Method for separating CD34 + hematopoietic stem cells from umbilical cord blood

Abstract

According to the method for separating the CD34 < + > hematopoietic stem cells from the umbilical cord blood, the nonspecific clearance rate of the CD34 < + > cells is reduced by reducing the dosage of a RosetteSep antibody; the ratio of the RosetteSep antibody to the CD34 positive antibody to the magnetic beads is optimized; the albuginea layer containing CD34 + cells and a part of density gradient centrifugate below the albuginea layer are absorbed in a whole interval, and loss of CD34 + hematopoietic stem cells is avoided; determining the standardized dilution ratio of the phosphate buffer saline, and eliminating the influence of the lymphocyte separation medium with high buoyancy density on the sedimentation separation of the stem cells; the centrifugal rate after the phosphate buffer saline is diluted and the acceleration of speed increase and speed reduction in the centrifugal process are optimized, so that complete precipitation of CD34 + cells is ensured, and mechanical damage is reduced; and multiple measures are coordinated and matched, so that the separation efficiency of the CD34 + hematopoietic stem cells is improved, the purity is improved, and the separation cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of cell separation technology, specifically to a method for isolating CD34 from umbilical cord blood. + Methods involving hematopoietic stem cells. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] CD34 is a transmembrane salivary mucin expressed on the surface of 1%–5% of human bone marrow cells. In vitro detection has revealed the presence of CD34 in bone marrow. + Cell populations bear the majority of hematopoietic activity, and after transplantation into immunodeficient mice, they can differentiate into various types of blood cells in the recipient's body. Therefore, CD34 molecules are considered a positive marker for hematopoietic stem cells.

[0004] Umbilical cord blood (including umbilical cord tissue) has become a source of CD34. + Umbilical cord blood CD34 is an important source of hematopoietic stem cells. Compared with traditional sources (mainly bone marrow and peripheral blood), it has the following significant advantages: the procedure is convenient, it does not harm the donor, and it has lower immunogenicity, significantly reducing the risk of graft-versus-host disease (GVHD) during transplantation. These characteristics make umbilical cord blood CD34... + Hematopoietic stem cells have shown great potential in basic research and clinical translation, driving the in-depth development of regenerative medicine, cell therapy and other fields.

[0005] However, due to CD34 in umbilical cord blood + Hematopoietic stem cells have a low content (typically accounting for 0.1%-1% of nucleated cells), limited sample blood volume, and significant differences in cell quality and viability among umbilical cord blood from different sources. Therefore, obtaining high-purity and high-recovery CD34 from umbilical cord blood is challenging. + Hematopoietic stem cells are crucial.

[0006] Currently available CD34 extraction methods on the market + There are various reagent kits for hematopoietic stem cell sorting, with the most commonly used magnetic bead sorting mainly divided into column-based magnetic bead sorting and non-column-based magnetic bead sorting. While column-based magnetic bead sorting achieves minimal labeling without affecting cell surface antigen epitopes, allowing direct flow cytometry analysis with high purity and recovery, it is complex, involving multiple washing and column passes. The high cost of magnets, consumables, and reagent kits limits its widespread application. Non-column-based magnetic bead sorting, while reducing steps, saving time, and lowering costs, requires more magnetic beads, potentially affecting cell surface antigen epitopes; it also results in lower purity and sorting efficiency.

[0007] Therefore, developing a method for isolating CD34+ hematopoietic stem cells from umbilical cord blood that balances low cost and high separation efficiency has become a key need to break through current technological bottlenecks and promote the rapid development of related research fields. Summary of the Invention

