A method for producing platelets based on biomimetic microcarriers

Abstract

This invention relates to the field of platelet production technology, and in particular to a method for producing platelets based on biomimetic microcarriers. The method overcomes the key bottlenecks of low in vitro platelet yield, incomplete function, and difficulty in large-scale production in the prior art. By constructing a biomimetic bone marrow-vascular wall microenvironment, it systematically solves the technical problem of low yield of megakaryocytes in in vitro culture systems, and provides a brand-new solution for the safe, efficient, and economical in vitro preparation of platelets.

Description

Technical Field

[0001] This invention relates to the field of platelet production technology, and in particular to a method for producing platelets based on biomimetic microcarriers. Background Technology

[0002] Platelet transfusion is a key treatment for malignant hematological diseases, postoperative massive bleeding, and thrombocytopenia after chemotherapy. However, natural platelets have risks such as donor limitations, short storage period (5-7 days), infection risk, and immune reactions. To overcome these drawbacks, in vitro artificial platelet synthesis technology has emerged, aiming to create a readily available, quality-controlled, and functionally safe alternative product through bioengineering methods.

[0003] Among these, artificial platelet technology based on stem cell technology is considered the most promising direction. This technology utilizes human pluripotent stem cells (such as human induced pluripotent stem cells, iPSCs) to produce platelets with complete biological functions on a large scale through directed differentiation, highly mimicking the in vivo platelet generation process. Specifically, firstly, specific regulatory genes are introduced into hematopoietic progenitor cells differentiated from iPSCs, causing them to differentiate into megakaryocyte progenitor cells, and further establishing an immortalized megakaryocyte precursor cell line (imMK) that can proliferate indefinitely. During culture, mature imMK cells extend slender proplatelet protrusions. These cells are placed in a bioreactor, and by applying external fluid shear force, the protrusions break off and release platelet-like particles, achieving in vitro generation.

[0004] Although the production system has been initially established, it still faces technical challenges in areas such as scaling up and cost control, functional integrity, safety, and regulation. Particularly concerning economic feasibility, a single treatment dose requires 3 × 10⁻⁶ doses. 11 Artificial platelets require large bioreactors and expensive culture media, resulting in high costs. Therefore, significantly improving yield and effectively controlling costs to make artificial platelets price-competitive is the biggest challenge for industrialization.

[0005] Unlike traditional cell culture, which typically requires a mild, low-shear environment, imMK platelet production relies on mechanical stimulation from an external turbulent environment. This means that conventional cell production processes cannot be directly applied. The key to commercialization lies in scaling up. Although cell lines can be expanded indefinitely, achieving industrial-scale production of trillions of platelets while keeping costs within acceptable limits is the core challenge for its success. Megakaryon, a leading company in the field, has completed safety testing of its artificial platelets and is currently actively developing large-scale bioreactors to optimize the platelet production process. Their Versus bioreactor generates turbulence to promote platelet production through the axial reciprocating motion of baffles. While it has verified its feasibility at the laboratory scale, significant yield improvements are still difficult to achieve when scaled up to production.

[0006] It is noteworthy that imMK is cultured in suspension in the reactor, while the development of natural MK primarily occurs within the complex bone marrow microenvironment. In vivo, megakaryocyte progenitors undergo multi-stage differentiation, migrate to the perivascular region, interact with endothelial cells, and elongate their cytoplasm to form platelet precursors. These platelet precursors extend into the bone marrow sinusoids, where they break down under the influence of blood turbulence and shear forces, ultimately releasing mature platelets into the bloodstream. This difference between in vivo and in vitro microenvironments may be the reason for the significant differences in platelet yield. Therefore, there is an urgent need to develop a method to simultaneously improve platelet quality and yield during mass production to overcome current technological bottlenecks. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for producing platelets based on biomimetic microcarriers, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention provides a method for producing platelets based on biomimetic microcarriers, the method comprising the following steps:

[0009] 1) Microcarriers and attachment factors were co-incubated in buffer solution to functionalize the surface of the microcarriers and obtain biomimetic microcarriers;

[0010] 2) Immortalized megakaryocyte precursor cells (imMK) were seeded onto biomimetic microcarriers and cultured to allow the cells to adhere, mature, and form an imMK cell-microcarrier complex.

[0011] 3) The imMK cell-microcarrier complex is placed in a bioreactor, and fluid shear force is applied to induce megakaryocytes to release platelet-like particles, thereby obtaining the platelets.

