Method for culturing novel artificial blood of nucleated red blood cells

Abstract

A method for culturing artificial blood of nucleated red blood cells. Firstly, nucleated red blood cells are obtained from the blood of a pregnant woman by a density gradient centrifugation method and an immunomagnetic bead reuse method, and the nucleated red blood cells can be replicated and amplified under appropriate conditions to form more nucleated red blood cells, and then tend to be stable. In terms of cell morphology, in the first few days of cell culture, the cells are kept in the state of nucleated red blood cells, and then the nucleated red blood cells are enucleated to form enucleated red blood cells. Hemoglobin content detection shows that there is no obvious change in the quality and function of hemoglobin before and after red blood cell enucleation, which means that the red blood cells maintain the same function of oxygen delivery before and after the enucleation. In the direct use of nucleated red blood cells for artificial blood transfusion, the red blood cell nuclei are retained, which can not only retain the replication and proliferation functions of red blood cells in the blood system, but also avoid the lengthy enucleation procedure in the process of culturing artificial red blood cells, thereby increasing hematopoiesis speed, reducing hematopoiesis cost, and solving the problem in artificial blood.

Description

A novel method for culturing artificial blood containing nucleated red blood cells Technical Field

[0001] The present invention relates to the field of artificial hemolysis technology, in particular to a method for culturing a novel artificial hemolysis containing nucleated red blood cells. Background Art

[0002] There are over thirty human blood group systems, including ABO and Rh. Currently, despite the increasing demand for blood for medical treatment, the blood supply still relies primarily on volunteer donations, which are often contaminated by bacteria and viruses. Whole human blood is extremely complex, making artificial whole blood virtually impossible. There are two approaches to artificial blood production: the first involves the biosynthesis of fully biologically functional red blood cells, white blood cells, and platelets; the second involves the artificial synthesis of substitutes that retain the primary functions of red blood cells or platelets. Artificial blood production remains in the laboratory due to its long development time and high cost. Researching the mechanisms and functions of red blood cell and platelet formation and simplifying the production of artificial blood have become pressing issues.

[0003] To address the above problems, we provide a novel method for culturing artificial blood with nucleated red blood cells.

[0004] Summary of the Invention

[0005] The object of the present invention is to provide a novel method for culturing artificial blood with nucleated red blood cells to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A novel method for culturing artificial blood containing nucleated red blood cells comprises the following steps:

[0008] Step 1: Under aseptic operation, collect 10 × 10 mL of peripheral blood from pregnant women (150 days) using 10 15 mL vacuum EDTA-K2 anticoagulant blood collection tubes and shake well to avoid hemolysis.

[0009] Step 2: Gently put the blood collection tube in step 1 into the centrifuge cup, balance it, centrifuge it at 4℃, 2000r / min for 10min, discard the supernatant, transfer the cell sediment in each blood collection tube into a centrifuge tube with 10mL culture medium, mix it thoroughly, incubate it at 4℃ for 10min, slowly add 8mL cell separation solution to the bottom of 10 centrifuge tubes, transfer the above blood cell culture solution into the cell separation solution centrifuge tube, centrifuge it at 1000r / min at room temperature for 15min, collect the red blood cell layer, transfer the cell sediment in each blood collection tube into a centrifuge tube with 5mL of Hank's solution, wash the cells after centrifugation; add 5mL PBS solution respectively, mix it gently, centrifuge it at 500r / min at room temperature for 10 minutes, discard the supernatant, then mix the cells with 10mL culture solution, and incubate it at 4℃ for 10min;

[0010] Step 3: Add 100 μL of CD71 monoclonal antibody-coated magnetic beads to the cell centrifuge tubes prepared in step 2 and incubate at 4°C for 30 minutes. After incubation, add 40 mL of buffer, centrifuge and discard the supernatant, then add 5 mL of buffer to resuspend the red blood cells.

[0011] Step 4: Place the separation column on a magnetic stand, first equilibrate the column with 2 mL of buffer, then add the cell suspension. When the cell suspension in the separation column is minimal, add another 2 mL of buffer to wash the separation column three times.

[0012] Step 5: Remove the separation column and place it in a new 15 mL centrifuge tube. Add 2 mL of buffer, blow off the cells, and then mix the cells with 10 mL of culture medium and incubate at 4°C for 10 minutes.

[0013] Step 6: The cells obtained in step 5 were treated with 100 μL of CD71 monoclonal antibody-coated magnetic beads, and purification steps 3-5 were repeated. The collected cells were identified as nucleated red blood cells using four morphological criteria to obtain seed cells;

[0014] Step 7: The seed cells are cultured and expanded in vitro in a suspension culture system to obtain nucleated artificial hemorrhage red blood cells that do not require denucleation, thereby completing the preparation of the new artificial blood.