[0008] To overcome the above problems, the present invention provides a method for isolating CD34 from umbilical cord blood. + Methods involving hematopoietic stem cells.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution: CD34 isolated from umbilical cord blood + The hematopoietic stem cell method includes the following steps: (1) Add RosetteSep™ antibody to umbilical cord blood and incubate at room temperature; (2) Add phosphate-buffered saline (PBS) to the incubated mixture to dilute it, and stack the diluted mixture on the surface of the density gradient centrifuge liquid, centrifuge, and discard the upper plasma layer; (3) Collect the white membrane layer and the density gradient centrifuge fluid below the white membrane layer, dilute with phosphate-buffered saline (PBS buffer), centrifuge, and discard the supernatant; resuspend with phosphate-buffered saline to obtain the pre-enriched hematopoietic stem cell solution; (4) Add CD34 positive antibody to the pre-enriched hematopoietic stem cell solution, incubate, add magnetic beads, and incubate again; (5) Use magnetic poles for magnetic separation and discard the supernatant; centrifuge to obtain CD34+ hematopoietic stem cells; In step (1), the volume ratio of RosetteSep™ antibody to umbilical cord blood is (0.1~4.5):1000; In step (3), the volume ratio of the white film layer and the density gradient centrifuge liquid below the white film layer to the incubated mixture is (0.5~2):1.

[0010] In one or more embodiments, in step (1), the volume ratio of RosetteSep™ antibody to umbilical cord blood is (0.5~4.5):1000, preferably (0.5~3):1000, and more preferably 2.5:1000.

[0011] In one or more embodiments, in step (1), the incubation time at room temperature is 15 to 30 minutes, preferably 20 minutes.

[0012] In one or more embodiments, in step (2), the volume ratio of the incubated mixture to phosphate-buffered saline (PBS) is 1:(0.5~3), preferably 1:1. Diluting with PBS reduces the viscosity of umbilical cord blood, ensuring the separation and acquisition of CD34. + Hematopoietic stem cells.

[0013] In one or more embodiments, in step (2), the density gradient centrifugation fluid is a lymphocyte separation fluid, and the specific gravity of the lymphocyte separation fluid at 20 °C is 1.06~1.08, preferably 1.077.

[0014] Preferably, the volume ratio of lymphocyte separation fluid to umbilical cord blood is (0.1~0.3):1, more preferably 0.15:1.

[0015] In one or more embodiments, in step (2), the centrifugation rate is 400~1400×g, preferably 900×g; the centrifugation time is 15~30 min, preferably 20 min; and the acceleration during centrifugation is 0.5-1 rad / s. 2 The preferred value is 0.8 rad / s 2 The acceleration during deceleration is 0.1-0.5 rad / s². 2 The preferred value is 0.2 rad / s 2 .

[0016] In one or more embodiments, in step (3), the volume ratio of the white film layer and the density gradient centrifuge solution below the white film layer to the phosphate buffer saline is 1:(0.5~5), preferably 1:3.

[0017] In one or more embodiments, in step (3), the centrifugation rate is 500~1200×g, preferably 900×g; the centrifugation time is 5~15 min, preferably 10 min; and the acceleration during centrifugation is 0.5-4 rad / s. 2 The preferred value is 0.8 rad / s 2 The acceleration during deceleration is 0.1-1.0 rad / s². 2 The preferred value is 0.2 rad / s 2 .

[0018] In one or more embodiments, in step (3), the volume ratio of phosphate-buffered saline used to resuspend the cell precipitate after centrifugation to the initial umbilical cord blood is (0.005~0.02):1, preferably (0.008~0.012):1, and more preferably 0.01:1.

[0019] In one or more embodiments, in step (4), the CD34 positive antibody includes one of EasySep™ Human CD34 Positive Selection Cocktail, CD34 MicroBead Kit-human, Anti-CD34 Reference Antibody (ITRI patent anti-CD34), CD34 Monoclonal antibody, and Purified anti-human CD34 Antibody, preferably EasySep™ Human CD34 Positive Selection Cocktail.

[0020] Preferably, the volume ratio of CD34 positive antibody to pre-enriched hematopoietic stem cell solution is (0.05~0.2):1, more preferably (0.05~0.2):1, and even more preferably 0.1:1.

[0021] Preferably, the incubation time for adding CD34 positive antibody is 5-30 min, more preferably 9-12 min, and even more preferably 10 min.

