A method of promoting platelet production from megakaryocytes in vitro

Abstract

The present application relates to a method for promoting megakaryocytes to produce platelets in vitro. Specifically, the present application provides a method for producing platelets by culturing megakaryocytes in stages, using two culture media with different compositions for maturation and differentiation of megakaryocytes, which significantly improves the number of platelets produced by megakaryocytes in vitro, and improves the activity and CD41+ / CD42b+ positive proportion of megakaryocytes producing platelets in vitro. In addition, the composition of the first stage culture medium is explored and optimized to obtain a culture medium that can significantly promote the maturation of megakaryocytes. The culture method of the present application can be used to produce platelets in vitro with high quality and high yield, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically to a method for promoting platelet production by megakaryocytes in vitro. Background Technology

[0002] Platelets obtained through traditional donation methods only maintain their activity for 5-7 days and require continuous shaking for preservation, resulting in high transportation costs. The contamination rate of pathogens such as bacteria and viruses is about 0.02%, and screening technologies cannot completely avoid them.

[0003] In vitro megakaryocyte differentiation technology, through the directed differentiation of umbilical cord blood hematopoietic stem cells or iPSCs, can overcome the limitations of time and space, achieve sterile and traceable production, and fundamentally solve the problems of supply shortages and safety hazards.

[0004] However, in vitro megakaryocyte differentiation suffers from low platelet production. Under static culture conditions, each megakaryocyte can only produce a single-digit number of platelets, far lower than the thousands produced by megakaryocytes in vivo. This is related to various factors such as the maturation environment of mature megakaryocytes and fluid shear stress.

[0005] One of the main reasons is the low degree of polyploidy of megakaryocytes in vitro, that is, insufficient replication of nuclear DNA. Polyploidy is a key marker of megakaryocyte maturation and efficient platelet production. In vivo, megakaryocytes form high-ploidy (32N-64N) cells through intranuclear mitosis, and a single cell can produce 2,000-5,000 platelets. However, the degree of polyploidy in vitro is significantly reduced, resulting in each megakaryocyte producing less than 20 platelets (less than 1% of the in vivo rate).

[0006] Furthermore, while platelets have a short natural lifespan (approximately 7-10 days), the time from differentiation to platelet release in vitro from cultured megakaryocytes is much longer, typically requiring several weeks, resulting in partially aged or inactive platelets at harvest. These challenges highlight the urgent need to optimize megakaryocyte culture systems to improve the yield, viability, and functional integrity of platelets produced in vitro.

[0007] Therefore, there is an urgent need in this field to develop methods for promoting high-yield and high-quality platelet production from megakaryocytes in vitro. Summary of the Invention

[0008] The purpose of this invention is to provide a method for promoting platelet production by megakaryocytes in vitro.

[0009] In a first aspect of the invention, a method for producing platelets by culturing megakaryocytes in vitro is provided, comprising the steps of: S1. Provide megakaryocytes and culture the megakaryocytes in culture medium I; the components of culture medium I include: a basic culture component for promoting megakaryocyte maturation, Stiripentol, and Romiplostim; S2. The cells cultured in step S1 are transferred to culture medium II for further culture; the components of culture medium II include: basic culture components for promoting platelet production; S3. Harvest the culture after step S2 and separate the platelets from it.

[0010] In another preferred embodiment, the concentration of Stiripentol in the culture medium I is 1-50 μM, preferably 2-20 μM, more preferably 5-15 μM, for example 8-12 μM, for example 10 μM.

[0011] In another preferred embodiment, the concentration of Romiplostim in the culture medium I is 10-100 ng / ml, preferably 20-80 ng / ml, more preferably 40-60 ng / ml, for example 50 ng / ml.

[0012] In another preferred embodiment, the basic culture components for promoting megakaryocyte maturation in culture medium I include: basic culture medium, nutritional and metabolic components, and megakaryocyte maturation factors.

[0013] 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.

[0014] In another preferred embodiment, the nutritional and metabolic components include: serum or serum substitutes, L-glutamine or substitutes thereof, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, and ascorbic acid.

[0015] In another preferred embodiment, the megakaryocyte maturation factors include: stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), thrombopoietin (TPO), IL-6, SR-1, and valproic acid (VPA).

