Stem cell extraction process

Abstract

The invention relates to the technical field of stem cell extraction, and discloses a stem cell extraction process which comprises the following steps: S1, collecting umbilical cord blood; s2, transporting the umbilical cord blood; s3, counting nucleated cells; s4, centrifuging the umbilical cord blood bag; according to the method, the umbilical cord blood bag is directly centrifuged through the density gradient centrifugation technology, the umbilical cord blood bag is vertically arranged on the rotor, centrifugal tube transfer of the umbilical cord blood is not needed, the pollution danger caused in the transfer process is reduced, meanwhile, the umbilical cord blood bag is convenient to fix, the centrifugation process is stable, the operation is simple, and the efficiency is high. The method has the characteristics of high practicability and convenience in leukocyte layer extraction.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell extraction, in particular to a stem cell extraction process. Background Art

[0002] Stem cell extraction is a complex and sophisticated technology involving a variety of methods and steps aimed at efficiently isolating stem cells from different tissues. These methods not only depend on the source of the stem cells, but are also affected by the type of stem cells required. Stem cells are a type of cell with self-replication and multidirectional differentiation potential. Hematopoietic stem cells can differentiate into various blood cell types, including red blood cells, white blood cells and platelets. Hematopoietic stem cells can be divided into umbilical cord blood hematopoietic stem cells, bone marrow hematopoietic stem cells and peripheral blood hematopoietic stem cells, and their sources are umbilical cord blood, bone marrow and peripheral blood, respectively.

[0003] Hematopoietic stem cells in umbilical cord blood are widely used in allogeneic transplantation due to their low immunogenicity, which reduces the risk of graft-versus-host disease. The extraction of hematopoietic stem cells from umbilical cord blood is a complex and precise process that requires strict aseptic operation and professional technology. The extraction of hematopoietic stem cells from umbilical cord blood usually uses blood sorting. This process first uses a connecting tube to draw out the umbilical cord blood in the umbilical cord vein under a sterile condition. The blood collector then returns the cord blood to the cord blood bank within 24 hours. In a GMP (Good Manufacturing Practice) standard laboratory, the separation of hematopoietic stem cells from umbilical cord blood is completed by centrifugation in a closed state, and the number of cells in the prepared umbilical cord blood is tested. In addition, centrifugation can also be used to stratify the components in the umbilical cord blood, ultimately leaving the nucleated cells.

[0004] At present, in the process of centrifuging the umbilical cord blood bag, the umbilical cord blood bag wall needs to be filled with an air bag to keep the umbilical cord blood bag vertical. The operation is cumbersome, and the umbilical cord blood bag is prone to deviation during the centrifugation process, resulting in mixing of the interface solutions of the plasma layer, the white blood cell layer and the red blood cell layer, which in turn hinders the extraction of the white blood cell layer. Therefore, it is necessary to design a stem cell extraction process that is highly practical and convenient for white blood cell layer extraction. Summary of the invention

[0005] The purpose of the present invention is to provide a stem cell extraction process to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: a stem cell extraction process, comprising the following steps: S1. Collection of umbilical cord blood: After the baby is delivered, before the umbilical cord is cut, clamp the two ends of the umbilical cord with a clamp, and use a sterile needle to draw blood from the umbilical vein into an umbilical cord blood bag containing an anticoagulant. The umbilical cord blood bag should be open at the top and bottom. The collection volume is usually 50 to 200 ml, depending on the blood flow of the umbilical cord. After the collection is completed, the umbilical cord blood bag is sealed and labeled, and relevant information such as the collection time, maternal information, etc. is recorded; S2, Transportation of umbilical cord blood: The collected umbilical cord blood is quickly sent to the laboratory, and the temperature is kept low (2-8°C) to prevent cell damage. After arriving at the laboratory, the sample is registered, and the packaging is checked to see if it is intact and the label information is confirmed to be accurate; S3, nucleated cell count: microbiological testing of the cord blood to ensure that it is free of contamination, determination of total blood volume and nucleated cell count, and assessment of suitability for further processing; S4, centrifugation of umbilical cord blood bag: The umbilical cord blood bag is directly centrifuged through density gradient centrifugation technology. The umbilical cord blood bag is vertically set on the rotor, and the umbilical cord blood does not need to be transferred, which reduces the risk of contamination during the transfer process. At the same time, the umbilical cord blood bag is easy to fix and the centrifugation process is stable. By using media such as Ficoll or 6% hydroxyethyl starch, the density difference of different cell components is used for separation, which can effectively remove red blood cells and other non-mononuclear cells, thereby enriching mononuclear cells (MNC), including hematopoietic stem cells; S5, CD34+ cell enrichment: CD34+ cells were collected by immunomagnetic sorting, using magnetic beads combined with specific antibodies to capture CD34+ cells, and then separated from other cells by a magnetic field; S6, cryopreservation: The cells were slowly cooled to -80°C for precooling in a refrigerator, and then transferred to a liquid nitrogen tank for long-term storage at -196°C.

