Method for preparing hematopoietic stem cells from umbilical cord blood

Abstract

The invention relates to a cell preparation technology in biomedical engineering, and particularly discloses a preparation method of umbilical cord blood hematopoietic stem cells. According to the method, an umbilical cord blood sample is transferred to a blood collection bag system which is in sterile connection, erythrocyte sedimentation is promoted through inverted centrifugation, an erythrocyte layer is removed through a constant-speed plasma separation technology, separation of plasma and target cell components is achieved through upright centrifugation, and finally the high-purity hematopoietic stem cell product is obtained. The whole preparation process is completed under the condition that no sedimentation reagent is added in the whole process, and cell activity and functional integrity are kept in a low-temperature environment in the whole process through the combined action of specific centrifugal force and proper duration in cooperation with layered control operation. According to the pure physical separation system established in the invention, clinical potential safety hazards caused by exogenous additives are effectively eliminated, synchronous improvement of cell recovery efficiency and product safety is realized through optimized process parameters and operation specifications, and a more reliable cell preparation solution is provided for clinical application.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering, and more specifically, to a method for preparing hematopoietic stem cells from umbilical cord blood. Background Technology

[0002] Umbilical cord blood hematopoietic stem cells, due to their low immunogenicity and strong proliferative and differentiation capabilities, have become an important resource for clinical hematopoietic stem cell transplantation. They are widely used to treat hematological diseases such as leukemia, aplastic anemia, and lymphoma, as well as some genetic and immune system diseases, providing hope for cure to countless patients. During the preparation of umbilical cord blood hematopoietic stem cells, the densities of various blood components are similar, making the separation of red blood cells from hematopoietic stem cells quite challenging. Current techniques typically add hydroxyethyl starch as a sedimentation agent to whole blood, promoting the formation of rouleaux formation of red blood cells to accelerate sedimentation, thereby improving the efficiency of blood component separation, ensuring the recovery rate of hematopoietic stem cells, and meeting the basic requirements for cell quantity and quality in clinical transplantation.

[0003] However, clinical studies have found that hydroxyethyl starch infusion may trigger potential adverse reactions such as allergic reactions, kidney damage, and coagulation abnormalities. These risks are particularly pronounced in transplant patients with weakened immune systems or liver and kidney dysfunction, seriously impacting the safety of umbilical cord blood products in clinical application. This safety concern contradicts the core clinical requirement of prioritizing the safety of cell products used in transplantation, becoming a key bottleneck restricting the wider and safer application of umbilical cord blood hematopoietic stem cells. Summary of the Invention

[0004] To address the issue that existing technologies require the addition of hydroxyethyl starch (HES) in the preparation of umbilical cord blood hematopoietic stem cells, and that HES infusion poses potential adverse reactions and affects the safety of clinical applications, this application provides a method for preparing hematopoietic stem cells from umbilical cord blood.

[0005] The method for preparing hematopoietic stem cells from umbilical cord blood provided in this application adopts the following technical solution: A method for preparing hematopoietic stem cells from umbilical cord blood includes the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood into a blood collection bag, which is connected to the two-piece bag via sterile tubing. S2, Inverted centrifugation: The blood collection bag is inverted and fixed in the centrifuge device, and centrifuged at a centrifugal force of 1300-1500g for 9-11 minutes; S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure. S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge with a centrifugal force of 350-450g for 8-12 minutes; S5. Plasma Removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components.

[0006] The entire preparation process does not involve the addition of hydroxyethyl starch or any other settling agents.

[0007] By employing the above technical solution, and without adding any settling agents, setting the centrifugal force of inverted centrifugation to 1300-1500g and maintaining it for 9-11 minutes effectively promotes the full sedimentation of red blood cells under gravity, while preventing premature precipitation of nucleated cells. Subsequently, the red blood cell layer is removed at a uniform speed using a separatory clamp, maintaining a stable cell separation interface. A final step of upright centrifugation at 350-450g for 8-12 minutes achieves the separation of plasma from the target cell components. This series of parameters and control methods, combined, constructs a complete physical separation system capable of effectively enriching hematopoietic stem cells in umbilical cord blood without relying on chemical settling agents.

