A reagent tube for detecting mesenchymal stem cell phenotypes

Abstract

This invention relates to the field of cell detection technology, specifically to a reagent tube for detecting mesenchymal stem cell phenotypes. The tube includes a reagent tube body and pre-encapsulated fluorescent antibody lyophilized microspheres. The lyophilized microspheres are formed by lyophilization of a mixture of lyophilization protection solution and fluorescently labeled antibody. The fluorescently labeled antibody is a combination of antibodies targeting positive markers of mesenchymal stem cell phenotypes CD73, CD90, and CD105, or a combination of antibodies targeting negative markers CD45, CD34, CD11b, CD19, and HLA-DR. This invention offers high efficiency and convenience: by pre-encapsulating the fluorescent antibody lyophilized microspheres, the traditional three-step process of "antibody preparation-sample addition-mixing" is simplified to direct addition of cell suspension. The single-sample detection time is reduced from 120 minutes to 30 minutes, the number of pipetting operations is reduced by 80%, and human error is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of cell detection technology, and in particular to a reagent tube for detecting the phenotype of mesenchymal stem cells. Background Technology

[0002] Phenotypic identification of mesenchymal stem cells (MSCs) is a core technical step in cell therapy, tissue engineering, and other fields. According to the standards established by the International Society for Cell Therapy (ISCT) in 2006, MSCs must meet the following criteria: ≥90% of cells express CD73, CD90, and CD105, and ≤2% of cells express hematopoietic markers CD45, CD34, CD11b, CD19, and MHC class II molecules HLA-DR. Currently, the mainstream detection method is flow cytometry, which uses fluorescently labeled antibodies to bind to cell surface antigens, and analyzes the fluorescence signals using a flow cytometer to achieve phenotypic identification. Traditional testing procedures involve multiple steps, including antibody preparation, cell processing, antibody addition, incubation, and washing, which have several drawbacks: low operational efficiency (approximately 120 minutes per sample), frequent pipetting with a high risk of human error, and a CV value exceeding 15% in the test results; significant reagent waste (commercial antibody packaging specifications do not match the amount needed for a single test, and approximately 60% of antibodies are discarded after opening due to expiration), and high costs due to the need for -20°C frozen storage and cold chain transportation; insufficient stability of liquid antibodies (fluorescent label detachment rate can reach 25% after 3 months of storage at 4°C), and activity easily affected by temperature during transportation; and difficulty in standardization (variations in operation between different laboratories and operators lead to significant deviations in test results, making it difficult to meet the quality control requirements of GMP-grade stem cell preparations). Therefore, there is an urgent need for an integrated, standardized, and highly stable test reagent tube to solve these problems. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and to propose a reagent tube for detecting the phenotype of mesenchymal stem cells.

[0004] To achieve the above objectives, the present invention employs the following technical solution: a reagent tube for detecting mesenchymal stem cell phenotypes, comprising a reagent tube body and pre-encapsulated fluorescent antibody lyophilized microspheres within the tube; the lyophilized microspheres are formed by lyophilization of a mixture of lyophilization protective solution and fluorescently labeled antibody solution; the fluorescently labeled antibody is an antibody combination targeting positive markers of mesenchymal stem cell phenotypes CD73, CD90, and CD105, or an antibody combination targeting negative markers CD45, CD34, CD11b, CD19, and HLA-DR; the reagent tube body is made of medical-grade polypropylene, the bottom of the tube is a truncated cone shape with a 15° inclination, the connection between the bottom of the tube and the tube body is a rounded transition with R=3mm, and laser-etched graduation lines are provided on the outer side of the tube body.

[0005] Preferably, the lyophilization protectant contains the following components: 10%-20% trehalose, 5%-10% bovine serum albumin, 3%-8% mannitol, 1%-3% polyvinylpyrrolidone, and 20mM Tris-HCl buffer at pH 7.4.

[0006] Preferably, the composition and concentration of the freeze-drying protective solution are: 15% trehalose, 8% bovine serum albumin, 5% mannitol, 2% polyvinylpyrrolidone K30, and 20 mM Tris-HCl buffer at pH 7.4.