[0015] As a further solution of the present invention: the culture medium formula in step 2 includes: 500 mL 1×PBS, 2.5 g BSA, 0.5 mol / L EDTA (pH 8.0), 5% fetal bovine serum and 0.2% glucose.

[0016] As a further solution of the present invention: the separation solution in step 2 comprises: 20 parts of 9% polysucrose and 11 parts of 33.9% diatrizoate.

[0017] As a further solution of the present invention: in the step 3, the centrifugation temperature is 4° C., and the centrifugation speed is maintained at 2000 r / min for 10 minutes.

[0018] As a further solution of the present invention: the centrifugation operation in step 2 includes centrifugation at room temperature at 500 r / min for 10 minutes.

[0019] As a further embodiment of the present invention, the active ingredient of the prepared new artificial blood is nucleated red blood cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method of enriching nucleated red blood cells of the present invention is to obtain nucleated red blood cells from the blood of pregnant women through density gradient centrifugation and repeated immunomagnetic beads. From the perspective of cell proliferation, nucleated red blood cells can replicate and proliferate under appropriate conditions to form more nucleated red blood cells. As the number of culture days increases, the number of cells tends to increase, reaching a peak on the 5th day and then tending to stabilize. From the perspective of cell morphology, the cells remain in the state of nucleated red blood cells in the first few days of cell culture. After proper culture, nucleated red blood cells can be denucleated to form anucleated red blood cells, or mature red blood cells. After 11 days of culture, almost all of them are differentiated into denucleated red blood cells. The denucleation rate of the entire culture system reaches more than 90%, and at the same time, almost the same amount of platelets as enucleated red blood cells are produced. The hemoglobin content test of nucleated red blood cells and enucleated red blood cells shows that there is no obvious change in the quality and function of hemoglobin before and after the denucleation of red blood cells, which means that nucleated red blood cells and enucleated red blood cells have the same oxygen transport function. The paper also explains the relationship between nucleated red blood cells (RBCs), denucleated RBCs, and platelets. Nucleated RBCs can replicate and proliferate within the blood system, facilitating hematopoiesis, rendering platelets dispensable. Although the capacity and speed of RBC expansion and denucleation in vitro are limited, directly using RBCs for artificial transfusions, while preserving the nucleus, preserves their replication and proliferation within the blood system. It also eliminates the denucleation step during the artificial hematopoietic red blood cell culture process, accelerating hematopoiesis and reducing costs. This addresses the challenges of artificial hematopoiesis and represents a significant innovation in artificial hematopoiesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a schematic diagram showing the formation of enucleated erythrocytes and platelets after denucleation of nucleated erythrocytes.

[0023] Figure 2 is a schematic diagram of the number of nucleated red blood cells, non-nucleated red blood cells and platelets.

[0024] FIG3 is a schematic diagram of red blood cell culture expansion. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0026] 1.1 Materials

[0027] Experimental instruments and reagents: CO2 incubator (ThermoScientific, USA); inverted microscope (Zeiss, Germany); MACS cell sorting column (Column LS) (Miltenyi Biotec, USA); MACS cell sorter (MACS Separator) (Miltenyi Biotec, USA); CD71 MicroBeads, human (Miltenyi Biotec, USA); culture medium (M-199, Sigma-Aldrich, USA); fetal bovine serum (Hyclone, USA); human AB plasma (Sigma-Aldrich, USA); human transferrin (Sigma-Aldrich, USA); PBS buffer (Sigma-Aldrich, USA), etc.

[0028] 1.2 Methods: With approval from the Ethics Committee of Ascrip Pharmaceuticals, we used peripheral blood from pregnant women (150 days) (informed consent was obtained from all donors) to isolate nucleated erythrocytes (RBCs). These RBCs served as seed cells and were expanded in vitro using a suspension culture system. Enucleated RBCs and platelets were then removed and their functions were evaluated by measuring hemoglobin concentrations.

[0029] Main outcome measures: 1) cell growth curve; 2) cell morphology; 3) rate of nucleated erythrocyte denucleation; 4) hemoglobin concentration.

[0030] Example 1: A method for separating fetal nucleated red blood cells from the peripheral blood of pregnant women, comprising the following steps:

[0031] (1) Under aseptic operation, collect 10 × 10 mL of peripheral blood from pregnant women (150 days) using 10 15 mL vacuum EDTA-K2 anticoagulant blood collection tubes and shake well to avoid hemolysis.