[0008] Preferably, in step S1, the connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

[0009] By employing the above technical solution, and ensuring that the connecting tubing between the blood collection bag and the two-piece bag is free from twisting or folding, the flow path of the cell suspension is maintained unobstructed during transfer and centrifugation. This specific technical approach effectively avoids increased flow resistance or cell retention caused by tubing bends, providing a stable fluid environment for subsequent centrifugation separation steps, thereby ensuring the integrity of cell components and transfer efficiency during the separation process.

[0010] Preferably, in step S2, the centrifugal force is 1400g and the centrifugation time is 10min.

[0011] By employing the above technical solution, and precisely controlling the centrifugal force of inverted centrifugation to 1400g and maintaining it for 10 minutes, this parameter combination creates optimal separation conditions for blood cells of different densities without the need for added sedimentation agents. This specific value setting ensures sufficient sedimentation of red blood cells while effectively preventing nucleated cells from prematurely precipitating due to excessive centrifugal force, providing a crucial guarantee for achieving a clear cell stratification interface subsequently.

[0012] Preferably, in steps S2 and S4, the blood collection bag is kept upright without deformation or collapse during centrifugation.

[0013] By employing the above technical solution, the physical stability of the cell suspension system is maintained by keeping the blood collection bag upright and free from deformation or collapse throughout the centrifugation process. This specific operational method effectively avoids uneven distribution of centrifugal force or tilting of the cell layer interface caused by container deformation, creating stable spatial conditions for the orderly sedimentation of cells of different densities in a gravitational field, thereby ensuring the integrity and reproducibility of the cell layer structure.

[0014] Preferably, in step S3, the translation speed of the slurry separator is 1-3 mm / s.

[0015] By employing the above technical solution and controlling the translational speed of the separator within the range of 1-3 mm / s, precise control of the erythrocyte layer removal process is achieved. This specific low-speed, uniform operation effectively maintains the stability of the cell layering interface, preventing fluid disturbances caused by excessively rapid separation from disrupting the established cell layering structure, thereby ensuring complete separation of the nucleated cell layer from the erythrocyte layer.

[0016] Preferably, in step S3, the cell layering interface is kept clear during the removal of red blood cells to prevent nucleated cells from being lost with the red blood cell layer.

[0017] By employing the above technical solution, and by maintaining a clear and intact cell layer interface in real time during the red blood cell removal process, coupled with the precise spatial positioning of the separation clamp, operators can accurately control the separation interface based on the visible cell layer boundaries. This technique, by maintaining the physical separation between the red blood cell layer and the nucleated cell layer, avoids unnecessary loss of nucleated cells that may be caused by blurred or overlapping interfaces at the operational level.

[0018] Preferably, in step S4, the centrifugal force is 400g and the centrifugation time is 10min.

[0019] By adopting the above technical solution, and setting the centrifugal force parameter of upright centrifugation to 400g for 10 minutes, this technical condition provides a suitable sedimentation environment for the effective separation of plasma and hematopoietic stem cell components. This specific parameter combination ensures sufficient precipitation of hematopoietic stem cells while avoiding cell aggregation or damage that may be caused by excessive centrifugal force, creating ideal conditions for subsequently obtaining intact and effective target cell components.

[0020] Preferably, in steps S2 and S4, the centrifugation operation is carried out at an ambient temperature of 20-24°C.

[0021] By employing the above technical solution, and controlling the centrifugation environment temperature within the range of 20-24℃, a stable and physiologically close external condition is provided for the cell separation process. This temperature range avoids cell metabolic inhibition that may be caused by low temperatures and reduces changes in physical properties due to temperature fluctuations, helping to maintain the stability of the cell suspension system. This provides a suitable environmental basis for obtaining a clear cell stratification structure and maintaining cell viability.