[0012] As described above, the method for producing platelets based on biomimetic microcarriers of the present invention has the following beneficial effects:

[0013] (1) Commercial microcarrier raw materials are used, which are widely available, inexpensive, and easy to operate, providing a stable, repeatable and easily convertible engineering platform for the process scale-up and large-scale manufacturing of in vitro platelet production;

[0014] (2) By constructing a biomimetic three-dimensional microenvironment, the conversion efficiency of megakaryocytes to platelets and the product yield in vitro have been effectively improved, directly solving the bottleneck of low yield in existing technologies and laying a key foundation for achieving economically feasible large-scale production.

[0015] (3) The simulation of the natural bone marrow-vascular wall physiological microenvironment prompts imMK cells to complete the maturation and release process in a state closer to the physiological state in the body. The platelet-like particles produced show a higher similarity to natural platelets in terms of size distribution, morphology and structure and functional characteristics.

[0016] (4) This invention integrates cell expansion, functional maturation and product release into a coherent microcarrier culture system, which reduces cell transfer and loss in traditional multi-step processes, making the operation simpler and the process more stable. Attached Figure Description

[0017] Figure 1 The diagram shown is of the microcarrier used in the embodiments of the present invention.

[0018] Figure 2 This diagram illustrates how cells bind to microcarriers via adhesion factors.

[0019] Figure 3 The graph shows the attachment efficiency of ImMK cells and microcarriers within 24 hours of co-incubation.

[0020] Figure 4 The image shows DAPI staining characterizing the cell attachment status on the microcarrier surface.

[0021] Figure 5 The graph shows a comparison of platelet yield between the method of this invention and the traditional suspension culture method.

[0022] Figure 6 The diagram shows the particle size distribution of platelets produced by the method of this invention and the conventional suspension culture method.

[0023] Figure 7 The image shows a flow cytometry analysis of surface markers in platelets produced by the method of this invention and by a conventional suspension culture method.

[0024] Figure 8 The image shows a comparison of the contractile function of platelets produced by the method of this invention and the traditional suspension culture method after in vitro activation. Detailed Implementation

[0025] Traditionally, MK cells, due to their inherent suspension growth characteristics, are typically cultured in direct suspension. Microcarrier technology, as a standard method for large-scale culture of adherent cells, is primarily geared towards cell types that require surface attachment for proliferation. This invention, through in-depth research, is the first to discover and successfully apply a porous microcarrier system to the culture system of MK cells—a typical suspension cell type. This application, by providing MK cells with a three-dimensional biomimetic attachment interface, not only achieves high-density, large-scale cell culture, but more importantly, the porous structure and surface modification of the microcarrier actively guide the polarization maturation of MK cells and the directional formation of platelet precursors, thus fundamentally solving the bottleneck of low platelet yield in traditional suspension culture.

[0026] Based on the above, the present invention first provides a method for producing platelets based on biomimetic microcarriers, the method comprising the following steps:

[0027] 1) Microcarriers and attachment factors were co-incubated in buffer solution to functionalize the surface of the microcarriers and obtain biomimetic microcarriers;

[0028] 2) Immortalized megakaryocyte precursor cells (imMK) were seeded onto biomimetic microcarriers and cultured to allow the cells to adhere, mature, and form an imMK cell-microcarrier complex.

[0029] 3) The imMK cell-microcarrier complex is placed in a bioreactor, and fluid shear force is applied to induce megakaryocytes to release platelet-like particles, thereby obtaining the platelets.

[0030] In some embodiments of the present invention, in step 1), the particle size of the microcarrier is 100~300 μm.

[0031] In some embodiments of the present invention, in step 1), the average pore size of the microcarrier is 10~100 μm.

[0032] In some embodiments of the present invention, in step 1), the porosity of the microcarrier is 80-99%.

[0033] In some embodiments of the present invention, in step 1), the microcarrier is selected from spherical microcarriers or sheet-like microcarriers. In a specific embodiment of the present invention, the microcarrier is a spherical microcarrier, purchased from Huakan Biotechnology Co., Ltd. (product number: W01-200), which can be found in the following details. Figure 1 .

[0034] The microcarriers described in this invention refer to particles with suitable three-dimensional porous structures that can be used for three-dimensional cell attachment culture. Their core functions are: 1) providing a biomimetic scaffold: providing a growth interface for cells (such as the imMK cells of this invention) that mimics the three-dimensional structure of in vivo tissues; 2) mimicking the physiological microenvironment: through their specific pore structure and specific chemical or biological properties after surface modification, simulating the natural microenvironment of target cells in vivo (such as simulating the microenvironment of bone marrow sinusoids / vascular walls in this application); 3) enabling large-scale culture: maintaining a suspended state in a bioreactor to achieve high-density, large-scale cell culture.