[0016] In another preferred embodiment, the components of the culture medium I are: IMDM medium, FBS, GlutaMAX, ITS, 1-thioglycerol, ascorbic acid, SCF, Flt3L, TPO, IL-6, SR-1, VPA, Stiripentol and Romiplostim.

[0017] In another preferred embodiment, the serum in the culture medium I is fetal bovine serum (FBS) at a volume ratio of 0.1-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

[0018] In another preferred embodiment, the L-glutamine derivative in the culture medium I is GlutaMAX, and its concentration is 0.5~5mM, preferably 1~3mM, and more preferably 2mM.

[0019] In another preferred embodiment, the volume ratio of the ITS in the culture medium I is 0.1-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

[0020] In another preferred embodiment, the concentration of 1-thioglycerol in the culture medium I is 0.2-0.8 mM, preferably 0.4-0.6 mM, and more preferably 0.45 mM.

[0021] In another preferred embodiment, the concentration of ascorbic acid in the culture medium I is 20-80 μg / ml, preferably 40-60 μg / ml, and more preferably 50 μg / ml.

[0022] In another preferred embodiment, the concentration of SCF in the culture medium I is 20-100 ng / ml, preferably 40-60 ng / ml, and more preferably 50 ng / ml.

[0023] In another preferred embodiment, the concentration of Flt3L in the culture medium I is 50-150 ng / ml, more preferably 80-120 ng / ml, and even more preferably 100 ng / ml.

[0024] In another preferred embodiment, the concentration of TPO in the culture medium I is 20-60 ng / ml, more preferably 30-50 ng / ml, and even more preferably 40 ng / ml.

[0025] In another preferred embodiment, the concentration of IL-6 in the culture medium I is 5-30 ng / ml, more preferably 10-20 ng / ml, and even more preferably 15 ng / ml.

[0026] In another preferred embodiment, the concentration of SR-1 in the culture medium I is 0.1-10 μM, preferably 0.5-5 μM, and more preferably 1 μM.

[0027] In another preferred embodiment, the concentration of VPA in the culture medium I is 100-300 μM, preferably 150-250 μM, and more preferably 200 μM.

[0028] In another preferred embodiment, the components of culture medium I are: IMDM medium, 0.1-5% (v / v) FBS, 0.5-5 mM GlutaMAX, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 50-150 ng / ml Flt3L, 20-60 ng / ml TPO, 5-30 ng / ml IL-6, 0.1-10 μM SR-1, 100-300 μM VPA, 1-50 μM Stiripentol, and 10-100 ng / ml Romiplostim.

[0029] In another preferred embodiment, the components of culture medium I are: IMDM medium, 1% (v / v) FBS, 2 mM GlutaMAX, 1% (v / v) ITS, 0.45 mM 1-thioglycerol, 50 μg / ml ascorbic acid, 50 ng / ml SCF, 100 ng / ml Flt3L, 40 ng / ml TPO, 15 ng / ml IL-6, 1 μM SR-1, 200 μM VPA, 10 μM Stiripentol and 50 ng / ml Romiplostim.

[0030] In another preferred embodiment, the platelet-promoting basic culture components in culture medium II include: basic culture medium, nutritional and metabolic components, and platelet-promoting factors.

[0031] In another preferred embodiment, the culture medium II does not contain Stiripentol and Romiplostim.

[0032] 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.

[0033] In another preferred embodiment, the nutritional and metabolic components include: serum or serum substitutes, L-glutamine or substitutes thereof, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, and ascorbic acid.

[0034] In another preferred embodiment, the thrombopoietin-promoting factors include: stem cell factor (SCF), thrombopoietin (TPO), Y27632, KP-457, and SR-1.

[0035] In another preferred embodiment, the components of the culture medium II are: IMDM medium, FBS, GlutaMAX, ITS, 1-thioglycerol, ascorbic acid, SCF, TPO, Y27632, KP-457, and SR-1.