[0007] According to the above technical solution, in step S4, the umbilical cord blood bag is fixed by the rotor, the rotor includes a central body and an edge frame, a universal docking port is provided in the middle of the central body, the edge frame is arranged in a circular array on the peripheral wall of the central body, the umbilical cord blood bag and the edge frame are arranged in a one-to-one correspondence, and a stabilizing clamp is symmetrically provided at one end of the edge frame away from the central body, and the stabilizing clamp stably clamps the upper and lower openings of the umbilical cord blood bag.

[0008] According to the above technical solution, the edge frame includes a fixed frame and a lifting body, the fixed frame has a hollow body structure with an isosceles trapezoidal cross-section, one end of the fixed frame is fixedly installed on the outer wall of the central body and the other end extends outward to form a lifting area, the lifting body is installed in the lifting area through a guide member for lifting and lowering, and an adjustment groove is symmetrically opened at one end of the lifting body away from the central body, and the stabilizing clip is correspondingly arranged in the adjustment groove, and an adjustment member is provided between the stabilizing clip and the adjustment groove, and the adjustment member appropriately adjusts the spacing between the stabilizing clips.

[0009] According to the above technical solution, a self-balancing member is provided between the central body and the lifting body, and the self-balancing member performs balance adjustment according to the mass of the umbilical cord blood bag to ensure the operation balance and operation stability of the rotor.

[0010] According to the above technical solution, the stabilizing clamp includes a fixed clamp seat, a movable groove, a micro motor, a bidirectional threaded rod, a movable block, an elbow and a clamp plate. The fixed clamp seat is movably installed in the adjustment groove, and the movable groove is arranged on the fixed clamp seat. The micro motor is fixedly installed at one end in the movable groove and the output end is fixedly installed with the bidirectional threaded rod. The bidirectional threaded rod is provided with opposite threads. The movable block is symmetrically threadedly connected to the bidirectional threaded rod. The clamp plate and the movable block are arranged in a one-to-one correspondence and the clamp plate and the movable block are fixedly connected by the elbow. The spacing between the clamp plates is smaller than the spacing between the movable blocks.

[0011] According to the above technical solution, the adjusting member includes a guide rod 1, a bidirectional threaded rod 2 and a screw handle, the guide rod 1 and the bidirectional threaded rod 2 are respectively arranged between the adjusting grooves, the bidirectional threaded rod 2 is mirror-imaged on both sides of the guide rod 1, both ends of the guide rod 1 extend into the adjusting groove and penetrate and are movably installed in the corresponding stable clamp seat, both ends of the bidirectional threaded rod 2 extend into the adjusting groove and are provided with opposite threads, the stable clamp seat is threadedly connected to the bidirectional threaded rod 2, and the upper end of the bidirectional threaded rod 2 penetrates the lifting body and is fixedly installed with the screw handle.

[0012] According to the above technical solution, the self-balancing member includes a tension sensor and a hydraulic balance member, the tension sensor is arranged between the fixed frame and the lifting body and is located at the upper end of the lifting body, a water storage cavity is opened in the central body, and a balancing cavity is opened in the lifting body. The hydraulic balance members are respectively arranged between the lifting body and the central body. The hydraulic balance member pumps or delivers water in the balancing cavity through the measurement of the tension sensor to maintain the mass of each lifting body the same. The uniform mass distribution helps to maintain the balance of the rotor.

[0013] According to the above technical solution, the hydraulic balance member includes a pumping member and a water delivery member, the pumping member includes a delivery pump 1, a delivery pipeline 1 and a flow control valve 1, the delivery pump 1 is fixedly installed on the side wall of the central body, one end of the delivery pump 1 is connected to the balance chamber through the delivery pipeline 1, and the other end of the delivery pump 1 is connected to the water storage chamber through another section of the delivery pipeline 1, the delivery pump 1 extracts water inside the balance chamber and delivers it to the water storage chamber, the flow control valve 1 is arranged on the delivery pipeline 1, the water delivery member includes a delivery pump 2, a delivery pipeline 2 and a flow control valve 2, the delivery pump 2 is fixedly installed on the side wall of the central body and is located at the upper end of the delivery pump 1, one end of the delivery pump 2 is connected to the balance chamber through the delivery pipeline 2, and the other end of the delivery pump 2 is connected to the water storage chamber through another section of the delivery pipeline 2, the delivery pump 2 extracts water inside the water storage chamber and delivers it back to the balance chamber, and the flow control valve 2 is arranged on the delivery pipeline 2.

[0014] According to the above technical solution, the guide member includes a guide groove, a guide rod 2 and a guide block. The guide groove is symmetrically arranged in the lifting area, the guide rod 2 is fixedly installed in the guide groove, the guide block is symmetrically fixedly installed on the side wall of the lifting body, and the guide block is movably installed on the corresponding guide rod 2.