[0035] In addition, in specific embodiments of the present invention, the microcarrier preferably has one or more of the following characteristics: 1) Material: It can be a natural or synthetic polymer material, such as gelatin, dextran, chitosan, polylactic acid-glycolic acid copolymer (PLGA), polystyrene, etc.; 2) Structure: It has a porous structure with an average pore size of 10~100 μm; 3) Size: The particle size is 100~300 μm; 4) Surface properties: The surface can be chemically or biologically modified.

[0036] It should be noted that the specific parameters of the above features (such as pore size and material) are exemplary. Any microcarrier capable of achieving the above-described biomimetic bone marrow vascular microenvironment function falls within the scope of this invention.

[0037] In some embodiments of the present invention, in step 1), the ratio of the microcarrier to the adhesion factor is 10~100 mg adhesion factor / g microcarrier.

[0038] In some embodiments of the present invention, in step 1), the buffer solution is selected from PBS buffer, DPBS buffer or HBSS buffer.

[0039] Furthermore, the pH of the PBS buffer is 7.0 to 7.6.

[0040] Furthermore, the pH of the DPBS buffer is 7.0 to 7.6.

[0041] Furthermore, the pH of the HBSS buffer solution is 7.0 to 7.6.

[0042] In some embodiments of the present invention, in step 1), the concentration of the microcarrier is 2-5 g / L based on the volume of the buffer solution. The concentration of the microcarrier is selected from any of the following ranges based on the volume of the buffer solution: 2-3 g / L, 3-4 g / L, or 4-5 g / L. In a specific embodiment of the present invention, the concentration of the microcarrier is 4 g / L based on the volume of the buffer solution.

[0043] In some embodiments of the present invention, in step 1), the adhesion factor is selected from one or more of fibrinogen, poly-L-lysine, laminin, hyoidin, or von Willebrand factor.

[0044] In some embodiments of the present invention, in step 1), the final concentration of fibrinogen is 150-250 μg / mL, based on the volume of the buffer solution. The final concentration of fibrinogen, based on the volume of the buffer solution, is selected from any of the following ranges: 150-170 μg / mL, 170-190 μg / mL, 190-210 μg / mL, 210-230 μg / mL, or 230-250 μg / mL. In a specific embodiment of the present invention, the final concentration of fibrinogen is 200 μg / mL, based on the volume of the buffer solution.

[0045] In some embodiments of the present invention, in step 1), the final concentration of poly-L-lysine is 50~200 μg / mL, based on the volume of the buffer solution. In a specific embodiment of the present invention, the final concentration of poly-L-lysine is 100 μg / mL, based on the volume of the buffer solution.

[0046] In some embodiments of the present invention, in step 1), the co-incubation temperature is 0~8°C. In a specific embodiment of the present invention, the co-incubation temperature is 4°C.

[0047] In some embodiments of the present invention, in step 1), the co-incubation time is 10 to 14 hours. In a specific embodiment of the present invention, the co-incubation time is 12 hours.

[0048] In some embodiments of the present invention, in step 2), the ratio of imMK cells to microcarriers is 5 × 10⁻⁶. 7 ~5×10 8 cells / g microcarriers.

[0049] In some embodiments of the present invention, in step 2), the culture is carried out in an amplification medium. Further, based on the volume of the amplification medium, the inoculation density is 3~8 × 10⁻⁶. 5 cells / mL.

[0050] In some embodiments of the present invention, the amplification medium comprises RPMI 1640 basal medium + 5%~20% fetal bovine serum (FBS).

[0051] In a specific embodiment of the present invention, the amplification culture medium comprises RPMI 1640 basal medium + 10% fetal bovine serum (FBS).

[0052] In some embodiments of the present invention, in step 2), a cycle of alternating stirring and settling is used to allow cells to adhere. Further, the stirring speed is 30-45 rpm, the stirring time is 10-20 minutes, and the settling time is 40-50 minutes. In a specific embodiment of the present invention, one cycle consists of stirring at 40 rpm for 15 minutes followed by settling for 45 minutes.

