Quantitative determination method for osteogenic differentiation capacity of mesenchymal stem cells based on calcium concentration

Through a quantitative method based on OCPC, the calcium ion concentration after osteogenesis and differentiation of hMSCs is determined by using formic acid to dissolve calcium nodules, which solves the problem of difficulty in accurately evaluating the osteogenesis and different differentiation capabilities of hMSCs in the prior art, and achieves accurate quantitative evaluation of different tissue sources and different generations of MSCs, with high sensitivity and specificity.

CN120028321AActive Publication Date: 2025-05-23NAT INST FOR FOOD & DRUG CONTROL
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Patent Information

Application Number
CN202510214582.1
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-02-26
Publication Date
2025-05-23
Estimated Expiration
2045-02-26

AI Technical Summary

Technical Problem

The prior art is difficult to accurately evaluate the osteogenic differentiation ability of mesenchymal stem cells (hMSCs), especially in different tissue sources and different generations, making it difficult to compare and compare the research results.

Method used

Through a quantitative method based on o-cresolphthalein complex ketone (OCPC), the concentration of calcium ions in the cell fluid of MSCs after osteogenesis differentiation was determined, and calcium nodules were dissolved by formic acid, combined with specific working fluid formulations and detection steps, the accurate quantitative evaluation of the osteogenesis differentiation ability of hMSCs was achieved.

Benefits of technology

This method can significantly improve detection sensitivity, is suitable for quantitative detection of MSCs with different osteogenic differentiation abilities, has high expertise and specificity, and has excellent accuracy and precision. It is suitable for quantitative evaluation of osteogenic induced differentiation of MSCs from different tissue sources and different generations.

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Abstract

The invention relates to the technical field of biological detection, in particular to a method for quantitatively determining the osteogenic differentiation capacity of mesenchymal stem cells based on calcium concentration and application. The osteogenic induction differentiation capacity of the MSCs is evaluated by determining the calcium content through mesenchymal stem cell osteogenic induction in combination with o-cresolphthalein complex ketone, the osteogenic induction differentiation capacity of the MSCs with different tissue sources and different generations can be accurately and quantitatively detected, and the method has the advantages of being high in sensitivity, good in specificity, excellent in repeatability, good in durability and the like.
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Claims

1. A method for quantitatively determining the osteogenic differentiation ability of mesenchymal stem cells MSCs, characterized in that: The method is based on o-cresolphthalein complexone OCPC to quantitatively determine the osteogenic differentiation ability of MSCs.

2. The quantitative determination method according to claim 1, characterized in that The method specifically comprises the following steps: 1) Adding formic acid solution to the cell fluid of osteogenic differentiated MSCs to dissolve them; 2) After dissolution, add o-cresolphthalein chelate ketone (OCPC) working solution and quantify based on color development.

3. The quantitative determination method according to claim 2, characterized in that: The step 1) specifically includes: adding 10-20% (v / v) formic acid solution to the osteogenic differentiated MSCs cell fluid, and dissolving the cell fluid at 35-37° C. for 30-60 minutes.

4. The quantitative determination method according to claim 3, characterized in that: The MSCs cell fluid is specifically prepared by the following method: reviving the MSC cells to be tested, digesting them when the cell fusion rate reaches 80%-90%, then inoculating them on a culture plate, adding differentiation induction culture fluid for differentiation induction culture, and obtaining osteogenic differentiated MSCs cell fluid.

5. The quantitative determination method according to claim 2, characterized in that: The working solution in step 2) comprises equal volumes of working solution A and working solution B; the working solution A comprises ethanolamine, guanidine hydrochloride and methanol; the working solution B comprises OCPC, 8-hydroxyquinoline, ethanol, glacial acetic acid and guanidine hydrochloride.

6. The quantitative determination method according to claim 5, characterized in that: The working solution A comprises 100-125 mL / L of ethanolamine, 100-150 g / L of guanidine hydrochloride, 200-250 mL / L of methanol, and glacial acetic acid is used to adjust the pH to 11-12; the working solution B comprises 60-70 mg / L of OCPC, 1-2 g / L of 8-hydroxyquinoline, 200-250 ml / L of ethanol, 0.5-2.0 mL / L of glacial acetic acid, and 100-150 g / L of guanidine hydrochloride.

7. The quantitative determination method according to claim 2, characterized in that: The step 2) is specifically as follows: taking the supernatant of the solution obtained in step 1) and centrifuging it, adding working solution A and working solution B respectively, mixing them and adding them to an ELISA plate, and performing color development and quantitative analysis at a visible light wavelength of 570-580nm to determine the osteogenic differentiation ability.

8. The quantitative determination method according to any one of claims 1 to 7, characterized in that: The mesenchymal stem cells MSCs include but are not limited to the following sources: fat, bone marrow, dental pulp, hair follicles, umbilical cord and placenta amniotic membrane.

9. A quantitative reagent or kit for the osteogenic differentiation ability of mesenchymal stem cells MSCs, characterized in that: The reagent or kit comprises a formic acid solution and an o-cresolphthalein complex ketone OCPC working solution; Preferably, the o-cresolphthalein chelate OCPC working solution comprises working solution A and working solution B; the working solution A comprises ethanolamine, guanidine hydrochloride and methanol; the working solution B comprises OCPC, 8-hydroxyquinoline, ethanol, glacial acetic acid and guanidine hydrochloride; More preferably, the formic acid solution is a 10-20% (v / v) formic acid solution; the working solution A comprises 100-125 mL / L of ethanolamine, 100-150 g / L of guanidine hydrochloride, 200-250 mL / L of methanol, and glacial acetic acid is used to adjust the pH to 11-12; the working solution B comprises 60-70 mg / L of OCPC, 1-2 g / L of 8-hydroxyquinoline, 200-250 ml / L of ethanol, 0.5-2.0 mL / L of glacial acetic acid, and 100-150 g / L of guanidine hydrochloride.

10. Use of the reagent or kit according to claim 9 in quantifying the osteogenic differentiation ability of mesenchymal stem cells MSCs.

Citation Information

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