[0015] According to the above technical solution, a resistance member is symmetrically provided at the lower end of the lifting body, and the resistance member includes a fixed plate, a cylinder and a resistance plate. The cylinder is fixedly installed at the lower end of the lifting body through the fixed plate, and the resistance plate is fixedly installed at the telescopic end of the cylinder. A notch is provided at one end of the resistance plate adjacent to the fixed frame, and the notch and the edge of the fixed frame fit together to increase the friction between the two parties, thereby ensuring the placement stability of the lifting body.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This application is applied to the extraction of hematopoietic stem cells from umbilical cord blood. The umbilical cord blood bag is directly centrifuged by density gradient centrifugation technology. The umbilical cord blood bag is vertically arranged on the rotor, and there is no need to transfer the centrifuge tube of the umbilical cord blood, which reduces the risk of contamination during the transfer process. At the same time, the umbilical cord blood bag is easy to fix and the centrifugation process is stable. (2) The universal docking port in the middle of the central body is convenient for matching with the rotating shaft of the centrifuge. The edge racks are arranged in a circular array on the peripheral wall of the central body. In the present application, the number of edge racks is set to six, and up to six umbilical cord blood bags can be centrifuged simultaneously. The upper and lower openings of the umbilical cord blood bag are clamped and fixed at the same time by the stabilizing clamp. The umbilical cord blood bag has high stability and is convenient and easy to fix. The umbilical cord blood bag can be directly placed in the centrifuge for processing without the need for additional air bag filling preparation steps, which speeds up the entire experimental process, simplifies the sample loading and unloading process, saves operation time, and improves work efficiency; (3) An anti-slip pad is arranged inside the stabilizing clamp to increase the stable clamping force of the umbilical cord blood bag. The clamping and loosening of the umbilical cord blood bag is realized by driving the micro motor. The elbow is arranged to shorten the distance between the clamps, which is convenient for clamping and fixing the opening of the umbilical cord blood bag. The screw handle is arranged at the upper end of the lifting body, which is convenient for operation. The adjusting part can appropriately adjust the distance between the stabilizing clamps, which can adapt to different types of umbilical cord blood bags, facilitate stable clamping and fixing of different types of umbilical cord blood bags, and adapt to various types of umbilical cord blood bags; (4) The self-balancing parts built into the rotor can help automatically detect and adjust the imbalance state, thereby reducing this risk, and can reduce the error caused by manual adjustment, improving the accuracy of the experimental results. The self-balancing parts balance and adjust according to the mass of the umbilical cord blood bag to ensure the operating balance and stability of the rotor. A well-balanced rotor can provide a more stable operating state and reduce the possibility of sudden failures, thereby improving the reliability and stability of the entire system. A well-balanced rotor can ensure uniform distribution of blood samples during centrifugation, thereby achieving clearer cell stratification, which is conducive to the separation of mononuclear cells (MNC) and further enrichment of CD34+ hematopoietic stem cells; BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 It is the overall process schematic diagram of the present invention; Figure 2 is a first stereoscopic schematic diagram of the present invention; Figure 3 is a second stereoscopic schematic diagram of the present invention; Figure 4 is a third stereoscopic schematic diagram of the present invention; Figure 5 is a first partial stereoscopic schematic diagram of the present invention; Figure 6 is a second partial stereoscopic schematic diagram of the present invention; Figure 7 is a third partial stereoscopic schematic diagram of the present invention; Figure 8 is a fourth partial stereoscopic schematic diagram of the present invention; Fig. 9 The present invention Figure 4 The enlarged schematic diagram of point A in the middle; Fig.10 The present invention Figure 7 The enlarged schematic diagram of point B in the middle; Fig.11 The present invention Figure 8 The enlarged schematic diagram of the center C; Fig.12 The present invention Figure 8 The enlarged schematic diagram of point D in the middle; In the figure: 1-central body, 11-water storage chamber, 2-edge frame, 21-fixed frame, 22-lifting body, 221-adjusting groove, 222-balance chamber, 3-universal docking port, 4-stabilizing clamp, 41-fixed clamp seat, 42-movable groove, 43-micro motor, 44-bidirectional threaded rod 1, 45-movable block, 46-elbow, 47-clamp, 5-guide member, 51-guide groove, 52-guide rod 2, 53-guide block, 6-adjusting member, 61-guide rod 1. 62-bidirectional threaded rod 2. 63-screwing handle 7-self-balancing part 71-tension sensor 72-hydraulic balancing part 721-pumping part 7211-delivery pump 1. 7212-delivery pipeline 1. 7213-flow control valve 1. 722-water delivery part 7221-delivery pump 2. 7222-delivery pipeline 2. 7223-flow control valve 2. 8-resistance part 81-fixed plate 82-cylinder 83-resistance plate 831-notch. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] See also Figure 1-12 The present invention provides a technical solution: a stem cell extraction process, comprising the following steps: S1. Collection of umbilical cord blood: After the baby is delivered, before the umbilical cord is cut, clamp the two ends of the umbilical cord with a clamp, and use a sterile needle to draw blood from the umbilical vein into an umbilical cord blood bag containing an anticoagulant. The umbilical cord blood bag should be open at the top and bottom. The collection volume is usually 50 to 200 ml, depending on the blood flow of the umbilical cord. After the collection is completed, the umbilical cord blood bag is sealed and labeled, and relevant information such as the collection time, maternal information, etc. is recorded; S2, Transportation of umbilical cord blood: The collected umbilical cord blood is quickly sent to the laboratory, and the temperature is kept low (2-8°C) to prevent cell damage. After arriving at the laboratory, the sample is registered, and the packaging is checked to see if it is intact and the label information is confirmed to be accurate; S3, nucleated cell count: microbiological testing of the cord blood to ensure that it is free of contamination, determination of total blood volume and nucleated cell count, and assessment of suitability for further processing; S4, centrifugation of umbilical cord blood bag: The umbilical cord blood bag is directly centrifuged through density gradient centrifugation technology. The umbilical cord blood bag is vertically set on the rotor, and the umbilical cord blood does not need to be transferred, which reduces the risk of contamination during the transfer process. At the same time, the umbilical cord blood bag is easy to fix and the centrifugation process is stable. By using media such as Ficoll or 6% hydroxyethyl starch, the density difference of different cell components is used for separation, which can effectively remove red blood cells and other non-mononuclear cells, thereby enriching mononuclear cells (MNC), including hematopoietic stem cells; S5, CD34+ cell enrichment: CD34+ cells were collected by immunomagnetic sorting, using magnetic beads combined with specific antibodies to capture CD34+ cells, and then separated from other cells by a magnetic field; S6, cryopreservation: the cells were slowly cooled to -80°C in a refrigerator for precooling, and then transferred to a liquid nitrogen tank for long-term storage at -196°C; Specifically, in step S4, the umbilical cord blood bag is fixed by the rotor, the rotor comprises a central body 1 and an edge frame 2, a universal docking port 3 is provided in the middle of the central body 1, the edge frame 2 is arranged in a circular array on the peripheral wall of the central body 1, the umbilical cord blood bag and the edge frame 2 are arranged one by one, and a stabilizing clamp 4 is symmetrically provided at one end of the edge frame 2 away from the central body 1, and the stabilizing clamp 4 stably clamps the upper and lower openings of the umbilical cord blood bag; The central body 1 and the edge frame 2 in the present application can be made of aluminum alloy. Aluminum alloy is one of the most commonly used materials, with good mechanical strength, lightness and good thermal conductivity. The aluminum alloy rotor can well withstand the stress generated during high-speed rotation and help to quickly reach the set temperature. The universal docking port 3 in the middle of the central body 1 is convenient for matching on the rotating shaft of the centrifuge. The edge frame 2 is arranged in a circular array on the peripheral wall of the central body 1. In the present application, the number of edge frames 2 is set to six, and up to six umbilical cord blood bags can be centrifuged simultaneously. By stably clamping 4 pairs of umbilical cords The upper and lower openings of the blood bag are clamped and fixed at the same time. The umbilical cord blood bag has high stability and is easy to fix. The umbilical cord blood bag can be directly placed in the centrifuge for processing without the need for additional air bag filling preparation steps, which speeds up the entire experimental process, simplifies the sample loading and unloading process, saves operation time, and improves work efficiency. After the umbilical cord blood bag is centrifuged, the white blood cell layer (MNCs) in the umbilical cord blood bag can be extracted through a slurry separation device for an umbilical cord blood hematopoietic stem cell inverted separation process with a publication number of CN114907943B, thereby realizing automatic slurry separation of the umbilical cord blood; Specifically, the edge frame 2 includes a fixed frame 21 and a lifting body 22. The fixed frame 21 has a hollow body structure with an isosceles trapezoidal cross section. One end of the fixed frame 21 is fixedly installed on the outer wall of the central body 1 and the other end extends outward to form a lifting area. The lifting body 22 is installed in the lifting area through a guide member 5. An adjustment groove 221 is symmetrically opened at one end of the lifting body 22 away from the central body 1. The stabilizing clip 4 is correspondingly arranged in the adjustment groove 221. An adjustment member 6 is provided between the stabilizing clip 4 and the