A method for improving the quality of platelets produced in vitro

Abstract

The present application relates to a method for improving the quality of platelets produced in vitro. Specifically, the present inventors provide a platelet culture medium for in vitro culture, which can significantly improve the yield and quality of platelets cultured in vitro by optimizing the ingredients and adding new ingredients. Specifically, the viability of platelets obtained by culturing megakaryocytes in the culture medium of the present application and the proportion of CD42b+ cells are significantly increased, and the number of platelets produced by a single megakaryocyte is also significantly increased. The present application improves the problems of imperfect platelet function and unstable quality in existing in vitro platelet production technology, provides a stable platelet source for clinical transfusion, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of in vitro cell culture, and more specifically to a method for improving the quality of platelet production in vitro. Background Technology

[0002] Platelets, as an important component of blood, play an irreplaceable core role in maintaining normal hemostasis, regulating thrombus formation, and promoting tissue repair and wound healing. However, the platelet products currently relied upon in clinical treatment mainly come from voluntary donations, which has several inherent limitations: platelets can be stored in vitro for a very short time, usually only 5-7 days; the supply is unstable and often in short supply; repeated transfusions may also trigger alloimmune reactions, leading to ineffective platelet transfusions and other risks.

[0003] To overcome these challenges, producing functional platelets from megakaryocytes using in vitro culture systems has become a promising potential solution, offering a stable, quality-controlled, and less immunogenic source of platelets. Researchers have conducted extensive explorations in the field of in vitro platelet production; however, in vitro-produced platelets still suffer from deficiencies in quality and yield.

[0004] One of the technical challenges in in vitro platelet production lies in the insufficient expression level and stability of the key functional receptor CD42b on the surface of in vitro-generated platelets. This receptor is a core component for platelets to initiate vascular adhesion and is crucial for hemostasis. However, with current techniques, the CD42b expression level of in vitro platelets is typically only 60%-70% of the in vivo level, making it difficult for the function and quality of in vitro platelets to meet clinical needs.

[0005] Therefore, there is an urgent need in this field to develop in vitro culture methods and culture media that can improve platelet quality. Summary of the Invention

[0006] The purpose of this invention is to provide an in vitro culture method and culture medium for improving platelet quality.

[0007] In a first aspect of the invention, a culture medium for inducing megakaryocyte differentiation to produce platelets is provided, the culture medium comprising: (i) a basal culture component; (ii) Birb796; and (iii) GM6001.

[0008] In another preferred embodiment, the concentration of Birb796 in the culture medium is 1-50 μM, preferably 2-20 μM, more preferably 5-15 μM, for example 8-12 μM, for example 10 μM.

[0009] In another preferred embodiment, the concentration of GM6001 in the culture medium is 1-100 μM, preferably 10-80 μM, more preferably 30-70 μM, for example 40-60 μM, for example 50 μM.

[0010] In another preferred embodiment, the culture medium consists of the following components: (i) a basic culture component; (ii) Birb796; and (iii) GM6001.

[0011] In another preferred embodiment, the basic culture components include: basic culture medium, nutritional and metabolic components, cytokines, signaling pathway regulatory molecules, or combinations thereof.

[0012] In another preferred embodiment, the basal culture medium is selected from the group consisting of IMDM, α-MEM, DMEM / F12, and RPMI-1640; preferably IMDM culture medium.

[0013] In another preferred embodiment, the nutritional and metabolic components include: L-glutamine or a derivative thereof, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, ascorbic acid, serum or serum substitutes, or combinations thereof.

[0014] In another preferred embodiment, the L-glutamine derivative in the culture medium is GlutaMAX, with a concentration of 0.5-5 mM, preferably 1-3 mM, and more preferably 2 mM.

[0015] In another preferred embodiment, the volume ratio of the ITS in the culture medium is 0.1-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

[0016] In another preferred embodiment, the concentration of 1-thioglycerol in the culture medium is 0.2-0.8 mM, preferably 0.4-0.6 mM, and more preferably 0.45 mM.

[0017] In another preferred embodiment, the concentration of ascorbic acid in the culture medium is 20-80 μg / ml, preferably 40-60 μg / ml, and more preferably 50 μg / ml.

