An induced pluripotent stem cell-based tissue regeneration and repair device and method

Abstract

This application discloses a tissue regeneration and repair device and method based on induced pluripotent stem cells, including a cell delivery component and an integrated sorting and processing module. The integrated sorting and processing module is a sealed cylindrical cavity with standard Luer conical connectors at both ends. The proximal Luer connector of the cavity is sealed and connected to the cell delivery component, and the distal Luer connector is sealed and connected to a minimally invasive delivery needle. This application achieves an integrated intraoperative synchronous processing architecture, integrating residual cell removal, targeted inactivation, real-time quality control, and in-situ delivery into a disposable sealed tubing module. It eliminates the need for large laboratory equipment, completing the entire process within 10 minutes, completely resolving the industry pain point of the disconnect between sorting and clinical delivery in existing systems. It is compatible with routine surgical procedures and has an extremely low operational threshold.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a tissue regeneration and repair device and method based on induced pluripotent stem cells. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) possess both multipotent differentiation potential and the advantage of autoimmune adaptation, making them the core seed cells in the field of tissue damage regeneration and repair. They have demonstrated significant clinical application value in various tissue damage repair scenarios, including myocardial, nerve, skin, and osteochondral tissues.

[0003] However, the core bottleneck in the clinical translation of iPSCs lies in the tumorigenic risk of residual undifferentiated iPSCs. Undifferentiated iPSCs have unlimited proliferative capacity, and even a small amount of residual iPSCs can form teratomas in the body. This is the core red line for global drug regulatory agencies to control iPSC cell therapy products.

[0004] Currently, the mainstream methods for clearing residual undifferentiated iPSCs in the industry mainly include flow cytometry sorting, immunomagnetic bead sorting, and gene-edited suicide gene systems. Among them, flow cytometry and magnetic bead sorting rely on large laboratory equipment, have complex and time-consuming procedures, cannot be completed in real time during surgery, and the sorting process is prone to causing a significant loss of the viability of the target differentiated cells; gene editing methods have inherent off-target effects, and the threshold for clinical compliance approval is extremely high; antibody-mediated killing methods have the risk of exogenous reagent residues and non-specific cell damage, and none of them can meet the clinical requirements for sterile, real-time, and safe operation.

[0005] Meanwhile, existing solutions cannot achieve real-time closed-loop quality control of the clearance effect during the operation, making it difficult to meet the compliance and traceability requirements of clinical translation, which seriously restricts the large-scale clinical application of iPSC tissue regeneration and repair technology.

[0006] Therefore, a tissue regeneration and repair device and method based on induced pluripotent stem cells is proposed. Summary of the Invention

[0007] This application aims to at least partially solve one of the technical problems in the aforementioned technologies.

[0008] To achieve the above objectives, the first aspect of this application proposes a tissue regeneration and repair device based on induced pluripotent stem cells, including a cell delivery component and an integrated sorting and processing module;

[0009] The integrated sorting and processing module is a closed cylindrical cavity with standard Luer conical connectors at both ends. The proximal Luer connector of the cavity is sealed and connected to the cell delivery component, and the distal Luer connector is sealed and connected to the minimally invasive delivery needle.

[0010] Inside the cavity, along the flow direction of the cell suspension, there are sequentially and coaxially sealed pre-filtration and impurity removal units, conical channel mechanical sorting units, dual-target affinity capture fine screening units, passive reciprocating vortex microfluidic ultimate capture units, targeted dry blockade in situ inactivation units, and label-free intraoperative visualization quality control units, with no dead zones of fluid flow stagnation between adjacent units.

[0011] The above-mentioned solution achieves integrated and simultaneous completion of iPSC cell sorting, safety management, and intraoperative delivery. It requires no large-scale laboratory equipment or specialized cell manipulation personnel, and can be performed independently by surgeons. It has extremely high clinical adaptability and constructs a full-chain tumor risk management system from physical interception to chemical inactivation. It completely solves the core regulatory bottlenecks in the clinical translation of iPSCs. Its innovation is far superior to existing solutions that only involve sorting. The closed sterile structure eliminates the need for open operation throughout the process, significantly reducing the risk of cell contamination. It complies with the clinical sterile operation specifications for Class III medical devices and cell therapy. At the same time, the standard Luer connectors at both ends are compatible with clinically routine syringes and minimally invasive delivery needles, without requiring any changes to existing clinical surgical procedures.

[0012] In addition, the tissue regeneration and repair device based on induced pluripotent stem cells proposed in this application may also have the following additional technical features:

[0013] As a further description of the above technical solution:

[0014] The pre-filtration and impurity removal unit uses a hydrophilic modified medical-grade polyethersulfone flat sheet filter membrane, and the liquid-facing surface of the filter membrane is provided with an integrated mesh-like polypropylene support skeleton. The nominal pore size of the filter membrane is 20μm, the porosity is ≥80%, and the water contact angle is ≤35°.

[0015] The tapered pore mechanical sorting unit uses a medical-grade polycarbonate core-pore membrane, which has uniformly connected tapered gradient pores. The nominal pore diameter at the inlet end of the tapered gradient pores is 16μm, and the nominal pore diameter at the outlet end is 12μm. The coefficient of variation of pore diameter uniformity is ≤2%.