adjustment groove 221. The adjustment member 6 is used to properly adjust the spacing between the stabilizing clips 4. The interior of the fixing frame 21 is hollow, which can reduce the overall mass of the rotor. A lighter rotor has a smaller mass, so it can reach the set speed faster when starting, and can also decelerate faster when stopping, which helps to shorten the entire experimental cycle and improve work efficiency. A lighter rotor generates less centrifugal force when unbalanced. Even if a slight imbalance occurs, its impact on the overall structure of the centrifuge is relatively small, thereby improving the safety of operation. The adjustment member 6 appropriately adjusts the spacing between the stabilizing clamps 4, which can adapt to different types of umbilical cord blood bags and facilitate the stable clamping and fixing of different types of umbilical cord blood bags; Specifically, a self-balancing member 7 is provided between the central body 1 and the lifting body 22, and the self-balancing member 7 performs balance adjustment according to the mass of the umbilical cord blood bag to ensure the running balance and running stability of the rotor; When the umbilical cord blood bag is installed, the overall mass of the rotor is uneven due to the different amounts of umbilical cord blood collected. The unbalanced rotor will generate huge centrifugal force when rotating at high speed, which may cause serious mechanical damage or accidents. The built-in self-balancing part 7 can help automatically detect and adjust the unbalanced state, thereby reducing this risk, and can reduce the error caused by manual adjustment, and improve the accuracy of the experimental results. The self-balancing part balances and adjusts according to the mass of the umbilical cord blood bag to ensure the running balance and running stability of the rotor. A well-balanced rotor can provide a more stable running state and reduce the possibility of sudden failures, thereby improving the reliability and stability of the entire system. A well-balanced rotor can ensure uniform distribution in the blood sample during centrifugation, thereby achieving clearer cell stratification, which is conducive to the separation of mononuclear cells (MNC) and further enrichment of CD34+ hematopoietic stem cells; Specifically, the stabilizing clamp 4 includes a fixed clamp seat 41, a movable groove 42, a micro motor 43, a bidirectional threaded rod 44, a movable block 45, an elbow 46 and a clamp plate 47. The fixed clamp seat 41 is movably installed in the adjusting groove 221. The movable groove 42 is arranged on the fixed clamp seat 41. The micro motor 43 is fixedly installed at one end in the movable groove 42 and the output end is fixedly installed with the bidirectional threaded rod 44. The bidirectional threaded rod 44 is provided with opposite threads. The movable block 45 is symmetrically threadedly connected to the bidirectional threaded rod 44. The clamp plate 47 and the movable block 45 are arranged in a one-to-one correspondence and the clamp plate 47 and the movable block 45 are fixedly connected through the elbow 46. The spacing between the clamp plates 47 is smaller than the spacing between the movable blocks 45. The stabilizing clamp 4 comprises a fixed clamp seat 41, a movable groove 42, a micro motor 43, a bidirectional threaded rod 44, a movable block 45, an elbow 46 and a clamping plate 47. The clamping plate 47 is arranged in an arc-shaped plate, and an anti-skid pad is arranged on the inner arc surface, which can increase the stable clamping force of the umbilical cord blood bag. The bidirectional threaded rod 44 is driven by the micro motor 43 to rotate, and then the movable block 45, the elbow 46 and the clamping plate 47 are driven to move towards each other, so as to achieve the clamping and loosening of the umbilical cord blood bag. The elbow 46 is arranged to shorten the distance between the clamping plates 47, so as to facilitate the clamping and fixing of the opening of the umbilical cord blood bag. Specifically, the adjusting member 6 includes a guide rod 1 61, a bidirectional threaded rod 2 62 and a screw handle 63. The guide rod 1 61 and the bidirectional threaded rod 2 62 are respectively arranged between the adjusting groove 221. The bidirectional threaded rod 2 62 is arranged on both sides of the guide rod 1 61 in a mirror image. Both ends of the guide rod 1 61 extend into the adjusting groove 221 and penetrate and are movably installed in the corresponding stable clamp seat 41. Both ends of the bidirectional threaded rod 2 62 extend into the adjusting groove 221 and are provided with opposite threads. The stable clamp seat 41 is threadedly connected to the bidirectional threaded rod 2 62. The upper end of the bidirectional threaded rod 2 62 penetrates the lifting body 22 and is fixedly installed with the screw handle 63. The screwing handle 63 is arranged at the upper end of the lifting body 22, which is convenient for operation. The screwing handle 63 drives the bidirectional threaded rod 2 62 to rotate, and then drives the stable clamp seat 41 to move toward each other along the direction of the guide rod 1 61, which helps to adjust the distance between the stable clamp seats 41 and adapt to various types of umbilical cord blood bags. The guide rod 1 61 plays a role of limiting and guiding the stable clamp seat 41; Specifically, the self-balancing member 7 includes a tension sensor 71 and a hydraulic balance member 72. The tension sensor 71 is arranged between the fixing frame 21 and the lifting body 22 and is located at the upper end of the lifting body 22. A water storage chamber 11 is provided in the central body 1, and a balance chamber 222 is provided in the lifting body 22. The hydraulic balance member 72 is respectively arranged between the lifting body 22 and the central body 1. The hydraulic balance member 72 pumps or delivers water in the balance chamber 222 through the measurement of the tension sensor 71, so as to maintain the same mass of each lifting body 22. The uniform mass distribution helps to maintain the balance state of the rotor. When the umbilical cord blood bag is fixed on the lifting body 22, the tension sensor 71 detects the mass of the lifting body 22. The tension sensor 71 and the control system are connected wirelessly (such as Wi-Fi, Bluetooth). The hydraulic balance member 72 pumps or delivers water in the balance chamber 222 through the measurement of the tension sensor 71 to maintain the same mass of each lifting body 22. The uniform mass distribution helps to maintain the balance state of the rotor. The water storage chamber 11 is set as a main water tank, which stores water for delivery. The balance chamber 222 is set as a water distribution tank. The amount of water in the water distribution tank is adjusted by the umbilical cord blood bag to keep the same mass of each lifting body 22. Specifically, the hydraulic balance member 72 includes a pumping member 721 and a water delivery member 722, the pumping member 721 includes a delivery pump 7211, a delivery pipeline 7212 and a flow control valve 7213, the delivery pump 7211 is fixedly installed on the side wall of the central body 1, one end of the delivery pump 7211 is connected to the balance chamber 222 through the delivery pipeline 7212, and the other end of the delivery pump 7211 is connected to the water storage chamber 11 through another section of the delivery pipeline 7212, the delivery pump 7211 extracts water from the balance chamber 222 and delivers it to the water storage chamber 11, and the flow control valve 7213 is arranged on the delivery pipeline. On the first 7212, the water delivery member 722 includes a delivery pump 2 7221, a delivery pipeline 2 7222 and a flow control valve 2 7223. The delivery pump 2 7221 is fixedly mounted on the side wall of the central body 1 and is located at the upper end of the delivery pump 1 7211. One end of the delivery pump 2 7221 is connected to the balance chamber 222 through the delivery pipeline 2 7222. The other end of the delivery pump 2 7221 is connected to the water storage chamber 11 through another section of the delivery pipeline 2 7222. The delivery pump 2 7221 extracts water from the water storage chamber 11 and delivers it to the balance chamber 222 in reverse. The flow control valve 2 7223 is arranged on the delivery pipeline 2 7222. The hydraulic balance member 72 includes a water pumping member 721 and a water delivery member 722. The delivery pump 1 7211 extracts water from the balance chamber 222 and delivers it to the water storage chamber 11 to reduce the mass of the corresponding lifting body 22. The delivery pump 2 7221 extracts water from the water storage chamber 11 and delivers it back to the balance chamber 222 to increase the mass of the corresponding lifting body 22, so as to keep the mass of each lifting body 22 the same and ensure that the mass of the rotor is evenly distributed. The flow control valve 1 7213 and the flow control valve 2 7223 are used to accurately control the amount of water flow in the balance chamber 222 and the water storage chamber 11, so as to ensure that the mass of each lifting body 22 is the same. The delivery pump 1 7211 and the delivery pump 2 7221 and the control system are wirelessly connected (such as Wi-Fi, Bluetooth); Specifically, the guide member 5 includes a guide groove 51, a second guide rod 52 and a guide block 53. The guide groove 51 is symmetrically arranged in the lifting area, the second guide rod 52 is fixedly installed in the guide groove 51, and the guide block 53 is symmetrically fixedly installed on the side wall of the lifting body 22. The guide block 53 is movably installed on the corresponding second guide rod 52; The lifting body 22 is movably arranged on the guide rod 2 52 through the guide block 53, and the lifting body 22 can only move up and down along the direction of the guide rod 52; Specifically, a resistance member 8 is symmetrically provided at the lower end of the lifting body 22, and the resistance member 8 includes a fixing plate 81, a cylinder 82, and a resistance plate 83. The cylinder 82 is fixedly installed at the lower end of the lifting body 22 through the fixing plate 81, and the resistance plate 83 is fixedly installed at the telescopic end of the cylinder 82. A notch 831 is provided at one end of the resistance plate 83 adjacent to the fixing frame 21, and the notch 831 and the edge of the fixing frame 21 are matched to increase the friction between the two sides, so as to ensure the placement stability of the lifting body 22; After the hydraulic balance adjustment is completed and the masses of each lifting body 22 are the same, the lifting body 22 is securely locked in the fixing frame 21 through the resistance member 8 to ensure the stability and balance of the rotor during rotation. The cylinder 82 and the control system are connected wirelessly (such as Wi-Fi, Bluetooth).