[0022] Preferably, in steps S2 and S4, after the centrifugation operation is completed, the blood collection bag is removed smoothly to avoid violent shaking.

[0023] By employing the above-mentioned technical solution, the mechanical stability of the cell stratification structure formed after centrifugation is effectively maintained by removing the blood collection bag smoothly and strictly avoiding violent shaking after centrifugation. This operational method, by eliminating external disturbances, prevents relative displacement and secondary mixing between cell layers due to inertia, thereby ensuring the integrity and availability of target cell components in subsequent separation steps.

[0024] Preferably, in step S5, the removal of plasma is accomplished by uniformly translating the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

[0025] By employing the above-mentioned technical solution, the plasma removal step is completed through uniformly shifting the plasma separator, which maintains the fluid stability of the separation interface. This controlled linear movement effectively avoids eddies or pressure fluctuations caused by sudden changes in flow rate, thereby ensuring the integrity of the interface between the plasma layer and the hematopoietic stem cell layer, and ensuring that the target cell components maintain their original physical distribution during the separation process.

[0026] In summary, this application has the following beneficial effects: 1. In this application, by not adding hydroxyethyl starch or any other settling agents throughout the entire process, the potential risks of adverse reactions caused by HES infusion are fundamentally avoided. Combined with centrifugation at low temperatures, this helps maintain the natural state and activity of cells, thereby improving the safety of umbilical cord blood products for clinical use from the source and ensuring the quality of basic cells.

[0027] 2. In this application, a stable physical separation environment is created by ensuring that the tubing is free from twisting or folding, that the blood collection bag remains upright and undeformed during centrifugation, and that the bag is removed smoothly after the operation. These measures effectively prevent cell layer mixing caused by tubing blockage or container shaking, creating the necessary conditions for effective sedimentation and removal of red blood cells.

[0028] 3. In this application, a specific combination of centrifugal force and time parameters is used, and the plasma separator is controlled to move at a uniform speed, working synergistically on the cell suspension system. This operating mode promotes the formation of a clear and stable stratified interface between cells of different densities, thereby maximizing the retention of nucleated cells, especially mononuclear cells, and minimizing the loss of target cells when removing red blood cells and plasma.

[0029] 4. In this application, by precisely controlling the translational speed of the plasma separation clamp and maintaining a clear layered interface, precise control is achieved over the separation process between the red blood cell and nucleated cell layers, and between the plasma and hematopoietic stem cell layers. This technique effectively suppresses interface disturbances and the generation of funnel-shaped eddies, thereby minimizing the non-targeted loss of nucleated cells and hematopoietic stem cells during supernatant removal at the physical level, ensuring the high purity of the final product.

[0030] 5. In this application, by setting the centrifugation operating environment temperature within the range of 20-24℃, this technical solution provides mild conditions compatible with physiological states for the cell separation process. This temperature range effectively avoids the potential impact of low-temperature environments on cell membrane fluidity and enzyme activity, which is conducive to maintaining the integrity of the cell's natural structure and function. Thus, it creates a stable and reliable operating basis for obtaining high-activity, high-quality final products without the need for additional temperature control energy consumption and equipment burden. Attached Figure Description

[0031] Figure 1 This is a flowchart of the method for preparing hematopoietic stem cells from umbilical cord blood provided in this application. Detailed Implementation

[0032] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0033] Technical concept: In existing umbilical cord blood hematopoietic stem cell preparation technologies, effective separation of erythrocytes and hematopoietic stem cells relies on sedimentation reagents such as hydroxyethyl starch (HES) to induce erythrocyte aggregation and sedimentation. However, HES infusion carries potential adverse reactions such as allergies and kidney damage, directly threatening the safety of clinical applications. Furthermore, since the densities of various blood components are similar, improper centrifugation parameters or non-standard operation without the aid of sedimentation reagents can severely affect the separation effect. This creates a contradiction between the reliance on reagents to ensure separation effectiveness and the potential safety risks associated with those reagents, making it difficult to balance safety and effectiveness.