[0022] In one or more embodiments, in step (4), the magnetic bead is a Rapid Spheres™ magnetic bead; Preferably, the volume ratio of magnetic beads to pre-enriched CD34+ hematopoietic stem cell solution is (0.01~0.1):1, more preferably (0.045~0.055):1, and even more preferably 0.05:1.

[0023] Preferably, the incubation time after adding magnetic beads is 2 to 5 minutes, more preferably 3 minutes.

[0024] In one or more embodiments, in step (5), the centrifugation rate is 300~900×g, preferably 400×g; the centrifugation time is 5~20 min, preferably 10 min; and the acceleration during centrifugation is 3-4 rad / s. 2 Preferably 3.5 rad / s 2 The acceleration during deceleration is 0.5-1.0 rad / s². 2 The preferred value is 0.8 rad / s 2 .

[0025] The beneficial effects of this invention are as follows: This invention provides a method for isolating CD34 from umbilical cord blood. + In this invention, a method for treating hematopoietic stem cells involves reducing the dosage of RosetteSep™ antibody to decrease CD34 levels. +Cell non-specific clearance rate; optimized ratio of RosetteSep™ antibody, CD34-positive antibody, and magnetic beads; full-range absorption of CD34-containing antibodies. + Centrifuge the white membrane layer of cells and the density gradient solution below the white membrane layer to avoid CD34. + Loss of hematopoietic stem cells; determination of the standardized dilution factor of phosphate-buffered saline to eliminate the influence of high buoyancy density lymphocyte separation medium on stem cell sedimentation and separation; optimization of the centrifugation rate, ascent and descent accelerations during centrifugation after dilution with phosphate-buffered saline to ensure CD34 + Complete cell precipitation reduces mechanical damage; multiple measures work synergistically to not only increase CD34 + The separation efficiency of hematopoietic stem cells is improved, the purity is increased, and the separation cost is reduced. Attached Figure Description

[0026] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 CD34 in Example 1 and Comparative Example 1 + Recovery rate of hematopoietic stem cells; Figure 2 CD34 in Example 1 and Comparative Example 2 + Recovery rate of hematopoietic stem cells; Figure 3 CD34 in Example 1 and Comparative Example 3 + Recovery rate of hematopoietic stem cells; Figure 4 CD34 in Example 1 and Comparative Example 4 + Recovery rate of hematopoietic stem cells; Figure 5 The graph shows a comparison of cell purity and activity between Example 1 and Comparative Example 6, where A represents the results of Comparative Example 6 and B represents the results of Example 1. Figure 6 CD34 in Example 1 and Comparative Example 6 + Recovery rate of hematopoietic stem cells; Figure 7 Compared to CD34 in Comparative Example 6, Example 1 is an example of this. + Statistical chart showing the rate of increase in hematopoietic stem cell recovery. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] Stem Cell's EasySep™ Human Cord Blood CD34 Positive Selection Kit II is widely used due to its simple operation. However, this kit is not suitable for umbilical cord blood CD34. + The recovery rate of hematopoietic stem cells has always been low, ranging from 30% to 50%, which increases the cost of large-scale stem cell extraction.