[0016] In the above scheme, the pre-filtration unit can reduce the risk of downstream unit pore blockage to zero, the non-specific adsorption rate of target cells is ≤0.5%, minimizing cell loss. The tapered gradient pore channel has a primary retention rate of ≥99% for undifferentiated iPSCs, which is much higher than that of nuclear pore membranes with fixed pore size. At the same time, there is no high shear force, so it does not damage the activity and function of terminally differentiated cells. The precisely defined materials and parameters ensure the performance stability between product batches and meet the mass production requirements of medical disposable consumables.

[0017] As a further description of the above technical solution:

[0018] The dual-target affinity capture screening unit uses a carboxylated three-dimensional porous medical-grade polyethersulfone sponge membrane. The inner wall of the sponge membrane is covalently cross-linked with amide bonds to fix a combination of undifferentiated induced pluripotent stem cell-specific dual-target nucleic acid aptamers. The dual-target nucleic acid aptamer combination is an equimolar ratio of Tra-1-60-specific nucleic acid aptamer and SSEA-4-specific nucleic acid aptamer, with an aptamer modification density of 500 pmol / cm².

[0019] In the above scheme, the three-dimensional porous structure achieves a single-pass capture rate of ≥99.9%, which is far higher than the efficiency of planar membrane affinity capture. The dual-target design completely solves the problem of single-target missed detection, with stronger specificity and no off-target binding. Compared with antibodies, nucleic acid aptamers have higher stability, no immunogenicity, and smaller batch-to-batch differences. Covalent cross-linking modification has no shedding and no exogenous reagent residue, and its clinical compliance is far higher than that of antibody modification schemes. The precise modification density parameters balance capture efficiency and manufacturing cost, taking into account both performance and mass production feasibility.

[0020] As a further description of the above technical solution:

[0021] The passive reciprocating vortex microfluidic ultimate capture unit is a flat, closed microfluidic chamber with multiple sets of symmetrical arc-shaped flow guide baffles on the inner wall of the chamber. The flow guide baffles are integrally formed with the bottom surface of the chamber. The entire surface of the inner wall of the chamber is covalently cross-linked with a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture fine screening unit, and the aptamer modification density is 300 pmol / cm².

[0022] In the above scheme, the purely passive structure without moving parts has stable performance and good sterility, which is fully compatible with the design requirements of disposable medical consumables. The vortex design can increase the capture rate of trace amounts of residual undifferentiated iPSCs to more than 99.9%, further increasing the overall clearance rate by two orders of magnitude. The wide channel design has no high shear force, and the loss of activity of terminally differentiated cells is ≤0.5%, which does not affect the subsequent tissue repair effect at all. It forms a double affinity screening with the affinity capture unit, forming a double redundancy of fine screening and final capture, and completely eliminating the missed detection of physical interception links.

[0023] As a further description of the above technical solution:

[0024] The targeted dry blockade in situ inactivation unit uses a three-dimensional porous medical-grade collagen polycaprolactone composite sponge membrane, and the inner wall of the sponge membrane is fixed with a dual-target nucleic acid aptamer dry inhibitor conjugate through reducible disulfide covalent cross-linking.

[0025] The targeting aptamer of the conjugate is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture screening unit, the stem inhibitor is a small molecule inhibitor specific to the Wnt / β-catenin pathway, and the conjugate modification density is 200 pmol / cm².

[0026] The above-mentioned scheme, through a targeted release mechanism, has no effect on the activity and function of terminally differentiated cells, no systemic side effects, and no gene editing operations. It completely avoids the off-target risks and extremely high regulatory approval thresholds of suicide gene editing schemes. The clinical translation path is clear, and the disulfide bond cross-linking structure is extremely stable in the extracellular environment, will not release inhibitors prematurely, and the safety is controllable.

[0027] As a further description of the above technical solution:

[0028] The label-free intraoperative visualization quality control unit is a flat, straight-through visualization window made of high-transmittance medical-grade polycarbonate material. The inner wall of the visualization window is pre-coated with a fluorescence-quenched dual-target nucleic acid aptamer on the liquid-facing side.

[0029] The fluorescent quenching dual-target nucleic acid aptamer is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture fine screening unit. The aptamer is modified with a fluorescent group at the 5' end and a quenching group at the 3' end, with a modification density of 100 pmol / cm².

[0030] The above-mentioned solution achieves intraoperative closed-loop quality control of iPSC sorting and removal effects, completely solving the core pain point of existing solutions that cannot verify the removal effect intraoperatively and can only be tested in the laboratory after the operation. It meets the requirements of clinical compliance and traceability, is label-free, has no foreign reagent addition, no cell retention or activity damage, does not affect subsequent cell delivery and tissue repair, is simple to operate, does not require professional testing personnel and equipment, and can be quickly completed by surgeons to complete quality control verification. It is fully adapted to the rhythm of clinical surgery, and the high light transmittance material and precise aptamer modification have no background fluorescence interference, and the detection sensitivity can reach the single cell level.

[0031] The second aspect of this application proposes a tissue regeneration and repair method based on induced pluripotent stem cells, comprising the following steps:

[0032] 1) Standardized preparation of cell suspension: Take the original solution of terminally differentiated functional cells derived from induced pluripotent stem cells, resuspend them in physiological saline containing 5% medical-grade human serum albumin as the cell delivery buffer, and adjust the final cell concentration to 1×10⁻⁶. 6 ~1×10 7 cells / mL, gently pipet to prepare a single-cell suspension;

[0033] 2) Aseptic installation and venting of the device: Seal the proximal Luer connector of the integrated sorting module to the syringe with pre-drawn cell delivery buffer, and seal the distal Luer connector to the minimally invasive delivery needle. Slowly inject the buffer at a flow rate of 1 mL / min to expel air bubbles from the cavity and confirm that the fluid path is unobstructed and leak-free.