[0034] This technical solution employs a targeted, reagent-free, parameter-optimized, and standardized operational collaborative design: no HES or other sedimentation reagents are added throughout the process, thus avoiding reagent-related safety risks at the source; precise settings for inverted and upright centrifugation, coupled with the requirement for upright fixation of blood collection bags during centrifugation and stable handling after centrifugation, solve the problem of difficult separation of blood components when reagents are unavailable; furthermore, red blood cells and plasma are removed by uniformly shifting the plasma separator, avoiding cell layer mixing and loss of nucleated cells, ultimately achieving safe and efficient hematopoietic stem cell preparation without relying on sedimentation reagents.

[0035] In all examples and comparative examples of this experiment, the umbilical cord blood used was derived from the umbilical cord and placental blood of healthy full-term mothers. Collection was conducted after delivery of the fetus, before or after delivery of the placenta, within 1-2 minutes of umbilical cord clamping. The collection process strictly adhered to aseptic techniques, using a medical puncture needle to puncture the umbilical vein, with a collection volume of 50mL-200mL per sample. All donor mothers were clinically screened and confirmed to be free of infectious diseases such as hepatitis B, hepatitis C, HIV, and syphilis. Prenatal examinations of the fetuses showed no congenital malformations or genetic diseases. The collected umbilical cord blood was stored for a short period at low or room temperature, not exceeding 6 hours, to ensure sample freshness and the accuracy of experimental data.

[0036] Example 1 This application provides a method for preparing hematopoietic stem cells from umbilical cord blood, including the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood to the blood collection bag, and connect the blood collection bag to the two-piece bag through sterile tubing.

[0037] The connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

[0038] S2. Inverted centrifugation: Invert the blood collection bag and fix it in the centrifuge device, centrifuge with a centrifugal force of 1400g for 10 minutes.

[0039] The centrifugal force was 1400g and the centrifugation time was 10min.

[0040] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0041] The centrifugation operation was carried out at an ambient temperature of 22°C.

[0042] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0043] S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure.

[0044] The translation speed of the slurry distribution clamp is 2 mm / s.

[0045] During the removal of red blood cells, the cell stratification interface is kept clear to prevent nucleated cells from being lost along with the red blood cell layer.

[0046] S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge for 10 minutes with a centrifugal force of 400g.

[0047] The centrifugal force was 400g and the centrifugation time was 10min.

[0048] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0049] The centrifugation operation was carried out at an ambient temperature of 22°C.

[0050] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0051] S5. Plasma Removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components.

[0052] The removal of plasma is accomplished by uniformly shifting the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

[0053] The entire preparation process does not involve the addition of hydroxyethyl starch or any other settling agents.

[0054] Example 2 This application provides a method for preparing hematopoietic stem cells from umbilical cord blood, including the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood to the blood collection bag, and connect the blood collection bag to the two-piece bag through sterile tubing.

[0055] The connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

[0056] S2, Inverted Centrifugation: Invert the blood collection bag and fix it in the centrifuge device, centrifuge at 1300g for 9 minutes.

[0057] The centrifugal force was 1300g and the centrifugation time was 9min.

[0058] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0059] The centrifugation operation was carried out at an ambient temperature of 20°C.

[0060] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0061] S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure.

[0062] The translation speed of the slurry separator is 1 mm / s.

[0063] During the removal of red blood cells, the cell stratification interface is kept clear to prevent nucleated cells from being lost along with the red blood cell layer.

[0064] S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge for 8 minutes with a centrifugal force of 350g.

[0065] The centrifugal force was 350g and the centrifugation time was 8min.

[0066] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0067] The centrifugation operation was carried out at an ambient temperature of 20°C.

[0068] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0069] S5. Plasma Removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components.

[0070] The removal of plasma is accomplished by uniformly shifting the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

[0071] The entire preparation process does not involve the addition of hydroxyethyl starch or any other settling agents.