[0032] (2) Gently place the above blood collection tubes into a centrifuge cup, balance it, and centrifuge it at 4°C, 2000 r / min for 10 min. Discard the supernatant and transfer the cell pellets in each blood collection tube into a centrifuge tube with 10 mL of culture medium [culture medium formula: 500 mL 1×PBS + 2.5 g BSA + 0.5 mol / LEDTA (pH 8.0) + 5% fetal bovine serum and 0.2% glucose]. After thorough mixing, incubate at 4°C for 10 min. 8 mL of cell separation solution (cell separation solution ingredients: 20 parts of 9% polysucrose and 11 parts of 33.9% diatrizoate by mass) was slowly added to the bottom of 10 centrifuge tubes respectively, and the above blood cell culture medium was transferred to the cell separation solution centrifuge tubes respectively, and centrifuged at 1000 r / min at room temperature for 15 minutes to collect the red blood cell layer. The cell sediment in each blood collection tube was transferred to a centrifuge tube with 5 mL of Hank's solution, and centrifuged at 500 r / min at room temperature for 10 minutes to wash the cells; 5 mL of PBS solution was added respectively, and the mixture was gently mixed. The mixture was centrifuged at 500 r / min at room temperature for 10 minutes, and the supernatant was discarded. The cells were then mixed with 10 mL of culture medium and incubated at 4°C for 10 minutes.

[0033] (3) Add 100 μL of CD71 monoclonal antibody-coated magnetic beads (CD71 microbeads) to each of the above-mentioned cell centrifuge tubes and incubate at 4°C for 30 min. After incubation, add 40 mL of buffer, centrifuge at 4°C, 2000 rpm for 10 min, discard the supernatant, and add 5 mL of buffer to resuspend the red blood cells.

[0034] (4) Place the separation column on a magnetic stand, first equilibrate the column with 2 mL of buffer, then add the cell suspension. When the cell suspension in the separation column is minimal, add another 2 mL of buffer and wash the separation column three times.

[0035] (5) Remove the separation column and place it in a new 15 mL centrifuge tube. Add 2 mL of buffer, blow off the cells, and then mix the cells with 10 mL of culture medium and incubate at 4°C for 10 min.

[0036] (6) The cells obtained in step 5 were treated with 100 μL of CD71 monoclonal antibody-coated magnetic beads (CD71 microbeads), and purification steps 3-5 were repeated. The collected cells were identified as nucleated red blood cells using four morphological criteria. The collected nucleated red blood cells were further used for the following studies.

[0037] Example 2: The nucleated erythrocytes obtained above were used for replication culture and enucleation culture of nucleated erythrocytes.

[0038] (1) The basal culture medium is M-199 with the addition of 3% fetal bovine serum, 2% human AB plasma, 10 mg / L insulin, and 1% penicillin-streptomycin.

[0039] The nucleated red blood cells obtained in step (6) above were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 6 mL of culture medium (basal culture medium plus 3 IU / mL heparin, 200 mg / L human transferrin, 3 IU / mL erythropoietin, 10 μg / L stem cell factor, and 1 μg / L interleukin) was added and mixed uniformly. Centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 10 mL of culture medium was added and blown uniformly. All the cell suspensions were then added to a culture bottle and cultured at 37° C. in an incubator containing 5% CO2 by volume. Cell counts were performed under an inverted microscope at regular intervals every day, and cell growth curves were drawn to understand cell growth.

[0040] (2) On the fourth day, after counting the cells under an inverted microscope, all cell suspensions were centrifuged at 1000 rpm for 5 minutes, the culture supernatant was discarded, and the cells were rinsed twice with PBS. 1 mL of digestion solution (0.25% Trypsin-0.02% EDTA) was added to the culture flask to allow the digestion solution to infiltrate all cells. The culture flask was placed in a 37°C incubator for digestion for 1-3 minutes, and then the cell digestion was observed under a microscope. When most of the cells fell off, 3 mL of complete culture medium was added to terminate the digestion. After gently mixing, the suspension was placed in a sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were rinsed twice with PBS. PBS was added to 10 mL, and 0.150 mL was taken for hemoglobin content test (nucleated red blood cell sample). The sample was taken for cell smear, Wright-Giemsa staining, and observed and photographed under a microscope.

