A method for preparing artificial platelets based on microfluidic technology

Abstract

The application belongs to the technical field of blood products, and particularly relates to a method for preparing artificial platelets based on microfluidic technology, wherein the artificial platelets are prepared by iPSCs after being differentiated into megakaryocytes, the HLA gene of the iPSCs is knocked out by Cas9 D10A nicking enzyme technology to reduce the probability of immune rejection, a series of optimized culture media are used to induce the differentiation of stem cells into EMPs and megakaryocytes, the megakaryocytes are efficiently driven to generate and release functional platelets in a microfluidic bioreactor simulating the physiological shear force of the body marrow sinusoidal space, and a general platelet product which can be produced on a large scale, has controllable quality and can be used on demand is provided.

Description

Technical Field

[0001] This invention belongs to the field of blood products technology, specifically relating to a method for preparing artificial platelets based on microfluidic technology. Background Technology

[0002] Platelets, a key component of blood, play an indispensable role in maintaining coagulation function and immune regulation. Especially in emergency medical treatments such as leukemia and massive blood loss, timely platelet transfusions directly impact patient survival rates. Currently, clinical platelet supply relies entirely on donations. This traditional method of platelet acquisition is constrained by donor willingness, physical condition, and seasonal variations, leading to unstable supply. Furthermore, platelet units have a short shelf life due to bacterial contamination and storage issues, and are easily wasted during transportation and storage, further exacerbating the shortage. With rising demand for blood products, the existing supply system struggles to balance the increasing demand for platelets. To overcome this supply-demand bottleneck, more and more research is exploring artificial platelet substitutes or in vitro culture methods, but these attempts have yielded limited success. Traditional in vitro culture techniques cannot simulate the complex physiological environment of human bone marrow, particularly the crucial influence of vascular shear stress on platelet production, resulting in low platelet yield and functional deficiencies, failing to meet clinically required quantity and quality standards. Microfluidics, as an emerging tool, can simulate the in vivo shear stress environment by precisely controlling fluid dynamics conditions, providing a new approach to platelet production. At the same time, the emergence of iPSCs (induced pluripotent stem cells) technology makes it possible to obtain pluripotent stem cells from adult cells through reprogramming and differentiate them into megakaryocytes, theoretically providing an unlimited source of platelet cells.

[0003] However, current technologies have not been able to effectively integrate the microfluidic simulation environment with the iPSC differentiation process, lacking an efficient and controllable system for large-scale platelet production, thus failing to fundamentally solve the platelet shortage problem. Therefore, developing a novel preparation method based on microfluidic technology to overcome the shortcomings of existing technologies has become an urgent need for the industry. Summary of the Invention

[0004] To address the above issues, this invention provides a method for preparing artificial platelets based on microfluidic technology. The method utilizes Cas9 D10A nickase technology to knock out the HLA gene in iPSCs to resolve immune rejection problems. Optimized culture media are used to induce differentiation into erythroid-myeloid progenitor cells (EMPs) and megakaryocytes. In a constructed microfluidic bioreactor, megakaryocytes are efficiently driven to generate and release functional platelets, providing a scalable, quality-controlled, and on-demand universal platelet product for clinical transfusion.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for preparing artificial platelets based on microfluidic technology, wherein the artificial platelets are prepared by iPSCs differentiating into megakaryocytes via a microfluidic bioreactor, specifically including the following steps:

[0007] Step 1: After activating and culturing iPSCs, gene editing was performed on them using Cas9 D10A nicking enzyme technology to screen for HLA (human leukocyte antigen) knockout iPSCs. The HLA knockout iPSCs were then induced and cultured using culture medium 1 and culture medium 2 to obtain EMPs (erythroid-myeloid progenitor cells).

[0008] Step 2: Culture EMPs in differentiation medium and collect megakaryocytes;

[0009] Step 3: Megakaryocytes were cultured using mature culture medium and amplification culture medium to promote megakaryocyte proliferation and the generation and release of platelets. Platelets were collected in a microfluidic bioreactor to simulate the in vivo shear stress environment.

[0010] Step 4: Perform functional activity quality testing on the collected platelets to obtain artificial platelets that meet the usage standards.

[0011] Furthermore, the culture medium 1 comprises the following raw materials: BMP4 (bone morphogenetic protein 4), WNT3A, VEGF (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2) and IMDM basal medium, wherein the concentrations of BMP4, WNT3A, VEGF and FGF2 are 25 ng / mL, 25 ng / mL, 10 ng / mL and 10 ng / mL, respectively.