[0031] This invention is based on the EasySep™ Human Cord Blood CD34 Positive Selection Kit II, and reduces the amount of RosetteSep™ antibody used to lower CD34 levels. + Cell non-specific clearance rate; optimized ratio of RosetteSep™ antibody, CD34-positive antibody, and magnetic beads; full-range absorption of CD34-containing antibodies. + Centrifuge the white membrane layer of cells and the density gradient solution below the white membrane layer to avoid CD34. + Loss of hematopoietic stem cells; determination of the standardized dilution factor of phosphate-buffered saline to eliminate the influence of high buoyancy density lymphocyte separation medium on stem cell sedimentation and separation; optimization of the centrifugation rate, ascent and descent accelerations during centrifugation after dilution with phosphate-buffered saline to ensure CD34 + Complete cell precipitation reduces mechanical damage; multiple measures work synergistically to not only increase CD34 + This invention improves the isolation efficiency and purity of hematopoietic stem cells while reducing isolation costs. The optimized method provided not only increases the amount of umbilical cord blood processed per reagent kit but also increases the CD34 concentration in a single unit of umbilical cord blood. + The recovery rate of hematopoietic stem cells can reach over 65%, which is 50% higher than the original method. This reduces the extraction cost and makes it easier to promote on a large scale.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] In the following examples, the RosetteSep™ antibody, CD34 positive antibody, and magnetic beads are the corresponding antibodies and magnetic beads from the EasySep™ HumanCord Blood CD34 Positive Selection Kit II.

[0034] Example 1 CD34 isolated from umbilical cord blood + The hematopoietic stem cell method includes the following steps: (1) Add 250 μL of RosetteSep™ antibody to 100 mL of umbilical cord blood and incubate at room temperature for 20 min; add an equal volume of PBS dilution buffer to the incubated mixture and mix well.

[0035] (2) Add 15 mL of lymphocyte separation medium (specific gravity 1.077 at 20 °C) to a 50 mL centrifuge tube. Add the diluted umbilical cord blood mixture incubated in step (1) to the surface of the lymphocyte separation medium, with each tube containing no more than 36 mL. Centrifuge (900 × g, 20 min, with an acceleration of 0.8 rad / s during centrifugation). 2 The acceleration during deceleration is 0.2 rad / s². 2 ), discard the upper layer of blood plasma; (3) Collect 12 mL of the white membrane layer and the lymphocyte separation medium below the white membrane layer in each centrifuge tube. The volume of the white membrane layer is approximately 5 mL, and the volume of the lymphocyte separation medium adjacent to the white membrane layer is approximately 7 mL. Dilute each centrifuge tube with 36 mL of PBS buffer, mix gently, and centrifuge (900×g, 10 min, with an acceleration of 0.8 rad / s during centrifugation). 2 The acceleration during deceleration is 0.2 rad / s². 2 The supernatant was discarded; the cell pellet was resuspended in PBS buffer according to the standard of 1 mL PBS / 100 mL initial umbilical cord blood, and the cell solutions from all centrifuge tubes were transferred to the same centrifuge tube to obtain the pre-enriched hematopoietic stem cell solution. (4) According to the standard that the volume ratio of CD34 positive antibody to pre-enriched hematopoietic stem cell solution is 0.1:1, add CD34 positive antibody (EasySep™ Human CD34Positive Selection Cocktail) to the pre-enriched hematopoietic stem cell solution obtained in step (3) and incubate at room temperature for 10 min; then add magnetic beads, the volume ratio of magnetic beads to pre-enriched hematopoietic stem cell solution is 1:20. Before use, the magnetic beads need to be vortexed for 30 s to make the magnetic beads evenly dispersed in the solution to prevent aggregation, and incubate again for 3 min.

[0036] (5) Add 5 mL of PBS buffer. First, add 3 mL and mix well, then add 2 mL. Place the round-bottom flow cytometer on the magnetic pole and use EasySep to... TM Magnetic separation was performed using magnetic poles, followed by centrifugation (400×g, 10 min, acceleration at ramp rate 3.5 rad / s). 2 The acceleration during deceleration is 0.8 rad / s². 2 Obtain CD34 + Hematopoietic stem cells were resuspended in cryopreservation solution at a cell concentration of 0.5-2 × 10⁻⁶ cells / mL. 6 / mL.

[0037] Comparative Example 1 Compared to Example 1, the amount of RosetteSep™ antibody used in step (1) is doubled, and the specific process is as follows: (1) Add 500 μL of RosetteSep™ antibody to 100 mL of umbilical cord blood and incubate at room temperature for 20 min; The other experimental procedures are the same as in Example 1.