[0018] In another preferred embodiment, the serum in the culture medium is AB serum, with a volume ratio of 1-10% (v / v), preferably 2-8% (v / v), more preferably 4-6% (v / v), for example 5% (v / v).

[0019] In another preferred embodiment, the basic culture components are as follows: IMDM, AB serum, GlutMax, ITS, 1-thioglycerol, ascorbic acid, SCF, TPO, Y27632, KP457, and SR-1.

[0020] In another preferred embodiment, the cytokines include: SCF, TPO, or a combination thereof.

[0021] In another preferred embodiment, the concentration of SCF in the culture medium is 20-100 ng / ml, preferably 40-60 ng / ml, and more preferably 50 ng / ml.

[0022] In another preferred embodiment, the concentration of TPO in the culture medium is 20-80 ng / ml, more preferably 40-60 ng / ml, and even more preferably 50 ng / ml.

[0023] In another preferred embodiment, the signaling pathway regulating molecule includes Y27632, KP-457, SR-1, or a combination thereof.

[0024] In another preferred embodiment, the concentration of Y27632 in the culture medium is 5-15 μM, preferably 8-12 μM, and more preferably 10 μM.

[0025] In another preferred embodiment, the concentration of KP-457 in the culture medium is 10-20 μM, preferably 12-18 μM, and more preferably 15 μM.

[0026] In another preferred embodiment, the concentration of SR-1 in the culture medium is 0.25-1.25 μM, preferably 0.5-1 μM, and more preferably 0.75 μM.

[0027] In another preferred embodiment, the culture medium comprises: basal culture medium, nutrients and metabolic components, cytokines, signaling pathway regulatory molecules, Birb796, and GM6001, wherein the concentration of Birb796 is 1-50 μM and the concentration of GM6001 is 1-100 μM.

[0028] In another preferred embodiment, the culture medium is composed of the following: IMDM, AB serum, GlutMax, ITS, 1-thioglycerol, ascorbic acid, SCF, TPO, Y27632, KP457, SR-1, Birb796, and GM6001.

[0029] In another preferred embodiment, the culture medium comprises the following components: IMDM, 1-10% (v / v) AB serum, 0.5-5 mM GlutMax, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 20-80 ng / ml TPO, 5-15 μM Y27632, 10-20 μM KP457, 0.25-1.25 μM SR-1, 1-50 μM Birb796, and 1-100 mM GM6001.

[0030] In another preferred embodiment, the culture medium comprises the following components: IMDM, 5% (v / v) AB serum, 2 mM GlutMax, 1% (v / v) ITS, 0.45 mM 1-thioglycerol, 50 μg / ml ascorbic acid, 50 ng / ml SCF, 50 ng / ml TPO, 10 μM Y27632, 15 μM KP457, 0.75 μM SR-1, 10 μM Birb796, and 50 μM GM6001.

[0031] In a second aspect of the invention, a method for producing platelets in vitro is provided, comprising the steps of: culturing megakaryocytes in the presence of Birb796 and GM6001 to obtain a culture containing platelets.

[0032] In another preferred embodiment, the method includes the steps of:

[0033] (a) Providing a culture medium as described in the first aspect of the invention;

[0034] (b) The megakaryocytes are cultured in the culture medium to obtain a platelet-containing culture.

[0035] In another preferred embodiment, the method further includes the steps of:

[0036] (c) Isolate platelets from the culture.

[0037] In another preferred embodiment, the source of the megakaryocytes is not limited.

[0038] In another preferred embodiment, the megakaryocytes are generated from hematopoietic stem cells differentiated in vitro.

[0039] In another preferred embodiment, the megakaryocytes are generated from pluripotent stem cells differentiated in vitro.

[0040] In another preferred embodiment, in step (b), the number of days of cultivation is 4-14 days, preferably 5-10 days, and more preferably 6-8 days.

[0041] In another preferred embodiment, in step (b), the temperature during the cultivation process is 37±2℃, preferably 37±1℃, and more preferably 37℃.

[0042] In another preferred embodiment, in step (b), the CO2 content during the cultivation process is 5% ± 1%.

[0043] In another preferred embodiment, in step (b), the culture is a dynamic culture, preferably an oscillating culture.