[0041] (3) Centrifuge at 1000 rpm for 5 min, discard the PBS supernatant, add 10 mL of culture medium (basal culture medium plus 1000 mg / L human transferrin), blow the cells evenly, add them to a new culture flask, and culture them in an incubator for another 8 days. Count the cells every two days under an inverted microscope, draw a cell growth curve, and understand the cell growth status.

[0042] (4) On the 11th day, after counting the cells under an inverted microscope, all cell suspensions were centrifuged at 1000rpm for 5 minutes, the culture supernatant was discarded, and the cells were rinsed twice with PBS. 1mL of digestion solution (0.25% Trypsin-0.02% EDTA) was added to the culture flask to allow the digestion solution to infiltrate all cells. The culture flask was placed in a 37°C incubator for digestion for 1-3 minutes, and then the cell digestion was observed under a microscope. When most of the cells fell off, 3ml of complete culture medium was added to terminate the digestion. After gently mixing, the cells were placed in a sterile centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, the cells were rinsed twice with PBS, PBS was added to 10mL, and 0.150ml was taken for hemoglobin content test (enucleated red blood cell sample). The samples were taken for cell smear, Wright-Giemsa staining, observation under a microscope and photographing. The collected Microparticles were identified as enucleated erythrocytes and platelets by morphological criteria.

[0043] Example 3: The nucleated erythrocytes and enucleated erythrocytes obtained above were used for testing and comparison of hemoglobin.

[0044] (1) Add 0.15 mL of the sample obtained above into a 5 mL centrifuge tube.

[0045] (2) Add 0.1 mL of red blood cell lysis buffer (Sigma-Aldrich), gently pipette to mix, and lyse on ice for 4-5 minutes. Occasionally shake the tubes to promote red blood cell lysis. Add 0.4 mL of PBS to each tube and shake well for later use.

[0046] (3) Dilute the hemoglobin standard with PBS solution to different concentrations: 0, 5, 10, 15, 20, 25, 30 mg / L. Take 0.15 mL of each and add them to 5 mL centrifuge tubes. Add 0.1 mL of red blood cell lysis buffer and 0.4 mL of PBS respectively, shake well and set aside.

[0047] (4) Add 2.0 mL of dye (Trace free hemoglobin (FHb) assay kit (Sigma-Aldrich, Trace free hemoglobin assay kit) to each of the samples from steps 2 and 3 above, mix well, incubate in a 37°C water bath for 20 minutes, and measure the OD of each tube using a 510 nm, 1 cm optical path. Calculate the hemoglobin concentration of the sample.

[0048] Hemoglobin (mg / L) = 126.03 x (measured OD value - blank OD value) + 3.5041.

[0049] 2. Results

[0050] Nucleated red blood cells (RBCs) were obtained from maternal blood by centrifugation and double magnetic bead separation. Nucleated RBCs were replicated and expanded in a suspension culture system to form large numbers of RBCs. These RBCs were then denucleated to form enucleated RBCs and platelets (Figure 1). Cell morphology confirmed the presence of RBCs, enucleated RBCs, and platelets. Cell replication, expansion, and denucleation, as well as hemoglobin function, were shown in the figures (Figures 2 and 3, Table 1). By day 11 of culture, nearly all cells had differentiated into enucleated RBCs. The enucleation rate of the entire culture system exceeded 90%.

[0051] Hemoglobin is typically present in red blood cells, and its content can be measured using the cyanide methemoglobin (HiCN) assay. However, the red blood cells prepared in this patent have a low concentration, and the amount of hemoglobin is much lower than that in normal blood, making it unsuitable for HiCN measurement. This patent uses the plasma-free hemoglobin (FHb) method to measure the amount of hemoglobin in a sample by lysing a small amount of red blood cells to release hemoglobin. This method has a recovery rate of 102%, a minimum detection limit of 3.6 mg / L, and a detection range of 3.6-200 mg / L.

[0052] Table 1:

[0053] 3. Conclusion:

[0054] The present invention utilizes a method for enriching nucleated red blood cells (NRBCs) from maternal blood through density gradient centrifugation and repeated immunomagnetic bead analysis. Cell proliferation reveals that NRBCs can replicate and expand under appropriate conditions, forming more NRBCs. The number of cells increases with increasing culture days, reaching a peak on the fifth day and then plateauing. Cell morphology indicates that the cells remain in the NRBC state during the first few days of culture. With appropriate culture, NRBCs can denucleate to form anucleated NRBCs, or mature NRBCs. By day 11, almost all NRBCs have differentiated into denucleated NRBCs, achieving a denucleation rate exceeding 90% across the entire culture system. Platelets are produced in quantities nearly equal to those produced by enucleated NRBCs. Hemoglobin content in NRBCs and enucleated NRBCs showed no significant changes in hemoglobin quality or function before and after denucleation, indicating that NRBCs and enucleated NRBCs have the same oxygen transport function. The relationship between NRBCs, enucleated NRBCs, and platelets is also demonstrated.