[0038] To improve the purity and efficiency of subsequent magnetic bead sorting, RosetteSep™ antibodies are used to specifically bind to mature blood cell surface antigens before lymphocyte enrichment and magnetic bead sorting, forming an "antibody-cell" complex. The cells bound to the antibody cross-link with each other through the Fc segment of the antibody, forming large "cell-antibody" aggregates (similar to rosettes). During density gradient centrifugation, the rosette aggregates, due to their higher density, settle to the lower layer of the gradient medium, while unlabeled CD34+ hematopoietic stem cells, with a lower density, remain at the interface between the upper plasma and the medium, thus achieving pre-enrichment.

[0039] Although the kit instructions state that the RosetteSep™ antibody can effectively remove a large number of mature lymphocytes and myeloid cells from umbilical cord blood, thereby reducing CD34... +The proportion of cells increases from the initial 0.1%-1% to 5%-10%, providing a high-purity pre-enriched sample for subsequent magnetic bead sorting or other separation steps. However, this approach has certain serious drawbacks. CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD61, and CD66b are not expressed with absolute specificity in T cells, B cells, NK cells, and myeloid cells; they are also expressed in a small number of hematopoietic stem cells. This means that some hematopoietic stem cells will be eliminated by the rosette antibody approach. Furthermore, if the resulting cell-antibody aggregates (similar to rosettes) are too large, they can also non-specifically carry away some hematopoietic stem cells, leading to a final reduction in CD34 levels. + Reduced efficiency in the isolation of hematopoietic stem cells can even affect some functions of hematopoietic stem cells and the development and differentiation of human immune cells.

[0040] To reduce the adverse effects of rosette antibody mixtures and simultaneously improve the isolation efficiency of CD34 hematopoietic stem cells, the amount of RosetteSep™ antibody used should be reduced. Figure 1 CD34 in Example 1 and Comparative Example 1 + Recovery rate of hematopoietic stem cells. From Figure 1 As can be seen, with the reduction of RosetteSep™ antibody usage, CD34... + The recovery efficiency of hematopoietic stem cells was improved to varying degrees. When using 50% of the original dose, CD34... + The recovery rate of hematopoietic stem cells was significantly improved, and the cell purity was high. Therefore, the amount of RosetteSep™ antibody added was set at 50% of the amount recommended in the instructions, which significantly reduced costs while improving the efficiency of stem cell isolation and purification.

[0041] Comparative Example 2 Compared with Example 1, only the white film layer (volume 5 mL) was collected in step (3), and the specific process is as follows: (3) Collect 5 mL of the white film layer, add 15 mL of PBS buffer to dilute, mix gently, and centrifuge (900×g, 10 min, acceleration of 0.8 rad / s). 2 The acceleration during deceleration is 0.2 rad / s². 2 Discard the supernatant; resuspend in 1 mL PBS buffer to obtain pre-enriched CD34. + Hematopoietic stem cell solution; The other experimental procedures are the same as in Example 1.

[0042] To further increase CD34 + The efficiency of hematopoietic stem cell isolation, tracking and exploration of human CD34 + CD34 during the entire process of hematopoietic stem cell separation+ To determine at which step hematopoietic stem cell loss occurs, cell suspensions were collected at each stage of the separation process and analyzed by counting and flow cytometry. CD34 was found during density gradient centrifugation. + Hematopoietic stem cells are distributed not only in the traditionally believed alveolar membrane layer, but also in the separation fluid layer beneath it. Therefore, a portion of the lymphocyte separation fluid suspension beneath the alveolar membrane layer was aspirated, and CD34 was found... + The recovery rate of hematopoietic stem cells has also been significantly improved. Figure 2 The volume of the white membrane layer aspirated is related to the final cell recovery rate; however, aspirating too much of the lymphocyte separation fluid layer beneath the white membrane layer can also affect the purity of subsequent separation.