[0044] In another preferred embodiment, in step (b), the initial density of the megakaryocytes is 1 × 10⁻⁶. 5 -3×10 5 / ml.

[0045] In another preferred embodiment, the viability of platelets obtained in step (b) is ≥70%, preferably ≥75%, and more preferably ≥80%.

[0046] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets obtained in step (b) is ≥80%, preferably ≥85%, and more preferably ≥90%.

[0047] In another preferred embodiment, in the culture method, the number of platelets produced by a single megakaryocyte is n, where 20 ≤ n ≤ 50, and preferably 30 ≤ n ≤ 40.

[0048] In a third aspect of the invention, an isolated platelet is provided, the platelet being cultured by the method described in the second aspect of the invention.

[0049] In another preferred embodiment, the platelet viability is ≥70%, preferably ≥75%, and more preferably ≥80%.

[0050] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets is ≥80%, preferably ≥85%, and more preferably ≥90%.

[0051] In a fourth aspect of the invention, there is provided the use of a reagent combination for preparing a kit for in vitro culture to produce platelets, said reagent combination being Birb796 and GM6001.

[0052] In another preferred embodiment, the working concentration of Birb796 in the kit is 1-50 μM, preferably 2-20 μM, more preferably 5-15 μM, for example 8-12 μM, for example 10 μM.

[0053] In another preferred embodiment, the working concentration of GM6001 in the kit is 1-100 μM, preferably 10-80 μM, more preferably 30-70 μM, for example 40-60 μM, for example 50 μM.

[0054] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0055] The beneficial effects of this invention include:

[0056] 1. This invention provides a novel culture medium suitable for in vitro platelet production, and a culture method based on this culture medium.

[0057] 2. The culture medium and culture method of the present invention can significantly improve the yield / production rate of platelets generated in vitro, improve the viability of platelets, and increase the proportion of CD41+ / CD42b+ double-positive cells in platelets.

[0058] 3. The components Birb796 and GM6001 contained in the culture medium of the present invention have an unexpected synergistic effect on the in vitro platelet generation process. Attached Figure Description

[0059] Figure 1 The effects of the four culture regimens in Example 1 on platelet production by megakaryocytes are shown. A: Schematic diagram of megakaryocyte differentiation on day 6 in the four culture regimens (top), and the corresponding platelets produced (bottom), with platelets labeled with Calcein-AM. B: Statistical graph of the number of platelets produced by a single megakaryocyte in the four culture regimens.

[0060] Figure 2 The flow cytometry results of platelets produced by the four culture protocols in Example 1 are shown. A: Flow cytometry diagram of platelet viability produced by megakaryocytes under the four culture protocols on day 6 of culture. B: Statistical graph of A. C: Flow cytometry diagram of CD41 / CD42b expression levels in platelets produced by megakaryocytes under the four culture protocols on day 6 of culture. D: Statistical graph of CD41+ / CD42b+ in C. Detailed Implementation

[0061] Through extensive and in-depth research, the inventors have provided an in vitro culture method and culture medium for improving platelet quality. By adding two small molecule compounds, Birb796 and GM6001, to a conventional megakaryocyte platelet-producing culture medium, the yield and quality of platelets obtained in vitro can be significantly improved. This includes increasing the number of platelets produced by a single megakaryocyte, platelet viability, and the proportion of CD42b+ cells in the platelets. Furthermore, the combined effect of the two small molecule compounds is superior to the sum of the effects of using them individually, exhibiting an unexpected synergistic effect. Based on this, the present invention was completed.

[0062] definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0065] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0066] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0067] As used herein, the term “in vitro” refers to events that occur in an artificial environment (e.g., in test tubes or reaction vessels, in cell cultures, in petri dishes, etc.) rather than within a living organism (e.g., an animal, plant, or microorganism).

[0068] In vitro platelet production

[0069] In this invention, platelet production in vitro refers to the process of producing platelets from megakaryocytes under in vitro conditions (e.g., in a culture medium), a process also known as platelet release.

[0070] As used in this article, the term "platelet" refers to anucleate cells formed by the fragmentation of megakaryocyte cytoplasm. Platelets play key roles in blood circulation, such as hemostasis, thrombosis, and immune regulation. Platelet transfusion is crucial for patients with thrombocytopenia caused by surgical blood loss, chemotherapy, radiotherapy, or bone marrow hematopoietic dysfunction.