[0072] Example 3 This application provides a method for preparing hematopoietic stem cells from umbilical cord blood, including the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood to the blood collection bag, and connect the blood collection bag to the two-piece bag through sterile tubing.

[0073] The connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

[0074] S2, Inverted Centrifugation: Invert the blood collection bag and fix it in the centrifuge device, centrifuge with a centrifugal force of 1500g for 11 minutes.

[0075] The centrifugal force was 1500g and the centrifugation time was 11min.

[0076] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0077] The centrifugation operation was carried out at an ambient temperature of 24°C.

[0078] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0079] S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure.

[0080] The translation speed of the slurry distribution clamp is 3 mm / s.

[0081] During the removal of red blood cells, the cell stratification interface is kept clear to prevent nucleated cells from being lost along with the red blood cell layer.

[0082] S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge at 450g for 12 minutes.

[0083] The centrifugal force was 450g and the centrifugation time was 12min.

[0084] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0085] The centrifugation operation was carried out at an ambient temperature of 24°C.

[0086] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0087] S5. Plasma Removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components.

[0088] The removal of plasma is accomplished by uniformly shifting the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

[0089] The entire preparation process does not involve the addition of hydroxyethyl starch or any other settling agents.

[0090] Comparative Example 1 This comparative example provides a traditional method for preparing hematopoietic stem cells from umbilical cord blood. The only difference from Example 1 is the addition of hydroxyethyl starch (HES) as a settling agent during the preparation process, along with the use of lower centrifugal force parameters to match it. The specific steps are as follows: The method for preparing hematopoietic stem cells from umbilical cord blood includes the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood into a blood collection bag, which is connected to the two-piece bag via sterile tubing.

[0091] The connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

[0092] Meanwhile, hydroxyethyl starch (HES) is added to the whole blood in the blood collection bag and gently mixed to cause the red blood cells to form rouleaux.

[0093] S2. Inverted centrifugation: Invert the blood collection bag and fix it in the centrifuge device, and centrifuge it with a centrifugal force of 50g for 8 minutes.

[0094] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0095] The centrifugation operation was carried out at an ambient temperature of 5°C.

[0096] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0097] S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure.

[0098] The translational speed of the slurry separator is 2 mm / s.

[0099] During the removal of red blood cells, the cell stratification interface is kept clear to prevent nucleated cells from being lost along with the red blood cell layer.

[0100] S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge with a centrifugal force of 400g for 10 minutes.

[0101] During centrifugation, the blood collection bag should be kept upright without deformation or collapse.

[0102] The centrifugation operation was carried out at an ambient temperature of 5°C.

[0103] After centrifugation, remove the blood bag gently, avoiding violent shaking.

[0104] S5. Plasma Removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components.

[0105] The removal of plasma is accomplished by uniformly shifting the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

[0106] Comparative Example 2 The only difference between this comparative example and Example 1 is that in steps S2 and S4, the centrifugation operation is carried out at a low temperature of 2-8°C.

[0107] I. Cell Recovery Rate Test Experiment Thirty umbilical cord blood samples of consistent specifications and meeting the above-mentioned source standards were selected and divided into five groups. Three groups were prepared into hematopoietic stem cells using the methods described in Examples 1-3, the fourth group (Comparative Example 1) was prepared using the traditional method described above, and the fifth group (Comparative Example 2) was used as Comparative Example 2. After preparation, the samples from all five groups were tested according to the method described in Example 1. The recovery rate of TNCs, the recovery rate of MNCs, and the total number of prepared TNCs and MNCs were measured in each group. Statistical methods were used to analyze the differences in the test data of the five groups, and the results of each group were recorded and compared for effectiveness.