[0007] Preferably, the reagent tube body has the following dimensions: tube diameter 12mm, height 75mm, capacity 10mL, effective detection volume 5mL, and minimum inner diameter of the tube bottom 5mm; the graduation lines include 50μL, 100μL, 500μL, 2.5mL, and 5mL markings, with an accuracy of ±2%.

[0008] Preferably, the reagent tube body is equipped with a screw cap made of high-density polyethylene with an internal thread pitch of 1.5 mm and a silicone sealing ring with a depth of 2 mm. The sealing performance meets the requirement of no leakage for 30 minutes under a vacuum of -80 kPa.

[0009] Preferably, the fluorescent antibody lyophilized microspheres are divided into 3 positive tube microspheres and 5 negative tube microspheres: 3 positive tube microspheres: 20 μL of a mixed antibody solution containing CD90-FITC, CD105-PE, and CD73-APC is mixed with 80 μL of lyophilization protection solution and lyophilized, with each antibody having a final concentration of 1 mg / mL; 5 negative tube microspheres: 50 μL of a mixed antibody solution containing CD45-PerCP, CD11b-PE-Cy7, CD19-APC-Cy7, CD34-PE, and HLA-DR-FITC is mixed with 150 μL of lyophilization protection solution and lyophilized, with each antibody having a final concentration of 1 mg / mL.

[0010] Preferably, the fluorescent antibody lyophilized microspheres have a diameter of 1-3 mm, a water content of ≤3%, a surface pore size distribution of 1-5 μm, and an internal porosity of 70%.

[0011] A mesenchymal stem cell phenotype detection system includes reagent tubes and a matching sheath solution; the sheath solution is a PBS buffer containing 0.05%-0.2% Tween-20 and 0.1% sodium azide by volume, pH 7.4.

[0012] A method for detecting the phenotype of mesenchymal stem cells includes the following steps: S1: Sample pretreatment: Take 1 mL of mesenchymal stem cell suspension, add 2.5 mL of the matching sheath solution and mix well. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant and add 100 μL of sheath solution to resuspend the cells. S2: Fluorescent labeling reaction: Take out 3 positive tubes and 5 negative tubes, equilibrate at room temperature for 5 minutes, add 50 μL of resuspended cell suspension to each, shake at 2000 rpm for 5 seconds, and incubate at room temperature in the dark for 15 minutes, inverting and mixing once every 5 minutes during this period; S3: Test preparation: Add 2.5 mL of the matching sheath solution to each tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 500 μL of sheath solution to resuspend, filter through a 300-mesh nylon screen and then test.

[0013] The preferred option is...