[0043] Figure 2 CD34 in Example 1 and Comparative Example 2 + The recovery rate of hematopoietic stem cells, from Figure 2 It can be seen that aspirating a portion of the lymphocyte separation fluid layer cell suspension beneath the white membrane layer can increase CD34 levels. + Recovery rate of hematopoietic stem cells.

[0044] Comparative Example 3 Compared with Example 1, the dilution factor of the phosphate buffer solution used in step (3) was changed, and the specific process is as follows: (3) A total of 12 mL of the white membrane layer and the lymphocyte separation medium below the white membrane layer were collected, of which the volume of the white membrane layer was 5 mL and the volume of the lymphocyte separation medium adhering to the white membrane layer was 7 mL. 24 mL of PBS buffer was added for dilution, and the mixture was gently mixed and centrifuged (900×g, 10 min, with an acceleration of 0.8 rad / s). 2 The acceleration during deceleration is 0.2 rad / s². 2 Discard the supernatant; resuspend in 1 mL PBS buffer to obtain pre-enriched CD34. + Hematopoietic stem cell solution; The other experimental procedures are the same as in Example 1.

[0045] When aspirating white blood cell layer cells, lymphocyte separation fluid is inevitably aspirated. The optimized method in Example 1 intentionally aspirates an additional amount of lymphocyte separation fluid. However, the high buoyancy density of the lymphocyte separation fluid affects subsequent centrifugation efficiency. To allow more immune cells and stem cells to be centrifuged into the precipitate, PBS buffer is generally used to dilute the lymphocyte separation fluid contained in the white blood cell layer. However, in practice, there is no standard dosage of PBS buffer. Research shows that twice the volume of PBS buffer is generally used to dilute the white blood cell layer cells, but this ratio is insufficient to dilute the remaining high-density lymphocyte separation fluid. The dilution ratio needs to be increased, but in industrial production, blindly increasing the amount of PBS will increase unnecessary costs and reduce the return on investment. Figure 3 As shown, through experimentation, it was found that dilution with 3 times the volume of PBS buffer yielded the best results.

[0046] Comparative Example 4 Compared with Example 1, the centrifugation rate in step (3) is changed, and the specific process is as follows: (3) A total of 12 mL of the white membrane layer and the lymphocyte separation medium below the white membrane layer were collected, of which the volume of the white membrane layer was 5 mL and the volume of the lymphocyte separation medium adhering to the white membrane layer was 7 mL. 36 mL of PBS buffer was added for dilution, and the mixture was gently mixed. The mixture was then centrifuged (900×g, 10 min, with an acceleration of 0.8 rad / s). 2 The acceleration during deceleration is 0.2 rad / s². 2 Discard the supernatant; resuspend in 1 mL PBS buffer to obtain pre-enriched CD34. + Hematopoietic stem cell solution; The other experimental procedures are the same as in Example 1.

[0047] In absorbing CD34 + When separating the hematopoietic stem cells from the white membrane layer and some lymphocytes below it, the buoyant density of the lymphocyte separation fluid may be drawn in. This means that low-speed centrifugation may not be able to separate most of the white blood cells and the CD34 cells they contain. + Hematopoietic stem cells are centrifuged to the bottom of the tube, so a relatively high centrifugal force is recommended. Although a higher centrifugation speed will increase contamination by red blood cells and platelets, these contaminated cells will be removed during subsequent magnetic bead sorting processes. Results are as follows... Figure 4 As shown, in CD34 + Optimizing the centrifugation speed and acceleration / deceleration of hematopoietic stem cells can, to some extent, increase CD34 levels. + Recovery rate of hematopoietic stem cells.

[0048] Comparative Example 5 Compared with Example 1, the amount of PBS buffer used to resuspend the cells in step (3) was changed, while the other experimental procedures were the same as in Example 1.