[0053] In some embodiments of the present invention, in step 2), after the imMK cells are cultured on the biomimetic microcarrier for 20-28 hours, they are transferred to the bioreactor for further culture. The culture time of the imMK cells on the biomimetic microcarrier is selected from any of the following ranges: 20-22 hours, 22-24 hours, 24-26 hours, and 26-28 hours.

[0054] In some embodiments of the present invention, in step 3), the imMK cells are cultured in the bioreactor for 5 to 8 days. In a preferred embodiment of the present invention, the imMK cells are cultured in the bioreactor for 6 to 7 days.

[0055] In some embodiments of the present invention, in step 3), the fluid shear force is 0.05~2.0 Pa.

[0056] In some embodiments of the present invention, step 3) further includes collecting and purifying the platelet-like particles.

[0057] The collection process is carried out using gradient centrifugation. Specifically, low-speed centrifugation (e.g., 100-500g, 5-15 minutes) is used to precipitate microcarriers and larger cell debris, while platelet-like particles are retained in the supernatant; then the supernatant is centrifuged at a higher speed (e.g., 800-2000g, 15-30 minutes) to precipitate and obtain crude platelet-like particles.

[0058] The purification is performed using one or more methods, such as density gradient centrifugation, flow cytometry, or immunomagnetic bead sorting based on specific surface markers (e.g., CD41 / CD42b).

[0059] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0061] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0062] Example

[0063] 1.1 Preparation and Pretreatment of Bionic Microcarriers

[0064] 1) Weigh an appropriate amount of microcarriers with a pore size of about 10~50 μm and a porosity of >90% (purchased from Huakan Biotechnology Co., Ltd., product number: W01-200), and soak them in DPBS buffer (pH 7.2) at a concentration of 4 g / L to obtain a microcarrier suspension;

[0065] 2) Add fibrinogen and poly-L-lysine to the microcarrier suspension and incubate at 4°C for 12 h to coat the microcarrier. Based on the volume of the microcarrier suspension, the final concentration of fibrinogen is 200 μg / mL and the concentration of poly-L-lysine is 100 μg / mL.

[0066] 3) After incubation, centrifuge to remove the supernatant coating solution, and wash the coated microcarriers three times with DPBS buffer to fully remove unbound fibrinogen and poly-L-lysine.

[0067] 4) After cleaning, the microcarriers are resuspended in amplification medium (specifically, RPMI 1640 basal medium + 10% fetal bovine serum FBS) and transferred to a roller bottle for later use.

[0068] 1.2 Cell seeding and microcarrier attachment

[0069] 1) Take imMK cells, centrifuge, wash, and transfer them to a roller bottle containing pretreated microcarriers. The seeding density is 5 × 10⁶ cells / ml, based on the volume of the amplification medium in the roller bottle. 5cells / mL;

[0070] 2) Turn on the rotary bottle machine and use a cycle mode of stirring at 40 rpm for 15 min and then letting it stand for 45 min to promote uniform cell adhesion to the surface of the microcarrier.

[0071] 3) 24 h after inoculation, after confirming good cell adhesion, the imMK cell-microcarrier complex was transferred into the bioreactor and cultured for another 6 days for platelet production.

[0072] 1.3 Platelet production and collection

[0073] In a bioreactor, cells gradually mature under the three-dimensional structural support and biochemical signal induction provided by biomimetic microcarriers, extending protoplatelet processes. A fluid shear field (1.0 Pa) established within the bioreactor induces the processes to break down, releasing platelet-like particles. After culture, the culture supernatant containing platelet-like particles is collected and purified through steps such as low-speed centrifugation to obtain the platelet product.

[0074] Example 1

[0075] After inoculation, ImMK cells are linked to microcarriers via adhesion factors fibrinogen and poly-L-lysine, such as... Figure 2 As shown. The porous structure of the microcarrier provides structural support for cells while facilitating the production of platelet precursors. The adhesion efficiency of ImMK cells co-incubated with the microcarrier within 24 hours is as follows. Figure 3 As shown, after inoculation, the cells were stained with DAPI, as follows: Figure 4 As shown, imMK cells successfully adhered to the surface of the microcarrier.

[0076] Example 2

[0077] 2.1 To verify the effectiveness of this invention, the following two groups were established:

[0078] Experimental group (inoculated with microcarriers): The method described in the examples was used;

[0079] Control group (suspension culture): Traditional direct suspension culture method was used. Specifically, imMK cells were directly cultured at the same density (5 × 10⁶ cells / year). 5 (cells / mL) were inoculated into the bioreactor and cultured in suspension for 6 days under the same conditions.

