61K8/98A61K35/28A61K38/19A61K2800/10A61Q19/00C07K14/475
Inventor 李俊黄海森徐能飞李升岐
Owner 四川华皓生物科技有限公司
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