[0108] The total number of TNCs and MNCs were detected using a fully automated hematology analyzer. Before detection, the cell suspension was thoroughly mixed, and 20L of sample was mixed with an equal volume of diluent and injected into the detection channel. The instrument automatically counted the total number of nucleated cells (TNCs) and mononuclear cells (MNCs). The TNC recovery rate was calculated as: (Total number of TNCs after preparation / Initial number of TNCs before preparation) × 100%. The MNC recovery rate was calculated as: (Total number of MNCs after preparation / Initial number of MNCs before preparation) × 100%. The detection process required three parallel replicate experiments, and the average value was taken as the final result. The allowable error range for parallel experiments was no more than 3%.

[0109] II. Red Blood Cell Clearance Test Experiment Thirty-five umbilical cord blood samples collected at similar times and meeting the above-mentioned source criteria were selected and divided into five groups. Groups one through three were prepared using the methods described in Examples 1, 2, and 3, respectively. Group four, as Comparative Example 1, was prepared using the aforementioned traditional method. Group five served as Comparative Example 2. After preparation, the five groups of samples were tested according to the method in Example 1. The red blood cell volume of each group was measured, and the red blood cell clearance rate was calculated based on the initial red blood cell volume. The red blood cell volume and clearance rate data of the five groups were statistically compared, and the results were recorded and analyzed.

[0110] The initial red blood cell volume was directly measured using a sterile volumetric flask before umbilical cord blood sample preparation. The red blood cell volume after preparation was read through the graduation line on the blood collection bag. When reading, the blood collection bag was placed vertically, and the line of sight was aligned with the graduation line, accurate to 0.01 mL. Red blood cell clearance rate = (initial red blood cell volume - red blood cell volume after preparation) / initial red blood cell volume × 100%. All volume measurements were repeated 3 times, and the average value was used for calculation. The measurement error did not exceed 0.1 mL.

[0111] III. Hematopoietic Stem / Progenitor Cell Detection Experiment Forty umbilical cord blood samples meeting the above-mentioned source standards and testing requirements were selected and divided into five groups. Three experimental groups prepared hematopoietic stem cells according to the methods of Examples 1, 2, and 3, respectively. The fourth group served as Comparative Example 1, prepared using the traditional method described above. The fifth group served as Comparative Example 2. After the samples were prepared, they were tested according to the method in Example 1. The activity of the prepared cells, the total number of CD34+ cells, and the total number of CFU-GM were measured sequentially in each group. Data collection was performed using professional testing equipment, and statistical analysis was conducted on the results of the five groups to compare the differences in hematopoietic stem / progenitor cell-related indicators among the groups.

[0112] Cell viability was assessed using trypan blue staining. 10 L of cell suspension was mixed with 10 L of 0.4% trypan blue solution, allowed to stand at room temperature for 1-2 minutes, and then observed under a light microscope using a hemocytometer. The number of unstained viable cells was counted alongside the total number of cells. Cell viability was calculated as viable cell count / total cell count × 100%. The total CD34+ cell count was determined by flow cytometry. After CD34 monoclonal antibody labeling, fixation, and membrane perforation treatment, the percentage of CD34+ cells was measured, and the total CD34+ cell count was calculated based on the total number of MNCs. The total CFU-GM count was determined using colony culture. Cell suspension was seeded onto methylcellulose medium and cultured for 14 days in a 37℃, 5% CO2, saturated humidity incubator. The number of colonies with more than 50 cells was counted under a microscope; this was the total CFU-GM count. All tests were performed with three parallel samples, and the average value was used as the final result.

[0113] IV. Post-thawing testing experiment Thirty umbilical cord blood samples of uniform specifications and meeting the above-mentioned source standards were selected and divided into five groups. Three groups were prepared into hematopoietic stem cell components using the methods described in Examples 1, 2, and 3, respectively. The fourth group served as Comparative Example 1, prepared using the aforementioned traditional method. The fifth group served as Comparative Example 2. After preparation, the hematopoietic stem cell components of all five groups were cryopreserved according to the same cryopreservation protocol. After 6 months of preservation, they were uniformly thawed. After thawing, tests were performed according to the method described in Example 1. The recovery rates of nucleated cells, CD34+ cells, CFU-GM, CD34+ cell activity, and nucleated cell activity were measured in each group. All test data were recorded, and the differences in various indicators among the five groups after thawing were analyzed using statistical methods.