[0034] 3) Synchronous clearance, quality control, and delivery: Replace the syringe pre-loaded with single-cell suspension and inject the cell suspension at a constant flow rate of 0.5~2mL / min. The cell suspension passes through the functional units inside the cavity in sequence, simultaneously completing the gradient clearance, targeted inactivation, and intraoperative quality control of residual undifferentiated induced pluripotent stem cells. The processed cell suspension is then precisely delivered to the target tissue injury site through a minimally invasive delivery needle.

[0035] 4) Intraoperative quality control confirmation: The label-free intraoperative visual quality control unit is illuminated using a handheld fluorescent navigation device in the operating room to confirm that there is no specific fluorescent illumination, thus completing the closed-loop quality control process.

[0036] In the above steps, the total time of the entire process is ≤10min, which is much faster than the 1-2 hours of operation time of flow cytometry and magnetic bead sorting. It does not prolong the clinical operation time at all. The entire process is carried out in a closed and sterile environment with no open links, which greatly reduces the risk of cell contamination and exogenous contamination. It meets the clinical aseptic operation standards, and the standardized steps are highly repeatable, eliminating the differences in operation between different operators and ensuring the stability of clinical application effects.

[0037] In addition, the tissue regeneration and repair method based on induced pluripotent stem cells proposed in this application may also have the following additional technical features:

[0038] As a further description of the above technical solution:

[0039] In step 3), the constant flow rate is 1 mL / min, and the total processing time is ≤10 min.

[0040] In the above steps, the precisely defined flow rate parameters ensure the stable performance of each functional unit, ensure the consistency of the clearance effect between batches, strictly control the entire process duration, avoid the decrease in cell viability and phenotypic drift caused by prolonged in vitro cell placement, and ensure the cell repair function.

[0041] As a further description of the above technical solution:

[0042] In step 1), the volume of a single treatment of the single-cell suspension is ≤10mL, the entire preparation process is completed in a sterile environment at 25℃, and the interval between preparation and injection delivery is ≤5min.

[0043] The above steps, with their strict time limits, maximize the preservation of the viability and repair function of terminally differentiated iPSC-derived cells, ensuring clinical repair efficacy. The clear environmental requirements comply with the operating procedures for GMP-grade cell clinical applications, reducing the risk of infection and contamination.

[0044] Advantages of this invention:

[0045] According to the present application, a tissue regeneration and repair device and method based on induced pluripotent stem cells realizes an integrated synchronous processing architecture during surgery, integrating residual cell removal, targeted inactivation, real-time quality control and in-situ delivery into a disposable closed tubing module. No large laboratory equipment is required, and the entire process can be completed within 10 minutes. This completely solves the industry pain point of the disconnect between sorting and clinical delivery, is compatible with routine surgical procedures, and has a very low operating threshold.

[0046] We have constructed a complete safety system that integrates gradient physical sorting, dual-target affinity capture, and targeted dry inactivation. The residual undifferentiated iPSC clearance rate is ≥99.9999%, completely eliminating the risk of teratoma. At the same time, the target cell viability retention rate is ≥93%, which far exceeds the performance of mainstream solutions such as flow cytometry and magnetic bead sorting.

[0047] The accompanying intraoperative label-free closed-loop quality control enables real-time visual verification of the clearance effect, meeting clinical compliance and traceability requirements.

[0048] The entire process involves no gene editing and no exogenous magnetic beads / antibodies, complies with global cell therapy and Class III medical device regulatory standards, and has a clear clinical translation pathway.

[0049] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0050] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0051] Figure 1 This is a schematic diagram of a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application;

[0052] Figure 2 This is a schematic diagram illustrating the principle of a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application.

[0053] Figure 3 This is a comparison chart of residual undifferentiated iPSC clearance rates in a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application;

[0054] Figure 4 This is a comparison chart of the viability retention rate of terminally differentiated cells derived from iPSCs in a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application.

[0055] Figure 5This is a graph showing the verification results of the subcutaneous tumorigenesis rate in nude mice after 12 weeks of a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application.

[0056] Figure 6 This is a comparison chart of the entire process processing time of a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application. Detailed Implementation

[0057] The embodiments of this application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0058] The following description, in conjunction with the accompanying drawings, describes a tissue regeneration and repair device and method based on induced pluripotent stem cells according to an embodiment of this application.

[0059] like Figure 1-2 As shown in the figure, a tissue regeneration and repair device based on induced pluripotent stem cells according to an embodiment of this application includes a cell delivery component and an integrated sorting and processing module;

[0060] The integrated sorting and processing module is a closed cylindrical cavity with standard Luer conical connectors at both ends. The proximal Luer connector of the cavity is sealed and connected to the cell delivery component, and the distal Luer connector is sealed and connected to the minimally invasive delivery needle.

[0061] Inside the cavity, along the flow direction of the cell suspension, there are sequentially and coaxially sealed pre-filtration and impurity removal units, conical channel mechanical sorting units, dual-target affinity capture fine screening units, passive reciprocating vortex microfluidic ultimate capture units, targeted dry blockade in situ inactivation units, and label-free intraoperative visualization quality control units, with no dead zones of fluid flow stagnation between adjacent units.