[0036] In another preferred embodiment, the serum in the culture medium II is fetal bovine serum (FBS) at a volume ratio of 0.1-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

[0037] In another preferred embodiment, the L-glutamine derivative in culture medium II is GlutaMAX, with a concentration of 0.5-5 mM, preferably 1-3 mM, and more preferably 2 mM.

[0038] In another preferred embodiment, the volume ratio of the ITS in the culture medium II is 0.1-5% (v / v), preferably 0.5-2% (v / v), and more preferably 1% (v / v).

[0039] In another preferred embodiment, the concentration of 1-thioglycerol in the culture medium II is 0.2-0.8 mM, preferably 0.4-0.6 mM, and more preferably 0.45 mM.

[0040] In another preferred embodiment, the concentration of ascorbic acid in culture medium II is 20-80 μg / ml, preferably 40-60 μg / ml, and more preferably 50 μg / ml.

[0041] In another preferred embodiment, the concentration of SCF in the culture medium II is 20-100 ng / ml, preferably 40-60 ng / ml, and more preferably 50 ng / ml.

[0042] In another preferred embodiment, the concentration of TPO in the culture medium II is 0.1-0.5 μg / ml, more preferably 0.1-0.3 μg / ml, and even more preferably 0.2 μg / ml.

[0043] In another preferred embodiment, the concentration of Y27632 in the culture medium II is 1-20 μM, preferably 5-15 μM, and more preferably 10 μM.

[0044] In another preferred embodiment, the concentration of KP-457 in the culture medium II is 5-25 μM, preferably 10-20 μM, and more preferably 15 μM.

[0045] In another preferred embodiment, the concentration of SR-1 in the culture medium II is 0.1-2 μM, preferably 0.5-1 μM, and more preferably 0.75 μM.

[0046] In another preferred embodiment, the components of culture medium II are: IMDM medium, 0.1-5% (v / v) FBS, 0.5-5 mM GlutaMAX, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 0.1-0.5 μg / ml TPO, 1-20 μM Y27632, 5-25 μM KP-457, and 0.1-2 μM SR-1.

[0047] In another preferred embodiment, the components of culture medium II are: IMDM medium, 1% (v / v) FBS, 2 mM GlutaMAX, 1% (v / v) ITS, 0.45 mM 1-thioglycerol, 50 μg / ml ascorbic acid, 50 ng / ml SCF, 0.2 μg / ml TPO, 10 μM Y27632, 15 μM KP-457, and 0.75 μM SR-1.

[0048] In another preferred embodiment, the source of the megakaryocytes is not limited.

[0049] In another preferred embodiment, the megakaryocytes are generated by in vitro differentiation of induced pluripotent stem cells (iPSCs).

[0050] In another preferred embodiment, the culture period in step S1 is 1-5 days, preferably 2-4 days, and more preferably 3 days.

[0051] In another preferred embodiment, the culture period in step S2 is 2-8 days, preferably 3-6 days, and more preferably 3 days.

[0052] In another preferred embodiment, in steps S1 and S2, the culture is a dynamic culture, preferably a culture on a shaker.

[0053] In another preferred embodiment, step S1 is carried out under low shear conditions; the low shear conditions are: the rotation speed of the shaker is 10~30 rpm, preferably 20 rpm.

[0054] In another preferred embodiment, step S2 is performed under high shear stress conditions; the high shear stress conditions are: the rotation speed of the shaker is 50~70 rpm, preferably 60 rpm.

[0055] In another preferred embodiment, in step S1, the initial density of the megakaryocytes is 1 × 10⁻⁶. 5 -3×10 5 / ml.

[0056] In another preferred embodiment, during the cultivation process in steps S1 and S2, the cultivation temperature is 37±2℃, preferably 37±1℃, and more preferably 37℃.

[0057] In another preferred embodiment, the CO2 content is 5% ± 1% during the cultivation process in steps S1 and S2.

[0058] In another preferred embodiment, in step S3, the method for separating platelets is centrifugation.

[0059] In another preferred embodiment, step S3 further includes: detecting platelet yield and quality.

[0060] In another preferred embodiment, the platelet viability obtained in step S3 is ≥60%, preferably ≥65%, and more preferably ≥70%.

[0061] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets obtained in step S3 is ≥70%, preferably ≥75%, and more preferably ≥80%.