[0012] Furthermore, the culture medium 2 comprises the following raw materials: SCF (stem cell factor), TPO (thrombopoietin), Flt3L (Fms-like tyrosine kinase 3 ligand), VEGF, FGF2 and IMDM basal medium, wherein the concentrations of SCF, TPO, Flt3L, VEGF and FGF2 are 60 ng / mL, 30 ng / mL, 10 ng / mL, 50 ng / mL and 10 ng / mL, respectively.

[0013] Furthermore, the differentiation medium includes the following raw materials: SCF, TPO, IL-11 (interleukin-11) and IMDM basal medium, wherein the concentrations of SCF, TPO and IL-11 are 20 ng / mL, 50 ng / mL and 20 ng / mL, respectively.

[0014] Furthermore, the maturation culture medium comprises the following raw materials: 2-mercaptoethanol, bovine insulin, iron-free human transferrin, sodium selenite, bovine serum albumin, linoleic acid, SCF, TPO, and IMDM basal medium, wherein the concentrations of 2-mercaptoethanol, bovine insulin, iron-free human transferrin, sodium selenite, bovine serum albumin, linoleic acid, SCF, and TPO are 50 μM, 10 μg / mL, 5.5 μg / mL, 5 ng / mL, 0.5 mg / mL, 4.7 μg / mL, 50 ng / mL, and 20 ng / mL, respectively.

[0015] Furthermore, the amplification medium consists of PFHM-II, SCF, TPO, and StemDiff. TM APEL TM 2. The culture medium composition, wherein the concentrations of PFHM-II, SCF and TPO are 5% (volume fraction), 50 ng / mL and 50 ng / mL, respectively.

[0016] Furthermore, the microfluidic bioreactor includes two inlet channels, a fluid resistor, a central channel, and two outlet channels. The inlet channels are equipped with passive filters for capturing bubbles and dust.

[0017] Furthermore, the microfluidic bioreactor is made of PDMS (polydimethylsiloxane) and a glass slide bonded together.

[0018] The beneficial effects achieved by this invention are as follows:

[0019] This invention provides a method for preparing artificial platelets based on microfluidic technology. It deeply integrates CRISPR / Cas9 D10A nickase gene editing technology, iPSC-directed differentiation technology, and microfluidic bioreactor technology to achieve an industrial-scale preparation process from cell source to platelet product. β2-microglobulin, as the β-chain (light chain) portion of HLA, is a crucial factor influencing platelet immune rejection. Through targeted editing and precise knockout, low-immunogenic, HLA-knockout universal iPSC seed cells were successfully prepared, reducing the immune rejection barrier during infusion. Then, through a series of optimized cell culture media, EMPs and functionally mature megakaryocytes were obtained efficiently and stably. Furthermore, a self-designed microfluidic bioreactor precisely simulates the physiological shear stress microenvironment of the bone marrow sinusoidal space in vivo, which is impossible to achieve with traditional static culture. This greatly promotes the efficient extension and release of proplatelets, resulting in a qualitative leap in platelet production and function.

[0020] The technical solution for industrial platelet preparation provided by this invention eliminates the absolute dependence on traditional donors, avoids the quality risks and supply instability caused by collection and storage, and forms a new model for large-scale and controllable preparation of high-quality platelets. It provides an efficient solution for addressing the increasingly severe blood demand challenges and improving the emergency response capabilities for major public health events. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the microfluidic bioreactor designed according to the present invention;

[0022] Figure 2 This is a flowchart illustrating the preparation of artificial platelets based on microfluidic technology according to the present invention;

[0023] Figure 3 The results of optical turbidimetric assay for artificial platelets;

[0024] Figure 4 The results are from flow cytometry analysis of artificial platelets. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0027] Unless otherwise specified, all methods described in the following examples are conventional; unless otherwise specified, all materials used in the following examples are new materials purchased from the market. The structure of the microfluidic bioreactor is as follows: Figure 1 As shown; the microfluidic bioreactor is constructed by bonding customized PDMS and glass slides; the process of iPSCs differentiating into megakaryocytes and releasing and collecting platelets is as follows. Figure 2 As shown.

[0028] Example 1: iPSC gene editing

[0029] A1: Using the Cas9 Nickase (D10A) NLS gene editing kit, take a Matrigel-coated 24-well culture plate and incubate it at room temperature for 30 min. Add 1 mL of plating medium to each well and place it in a cell culture incubator to obtain an incubation plate.