[0114] The thawing process involves rapidly thawing the frozen hematopoietic stem cell components in a 37°C constant temperature water bath, and gently mixing them after complete thawing. The recovery rates of nucleated cells after thawing are calculated as follows: total number of nucleated cells after thawing / total number of nucleated cells before freezing × 100%; CD34+ cell recovery rate is calculated as follows: total number of CD34+ cells after thawing / total number of CD34+ cells before freezing × 100%; CFU-GM recovery rate is calculated as follows: total number of CFU-GM cells after thawing / total number of CFU-GM cells before freezing × 100%. The viability of nucleated cells after thawing is detected using trypan blue staining, and the viability of CD34+ cells is detected using flow cytometry combined with 7-AAD staining. The detection steps and parallel experiment requirements are consistent with the standards for hematopoietic stem / progenitor cell detection, and the allowable error range for parallel experiments is no more than 5%.

[0115] The experimental data for cell recovery rate testing are shown in Table 1.

[0116] Table 1: ; The experimental data for the red blood cell clearance rate test are shown in Table 2.

[0117] Table 2: ; The experimental data for hematopoietic stem / progenitor cell detection are shown in Table 3.

[0118] Table 3: ; Nucleated cells and CD34 were observed after thawing. + The cell recovery rate data are shown in Table 4.

[0119] Table 4: ; The data on cell viability and CFU-GM recovery rate after thawing are shown in Table 5.

[0120] Table 5: ; Combining Examples 1-3 and Comparative Example 1 with Table 1, it can be seen that whether or not hydroxyethyl starch is added during the preparation of hematopoietic stem cells from umbilical cord blood, as well as the selection of centrifugation parameters, significantly affects the cell recovery rate. Without the addition of hydroxyethyl starch, efficient recovery of mononuclear cells can be achieved by adjusting the centrifugal force and time during inverted centrifugation, along with standardized operating procedures. A proper match between centrifugal force and time allows red blood cells to settle sufficiently for subsequent separation while avoiding the loss or sedimentation of nucleated cells due to improper parameters. Traditional methods with added hydroxyethyl starch struggle to achieve the same level of mononuclear cell recovery efficiency. This confirms that ideal cell recovery can be achieved simply by optimizing centrifugation parameters and operating procedures, without relying on sedimentation reagents.

[0121] As can be seen from Examples 1-3 and Comparative Example 1, and Table 2, the red blood cell removal effect is closely related to whether hydroxyethyl starch is added, the centrifugation parameters, and the standardization of the separation operation. Without adding hydroxyethyl starch, appropriately adjusting the centrifugal force and time during inverted centrifugation can promote sufficient sedimentation of red blood cells. Combined with uniform displacement of the separator clamp, this can effectively reduce residual red blood cells. Traditional methods rely on hydroxyethyl starch to cause red blood cell aggregation and sedimentation, but the removal effect is still inferior to the reagent-free method with optimized centrifugation parameters. This indicates that by optimizing centrifugation parameters to enhance red blood cell sedimentation and controlling the separation operation to avoid cell layer mixing, a better red blood cell removal effect can be achieved without using sedimentation reagents, demonstrating the synergistic effect of parameter adjustment and standardized operation.

[0122] Combining Examples 1-3 and Comparative Example 1 with Table 3, it can be seen that the preparation method without adding hydroxyethyl starch, and with optimized centrifugation parameters and standardized operation, does not adversely affect the activity and quantity of hematopoietic stem / progenitor cells. Maintaining the blood collection bag upright without deformation and ensuring stable handling during centrifugation, along with appropriate centrifugal force and temperature control, can reduce damage to hematopoietic stem / progenitor cells, ensuring their stable activity and quantity. The traditional method with added hydroxyethyl starch and the optimized method without reagent addition showed no significant difference in hematopoietic stem / progenitor cell-related indicators, indicating that it is not necessary to rely on hydroxyethyl starch. Simply by setting reasonable preparation parameters and standardizing the operation process, the preparation can be completed while ensuring the quality of hematopoietic stem / progenitor cells, verifying the reliability of this technical solution in preserving the function of the target cells.