[0062] This solution utilizes a coaxial, cascaded structure integrating gradient clearance, targeted inactivation, closed-loop quality control, and in-situ delivery. This allows cell suspensions to complete the entire process—impurity removal, primary sorting, fine screening and capture, final clearance, tumorigenic risk inactivation, and intraoperative quality control—with a single injection. The entire process is seamless, with no dead zones or additional operational steps. By leveraging the progressive synergistic effect of multiple units, the solution integrates the laboratory-level iPSC safety management process into a closed tubing module that can be used immediately during surgery, thus resolving the disconnect between current sorting technologies and clinical delivery.

[0063] like Figure 1 As shown:

[0064] The pre-filtration and impurity removal unit uses a hydrophilic modified medical-grade polyethersulfone flat sheet filter membrane, and the liquid-facing surface of the filter membrane is equipped with an integrated mesh-like polypropylene support skeleton. The nominal pore size of the filter membrane is 20μm, the porosity is ≥80%, and the water contact angle is ≤35°. The hydrophilic modified medical-grade polyethersulfone flat sheet filter membrane can reduce non-specific cell adsorption, and the precise 20μm pore size can completely intercept cell clumps and dead cell fragments. The matching mesh support skeleton can avoid membrane deformation during high flow rate injection, ensure downstream liquid flow stability, and prevent pore blockage.

[0065] The tapered pore mechanical sorting unit uses a medical-grade polycarbonate nuclear pore membrane, which has uniformly continuous tapered gradient pores. The nominal pore diameter at the inlet end of the tapered gradient pores is 16μm, and the nominal pore diameter at the outlet end is 12μm. The coefficient of variation of pore diameter uniformity is ≤2%. It uses the inherent differences in size and cell rigidity between undifferentiated iPSCs and terminally differentiated cells to achieve sorting. This makes undifferentiated iPSCs spherical, extremely rigid, and almost non-deformable, and unable to pass through the 12μm outlet narrowing. Terminally differentiated cells have strong cell membrane fluidity and excellent deformability, and can pass through the gradient pores without damage. This solves the inherent defects of fixed pore diameter membranes, which are prone to missed detection and pore blockage.

[0066] like Figure 1 As shown:

[0067] The dual-target affinity capture screening unit uses a carboxylated three-dimensional porous medical-grade polyethersulfone sponge membrane. The inner wall of the sponge membrane is covalently cross-linked with undifferentiated induced pluripotent stem cell-specific dual-target nucleic acid aptamers via amide bonds. The dual-target nucleic acid aptamer combination is an equimolar ratio of Tra-1-60-specific nucleic acid aptamer and SSEA-4-specific nucleic acid aptamer, with an aptamer modification density of 500 pmol / cm². This scheme uses a three-dimensional porous sponge membrane to replace the traditional planar membrane, increasing the specific surface area by more than 50 times, significantly increasing the contact opportunities between cells and aptamers. The Tra-1-60 and SSEA-4 dual-target nucleic acid aptamers are covalently cross-linked via amide bonds. Both targets are stem markers that are highly expressed in undifferentiated iPSCs but not expressed in terminally differentiated cells. The equimolar ratio combination can completely avoid false negatives caused by single-target aptamers, specifically binding only to trace amounts of undifferentiated iPSCs that are missed in the sorting unit, with no risk of aptamer detachment.

[0068] like Figure 1 As shown:

[0069] The passive reciprocating vortex microfluidic ultimate capture unit is a flat, sealed microfluidic chamber with multiple sets of symmetrical arc-shaped flow guide baffles on the inner wall. The flow guide baffles are integrally formed with the bottom surface of the chamber. The entire surface of the inner wall of the chamber is covalently cross-linked with a combination of dual-target nucleic acid aptamers, consistent with the dual-target affinity capture fine screening unit. The aptamer modification density is 300 pmol / cm². Through the integrated symmetrical arc-shaped flow guide baffles in the chamber, the direct flow is transformed into a low-speed reciprocating vortex. No additional power source is required; it can be achieved solely by the injection pressure of the syringe. This extends the contact time between the cells and the aptamers on the inner wall of the chamber from 2 seconds to more than 12 seconds, completely solving the problem of missed detection of trace residual cells caused by excessively fast flow rate in the direct flow channel and insufficient contact between cells and aptamers, thus achieving ultimate capture.

[0070] like Figure 1 As shown:

[0071] The targeted dry blockade in situ inactivation unit uses a three-dimensional porous medical-grade collagen polycaprolactone composite sponge membrane, and the inner wall of the sponge membrane is fixed with a dual-target nucleic acid aptamer dry inhibitor conjugate through reducible disulfide covalent cross-linking.

[0072] The targeting aptamer of the conjugate is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture screening unit, the stem inhibitor is a small molecule inhibitor specific to the Wnt / β-catenin pathway, and the conjugate modification density is 200 pmol / cm².

[0073] A dual-target aptamer is covalently cross-linked with a Wnt / β-catenin pathway-specific stemness inhibitor via a reducible disulfide bond to form a targeted conjugate. The conjugate specifically binds only to undifferentiated iPSCs that have missed all capture units under extreme conditions. After binding, the disulfide bond is cleaved by high concentrations of glutathione in the cell, releasing the stemness inhibitor at the site. Within 10 minutes, the core pathway for maintaining iPSC stemness can be blocked, causing it to lose its unlimited proliferative capacity and tumorigenic potential. The inhibitor is released only within the target cells and does not diffuse freely.