[0030] A2: Using beta-2-microglobulin as the keyword, the amino acid sequence of HLA light chain β2-microglobulin and its encoding gene were searched on the NCIB search website. crRNA was designed and synthesized targeting the encoding gene. 200 μM crRNA (4 μL), 200 μM tracrRNA (4 μL) and annealing buffer (2 μL) were mixed in a microcentrifuge tube and incubated at 95℃ for 5 min in a thermal cycler, and then incubated at 60℃ for 1 min to obtain gRNA targeting β2-microglobulin at a concentration of 80 μM.

[0031] A3: Mix gRNA (0.56 μL), 1 μM Cas9 Nickase (D10A) NLS (0.90 μL) and resuspension buffer (3.54 μL), and incubate at room temperature for 20 min to obtain the RNP complex;

[0032] A4: Take ATCC-DYS0100 (iPSCs) in the logarithmic growth phase, digest and decellularize them, add cell buffer, gently pipette to prepare a cell suspension, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend the pellet in single-cell culture medium and count the cells, then dilute to a cell concentration of 3 × 10⁻⁶ cells / mL. 5 Single cells / mL were obtained to form a single-cell suspension.

[0033] A5: Centrifuge 1 mL of single-cell suspension at 800 rpm for 5 min, discard the supernatant, add 7.5 μL of resuspension buffer to the pellet to resuspend the cells evenly, then add 7.5 μL of RNP complex, mix well, and place in an electroporation chamber containing 3 mL of electrolysis buffer for electroporation to obtain transfected cells.

[0034] A6: Transfected cells were transferred to incubation plates and incubated in a cell culture incubator for 72 h. The medium was changed every 24 h using 0.5 mL of preheated plate-laying medium at room temperature. After incubation, cells were collected and cell gene sequencing was performed to obtain HLA knockout iPSCs.

[0035] Example 2: Megakaryocyte Differentiation

[0036] B1: Weigh 0.025 mg BMP4, 0.025 mg WNT3A, 0.01 mg EGF and 0.01 mg FGF2 and dissolve them in 1000 mL IMDM basal medium to prepare medium 1. Weigh 0.06 mg SCF, 0.03 mg TPO, 0.01 mg Flt3L, 0.05 mg VEGF and 0.01 mg FGF2 and dissolve them in 1000 mL IMDM basal medium to prepare medium 2.

[0037] B2: After centrifuging HLA knockout iPSCs at 800 rpm for 5 min, resuspend them in 5 mL of medium 1, seed them in cell culture flasks, and incubate them in a cell culture incubator for 2 days, changing half of the medium every day. On the 3rd day, change the medium to medium 2 for induction culture and continue culturing for 6 days, changing half of the medium every day to obtain EMPs.

[0038] B3: Weigh 0.02 mg SCF, 0.05 mg TPO and 0.02 mg IL-11 and dissolve them in 1000 mL IMDM basal medium to obtain differentiation medium. Culture EMPs in differentiation medium for 5 days, changing half the medium daily to obtain megakaryocytes.

[0039] Example 3: Platelet production

[0040] C1: Weigh 3.9 mg 2-mercaptoethanol, 10 mg bovine insulin, 5.5 mg iron-free human transferrin, 0.005 mg sodium selenite, 0.5 g bovine serum albumin, 4.7 mg linoleic acid, 0.05 mg SCF and 0.02 mg TPO and dissolve them in 1000 mL IMDM basal medium to prepare mature culture medium;

[0041] C2: Dissolve 5 mL of PFHM-II, 0.05 mg of SCF, and 0.05 mg of TPO in 995 mL of StemDiff solution. TM APEL TM 2. Prepare an amplification medium in the culture medium;

[0042] C3: Megakaryocytes were cultured in maturation medium for 11 days, with the medium changed daily. When the cells reached approximately 80% confluence with the flask wall, they were passaged. On day 12, the medium was replaced with amplification medium. The cells were then subjected to a microfluidic bioreactor simulating in vivo shear stress. The microfluidic bioreactor was pretreated with 0.22 mm filtration and 10% bovine serum albumin solution for 30 min to prevent direct contact between the cells and the glass slide. Megakaryocytes were injected into the microfluidic bioreactor at a rate of 12.5 mL / h through two inlet channels using a dual-injection microfluidic pump. After passing through a passive filter to capture air bubbles and dust, the cells passed through a fluid resistor used to suppress flow fluctuations during chip operation. The megakaryocyte solution injected from the two inlet channels converged at a central channel 1300 mm long and 130 mm wide. This central channel consisted of a series of 10 mm wide, 90 mm long, and spaced 2... The columnar separations at μm can simulate the in vivo shear stress environment. Megakaryocytes are trapped in the central channel when passing through, while the released platelets enter the outflow channel through the gaps and collect the outflow at the end of the outflow channel.