[0020] Working principle: The present application is applied in the extraction of hematopoietic stem cells from umbilical cord blood. The umbilical cord blood bag is directly centrifuged through density gradient centrifugation technology. The umbilical cord blood bag is vertically arranged on the rotor, and there is no need to transfer the centrifuge tube of the umbilical cord blood, which reduces the risk of contamination during the transfer process. At the same time, the umbilical cord blood bag is easy to fix and the centrifugation process is stable. The central body 1 and the edge frame 2 in the present application can be made of aluminum alloy. Aluminum alloy is one of the most commonly used materials with good mechanical strength, lightness and good thermal conductivity. The aluminum alloy rotor can well withstand the stress generated during high-speed rotation and help to quickly reach the set temperature. The universal docking port 3 in the middle of the central body 1 is convenient for matching with the rotating shaft of the centrifuge, and the edge frame 2 is arranged in a circular array. The edge racks 2 are arranged on the peripheral wall of the central body 1. In the present application, the number of the edge racks 2 is set to six, and a maximum of six umbilical cord blood bags can be centrifuged simultaneously. The upper and lower openings of the umbilical cord blood bags are clamped and fixed simultaneously by the stabilizing clamps 4. The umbilical cord blood bags have high stability and are convenient and easy to fix. The umbilical cord blood bags can be directly placed in the centrifuge for processing without the need for additional air bag filling preparation steps, which speeds up the entire experimental process, simplifies the sample loading and unloading process, saves operation time, and improves work efficiency. After the umbilical cord blood bags are centrifuged, the white blood cell layer (MNCs) in the umbilical cord blood bags can be extracted through a slurry separation device for an inverted separation process of umbilical cord blood hematopoietic stem cells with a publication number of CN114907943B, thereby realizing automatic slurry separation of the umbilical cord blood; When the umbilical cord blood bag is installed, the overall mass of the rotor is uneven due to the different amounts of umbilical cord blood collected. An unbalanced rotor will generate huge centrifugal force when rotating at high speed, which may cause serious mechanical damage or accidents. The built-in self-balancing part 7 can help automatically detect and adjust the unbalanced state, thereby reducing this risk, and can reduce the error caused by manual adjustment, and improve the accuracy of the experimental results. The self-balancing part balances and adjusts according to the mass of the umbilical cord blood bag to ensure the operating balance and stability of the rotor. A well-balanced rotor can provide a more stable operating state and reduce the possibility of sudden failures, thereby improving the reliability and stability of the entire system. A well-balanced rotor can ensure uniform distribution in the blood sample during centrifugation, thereby achieving clearer cell stratification, which is beneficial to the separation of mononuclear cells (MNC) and further enrichment of CD34+ hematopoietic stem cells.