[0049] Following the instructions in the EasySep™ Human Cord Blood CD34 Positive Selection Kit II, the volume of PBS buffer was calculated based on the initial cord blood volume and divided into four levels: ① <50 mL resuspend in 0.5 mL. When the initial umbilical cord blood volume is less than 50 mL, resuspend the pre-enriched cell pellet in 0.5 mL of the specified buffer solution. ② Resuspend 50-100 mL in 0.75 mL. When the initial umbilical cord blood volume is 50-100 mL, resuspend the pre-enriched cell pellet in 0.75 mL of the specified buffer. ③ Resuspend 100-150 mL in 1.0 mL. When the initial umbilical cord blood volume is 100-150 mL, resuspend the pre-enriched cell pellet in 1.0 mL of the specified buffer. ④>150mL resuspend in 1.5 mL. When the initial umbilical cord blood volume is greater than 150 mL, resuspend the pre-enriched cell pellet in 1.5 mL of the specified buffer.

[0050] While the aforementioned resuspension method simplifies operations to some extent, it lacks precision in matching the resuspension volume of pre-enriched cells, the amount of CD34-positive antibody, and the amount of magnetic beads based on the initial volume of umbilical cord blood. This leads to reduced sorting efficiency and waste of expensive reagents. A more precise and convenient resuspension method has been developed: when resuspending pre-enriched cells, add 0.01 mL of the recommended resuspension solution per mL of initial umbilical cord blood volume. This standardized approach ensures precise matching of the resuspension volume of pre-enriched cells, the amount of CD34-positive antibody, and the amount of RapidSpheres™ magnetic beads based on the specific initial umbilical cord blood volume. This not only saves a significant amount of expensive reagents but also results in higher sorting efficiency and greater ease of operation.

[0051] Comparative Example 6 Comparative Example 1 is based on the specific procedures performed according to the instructions in the EasySep™ Human Cord Blood CD34 Positive Selection Kit II. It serves as the original protocol. The key performance indicators compared to Example 1 are shown in Table 1. Figures 5-7 As shown.

[0052] Table 1. Results of key indicators in Example 1 and Comparative Example 1

[0053] From Table 1 and Figures 5-7 As can be seen, the optimized method provided by this invention not only increases the amount of umbilical cord blood processed by a single reagent kit, but also increases the CD34 content in a single unit of umbilical cord blood. + The recovery rate of hematopoietic stem cells can reach over 65%, which is 50% higher than the original method, and the extraction cost is reduced. The original method yielded an average of 1,000,000 CD34 cells per 100 mL of umbilical cord blood. + Based on cellular calculations, the original protocol allowed a single kit to process only 1000 mL of umbilical cord blood, and a single kit could only sort approximately 10,000,000 CD34 cells. + Umbilical cord blood stem cells; and the method provided by this invention allows a single kit to process 2000 mL of umbilical cord blood, and a single unit of umbilical cord blood CD34 + The cell recovery rate is 51.5% higher than the original method, meaning that a single kit provided by this invention can sort approximately 30,300,000 (10,000,000 × 2 × 151.5%) CD34 cells. + Umbilical cord blood stem cells, compared to the original protocol of 10,000,000 CD34 cells. + Umbilical cord blood stem cells showed an increase of approximately 203%.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of isolating CD34 + hematopoietic stem cells from umbilical cord blood, characterized in that, The method comprises the following steps: (1) adding RosetteSep™ antibody to the cord blood and incubating at room temperature; (2) adding phosphate buffered saline (PBS buffer) to dilute the incubated mixture, and then adding the diluted mixture to the surface of a density gradient centrifugation liquid, centrifuging, and discarding the upper plasma; (3) collecting the white membrane layer and part of the density gradient centrifugation liquid below the white membrane layer, adding phosphate buffered saline (PBS buffer) to dilute, centrifuging, and discarding the supernatant; and obtaining a pre-enriched hematopoietic stem cell solution after resuspension with phosphate buffered saline; (4) adding CD34 positive antibody to the pre-enriched hematopoietic stem cell solution, incubating, adding magnetic beads, and incubating again; (5) performing magnetic separation by using a magnetic pole, discarding the supernatant, and centrifuging to obtain CD34+ hematopoietic stem cells. In step (1), the volume ratio of RosetteSep™ antibody to cord blood is (0.1-4.5):1000. In step (3), the volume ratio of the white membrane layer and part of the density gradient centrifugation liquid below the white membrane layer to the incubated mixture is (0.5-2):

1.