[0043] C4: Platelets in the effluent are detected by optical turbidimetry and flow cytometry to obtain artificial platelets that meet clinical use standards.

[0044] Optical turbidimetric assay: Platelet aggregation ability was assessed using a platelet aggregometer. Functional activity was evaluated by monitoring changes in transmittance caused by platelet aggregation. Artificial platelets were formulated to a size of 2.5 × 10⁻⁶. 8 Standard suspensions of platelets / mL were used, with agonists selected as 2 μM ADP, 2 μg / mL collagen, and 5 μM TRAP-6 (thrombin receptor activating peptide). 450 μL of platelet suspension was incubated at 37℃ for 2 min, followed by the addition of 50 μL of each of the three agonists. Transmittance changes were continuously recorded, and the maximum aggregation rate was calculated. Results are shown below. Figure 3 .

[0045] Flow cytometry was used to investigate the effects of resting and ADP-activated artificial platelet samples. These samples were incubated with antibodies in the dark for 15 min, resuspended in PBS, and analyzed by flow cytometry. The detection parameters included CD62P, GPⅡb / Ⅲa, and [missing information - likely related to antibody incubation]. See results Figure 4 .

[0046] Figure 3 The results showed that the aggregation rate of artificial platelets was higher than 75% under the treatment of the three agonists, and the coefficient of variation was much lower than 15%, indicating that the platelet volume size had good dispersion and high uniformity.

[0047] Figure 4The results showed that GPIIb / IIIa and GPIb were expressed at high levels (>97%) with high yield and purity. CD62P was expressed in the resting state, indicating that platelets were not activated in advance, which is beneficial to post-infusion circulation life. CD62P increased significantly after ADP activation, indicating its excellent activation reserve.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0049] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing artificial platelets based on microfluidic technology, characterized in that, The artificial platelets are prepared from iPSCs differentiated into megakaryocytes via a microfluidic bioreactor, specifically including the following steps: Step 1: Gene editing was performed on iPSCs to obtain HLA knockout iPSCs. Culture media 1 and culture media 2 were used to induce the formation of EMPs. Step 2: Culture EMPs in differentiation medium and collect megakaryocytes; Step 3: After culturing megakaryocytes in mature culture medium and amplification culture medium, they are transferred to a microfluidic bioreactor and platelets are collected. Step 4: Perform quality testing on the collected platelets to obtain artificial platelets; The differentiation medium contains 20 ng / mL SCF, 50 ng / mL TPO, 20 ng / mL IL-11 and IMDM basal medium; The mature culture medium contains 50 μM 2-mercaptoethanol, 10 μg / mL bovine insulin, 5.5 μg / mL iron-free human transferrin, 5 ng / mL sodium selenite, 0.5 mg / mL bovine serum albumin, 4.7 μg / mL linoleic acid, 50 ng / mL SCF, 20 ng / mL TPO and IMDM basal medium; The amplification medium contained 5% PFHM-II, 50 ng / mL SCF, 50 ng / mL TPO, and StemDiff. TM APEL TM 2. Culture medium.

2. The method for preparing artificial platelets based on microfluidic technology according to claim 1, characterized in that, The culture medium 1 contains 25 ng / mL BMP4, 25 ng / mL WNT3A, 10 ng / mL VEGF, 10 ng / mL FGF2 and IMDM basal medium; The culture medium 2 contains 60 ng / mL SCF, 30 ng / mL TPO, 10 ng / mL Flt3L, 50 ng / mL VEGF, 10 ng / mL FGF2 and IMDM basal medium.

3. The method for preparing artificial platelets based on microfluidic technology according to claim 1, characterized in that, The microfluidic bioreactor includes two inlet channels, a fluid resistor, a central channel, and two outlet channels. The inlet channels are equipped with passive filters for capturing bubbles and dust. The microfluidic bioreactor is made of PDMS and glass slides bonded together.

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