[0062] 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 25 ≤ n ≤ 40.

[0063] In a second aspect of the invention, a culture medium for promoting megakaryocyte maturation is provided, the components of which include: a basic culture component for promoting megakaryocyte maturation, Stiripentol, and Romiplostim.

[0064] In another preferred embodiment, the concentration of Stiripentol 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.

[0065] In another preferred embodiment, the concentration of Romiplostim in the culture medium is 10-100 ng / ml, preferably 20-80 ng / ml, more preferably 40-60 ng / ml, for example 50 ng / ml.

[0066] In another preferred embodiment, the culture medium contains the following basic culture components for promoting megakaryocyte maturation: basic culture medium, nutritional and metabolic components, and megakaryocyte maturation factors.

[0067] In another preferred embodiment, the culture medium comprises: IMDM medium, FBS, GlutaMAX, ITS, 1-thioglycerol, ascorbic acid, SCF, Flt3L, TPO, IL-6, SR-1, VPA, Stiripentol, and Romiplostim.

[0068] In another preferred embodiment, the culture medium comprises: IMDM medium, 0.1-5% (v / v) FBS, 0.5-5 mM GlutaMAX, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 50-150 ng / ml Flt3L, 20-60 ng / ml TPO, 5-30 ng / ml IL-6, 0.1-10 μM SR-1, 100-300 μM VPA, 1-50 μM Stiripentol, and 10-100 ng / ml Romiplostim.

[0069] In another preferred embodiment, the culture medium comprises: IMDM medium, 1% (v / v) FBS, 2 mM GlutaMAX, 1% (v / v) ITS, 0.45 mM 1-thioglycerol, 50 μg / ml ascorbic acid, 50 ng / ml SCF, 100 ng / ml Flt3L, 40 ng / ml TPO, 15 ng / ml IL-6, 1 μM SR-1, 200 μM VPA, 10 μM Stiripentol, and 50 ng / ml Romiplostim.

[0070] In a third aspect of the invention, an isolated platelet is provided, said platelet being cultured by the method described in the first aspect of the invention.

[0071] In another preferred embodiment, the platelet viability obtained in step S3 is ≥60%, preferably ≥65%, and more preferably ≥70%.

[0072] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets obtained in step S3 is ≥70%, preferably ≥75%, and more preferably ≥80%.

[0073] 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.

[0074] The beneficial effects of this invention include: 1. This invention provides a staged megakaryocyte culture method, which adjusts and optimizes the composition of the culture medium and culture conditions according to the different stages of platelet production by megakaryocytes, thereby maximizing the yield and quality of platelet production in vitro.

[0075] 2. The culture method of the present invention can significantly promote megakaryocyte maturation, increase the yield / production rate of platelets generated in vitro, increase the platelet viability, and increase the proportion of CD41+ / CD42b+ double-positive cells in platelets.

[0076] 3. The components Stiripentol and Romiplostim contained in the culture medium of the first stage of this invention have an unexpected synergistic effect on promoting megakaryocyte maturation in vitro.

[0077] 4. The phased culture method of the present invention significantly shortens the culture time, and platelets can be produced in a total of 6 days for the two phases. Attached Figure Description

[0078] Figure 1 A schematic diagram of the phased cultivation method of the present invention is shown.

[0079] Figure 2 The effects of different culture media on megakaryocyte maturation are shown. A: Schematic diagram of megakaryocyte morphology in the four culture protocols. B: Statistical graph of megakaryocyte diameter after 0, 3, and 6 days of culture in the four culture protocols. C: Flow cytometry analysis of megakaryocyte polyploidy after 6 days of culture in the four culture protocols. D: Statistical graph of polyploidy ratio in C.

[0080] Figure 3 The effects of five culture regimens on platelet production by megakaryocytes are shown. A: Effects of the five culture regimens on platelet viability produced by megakaryocytes. B: Effects of the five culture regimens on the expression of CD41 and CD42b in platelets produced by megakaryocytes. Detailed Implementation

[0081] Through extensive and in-depth research, the inventors have provided a method for promoting platelet production from megakaryocytes in vitro. By employing a staged culture approach using two different culture media to mature and differentiate megakaryocytes, the method significantly increases the number of platelets produced by megakaryocytes in vitro, as well as the viability and CD41+ / CD42b+ positivity rate of the platelets. Furthermore, the inventors optimized the culture medium for the first stage by adding Stiripentol and Romiplostim, which synergistically promote megakaryocyte maturation. This invention is based on these findings.