[0021] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0022] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A stem cell extraction process, characterized in that: The following steps are involved: S1. Collection of umbilical cord blood: After the baby is delivered, before the umbilical cord is cut, clamp the two ends of the umbilical cord with a clamp, and use a sterile needle to draw blood from the umbilical vein into an umbilical cord blood bag containing an anticoagulant. The umbilical cord blood bag should be open at the top and bottom. The collection volume is usually 50 to 200 ml, depending on the blood flow of the umbilical cord. After the collection is completed, the umbilical cord blood bag is sealed and labeled, and relevant information such as the collection time, maternal information, etc. is recorded; S2, Transportation of umbilical cord blood: The collected umbilical cord blood is quickly sent to the laboratory, and the temperature is kept low (2-8°C) to prevent cell damage. After arriving at the laboratory, the sample is registered, and the packaging is checked to see if it is intact and the label information is confirmed to be accurate; S3, nucleated cell count: microbiological testing of the cord blood to ensure that it is free of contamination, determination of total blood volume and nucleated cell count, and assessment of suitability for further processing; S4, centrifugation of umbilical cord blood bag: The umbilical cord blood bag is directly centrifuged through density gradient centrifugation technology. The umbilical cord blood bag is vertically set on the rotor, and the umbilical cord blood does not need to be transferred, which reduces the risk of contamination during the transfer process. At the same time, the umbilical cord blood bag is easy to fix and the centrifugation process is stable. By using media such as Ficoll or 6% hydroxyethyl starch, the density difference of different cell components is used for separation, which can effectively remove red blood cells and other non-mononuclear cells, thereby enriching mononuclear cells (MNC), including hematopoietic stem cells; S5, CD34+ cell enrichment: CD34+ cells were collected by immunomagnetic sorting, using magnetic beads combined with specific antibodies to capture CD34+ cells, and then separated from other cells by a magnetic field; S6, cryopreservation: The cells were slowly cooled to -80°C for precooling in a refrigerator, and then transferred to a liquid nitrogen tank for long-term storage at -196°C.