[0080] 2.2 Detection and Characterization Methods

[0081] Platelet count (yield assay): The number of platelets is counted using a cell counter.

[0082] Particle size analysis: The diameter distribution of platelet-like particles was detected using a laser particle size analyzer.

[0083] Surface marker analysis: The expression of CD41 (platelet-specific marker) and CD42b (platelet activation-related marker) was detected by flow cytometry.

[0084] Functional verification: Platelet contraction function was observed after activation with thrombin.

[0085] 2.3 Experimental Results

[0086] like Figure 5 As shown, after 6 days of culture, the platelet yield in the control group was approximately 32 Plts / MKs, meaning that each imMK cell produced an average of 32 platelets. The platelet yield in the experimental group reached 70 Plts / MKs. The method of this invention can increase the in vitro platelet yield of imMK by more than 2 times.

[0087] like Figure 6 As shown, the platelet particles produced using the method of the present invention have a similar particle size distribution to platelets prepared by conventional methods, indicating that the method of the present invention does not affect the normal morphological formation of platelets.

[0088] like Figure 7 Flow cytometry showed that both groups of products had high CD41 levels. + CD42b + The expression rate showed a similar distribution pattern in the experimental group to that in the control group, indicating that the platelets produced by the method of the present invention maintained key surface membrane protein characteristics.

[0089] like Figure 8 As shown, after thrombin activation, platelets from both the experimental and control groups exhibited significant aggregation and contraction. This indicates that platelets prepared using the method of this invention possess complete activation and contraction biological functions, consistent with the key physiological characteristics of natural platelets.

[0090] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for producing platelets based on biomimetic microcarriers, characterized in that, The method includes the following steps: 1) Microcarriers and attachment factors were co-incubated in buffer solution to functionalize the surface of the microcarriers and obtain biomimetic microcarriers; 2) ImMK cells were seeded onto biomimetic microcarriers and cultured to allow the cells to adhere, mature, and form an imMK cell-microcarrier complex. 3) The imMK cell-microcarrier complex is placed in a bioreactor, and fluid shear force is applied to induce megakaryocytes to release platelet-like particles, thereby obtaining the platelets.

2. The method according to claim 1, characterized in that, In step 1), the average pore size of the microcarrier is 10~100 μm; And / or, the particle size of the microcarrier is 100~300 μm; And / or, the porosity of the microcarrier is 80-99%; And / or, the microcarrier is selected from spherical microcarriers or sheet-like microcarriers.

3. The method according to claim 1, characterized in that, In step 1), the ratio of the microcarrier to the adhesion factor is 10~100 mg adhesion factor / g microcarrier; And / or, the buffer is selected from PBS buffer, DPBS buffer or HBSS buffer; And / or, based on the volume of the buffer solution, the concentration of the microcarrier is 2~5 g / L.

4. The method according to claim 1, characterized in that, In step 1), the adhesion factor is selected from one or more of fibrinogen, poly-L-lysine, laminin, hyalin, or von Willebrand factor.

5. The method according to claim 4, characterized in that, Based on the volume of the buffer solution, the final concentration of fibrinogen is 150~250 μg / mL; And / or, based on the volume of the buffer solution, the final concentration of the poly-L-lysine is 50~200 μg / mL.

6. The method according to claim 1, characterized in that, In step 1), the co-incubation temperature is 0~8℃; And / or, the co-incubation time is 10 to 14 hours.

7. The method according to claim 1, characterized in that, In step 2), the ratio of imMK cells to microcarriers is 5 × 10⁻⁶. 7 ~5×10 8 cells / g microcarriers; And / or, the culture is carried out in an amplification medium; preferably, the inoculation density is 3~8×10⁻⁶ based on the volume of the amplification medium. 5 cells / mL; And / or, use a cyclical pattern of alternating stirring and settling to allow cells to adhere; And / or, after the imMK cells are cultured on the biomimetic microcarrier for 20-28 hours, they are transferred to the bioreactor for further culture.

8. The method according to claim 7, characterized in that, The stirring speed is 30~45 rpm; And / or, the stirring time is 10-20 minutes; And / or, the settling time is 40 to 50 minutes.

9. The method according to claim 1, characterized in that, In step 3), the imMK cells are cultured in the bioreactor for 5-8 days; And / or, the fluid shear force is 0.05~2.0 Pa.

10. The method according to claim 1, characterized in that, Step 3) also includes collecting and purifying the platelet-like particles.

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