[0082] definition 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.

[0083] 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”.

[0084] 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.

[0085] 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.

[0086] 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).

[0087] Platelet production from in vitro cultured megakaryocytes This invention provides a method for producing platelets by culturing megakaryocytes in vitro, the process comprising promoting megakaryocyte maturation and promoting the release of platelets from megakaryocytes.

[0088] As used in this article, the term "megakaryocyte (MK)" refers to a type of large, polyploid cell found in the bone marrow, which differentiates 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 proplatelets.

[0089] Immature megakaryocytes have a single nucleus (2N) and a diameter of 10-15µm, while mature megakaryocytes have multiple nuclei (8-64N) and a diameter >20µm. Megakaryocyte maturation is characterized by an increase in diameter and a higher proportion of polyploidy. Mature megakaryocytes produce / release platelets.

[0090] As used herein, the source of megakaryocytes is not limited; for example, the megakaryocytes may be hematopoietic stem cells induced to differentiate in vitro. In a preferred embodiment, the megakaryocytes are pluripotent stem cells differentiated in vitro. In a preferred embodiment, the megakaryocytes are induced pluripotent stem cells (iPSCs) differentiated in vitro. The method for differentiating iPSCs into megakaryocytes in vitro is not limited and may be any method known in the art.

[0091] 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.

[0092] 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.

[0093] Indicators for assessing the quality of in vitro-generated platelets include platelet viability and the positive rate of key surface markers. CD41 is one of the cell surface markers of platelets; CD42b is a key functional receptor on the platelet surface, and its extracellular domain is easily cleaved by enzymes. In particular, the positive rate 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.

[0094] The culture medium of the present invention In this invention, a culture medium for promoting megakaryocyte maturation is provided, namely culture medium I of this invention. This culture medium contains the compounds Stiripentol (CAS No.: 49763-96-4) and Romiplostim (Fc peptide fusion protein mimic of TPO), which can synergistically promote the increase in megakaryocyte volume and promote the polyploidization of megakaryocytes, thereby improving the ability of megakaryocytes to produce platelets.

[0095] The ingredients used in the culture medium of the present invention are not limited in origin; for example, they can be commercially available or homemade. In a preferred embodiment, all ingredients used in the culture medium of the present invention are commercially available.

[0096] The culture medium of the present invention further contains a basic culture component for promoting megakaryocyte maturation. In the present invention, the "basic culture component for promoting megakaryocyte maturation" refers to culture medium components known in the art suitable for in vitro culture of megakaryocytes, including basal culture medium, nutritional and metabolic components, and megakaryocyte maturation factors.

[0097] 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, RPMI-1640, as long as these media can provide a suitable nutritional environment for the growth and maturation of megakaryocytes. 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 media modified based thereon.

[0098] 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.

[0099] In this invention, the megakaryocyte maturation factors include cytokines and signaling pathway regulatory molecules required to promote megakaryocyte proliferation, survival, and maturation, including but not limited to: stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), thrombopoietin (TPO), IL-6, SR-1, and valproic acid (VPA). The phased cultivation method of the present invention This invention provides a method for producing platelets from megakaryocytes in vitro, comprising two stages, each using a different culture medium composition. Compared to continuous culture methods that do not differentiate between stages and use only the same culture medium, the staged culture method of this invention is more conducive to improving platelet yield and quality.

[0100] The first stage is primarily used to rapidly promote the maturation of megakaryocytes, and the culture medium used in this stage is referred to as Culture Medium I. In this invention, Culture Medium I is the megakaryocyte maturation-promoting culture medium described herein, containing Stiripentol and Romiplostim. The components of Culture Medium I are as previously described. The preferred culture period for the first stage is 3 days.