[0074] like Figure 1 As shown:

[0075] The intraoperative visualization quality control unit is a flat, straight-through visualization window made of high-transmittance medical-grade polycarbonate. The inner wall of the visualization window is pre-coated with a fluorescent quenched dual-target nucleic acid aptamer on the liquid-facing side. The fluorescent quenched dual-target nucleic acid aptamer is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture screening unit. The aptamer is modified with a fluorescent group at the 5' end and a quenching group at the 3' end, with a modification density of 100 pmol / cm². When using the fluorescent quenched dual-target aptamer, the fluorescent group and the quenching group are spatially adjacent when not bound to target cells, and the fluorescence is in a quenched state with no background signal. When residual undifferentiated iPSCs flow through, the aptamer specifically binds to the target cells, the conformation changes, the fluorescent group and the quenching group are spatially separated, and the fluorescence signal immediately illuminates. No exogenous fluorescent dyes are required. The clearance effect can be verified within 10 seconds using only a standard handheld fluorescence navigation device in the operating room. The straight-through structure prevents cell retention, and the verified cell suspension can be directly delivered.

[0076] like Figure 2 As shown:

[0077] A tissue regeneration and repair method based on induced pluripotent stem cells includes the following steps:

[0078] 1) Standardized preparation of cell suspension: Take the original solution of terminally differentiated functional cells derived from induced pluripotent stem cells, resuspend them in physiological saline containing 5% medical-grade human serum albumin as the cell delivery buffer, and adjust the final cell concentration to 1×10⁻⁶. 6 ~1×10 7 cells / mL, gently pipet to prepare a single-cell suspension;

[0079] 2) Aseptic installation and venting of the device: Seal the proximal Luer connector of the integrated sorting module to the syringe with pre-drawn cell delivery buffer, and seal the distal Luer connector to the minimally invasive delivery needle. Slowly inject the buffer at a flow rate of 1 mL / min to expel air bubbles from the cavity and confirm that the fluid path is unobstructed and leak-free.

[0080] 3) Synchronous clearance, quality control, and delivery: Replace the syringe pre-loaded with single-cell suspension and inject the cell suspension at a constant flow rate of 0.5~2mL / min. The cell suspension passes through the functional units inside the cavity in sequence, simultaneously completing the gradient clearance, targeted inactivation, and intraoperative quality control of residual undifferentiated induced pluripotent stem cells. The processed cell suspension is then precisely delivered to the target tissue injury site through a minimally invasive delivery needle.

[0081] 4) Intraoperative quality control confirmation: The label-free intraoperative visual quality control unit is illuminated using a handheld fluorescent navigation device in the operating room to confirm that there is no specific fluorescent illumination, thus completing the closed-loop quality control process.

[0082] This method integrates four steps in a closed loop: cell preparation, device installation and venting, cleaning, quality control, delivery, and intraoperative quality control confirmation. Under the sterile environment of the operating room, the entire process of residual undifferentiated iPSCs and in-situ delivery to the damaged site are completed simultaneously through a single uniform injection. This completely breaks the disconnect between the existing technology of pre-sorting in the laboratory and intraoperative transport and delivery, and avoids the risk of decreased activity and contamination during cell transport and storage after sorting.

[0083] like Figure 2 As shown:

[0084] In step 3), the constant flow rate is 1 mL / min, and the total processing time is ≤10 min. The constant injection flow rate of 1 mL / min is the optimal parameter for balancing the performance of each functional unit: too high a flow rate will result in insufficient contact time between cells and aptamers, leading to a decrease in capture efficiency; too low a flow rate will result in excessive in vitro cell residence time, leading to a decrease in viability. This flow rate ensures that the sorting, capture, and inactivation effects of each unit are fully met, while strictly controlling the total processing time to within 10 min, which meets the time requirements of clinical surgery.

[0085] like Figure 2 As shown:

[0086] In step 1), the single-cell suspension volume for each treatment is ≤10mL. The entire preparation process is completed under sterile conditions at 25℃. The interval between preparation and injection delivery is ≤5min. The single-treatment volume of ≤10mL fully covers the cell suspension volume requirements for repairing various tissue injuries in clinical practice. At the same time, it avoids the processing time exceeding the limit due to excessive volume. The time limit of ≤5min between preparation and injection delivery, as well as the requirement of a sterile environment at 25℃, can maintain the stability of the physiological state of cells to the greatest extent and avoid the decrease in activity, apoptosis and phenotypic changes caused by prolonged placement of cells in an in vitro room temperature environment, while eliminating the risk of contamination.

[0087] Example 2, illustrated below with a specific case:

[0088] The following materials were selected: medical-grade hydrophilic modified polyethersulfone (PES) flat sheet filter membrane, carboxylated PES sponge membrane (Sartorius, Germany), medical-grade polycarbonate (PC) core pore membrane (Whatman, UK), medical-grade collagen polycaprolactone (PCL) composite sponge membrane (Shandong Daigang Biotechnology), and high-transmittance medical-grade PC particles (Covestro, Germany).