[0071] As used in this article, the term "megakaryocyte (MK)" refers to a type of large, polyploid cell found in the bone marrow, derived from hematopoietic stem cells, and is the source cell of platelets. Its development involves differentiation from hematopoietic stem cells into megakaryotic progenitor cells (MkPs), followed by intranuclear replication to form polyploid cells, and finally, cytoplasmic extension to form platelets. Immature megakaryocytes are single-nucleated (2N) cells with a diameter of 10-15 µm, while mature megakaryocytes are multinucleated (8-64N) cells with a diameter >30 µm. Mature megakaryocytes rapidly produce platelets.

[0072] In this invention, the source of megakaryocytes is not limited; for example, the megakaryocytes are hematopoietic stem cells, induced to differentiate in vitro. In a preferred embodiment, the megakaryocytes are pluripotent stem cells (e.g., embryonic stem cells ESCs or induced pluripotent stem cells iPSCs) induced to differentiate in vitro.

[0073] The production of platelets generated in vitro can be indicated by the number of platelets released by a single megakaryocyte. The more platelets produced by a single megakaryocyte, the higher the production or efficiency of platelets generated in vitro.

[0074] Indicators for assessing the quality of in vitro-generated platelets include the proportion of key platelet surface markers obtained from cells. CD41 is one of the platelet cell surface markers; CD42b is a key functional receptor on the platelet surface, and its extracellular domain is easily cleaved by enzymes. In particular, the positive proportion of CD42b in in vitro cultured platelets is lower than that in vivo. Therefore, increasing the expression level of CD42b is one of the keys to improving the quality of in vitro-generated platelets.

[0075] The culture medium of the present invention

[0076] In this invention, a culture medium for producing platelets from megakaryocytes in vitro is provided. In addition to the basic culture components, it contains two small molecule compounds, Birb796 (CAS No.: 285983-48-4) and GM6001 (CAS No.: 142880-36-2), which can significantly improve the yield and quality of platelets.

[0077] The source of the ingredients used in the culture medium of the present invention is not limited; for example, they can be commercially available or homemade.

[0078] In this invention, the "basic culture components" refer to culture medium components known in the art that are suitable for the platelet-producing stage of megakaryocytes, including but not limited to: basic culture medium, nutritional and metabolic components, cytokines, and signaling pathway regulatory molecules.

[0079] In this invention, the type and source of the basal culture medium suitable for megakaryocyte platelet production are not limited, such as IMDM, α-MEM, DMEM / F12, and RPMI-1640, as long as these media can provide a suitable nutritional environment for the growth and maturation of megakaryocytes and promote their platelet production. Preferably, the basal culture medium used in this invention is IMDM medium. It should be understood that the basal culture medium mentioned in this invention also includes culture media modified based thereon.

[0080] In this invention, the nutritional and metabolic components refer to components that can provide nutritional and metabolic support for the in vitro culture of megakaryocytes, including but not limited to: L-glutamine or its derivatives, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, ascorbic acid, serum or serum substitutes.

[0081] In this invention, cytokines play a role in promoting the proliferation, survival and differentiation of megakaryocytes, including but not limited to: stem cell factor (SCF) and thrombopoietin (TPO).

[0082] In this invention, signaling pathway regulators promote platelet formation in megakaryocytes by regulating multiple signaling pathways, including inhibitors and promoters of signaling pathways. For example, the signaling pathway regulators used in this invention include, but are not limited to, Y27632, KP-457, and SR-1.

[0083] The cultivation method of the present invention

[0084] This invention provides a method for producing platelets in vitro, comprising culturing megakaryocytes in the presence of Birb796 and GM6001 to obtain a platelet-containing culture. Birb796 and GM6001 have a synergistic effect in improving the yield and quality of platelets in vitro.

[0085] In a preferred embodiment, the method for generating platelets in vitro according to the present invention includes culturing megakaryocytes in the culture medium provided by the present invention. The composition of the culture medium of the present invention is as described above.