[0101] The second stage primarily promotes the differentiation of mature megakaryocytes and the release of platelets. The culture medium used in this stage is referred to as Culture Medium II. Culture Medium II contains basic platelet-producing culture components, but does not contain Stiripentol or Romiplostim. In this invention, the "basic platelet-producing culture components" refer to culture medium components known in the art suitable for platelet production, including basal culture medium, nutritional and metabolic components, and platelet-producing factors. In a preferred embodiment, the platelet-producing factors include: stem cell factor (SCF), thrombopoietin (TPO), Y27632, KP-457, and SR-1.

[0102] 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, such as 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, for example, oscillation culture on a horizontal shaker.

[0103] Preferably, the first stage is cultured under low shear force conditions; the low shear force conditions are: the rotation speed of the shaker is 10~30 rpm, preferably 20 rpm; and / or the second stage is cultured under high shear force conditions; the high shear force conditions are: the rotation speed of the shaker is 50~70 rpm, preferably 60 rpm.

[0104] 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 ≥60%, preferably ≥65%, and more preferably ≥70%. In a preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the platelets obtained by the method of the present invention is ≥70%, preferably ≥75%, and more preferably ≥80%. 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.

[0105] 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.

[0106] 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.

[0107] Example 1: Culture Protocol Multiple culture protocols for platelet production from megakaryocytes are provided. The control group and culture groups 1-3 are staged culture groups, while culture group 4 is a continuous culture group. The specific culture protocols for each group are as follows: (1) Control Group The components of medium I are as follows: IMDM (basal medium), FBS (1%), GlutMax (2mM), 1% ITS, 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), Flt3L (100ng / ml), TPO (40ng / ml), IL-6 (15ng / ml), SR-1 (1μM), VPA (200μM).

[0108] The components of medium II are as follows: IMDM (basal medium), FBS (1%), GlutMax (2mM), 1% ITS, 0.45mM 1-thioglycerol (0.45mM), ascorbic acid (50ug / ml), SCF (50ng / ml), TPO (0.2μg / ml), Y27632 (10μM), KP-457 (15μM), SR-1 (0.75μM).

[0109] Culture steps: Stage 1: Megakaryocytes derived from iPSCs are cultured in a 75 cm³ culture medium containing culture medium I. 2 The cultures were incubated in shake flasks at an initial density of 2 × 10⁻⁶. 5 / ml, place the shake flask on a horizontal shaker, set the shaker speed to 20 rpm, and incubate for 3 days at 37℃, 5% CO2, and sufficient humidity; Second stage: centrifuge at 300g for 5min, discard the supernatant, and transfer to a 75cm medium containing medium II. 2 Continue culturing in shake flasks, place the shake flasks on a horizontal shaker, set the shaker speed to 60 rpm, and culture for 3 days at 37°C, 5% CO2, and sufficient humidity; then centrifuge at 300g for 5 min to obtain the supernatant; then centrifuge the supernatant at 2000g for 10 min to harvest platelets.

[0110] (2) Culture Group 1 (Group S) Based on the control group, Stiripentol (10 μM) was added to culture medium I, and this group was named Group S. Other culture medium components and culture conditions were the same as the control method.

[0111] (3) Culture group 2 (R group) Based on the control group, Romiplostim (50 ng / ml) was added to culture medium I, and this group was named R group. Other culture medium components and culture conditions were the same as the control group.

[0112] (4) Culture group 3 (S+R group) Based on the control group, Stiripentol (10 μM) and Romiplostim (50 ng / ml) were added to culture medium I, and this group was named S+R group. Other culture medium components and culture conditions were the same as the control method.

[0113] (5) Culture group 4 (S+R continuous culture group) The culture process used only one culture medium, the composition of which was as follows: Stiripentol (10 μM) and Romiplostim (50 ng / ml) were added to the control group's culture medium I.

[0114] Culture steps: Culture iPSC-derived megakaryocytes in the above culture medium, place the shake flask in a horizontal shaker, set the shaker speed to 20 rpm, and culture for 6 days at 37°C, 5% CO2, and sufficient humidity; centrifuge at 300g for 5 min to obtain the supernatant; then centrifuge the supernatant at 2000g for 10 min to harvest platelets.