[0089] Among them are Tra-1-60 specific nucleic acid aptamer (sequence: 5'-NH2-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'), SSEA-4 specific nucleic acid aptamer (sequence: 5'-NH2-TGGGGTTGGTGTGGTTGG-3'), and a fluorescence-quenching dual-target aptamer (5' end modified with FAM fluorescent group, 3' end modified with BHQ1 quenching group, synthesized by Sangon Biotech and purified by HPLC).

[0090] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), XAV939 (Wnt / β-catenin pathway inhibitor, Sigma-Aldrich); medical-grade UV-curing adhesive (Henkel, Germany), standard Luer tapered connector (Zhejiang KangKang Medical Devices).

[0091] The device prepared in this embodiment is a disposable sterile cylindrical cavity with a total length of 70 mm, an outer diameter of 10 mm, and an inner diameter of 6 mm. Both ends are injection-molded with standard Luer conical connectors. Inside the cavity, six functional units are coaxially sealed and fixed along the fluid flow direction. The specific preparation steps are as follows:

[0092] Preparation of the pre-filtration and impurity removal unit: Take a 20μm pore size hydrophilic modified PES flat sheet filter membrane, punch it into a 6mm diameter disc, and hot press the liquid-facing side into a grid-shaped polypropylene support skeleton (grid pore size 500μm). Use UV curing adhesive to seal and fix the filter membrane edge to the inner wall of the cavity. After curing, rinse three times with sterile water to remove residual impurities.

[0093] Fabrication of tapered channel mechanical sorting unit: A 20 μm thick PC core-pore film was used to fabricate tapered graded channels with an inlet diameter of 16 μm and an outlet diameter of 12 μm using laser etching, with a channel density of 4 × 10⁻⁶. 5 The pore size is ≤2% (per cm²), and the pore size uniformity (CV) is ≤2%. The pores are punched into 6 mm diameter discs and sealed and fixed 1 mm to the right of the pre-filtration and impurity removal unit.

[0094] Preparation of the dual-target affinity capture fine screening unit: A carboxylated PES sponge membrane with a thickness of 500 μm and an average pore size of 30 μm was cut into 6 mm diameter discs. An EDC / NHS mixture (EDC concentration 50 mmol / L, NHS concentration 25 mmol / L) was prepared using 0.1 mol / L LMES buffer (pH=5.5) and the carboxyl groups on the membrane surface were activated at room temperature for 2 h. After activation, an aptamer solution (total concentration 500 pmol / cm²) of Tra-1-60 and SSEA-4 mixed in an equimolar ratio was added and crosslinked overnight at 4 °C. The membrane was washed 3 times with PBST buffer to block unreacted active sites and sealed and fixed 1 mm to the right side of the conical channel sorting unit.

[0095] Fabrication of a passive reciprocating vortex microfluidic ultimate capture unit: A flat microfluidic chamber (2 mm thick, 6 mm inner diameter) was injection molded. Four sets of symmetrical arc-shaped flow guide baffles (1.5 mm radius of curvature, 3 mm spacing between adjacent baffles) were integrally formed on the inner wall of the chamber. The inner wall of the chamber was cross-linked with the aforementioned dual-target aptamer at a density of 300 pmol / cm² and sealed and fixed 1 mm to the right of the dual-target affinity capture unit.

[0096] Preparation of targeted dry blocking in situ inactivation unit: A collagen-PCL composite sponge membrane with a thickness of 300 μm and an average pore size of 50 μm was cut into 6 mm diameter discs. The dual-target aptamer and XAV939 were covalently cross-linked through disulfide bonds to prepare an aptamer-inhibitor conjugate. The conjugate was fixed on the membrane surface at a density of 200 pmol / cm² and sealed and fixed 1 mm to the right side of the microfluidic unit.

[0097] Preparation of label-free intraoperative visualization quality control unit: injection-molded high-transmittance PC material flat straight-through visualization window (thickness 1mm, length 10mm, light transmittance ≥92%), the inner wall of the window is pre-coated with fluorescent quenching dual-target aptamers at a density of 100pmol / cm², and sealed and fixed 1mm to the right side of the dry inactivation unit, inside the distal Luer connector.

[0098] After all modules are assembled, they are sterilized with ethylene oxide, aseptically sealed in packaging, and stored at 4°C away from light for later use.

[0099] Implementation and performance verification of tissue regeneration and repair methods based on the above-mentioned device:

[0100] Human iPSC cell line (WiCell, without foreign gene integration) was cultured in mTeSR1 medium to maintain an undifferentiated state. Human iPSC-derived dermal fibroblasts (iPSC-HDF) were prepared by directed differentiation. Flow cytometry confirmed that the stem cell markers Tra-1-60 and SSEA-4 were negative, while the fibroblast markers Vimentin and CollagenI were positive, with a differentiation purity of ≥99%.

[0101] To simulate a clinical scenario with residual undifferentiated cells, undifferentiated iPSCs and iPSC-HDF were mixed at a ratio of 1:100,000, resuspended in physiological saline containing 5% medical-grade human serum albumin, and the final cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 The cells / mL method was used to prepare single-cell suspensions, with a single treatment volume of 5mL. The entire process was carried out under sterile conditions at 25℃, and the interval between preparation and injection was ≤3min.

[0102] Standardized intraoperative operating procedures:

[0103] Aseptic installation and venting of the device: In the aseptic environment of the operating room, the proximal Luer connector of the sterilized integrated module is sealed and connected to a 10mL syringe pre-drawn buffer solution, and the distal Luer connector is sealed and connected to a 21G minimally invasive delivery needle. Buffer solution is injected at a flow rate of 1mL / min to vent the air and confirm that the fluid path is unobstructed and leak-free.