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows: This invention is highly efficient and convenient to operate: by pre-encapsulating fluorescent antibody lyophilized microspheres, the traditional three-step operation of "antibody preparation-sample addition-mixing" is simplified to direct addition to cell suspension, reducing the detection time of a single sample from 120 minutes to 30 minutes, reducing the number of pipetting operations by 80%, and significantly reducing human operation error; This invention significantly reduces costs: the independent packaging design allows for precise control of antibody dosage per tube, reducing antibody waste from 60% to <5%, and lowering the cost of a single test reagent from 25 yuan to 8 yuan, a reduction of 68%; the room temperature storage period is extended to 12 months, eliminating the need for -20℃ frozen storage and cold chain transportation, thus reducing storage and transportation costs; This invention improves stability and reliability: the freeze-drying process enables antibody activity recovery of ≥98%, and antibody activity loss of ≤3% after storage at 37°C for 3 months, which is far superior to traditional liquid antibodies; the uniform reagent tube specifications and standardized operating procedures ensure that the CV value of test results between different laboratories is ≤5%, guaranteeing the consistency and reliability of test results. This invention has a high degree of standardization: it integrates the eight testing indicators specified by ISCT, and through predefined antibody combinations and standardized operating procedures, it meets the standardized requirements for quality control of GMP-grade stem cell preparations, and is applicable to various scenarios such as stem cell preparation companies, hospital cell therapy centers and research institutes. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the overall structure of the reagent tube of the present invention; Figure 2 This is a schematic diagram of the longitudinal section structure of the reagent tube of the present invention; Figure 3 This is a diagram showing the internal structure of the screw cap of the reagent tube of the present invention; Figure 4 This is a flowchart of the detection method of the present invention; 1. Reagent tube body; 2. Fluorescent antibody lyophilized microspheres; 3. Screw cap; 4. Silicone sealing ring; 5. Graduation lines; 6. Tube body; 7. Frustum conical tube bottom; 8. Rounded transition structure. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] A reagent tube for detecting mesenchymal stem cell phenotypes is characterized by comprising a reagent tube body and pre-encapsulated fluorescent antibody lyophilized microspheres within the tube; the lyophilized microspheres are formed by lyophilization of a mixture of lyophilization protectant and fluorescently labeled antibody; the fluorescently labeled antibody is a combination of antibodies against positive markers of mesenchymal stem cell phenotypes CD73, CD90, and CD105, or a combination of antibodies against negative markers CD45, CD34, CD11b, CD19, and HLA-DR; the reagent tube body is made of medical-grade polypropylene, the bottom of the tube is a truncated cone shape with a 15° inclination, the connection between the bottom and the body of the tube is a rounded transition with R=3mm, and laser-etched graduation lines are provided on the outer side of the tube body; The lyophilization protectant contains the following components: trehalose 10%-20%, bovine serum albumin 5%-10%, mannitol 3%-8%, polyvinylpyrrolidone 1%-3%, and 20mM Tris-HCl buffer at pH 7.4. The composition and concentration of the lyophilization protectant are: trehalose 15%, bovine serum albumin 8%, mannitol 5%, polyvinylpyrrolidone K3O 2%, and 20mM Tris-HCl buffer at pH 7.4. The reagent tube body has a diameter of 12mm, a height of 75mm, a capacity of 10mL, an effective detection volume of 5mL, and a minimum inner diameter of 5mm at the bottom. The graduation marks include 50μL, 100μL, 500μL, 2.5mL, and 5mL markings with an accuracy of ±2%. The reagent tube body is equipped with a screw cap made of high-density polyethylene with an internal thread pitch of 1.5mm, fitted with a 2mm deep silicone sealing ring, ensuring a leak-free seal under a vacuum of -80kPa for 30 minutes. The fluorescent antibody lyophilized microspheres are divided into 3-positive microspheres and 5-negative microspheres: 3-positive microspheres: containing 20 μL of a mixed antibody solution of CD90-FITC, CD105-PE, and CD73-APC mixed with 80 μL of lyophilization protectant, and lyophilized, with each antibody having a final concentration of 1 mg / mL; 5-negative microspheres: containing 50 μL of a mixed antibody solution of CD45-PerCP, CD11b-PE-Cy7, CD19-APC-Cy7, CD34-PE, and HLA-DR-FITC mixed with 150 μL of lyophilization protectant, and lyophilized, with each antibody having a final concentration of 1 mg / mL. The diameter of the fluorescent antibody lyophilized microspheres is 1-3 mm, the water content is ≤3%, the surface pore size distribution is 1-5 μm, and the internal porosity reaches 70%. A mesenchymal stem cell phenotype detection system includes reagent tubes and a matching sheath solution; the sheath solution is a PBS buffer containing 0.05%-0.2% Tween-20 and 0.1% sodium azide by volume, pH 7.4; A method for detecting the phenotype of mesenchymal stem cells includes the following steps: S1: Sample pretreatment: Take 1 mL of mesenchymal stem cell suspension, add 2.5 mL of the matching sheath solution and mix well. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant and add 100 μL of sheath solution to resuspend the cells. S2: Fluorescent labeling reaction: Take out 3 positive tubes and 5 negative tubes, equilibrate at room temperature for 5 minutes, add 50 μL of resuspended cell suspension to each, shake at 2000 rpm for 5 seconds, and incubate at room temperature in the dark for 15 minutes, inverting and mixing once every 5 minutes during this period; S3: Test preparation: Add 2.5 mL of the matching sheath solution to each tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 500 μL of sheath solution to resuspend, filter through a 300-mesh nylon screen and then test. Example 1

[0018] Preparation of freeze-drying protective solution Weigh 15g of trehalose, 8g of bovine serum albumin, 5g of mannitol, and 2g of polyvinylpyrrolidone K30. Dissolve them in 80mL of 20mM pH 7.4 Tris-HCl buffer. Stir magnetically for 30 minutes until completely dissolved. After sterilization by filtration through a 0.22μm filter membrane, dispense into 50mL centrifuge tubes and store at 4℃ for later use. Shelf life is 1 month. Example 2