2. A stem cell extraction process according to claim 1, characterized in that: In step S4, the umbilical cord blood bag is fixed by the rotor. The rotor comprises a central body (1) and an edge frame (2). A universal docking port (3) is provided in the middle of the central body (1). The edge frames (2) are arranged in a circular array on the peripheral wall of the central body (1). The umbilical cord blood bag and the edge frames (2) are arranged in a one-to-one correspondence. A stabilizing clamp (4) is symmetrically provided at one end of the edge frame (2) away from the central body (1). The stabilizing clamp (4) stably clamps the upper and lower openings of the umbilical cord blood bag.

3. A stem cell extraction process according to claim 2, characterized in that: The edge frame (2) comprises a fixed frame (21) and a lifting body (22); the fixed frame (21) has a hollow body structure with an isosceles trapezoidal cross section; one end of the fixed frame (21) is fixedly mounted on the outer wall of the central body (1) and the other end extends outward to form a lifting area; the lifting body (22) is installed in the lifting area through a guide member (5) for lifting and movement; an adjustment groove (221) is symmetrically provided at one end of the lifting body (22) away from the central body (1); the stabilizing clamp (4) is correspondingly arranged in the adjustment groove (221); an adjustment member (6) is provided between the stabilizing clamp (4) and the adjustment groove (221); the adjustment member (6) is used to appropriately adjust the spacing between the stabilizing clamps (4).

4. A stem cell extraction process according to claim 3, characterized in that: A self-balancing member (7) is provided between the central body (1) and the lifting body (22), and the self-balancing member (7) performs balance adjustment according to the mass of the umbilical cord blood bag to ensure the running balance and running stability of the rotor.