[0086] In the in vitro platelet culture method of the present invention, the culture conditions are known to those skilled in the art or can be determined by those skilled in the art through conventional methods. For example, the choice of culture container in the present invention is not limited, and it can be a shake flask. In the present invention, the preferred culture temperature is 37±2℃, for example, 37℃. In the present invention, the CO2 content during the preferred culture process is 5%±1%, preferably 5%. In a preferred embodiment, the culture process of the present invention is dynamic culture, such as shaking culture.

[0087] The platelets obtained by the culture method of the present invention have high yield, viability, and a high proportion of CD41+ / CD42b+ double-positive cells. For example, the viability of platelets obtained by the method of the present invention is ≥70%, preferably ≥75%, and more preferably ≥80%. In a preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets obtained by the method of the present invention is ≥80%, preferably ≥85%, and more preferably ≥90%. In a preferred embodiment, in the culture method of the present invention, the number of platelets produced by a single megakaryocyte is n, where 20 ≤ n ≤ 50, preferably 30 ≤ n ≤ 40. It should be understood that the methods for detecting platelet yield, viability, and the proportion of positive cells can be any conventional methods known in the art.

[0088] This invention also provides platelets obtained in vitro by the culture medium or culture method of this invention and their applications. Platelets produced by the method of this invention can be used for transfusion in patients with thrombocytopenia, including but not limited to platelet transfusions for patients with thrombocytopenia caused by chemotherapy, radiotherapy, bone marrow hematopoietic dysfunction, etc., and for patients who require platelet replacement due to massive blood loss during surgery.

[0089] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0090] Example 1: Platelet Culture Method

[0091] In this embodiment, to investigate the effects of Birb796 and GM6001 on the differentiation of megakaryocytes into platelets, the following four different culture media were prepared:

[0092] The culture medium composition of the control group was as follows: IMDM (basal medium), AB serum (5%), GlutMax (2mM), ITS (1%), 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), TPO (50ng / ml), Y27632 (10μM), KP457 (15μM), SR-1 (0.75μM);

[0093] Group B: The group whose culture medium was supplemented with only Birb796 (10μM) on the basis of the control group was named Group B. The composition of its culture medium was as follows: IMDM (basal medium), AB serum (5%), GlutMax (2mM), ITS (1%), 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), TPO (50ng / ml), Y27632 (10μM), KP457 (15μM), SR-1 (0.75μM), Birb796 (10μM);

[0094] GM group: The group whose culture medium was supplemented with GM6001 (50μM) in addition to the control group culture medium was named GM group. Its culture medium composition is as follows: IMDM (basal culture medium), AB serum (5%), GlutMax (2mM), ITS (1%), 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), TPO (50ng / ml), Y27632 (10μM), KP457 (15μM), SR-1 (0.75μM), GM6001 (50μM);

[0095] B+GM group: The group whose culture medium was supplemented with Birb796 (10μM) and GM6001 (50μM) was named B+GM group. Its culture medium composition is as follows: IMDM (basal medium), AB serum (5%), GlutMax (2mM), ITS (1%), 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), TPO (50ng / ml), Y27632 (10μM), KP457 (15μM), SR-1 (0.75μM), Birb796 (10μM), GM6001 (50μM).

[0096] Megakaryocytes were cultured using the four culture media described above, and the steps are as follows:

[0097] Megakaryocytes differentiate from CD34+ hematopoietic stem cells. (The following appears to be a separate, unrelated sentence: Absorb 4 × 10⁻⁶)6 Transfer megakaryocytes to an EP tube, centrifuge to remove the supernatant. Resuspend the megakaryocytes in PBS, centrifuge again and discard the supernatant, repeating twice. Resuspend the megakaryocytes in 20 ml of platelet production medium, transfer to a shake flask, and place the flask on a shaker for incubation. Incubate at 37°C, 5% CO2, and adequate humidity. After 6 days, harvest the platelet culture product. Transfer to a centrifuge tube, centrifuge at 300g for 5 min, and collect the supernatant. Centrifuge the supernatant at 2000g for 10 min; the precipitate is the platelet.