[0104] Synchronous clearance, quality control, and delivery: Replace the syringe with one containing the mixed cell suspension and inject the mixture at a constant flow rate of 1 mL / min. The cell suspension passes through 6 functional units in sequence, simultaneously completing the gradient clearance, targeted inactivation, and quality control of residual undifferentiated iPSCs. The processed cell suspension is then used directly for subsequent detection and animal experiments. The total processing time is 5 min.

[0105] Intraoperative quality control confirmation: The visual quality control window was illuminated using a standard handheld fluorescence navigator (excitation wavelength 488nm) in the operating room. No specific fluorescence was observed within the window, confirming the absence of residual undifferentiated iPSCs.

[0106] In vitro residual cell clearance efficiency detection:

[0107] The proportion of Tra-1-60 / SSEA-4 double-positive undifferentiated iPSCs in cell suspensions before and after treatment was detected by flow cytometry. The results showed that the proportion of double-positive cells before treatment was 0.001% (1:100000), and the detection limit of double-positive cells after treatment was lower than the flow cytometry detection limit (1×10⁻⁶). -7 The clearance rate of residual undifferentiated iPSCs was ≥99.9999%.

[0108] Target cell viability and function verification: Trypan blue staining, CCK-8 proliferation assay, and immunofluorescence staining were used to detect the activity and function of iPSC-HDF after treatment. The results showed that the cell viability after treatment was 93.2±1.1%, which met the design target of ≥93%. There was no statistically significant difference in cell proliferation capacity and CollagenI secretion capacity between the treated and untreated groups (P>0.05), confirming that the device treatment did not affect the phenotype and repair function of the target differentiated cells.

[0109] In vivo tumorigenicity and safety verification (e.g.) Figure 5 (As shown): Six-week-old SPF-grade BALB / c immunodeficient nude mice were randomly divided into three groups of 10 mice each.

[0110] Experimental group: Subcutaneous injection of the mixed cell suspension treated by the device in this embodiment, with an injection volume of 1×10-1 per device. 6 cells;

[0111] Positive control group 1: Subcutaneous injection of untreated mixed cell suspension, 1×10⁻⁶ cells per animal. 6 cells;

[0112] Positive control group 2: Subcutaneous injection of undifferentiated iPSC suspension, 1×10⁻⁶ per animal. 4 cells;

[0113] The animals were fed and observed for 12 weeks, and the results showed that:

[0114] No teratomas formed in any of the 10 nude mice in the experimental group, with a tumorigenesis rate of 0%.

[0115] In positive control group 1, 8 nude mice developed subcutaneous teratomas, with a tumorigenesis rate of 80%; in positive control group 2, all 210 nude mice developed teratomas, with a tumorigenesis rate of 100%.

[0116] Pathological HE staining confirmed that the organs of the nude mice in the experimental group were normal and no tumors were formed, confirming that this regimen can completely eliminate the tumorigenic risk of residual iPSCs.

[0117] Compared with existing flow cytometry sorting methods (such as...) Figure 3 , 4 (as shown in Figure 6)

[0118] Using mixed cell suspensions from the same batch, the standard industry flow cytometry sorting protocol (Tra-1-60 / SSEA-4 double-negative sorting) was employed, and the BDFACSAriaIII flow cytometer was used for sorting. The results showed that the clearance rate of residual undifferentiated iPSCs after sorting was 99.92%, which was lower than that of the protocol in this embodiment. The viability of the target cells after sorting was 82.5±2.3%, which was significantly lower than that of the protocol in this embodiment (P<0.05). The total sorting time was 92 minutes, which requires professional operators and large equipment and cannot be completed intraoperatively.

[0119] Compared with existing immunomagnetic bead sorting methods (such as...) Figure 3 , 4 (as shown in Figure 6)

[0120] Using the same batch of mixed cell suspensions, a commercial Tra-1-60 immunomagnetic bead negative sorting protocol (Mitteni) was employed. The results showed that the clearance rate of residual undifferentiated iPSCs after sorting was 99.95%, which was lower than that of the protocol in this embodiment; the viability of target cells after sorting was 85.1±1.8%, which was significantly lower than that of the protocol in this embodiment (P<0.05); the total sorting time was 38 minutes, requiring a magnetic rack and exogenous antibody beads, posing a risk of reagent residue, and could not be completed intraoperatively.

[0121] In summary, the tissue regeneration and repair device and method based on induced pluripotent stem cells according to the embodiments of this application can efficiently remove residual undifferentiated iPSCs within 10 minutes, with a removal rate ≥99.9999% and a target cell viability retention rate ≥93%, completely eliminating the tumorigenic risk of teratoma, and its performance is significantly better than the prior art.

[0122] In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0123] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0124] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A tissue regeneration and repair device based on induced pluripotent stem cells, characterized in that, Includes cell delivery components and an integrated sorting and processing module; The integrated sorting and processing module is a closed cylindrical cavity with standard Luer conical connectors at both ends. The proximal Luer connector of the cavity is sealed and connected to the cell delivery component, and the distal Luer connector is sealed and connected to the minimally invasive delivery needle. Inside the cavity, along the flow direction of the cell suspension, there are sequentially and coaxially sealed pre-filtration and impurity removal units, conical channel mechanical sorting units, dual-target affinity capture fine screening units, passive reciprocating vortex microfluidic ultimate capture units, targeted dry blockade in situ inactivation units, and label-free intraoperative visualization quality control units, with no dead zones of fluid stagnation between adjacent units.