5. A stem cell extraction process according to claim 3, characterized in that: The stabilizing clamp (4) comprises a fixed clamp seat (41), a movable groove (42), a micro motor (43), a bidirectional threaded rod (44), a movable block (45), an elbow (46) and a clamping plate (47); the fixed clamp seat (41) is movably mounted in the adjusting groove (221); the movable groove (42) is provided on the fixed clamp seat (41); the micro motor (43) is fixedly mounted in one end of the movable groove (42) and the bidirectional threaded rod (44) is fixedly mounted on the output end; the bidirectional threaded rod (44) is provided with opposite threads; the movable block (45) is symmetrically threadedly connected to the bidirectional threaded rod (44); the clamping plate (47) and the movable block (45) are arranged in a one-to-one correspondence and the clamping plate (47) and the movable block (45) are fixedly connected via the elbow (46); the spacing between the clamping plates (47) is smaller than the spacing between the movable blocks (45).

6. A stem cell extraction process according to claim 5, characterized in that: The adjusting member (6) comprises a guide rod 1 (61), a bidirectional threaded rod 2 (62) and a screwing handle (63); the guide rod 1 (61) and the bidirectional threaded rod 2 (62) are respectively arranged between the adjusting groove (221); the bidirectional threaded rod 2 (62) is arranged on both sides of the guide rod 1 (61) in a mirror image; both ends of the guide rod 1 (61) extend into the adjusting groove (221) and penetrate and are movably installed in the corresponding stable clamping seat (41); both ends of the bidirectional threaded rod 2 (62) extend into the adjusting groove (221) and are provided with opposite threads; the stable clamping seat (41) is threadedly connected to the bidirectional threaded rod 2 (62); the upper end of the bidirectional threaded rod 2 (62) penetrates the lifting body (22) and is fixedly installed with the screwing handle (63).

7. A stem cell extraction process according to claim 4, characterized in that: The self-balancing member (7) comprises a tension sensor (71) and a hydraulic balance member (72); the tension sensor (71) is arranged between the fixing frame (21) and the lifting body (22) and is located at the upper end of the lifting body (22); a water storage chamber (11) is provided in the central body (1); a balance chamber (222) is provided in the lifting body (22); the hydraulic balance member (72) is arranged between the lifting body (22) and the central body (1); the hydraulic balance member (72) performs water pumping or water delivery operations in the balance chamber (222) based on the measurement of the tension sensor (71), thereby maintaining the same mass of each lifting body (22); and the uniform mass distribution helps to maintain the balance state of the rotor.

8. A stem cell extraction process according to claim 7, characterized in that: The hydraulic balancing component (72) comprises a water pumping component (721) and a water delivery component (722); the water pumping component (721) comprises a delivery pump (7211), a delivery pipeline (7212) and a flow control valve (7213); the delivery pump (7211) is fixedly mounted on the side wall of the central body (1); one end of the delivery pump (7211) is connected to the balancing chamber (222) via the delivery pipeline (7212); the other end of the delivery pump (7211) is connected to the water storage chamber (11) via another section of the delivery pipeline (7212); the delivery pump (7211) extracts water from the balancing chamber (222) and delivers it to the water storage chamber (11); the flow control valve (7213) is arranged on the delivery pipeline (7211). 212), the water delivery component (722) comprises a delivery pump (7221) and a delivery pipeline (7222) and a flow control valve (7223), the delivery pump (7221) is fixedly mounted on the side wall of the central body (1) and is located at the upper end of the delivery pump (7211), one end of the delivery pump (7221) is connected to the balance chamber (222) through the delivery pipeline (7222), and the other end of the delivery pump (7221) is connected to the water storage chamber (11) through another section of the delivery pipeline (7222), the delivery pump (7221) extracts water from the water storage chamber (11) and delivers it back to the balance chamber (222), and the flow control valve (7223) is arranged on the delivery pipeline (7222).

9. A stem cell extraction process according to claim 3, characterized in that: The guide member (5) comprises a guide groove (51), a second guide rod (52) and a guide block (53); the guide groove (51) is symmetrically arranged in the lifting area; the second guide rod (52) is fixedly mounted in the guide groove (51); the guide block (53) is symmetrically fixedly mounted on the side wall of the lifting body (22); and the guide block (53) is movably mounted on the corresponding second guide rod (52).

10. A stem cell extraction process according to claim 9, characterized in that: A resistance member (8) is symmetrically provided at the lower end of the lifting body (22), the resistance member (8) comprising a fixing plate (81), a cylinder (82) and a resistance plate (83), the cylinder (82) being fixedly mounted on the lower end of the lifting body (22) via the fixing plate (81), the resistance plate (83) being fixedly mounted on the telescopic end of the cylinder (82), a notch (831) being provided at one end of the resistance plate (83) adjacent to the fixing frame (21), the notch (831) being fitted with an edge of the fixing frame (21) to increase the friction between the two sides, thereby ensuring the placement stability of the lifting body (22).

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