[0115] Example 2: Effects of different culture medium components on megakaryocyte maturation In this embodiment, the effects of the culture protocols of the control group and culture groups 1-3 in Example 1 on megakaryocyte maturation were compared.

[0116] (1) Detection method During the culture process in the control group and culture groups 1-3, the morphology of megakaryocytes was analyzed by microscopic examination on days 0, 3 and 6, and the diameter of megakaryocytes was measured.

[0117] On day 6, the polyploidy rate of megakaryocytes was detected by flow cytometry. MK cells were treated with ribonuclease A and stained with CD41-APC, CD42b-PE, and Hoechst. Polyploidy of MK cells was then detected by flow cytometry. Data were analyzed using FlowJO software, with undifferentiated MK cells used as a diploid control.

[0118] (2) Test results During the culture process of the control group and culture groups 1-3, the morphology of megakaryocytes was as follows: Figure 2 As shown in Figure A, the diameter statistics of megakaryocytes on days 0, 3, and 6 are as follows. Figure 2 As shown in B.

[0119] The results of megakaryocyte diameter analysis showed that on day 3 of culture, the megakaryocyte diameter in the S+R group increased significantly, while there was no difference between the S or R groups and the control group, indicating that the S+R group was beneficial for the rapid maturation of megakaryocytes. On day 6, the megakaryocyte diameter in the S+R group was larger than that in the S and R groups, indicating that the S+R group promoted the full maturation of megakaryocytes.

[0120] Flow cytometry results of megakaryocyte polyploidy ratio are as follows Figure 2 As shown in Figure C, the statistical results of the three parallel experiments are as follows: Figure 2 As shown in D and Table 1.

[0121] Table 1. Statistical results of the proportion of polyploid megakaryocytes The polyploidy ratio test results showed that the S group increased by ΔS=28.9-10.3=18.6 compared with the control group, the R group increased by ΔR=29.7-10.3=19.4 compared with the control group, and the S+R group increased by ΔS+R=57-10.3=46.7 compared with the control group. The calculation shows that ΔS+R>ΔS+ΔR, indicating that the S+R combination group produced a synergistic effect in increasing the polyploidy ratio of megakaryocytes and promoting megakaryocyte maturation.

[0122] Example 3: Effects of different culture regimens on platelet production by megakaryocytes In this embodiment, the effects of the culture protocols of the control group and culture groups 1-4 on platelet production by megakaryocytes were compared.

[0123] (1) Detection method On day 6 of culture, the number of platelets produced by individual megakaryocytes was counted, the platelet diameter was measured, and the platelet viability and the proportion of CD42b+ / CD41+ double-positive cells were detected by flow cytometry.

[0124] The steps of flow cytometry detection are as follows: Cells were resuspended in 1 ml of PBS, and platelet counts were performed using a platelet counter. 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 plates were centrifuged at 2000 g for 10 min, the supernatant was discarded, and the platelets were washed twice with PBS. Platelets were resuspended in 200 μL of PBS, and platelet viability was assessed using flow cytometry (Calcein-AM negative indicated viable platelets), along with the proportion of CD41+ and CD42b+ cells.

[0125] (2) Test results Test results as follows Figure 3 As shown in Table 2.

[0126] Table 2. Platelet count results obtained from each culture group The statistical results of platelet production per megakaryocyte showed that the control group produced less than 15 platelets per megakaryocyte, the S or R group produced less than 20 platelets per megakaryocyte, while the S+R group produced 36.45 platelets per megakaryocyte. However, after six days of continuous culture in the first-stage S+R medium alone, the number of platelets produced per megakaryocyte was less than 10. This indicates that staged culture is beneficial for megakaryocyte maturation, leading to increased platelet production. Furthermore, in the staged culture groups, the S+R group was more conducive to megakaryocyte platelet production and produced a synergistic effect, consistent with the results of Example 2.

[0127] Flow cytometry results showed that, compared with the S group and the R group, the platelet viability and CD41+ / CD42b+ ratio of the S+R group were significantly higher, and both were higher than those of the S+R continuous culture group, indicating that the S+R group (staged culture) is more conducive to the production of high-quality platelets.