[0123] Combining Examples 1-3 and Comparative Example 1 with Tables 4 and 5, it can be seen that whether or not hydroxyethyl starch is added during the preparation process, as well as the standardization of centrifugation and separation operations, affects the recovery effect of hematopoietic stem cells after cryopreservation and thawing. The preparation method without hydroxyethyl starch, through optimized centrifugation parameters and operating procedures, can reduce latent damage to cells during preparation, allowing hematopoietic stem cells to maintain high activity and recovery rate after long-term cryopreservation. The traditional method with added hydroxyethyl starch showed significantly lower nucleated cell activity after thawing compared to the optimized method without added reagents. This indicates that the reagent-free preparation method is more conducive to protecting the integrity of cell structure and function. Combined with standardized freezing and thawing procedures, it can achieve good recovery results after long-term preservation of hematopoietic stem cells, demonstrating the correlation between the preparation process and subsequent preservation and recovery effects.

[0124] Based on Examples 1-3 and Comparative Example 2, and referring to Tables 1-5, it can be seen that the preparation method provided in this application, performed at room temperature centrifugation (20-24℃), exhibits superior cell recovery rate, erythrocyte clearance effect, and hematopoietic stem cell activity and functional indicators compared to Comparative Example 2, which was centrifuged at a low temperature (2-8℃). This indicates that the core advantage of this method does not rely on a low-temperature environment for cell protection. Through the combination of specific centrifugation parameters and a refined operational procedure, this technology can effectively maintain stable separation of cell layers at room temperature, ensuring efficient recovery and optimal condition of target cells. This reduces the stringent requirements for environmental temperature control during the preparation process, enhancing the practicality and operability of the process.

[0125] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing hematopoietic stem cells from umbilical cord blood, characterized in that: Includes the following steps: S1. Sample preparation: Transfer the collected umbilical cord blood into a blood collection bag, which is connected to the two-piece bag via sterile tubing. S2, Inverted centrifugation: The blood collection bag is inverted and fixed in the centrifuge device, and centrifuged at a centrifugal force of 1300-1500g for 9-11 minutes; S3. Red blood cell removal: After centrifugation, remove the red blood cell layer at a uniform speed in the separator to maintain cell stratification and avoid the formation of a funnel-shaped structure. S4. Upright centrifugation: Fix the blood collection bag upright in the centrifuge and centrifuge with a centrifugal force of 350-450g for 8-12 minutes; S5. Plasma removal: Remove most of the plasma and collect the lower layer hematopoietic stem cell components; The entire preparation process does not involve the addition of hydroxyethyl starch or any other settling agents.

2. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S1, the connection between the blood collection bag and the two-piece bag ensures that the tubing is free from twisting or folding.

3. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S2, the centrifugal force is 1400g and the centrifugation time is 10min.

4. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In steps S2 and S4, the blood collection bag is kept upright without deformation or collapse during centrifugation.

5. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S3, the translation speed of the slurry separator is 1-3 mm / s.

6. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S3, the cell layering interface is kept clear during the removal of red blood cells to prevent nucleated cells from being lost with the red blood cell layer.

7. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S4, the centrifugal force is 400g and the centrifugation time is 10min.

8. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In steps S2 and S4, the centrifugation operation is carried out at an ambient temperature of 20-24°C.

9. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In steps S2 and S4, after the centrifugation operation is completed, the blood collection bag is removed smoothly, avoiding violent shaking.

10. The method for preparing hematopoietic stem cells from umbilical cord blood according to claim 1, characterized in that: In step S5, the removal of plasma is accomplished by uniformly translating the plasma separator to avoid damaging the interface between the plasma layer and the hematopoietic stem cell layer.

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