2. The tissue regeneration and repair device based on induced pluripotent stem cells according to claim 1, characterized in that, The pre-filtration and impurity removal unit uses a hydrophilic modified medical-grade polyethersulfone flat sheet filter membrane, and the liquid-facing surface of the filter membrane is provided with an integrated mesh-like polypropylene support skeleton. The nominal pore size of the filter membrane is 20μm, the porosity is ≥80%, and the water contact angle is ≤35°. The tapered pore mechanical sorting unit uses a medical-grade polycarbonate core pore membrane, and the core pore membrane has uniformly connected tapered gradient pores. The nominal pore diameter at the inlet end of the tapered gradient pores is 16μm, the nominal pore diameter at the outlet end is 12μm, and the coefficient of variation of pore diameter uniformity is ≤2%.

3. The tissue regeneration and repair device based on induced pluripotent stem cells according to claim 1, characterized in that, The dual-target affinity capture screening unit uses a carboxylated three-dimensional porous medical-grade polyethersulfone sponge membrane. The inner wall of the sponge membrane is covalently cross-linked with amide bonds to fix a combination of undifferentiated induced pluripotent stem cell-specific dual-target nucleic acid aptamers. The dual-target nucleic acid aptamer combination is an equimolar ratio of Tra-1-60-specific nucleic acid aptamer and SSEA-4-specific nucleic acid aptamer, with an aptamer modification density of 500 pmol / cm².

4. The tissue regeneration and repair device based on induced pluripotent stem cells according to claim 1, characterized in that, The passive reciprocating vortex microfluidic ultimate capture unit is a flat, closed microfluidic chamber with multiple sets of symmetrical arc-shaped flow guide baffles on the inner wall of the chamber. The flow guide baffles are integrally formed with the bottom surface of the chamber. The entire surface of the inner wall of the chamber is covalently cross-linked with a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture fine screening unit, and the aptamer modification density is 300 pmol / cm².

5. The tissue regeneration and repair device based on induced pluripotent stem cells according to claim 1, characterized in that, The targeted dry blockade in situ inactivation unit uses a three-dimensional porous medical-grade collagen polycaprolactone composite sponge membrane, and the inner wall of the sponge membrane is fixed with a dual-target nucleic acid aptamer dry inhibitor conjugate through reducible disulfide covalent cross-linking. The targeting aptamer of the conjugate is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture screening unit, the stemness inhibitor is a small molecule inhibitor specific to the Wnt / β-catenin pathway, and the conjugate modification density is 200 pmol / cm².

6. The tissue regeneration and repair device based on induced pluripotent stem cells according to claim 1, characterized in that, The label-free intraoperative visualization quality control unit is a flat, straight-through visualization window made of high-transmittance medical-grade polycarbonate material. The inner wall of the visualization window is pre-coated with a fluorescence-quenched dual-target nucleic acid aptamer on the liquid-facing side. The fluorescent quenching dual-target nucleic acid aptamer is a combination of dual-target nucleic acid aptamers consistent with the dual-target affinity capture screening unit. The aptamer is modified with a fluorescent group at the 5' end and a quenching group at the 3' end, with a modification density of 100 pmol / cm².

7. A tissue regeneration and repair method based on induced pluripotent stem cells according to any one of claims 1-6, characterized in that, Includes the following steps: 1) Standardized preparation of cell suspension: Take the original solution of terminally differentiated functional cells derived from induced pluripotent stem cells, resuspend them in physiological saline containing 5% medical-grade human serum albumin as the cell delivery buffer, and adjust the final cell concentration to 1×10⁻⁶. 6 ~1×10 7 cells / mL, gently pipet to prepare a single-cell suspension; 2) Aseptic installation and venting of the device: Seal the proximal Luer connector of the integrated sorting module to the syringe with pre-drawn cell delivery buffer, and seal the distal Luer connector to the minimally invasive delivery needle. Slowly inject the buffer at a flow rate of 1 mL / min to expel air bubbles from the cavity and confirm that the fluid path is unobstructed and leak-free. 3) Synchronous clearance, quality control, and delivery: Replace the syringe pre-loaded with single-cell suspension and inject the cell suspension at a constant flow rate of 0.5~2mL / min. The cell suspension passes through the functional units inside the cavity in sequence, simultaneously completing the gradient clearance, targeted inactivation, and intraoperative quality control of residual undifferentiated induced pluripotent stem cells. The processed cell suspension is then precisely delivered to the target tissue injury site through a minimally invasive delivery needle. 4) Intraoperative quality control confirmation: The label-free intraoperative visual quality control unit is illuminated using a handheld fluorescent navigation device in the operating room to confirm that there is no specific fluorescence illumination, thus completing the closed-loop quality control process.

8. The method according to claim 7, characterized in that, In step 3), the constant flow rate is 1 mL / min, and the total processing time is ≤10 min.

9. The tissue regeneration and repair method based on induced pluripotent stem cells according to claim 7, characterized in that, In step 1), the volume of a single-cell suspension treated at one time is ≤10mL, the entire preparation process is completed in a sterile environment at 25℃, and the interval between preparation and injection delivery is ≤5min.

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