[0098] Example 2: Platelet Detection in Different Culture Groups

[0099] For the four groups of culture results in Example 1, the morphology of megakaryocytes was analyzed by microscopic examination, and the platelet yield, viability, and CD42b+ / CD41+ ratio obtained from the four groups of culture media were detected. The steps are as follows:

[0100] For the platelets harvested in Example 1, the cells were resuspended in 1 ml of PBS, and the platelet count was performed using a platelet counter. The harvested platelets were stained with Calcein-AM, anti-CD41-APC, and anti-CD42b-PE at room temperature for 30 min, with ISO and nc control tubes set. The platelets were centrifuged at 2000 g for 10 min, the supernatant was discarded, and the platelets were washed twice with PBS. The platelets were resuspended in 200 μL of PBS, and platelet viability was detected by flow cytometry (Calcein-AM negative indicated viable platelets), and the proportion of CD41+ and CD42b+ cells was also measured.

[0101] Test results as follows Figure 1 and Figure 2 As shown, Figure 1 B, Figure 2 China B and Figure 2 The statistical values ​​of D are shown in Table 1.

[0102] Table 1 Statistical Values

[0103]

[0104] Regarding the number of platelets produced by a single megakaryocyte, group B showed a △ increase compared to the control group. B =17.35-10.60=6.75, the GM group improved by Δ compared to the control group. GM =17.70-10.60=7.10, while the B+GM combination group improved by △ compared to the control group. B+GM =34.93-10.6=24.33, from which we can calculate △ B+GM >△ B +△ GMThis indicates that the B+GM combination group produced a significant synergistic effect in promoting platelet production by megakaryocytes.

[0105] Regarding platelet activity, group B showed an increase of △ compared to the control group. B =70.37-64.57=5.80, the GM group improved by △ compared to the control group. GM =67.73-64.57=3.17, while the B+GM combination group improved by △ compared to the control group. B+GM =79.40-64.57=14.83, from which we can calculate △ B+GM >△ B +△ GM This indicates that the B+GM combination therapy produced a significant synergistic effect in improving platelet activity.

[0106] Regarding the proportion of CD41+ / CD42b+ double-positive cells, group B showed a Δ% increase compared to the control group. B =78.03-74.03=4, the GM group improved by △ compared to the control group. GM =81.07-74.03=7.04, while the B+GM combination group improved by △ compared to the control group. B+GM =93.83-74.03=19.08, from which we can calculate △ B+GM >△ B +△ GM This indicates that the B+GM combination group produced a significant synergistic effect in increasing the proportion of CD41+ / CD42b+ double-positive cells.

[0107] The results above show that the B+GM group had the highest number of platelets produced by a single megakaryocyte, the highest platelet viability, and the highest CD41+ / CD42b+ cell ratio. Moreover, the increase in the B+GM group compared to the control group was greater than the sum of the increases in the B group and the GM group. This indicates that the simultaneous addition of Birb796 and GM6001 had a significant synergistic effect on improving the yield and quality of platelet culture.

[0108] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A culture medium for inducing megakaryocyte differentiation to produce platelets, characterized in that, The culture medium comprises: (i) a basic culture component; (ii) Birb796; and (iii) GM6001; The basic culture components are as follows: basal culture medium, AB serum, GlutMax, ITS, 1-thioglycerol, ascorbic acid, SCF, TPO, Y27632, KP457, and SR-1; wherein, the basal culture medium is selected from the following group: IMDM, α-MEM, DMEM / F12, RPMI-1640; In the culture medium, the concentration of Birb796 is 5-15 μM; and the concentration of GM6001 is 30-70 μM.

2. The culture medium as described in claim 1, characterized in that, The concentration of Birb796 is 8-12 μM; and the concentration of GM6001 is 40-60 μM.

3. The culture medium as described in claim 1, characterized in that, The basal culture medium is IMDM.

4. A method for generating platelets in vitro, characterized in that, The steps include: culturing megakaryocytes in the presence of Birb796 and GM6001 to obtain a culture containing platelets; The concentration of Birb796 is 5-15 μM; and the concentration of GM6001 is 30-70 μM. The method includes the following steps: (a) Providing the culture medium as described in claim 1; (b) The megakaryocytes are cultured in the culture medium to obtain a platelet-containing culture.

5. The method as described in claim 4, characterized in that, The concentration of Birb796 is 8-12 μM; and the concentration of GM6001 is 40-60 μM.

6. The method as described in claim 4, characterized in that, In step (b), the culture period is 4-14 days.

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