2. The method of claim 1, wherein, In step (1), the volume ratio of RosetteSep™ antibody to cord blood is (0.5-4.5):1000, preferably (0.5-3):1000, and further preferably 2.5:1000. In step (1), the incubation time at room temperature is 15-30 min, preferably 20 min.

3. The method of claim 1, wherein, In step (2), the volume ratio of the incubated mixture to phosphate buffered saline (PBS buffer) is 1:(0.5-3), preferably 1:

1. In step (2), the density gradient centrifugation liquid is a lymphocyte separation medium, and the specific gravity of the lymphocyte separation medium is 1.06-1.08, preferably 1.077, at 20°C. Preferably, the volume ratio of the lymphocyte separation medium to the cord blood is (0.1-0.3):1, preferably 0.15:

1.

4. The method of claim 1, wherein, In step (2), the rate of centrifugation is 400-1400 x g, preferably 900 x g; the time of centrifugation is 15-30 min, preferably 20 min; the acceleration of the rate increase during centrifugation is 0.5-1 rad / s 2 , preferably 0.8 rad / s 2 ; the acceleration of the rate decrease is 0.1-0.5 rad / s 2 , preferably 0.2 rad / s 2 .

5. The method of claim 1, wherein, In step (3), the volume ratio of the white membrane layer and part of the density gradient centrifugation liquid below the white membrane layer to phosphate buffered saline is 1:(0.5-5), preferably 1:

3.

6. The method of claim 1, wherein, In step (3), the rate of centrifugation is 500-1200 x g, preferably 900 x g; the time of centrifugation is 5-15 min, preferably 10 min; the acceleration of the rate increase during centrifugation is 0.5-4 rad / s 2 , preferably 0.8 rad / s 2 ; the acceleration of the rate decrease is 0.1-1.0 rad / s 2 , preferably 0.2 rad / s 2 .

7. The method of claim 1, wherein, In step (3), the volume ratio of phosphate buffered saline used for resuspending the cell precipitate after centrifugation to the initial cord blood is (0.005-0.02):1, preferably (0.008-0.012):1, and further preferably 0.01:

1.

8. The method of claim 1, wherein, In step (4), the CD34 positive antibody includes one of EasySep™ Human CD34 Positive Selection Cocktail, CD34 MicroBead Kit-human, Anti-CD34 Reference Antibody (ITRI patent anti-CD34), CD34 Monoclonal antibody and Purified anti-human CD34 Antibody, preferably EasySep™ Human CD34 Positive Selection Cocktail; Preferably, the ratio of the volume of the CD34 positive antibody to the pre-enriched hematopoietic stem cell solution is (0.05-0.2):1, preferably (0.05-0.2):1, and further preferably 0.1:

1. Preferably, the incubation time of the CD34 positive antibody is 5-30 min, preferably 9-12 min, and further preferably 10 min.

9. The method of claim 1, wherein, In step (4), the magnetic beads are Rapid Spheres™; Preferably, the ratio of the volume of the magnetic beads to the pre-enriched CD34+ hematopoietic stem cell solution is (0.01-0.1):1, preferably (0.045-0.055):1, and further preferably 0.05:

1. Preferably, the incubation time of the magnetic beads is 2-5 min, preferably 3 min.

10. The method of claim 1, wherein, In step (5), the rate of centrifugation is 300-900 x g, preferably 400 x g; the time of centrifugation is 5-20 min, preferably 10 min; the acceleration of the rate of increase during centrifugation is 3-4 rad / s 2 , preferably 3.5 rad / s 2 ; the acceleration of the rate of decrease is 0.5-1.0 rad / s 2 , preferably 0.8 rad / s 2 .

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