[0019] Preparation of fluorescent antibody mixture Triple positive antibody mixture: Take 10 μL of CD90-FITC (10 mg / mL), 10 μL of CD105-PE (10 mg / mL), and 10 μL of CD73-APC (10 mg / mL), add 70 μL of PBS buffer, vortex to mix, and prepare a mixture with a final concentration of 1 mg / mL for each antibody, with a total volume of 100 μL.

[0020] 5. Antibody-negative mixture: Take 10 μL of CD45-PerCP (10 mg / mL), 10 μL of CD11b-PE-Cy7 (10 mg / mL), 10 μL of CD19-APC-Cy7 (10 mg / mL), 10 μL of CD34-PE (10 mg / mL), and 10 μL of HLA-DR-FITC (10 mg / mL), add 150 μL of PBS buffer, vortex to mix, and prepare a mixture with a final concentration of 1 mg / mL for each antibody, with a total volume of 200 μL. Example 3

[0021] Preparation and encapsulation of fluorescent antibody lyophilized microspheres Mixing and dispensing: Add 20 μL of 3-positive antibody mixture and 80 μL of lyophilization protection solution to 3-positive tubes, and add 50 μL of 5-negative antibody mixture and 150 μL of lyophilization protection solution to 5-negative tubes. The volume of the mixture in each tube is 100 μL and 200 μL, respectively. Vortex to mix and ensure that the liquid is concentrated at the bottom of the tube.

[0022] Freeze-drying process: Place the reagent tubes into a freeze dryer and freeze-dry according to the following procedure: pre-freeze at -40℃ for 2 hours → under conditions of cold trap temperature of -50℃ and vacuum degree of 10mTorr, raise the temperature to -20℃ at 0.5℃ / min and hold for 12 hours (first drying) → raise the temperature to 25℃ and hold for 6 hours (second drying), and finally obtain freeze-dried microspheres with a water content of ≤3%.

[0023] Sealed packaging: After freeze-drying, immediately place the reagent tubes into aluminum foil bags with built-in silica gel desiccant, seal them with a heat sealer (temperature 180℃, pressure 50kPa, time 2s), and pack 10 tubes into a rigid polypropylene box. Example 4

[0024] Performance verification experiment Reconstitution time test: 50 μL of distilled water was added to 3 cation tubes and 5 anion tubes respectively, and the time for complete dissolution of microspheres was recorded with a stopwatch. The test was repeated 10 times and the average value was taken. The results showed that the reconstitution time was ≤65 seconds, which meets the requirements for rapid detection.

[0025] Antibody activity assay: The binding affinity of antibodies to corresponding antigens before and after lyophilization was determined by ELISA, and the activity recovery rate was calculated as (binding affinity after lyophilization / binding affinity before lyophilization × 100%). The results showed that the activity recovery rate of each antibody was ≥98%, ensuring the sensitivity of the assay.

[0026] Stability testing: The reagent tubes were stored at 25℃, 37℃, and 4℃ respectively, and samples were taken monthly. The positive and negative rates of the standard cell line (ATCCPCS-500-010) were determined by flow cytometry. The results showed that after storage at 25℃ for 12 months, 37℃ for 3 months, and 4℃ for 18 months, the positive rate changed by ≤2%, and the negative rate changed by ≤1%, demonstrating significantly better stability than traditional liquid antibodies.

[0027] Repeatability testing: Five different laboratories were invited to use the reagent tubes of this invention to test the same batch of mesenchymal stem cell samples. The CV values ​​of the positive rates of CD73, CD90, and CD105 and the negative rates of CD45 and CD34 were calculated. The results showed that the CV values ​​of all indicators were ≤5%, which met the requirements for standardized testing across laboratories.

[0028] This invention, through structural, process, and methodological innovations, has developed a standardized mesenchymal stem cell phenotypic detection reagent tube that is ready to use immediately. This simplifies the detection process, reduces detection costs, and improves detection stability and standardization. It provides a reliable technical means for stem cell quality control and has broad prospects for industrial application.