[0055] The research results of this invention provide a breakthrough for artificial hematopoiesis. Blood can contain nucleated red blood cells (RBCs), which can replicate and proliferate in the blood system, alleviating hematopoiesis and rendering platelets dispensable. Although the capacity and speed of RBC expansion and denucleation in vitro are limited, avoiding the denucleation step and directly using RBCs for artificial transfusions preserves the RBC's replication and proliferation function in the blood system. This also eliminates the denucleation step during the artificial hematopoietic red blood cell culture process, increasing the speed and reducing the cost of hematopoiesis. This addresses the challenges of artificial hematopoiesis and represents a significant technological innovation.

[0056] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Although this specification describes the embodiments, not every embodiment contains only one technical solution. This description is for clarity only. Those skilled in the art should read the specification as a whole. The technical solutions in the various embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for culturing a novel artificial blood containing nucleated red blood cells, characterized in that: The specific steps include: Step 1: Under sterile operation, use 10 15mL vacuum EDTA-K2 anticoagulation blood collection tubes to collect 10X10mL of whole peripheral blood from pregnant women and shake well to avoid hemolysis; Step 2: Gently put the blood collection tube in step 1 into the centrifuge cup, balance it, centrifuge it at 4℃, 2000r / min for 10min, discard the supernatant, transfer the cell sediment in each blood collection tube into the centrifuge tube with 10mL culture medium, mix it thoroughly, incubate it at 4℃ for 10min, slowly add 8mL cell separation solution to the bottom of 10 centrifuge tubes, transfer the above blood cell culture solution into the cell separation solution centrifuge tube, centrifuge it at room temperature at 1000r / min for 15min, collect the red blood cell layer, transfer the cell sediment in each blood collection tube into the centrifuge tube with 5mL Hank's solution, wash the cells after centrifugation, add 5mL PBS solution respectively, mix it gently, centrifuge it at 500r / min for 10min at room temperature, discard the supernatant, then mix the cells with 10mL culture solution, and incubate it at 4℃ for 10min; Step 3: Add 100 μL of CD71 monoclonal antibody-coated magnetic beads to the cell centrifuge tubes in step 2, incubate at 4°C for 30 min, add 40 mL of buffer, centrifuge and discard the supernatant, and add 5 mL of buffer to resuspend the red blood cells; Step 4: Place the separation column on the magnetic rack, first balance the column with 2 mL of buffer, then add the cell suspension, and when the cell suspension in the separation column is trace, add 2 mL of buffer to wash the separation column 3 times; Step 5: Remove the separation column, place it in a new 15 mL centrifuge tube, add 2 mL of buffer, blow off the cells, mix the cells with 10 mL of culture medium, and incubate at 4 °C for 10 min; Step 6: The cells obtained in step 5 were treated with 100 μL of CD71 monoclonal antibody-coated magnetic beads, and purification steps 3-5 were repeated. The collected cells were identified as nucleated red blood cells by quadruple morphological standards to obtain seed cells; Step 7: The seed cells are cultured and expanded in vitro in a suspension culture system to obtain nucleated artificial hemorrhage red blood cells that do not require denucleation, thereby completing the preparation of the new artificial blood.

2. The method for culturing a novel artificial blood with nucleated red blood cells according to claim 1, characterized in that: The culture medium formula of step 2 includes: 500 mL 1×PBS, 2.5 g BSA, 0.5 mol / L EDTA (pH 8.0), 5% fetal bovine serum and 0.2% glucose.

3. The method for culturing the novel artificial blood with nucleated red blood cells according to claim 2, characterized in that: The separation solution in step 2 comprises: 20 parts of 9% polysucrose and 11 parts of 33.9% diatrizoate.

4. The method for culturing the novel artificial blood with nucleated red blood cells according to claim 1, characterized in that: In step 3, the centrifugation temperature is 4° C. and the centrifugation speed is maintained at 2000 r / min for 10 min.

5. The method for culturing the novel artificial blood with nucleated red blood cells according to claim 4, characterized in that: The centrifugation operation in step 2 includes centrifugation at room temperature at 500 r / min for 10 minutes.

6. The method for culturing a novel artificial blood with nucleated red blood cells according to claim 1, characterized in that: The active ingredient of the new artificial blood prepared is nucleated red blood cells.

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