[0128] 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 method for producing platelets from megakaryocytes in vitro, characterized in that, Including the following steps: S1. Provide megakaryocytes, said megakaryocytes being generated in vitro from induced pluripotent stem cells (iPSCs), and cultured said megakaryocytes in culture medium I; said culture medium I comprises: a megakaryocyte maturation basal culture component, Stiripentol, and Romiplostim, wherein the concentration of Stiripentol is 5-15 μM; and the concentration of Romiplostim is 40-60 ng / ml; said megakaryocyte maturation basal culture component comprises: basal culture medium, nutrient and metabolic components, and megakaryocyte maturation factor; said basal culture medium is selected from the group consisting of: IMDM, α-MEM. The nutrients and metabolic components include: fetal bovine serum (FBS), GlutaMAX, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, and ascorbic acid; the megakaryocyte maturation factors are: stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), thrombopoietin (TPO), IL-6, SR-1, and valproic acid (VPA). S2. Cells cultured in step S1 are transferred to culture medium II for further culture. Culture medium II does not contain Stiripentol or Romiplostim. The components of culture medium II include: basal platelet-producing culture components, which are: basal culture medium, nutritional and metabolic components, and platelet-producing factors. The basal culture medium is selected from the following group: IMDM, α-MEM, DMEM / F12, RPMI-1640. The nutritional and metabolic components include: fetal bovine serum (FBS), GlutaMAX, insulin-transferrin-sodium selenite complex (ITS), 1-thioglycerol, and ascorbic acid. The platelet-producing factors are: stem cell factor (SCF), thrombopoietin (TPO), Y27632, KP-457, and SR-1. S3. Harvest the culture after step S2 and separate the platelets from it; Step S1 involves culturing under low shear stress conditions, wherein the low shear stress conditions are: the shaking speed is 10~30 rpm; and step S2 involves culturing under high shear stress conditions, wherein the high shear stress conditions are: the shaking speed is 50~70 rpm. The incubation period for step S1 is 1-5 days; and the incubation period for step S2 is 2-8 days.

2. The method as described in claim 1, characterized in that, In culture medium I, the concentration of Stiripentol is 10 μM; and the concentration of Romiplostim is 50 ng / ml.

3. The method as described in claim 1, characterized in that, The components of culture medium I are: IMDM medium, 0.1-5% (v / v) FBS, 0.5-5 mM GlutaMAX, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 50-150 ng / ml Flt3L, 20-60 ng / ml TPO, 5-30 ng / ml IL-6, 0.1-10 μM SR-1, 100-300 μM VPA, 5-15 μM Stiripentol, and 40-60 ng / ml Romiplostim.

4. The method as described in claim 3, characterized in that, The components of culture medium I are: IMDM medium, 1% (v / v) FBS, 2 mM GlutaMAX, 1% (v / v) ITS, 0.45 mM 1-thioglycerol, 50 μg / ml ascorbic acid, 50 ng / ml SCF, 100 ng / ml Flt3L, 40 ng / ml TPO, 15 ng / ml IL-6, 1 μM SR-1, 200 μM VPA, 10 μM Stiripentol and 50 ng / ml Romiplostim.

5. The method as described in claim 4, characterized in that, The components of culture medium II are: IMDM medium, 0.1-5% (v / v) FBS, 0.5-5 mM GlutaMAX, 0.1-5% (v / v) ITS, 0.2-0.8 mM 1-thioglycerol, 20-80 μg / ml ascorbic acid, 20-100 ng / ml SCF, 0.1-0.5 μg / ml TPO, 1-20 μM Y27632, 5-25 μM KP-457, and 0.1-2 μM SR-1.

6. The method as described in claim 5, characterized in that, The culture period for step S1 is 2-4 days; and / or the culture period for step S2 is 3-6 days.

7. The method as described in claim 1, characterized in that, The incubation period for step S1 is 3 days; and / or, the incubation period for step S2 is 3 days.

8. The method as described in claim 1, characterized in that, Step S1 involves culturing under low shear stress conditions, wherein the low shear stress conditions are: the shaking speed is 20 rpm; and step S2 involves culturing under high shear stress conditions, wherein the high shear stress conditions are: the shaking speed is 60 rpm.

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