[0029] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A reagent tube for detecting mesenchymal stem cell phenotypes, characterized in that, The reagent tube includes a reagent tube body and pre-encapsulated fluorescent antibody lyophilized microspheres within the tube. The lyophilized microspheres are formed by lyophilization of a mixture of lyophilization protection solution and fluorescently labeled antibody solution. The fluorescently labeled antibody is a combination of antibodies against mesenchymal stem cell phenotypic positive markers CD73, CD90, and CD105, or a combination of antibodies against negative markers CD45, CD34, CD11b, CD19, and HLA-DR. The reagent tube body is made of medical-grade polypropylene, with a 15° angled frustum-shaped bottom. The connection between the bottom and the body is achieved with a 3mm radius arc transition, and laser-etched graduation lines are provided on the outer side of the body.

2. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 1, characterized in that: The lyophilization protective solution contains the following components: trehalose 10%-20%, bovine serum albumin 5%-10%, mannitol 3%-8%, polyvinylpyrrolidone 1%-3%, and 20mM Tris-HCl buffer at pH 7.

4.

3. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 2, characterized in that: The composition and concentration of the freeze-drying protective solution are as follows: trehalose 15%, bovine serum albumin 8%, mannitol 5%, polyvinylpyrrolidone K30 2%, and 20mM Tris-HCl buffer at pH 7.

4.

4. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 1, characterized in that: The reagent tube body has the following dimensions: tube diameter 12mm, height 75mm, capacity 10mL, effective detection volume 5mL, and minimum inner diameter of the tube bottom 5mm; the graduation lines include 50μL, 100μL, 500μL, 2.5mL, and 5mL markings, with an accuracy of ±2%.

5. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 1, characterized in that: The reagent tube body is equipped with a screw cap made of high-density polyethylene with an internal thread pitch of 1.5mm. It is fitted with a silicone sealing ring with a depth of 2mm, and the sealing performance meets the requirement of no leakage for 30 minutes under a vacuum of -80kPa.

6. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 1, characterized in that: The fluorescent antibody lyophilized microspheres are divided into 3-positive-tube microspheres and 5-negative-tube microspheres: 3-positive-tube microspheres: 20 μL of a mixed antibody solution containing CD90-FITC, CD105-PE, and CD73-APC is mixed with 80 μL of lyophilization protection solution and lyophilized, with each antibody having a final concentration of 1 mg / mL; 5-negative-tube microspheres: 50 μL of a mixed antibody solution containing CD45-PerCP, CD11b-PE-Cy7, CD19-APC-Cy7, CD34-PE, and HLA-DR-FITC is mixed with 150 μL of lyophilization protection solution and lyophilized, with each antibody having a final concentration of 1 mg / mL.

7. The reagent tube for detecting mesenchymal stem cell phenotypes according to claim 1, characterized in that: The fluorescent antibody lyophilized microspheres have a diameter of 1-3 mm, a water content of ≤3%, a surface pore size distribution of 1-5 μm, and an internal porosity of up to 70%.

8. A mesenchymal stem cell phenotype detection system, characterized in that, The reagent tube includes any one of claims 1-7, and a matching sheath solution; the sheath solution is a PBS buffer containing 0.05%-0.2% Tween-20 and 0.1% sodium azide by volume, at pH 7.

4.

9. A method for detecting mesenchymal stem cell phenotypes using the reagent tube described in any one of claims 1-7, characterized in that, Includes the following steps: S1: Sample pretreatment: Take 1 mL of mesenchymal stem cell suspension, add 2.5 mL of the matching sheath solution and mix well. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant and add 100 μL of sheath solution to resuspend the cells. S2: Fluorescent labeling reaction: Take out 3 positive tubes and 5 negative tubes, equilibrate at room temperature for 5 minutes, add 50 μL of resuspended cell suspension to each, shake at 2000 rpm for 5 seconds, and incubate at room temperature in the dark for 15 minutes, inverting and mixing once every 5 minutes during this period; S3: Test preparation: Add 2.5 mL of the matching sheath solution to each tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 500 μL of sheath solution to resuspend, filter through a 300-mesh nylon screen and then test.

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