Membrane manufacturing process

Abstract

The present invention relates to a membrane for separating target stem cells from a biological sample, more specifically, a membrane for separating target stem cells from a single-cell suspension prepared from a biological sample. As a result, sterile target stem cells are obtained in a physiological buffer solution. The membrane of the present invention is a 3D support structure consisting of at least one layer of a biocompatible polymer with a specific pore size as a support material, and a target molecule covalently bound to its surface and / or within the pores. The target molecule is preferably a target antibody, which recognizes a specific antigen bound to the surface of the target stem cells and thereby binds the target stem cells to the membrane. The target molecule is bound to the surface and / or pores of the support structure directly, or by specific functionalized nanoparticles bound to or incorporated into the 3D support structure of the membrane. Furthermore, the present invention also includes processes and devices for separating target stem cells from a biological sample, in addition to the membrane manufacturing process, which include the above-described membrane as a component.

Description

[Technical Field]

[0001] This invention ,film Manufacturing process S To relate to. [Background technology]

[0002] Cutting-edge technology, technical problems, and defects that are solved by the present invention. Patent Document 1 refers to a method of separating cells by binding magnetic particles to them. The solution disclosed in this document uses a cell separation technique based on the magnetic attraction of a field and is therefore incomparable to the solution of the present invention, which exclusively induces cell separation based on the reaction of antibodies with cells.

[0003] Patent Document 2 describes a biocompatible support structure and a method using activation of surface markers. reg This section discusses methods for separating cells. Related literature is T reg It does not suggest sufficiently specific markers in cells. In other words, the process by which the invention was made is a different approach, T reg This is based on the differentiation of stem cells into other cells.

[0004] Patent Document 3 describes obtaining cells using a corresponding marker. The authors did not specify the type of tissue to be introduced. The procedure is performed manually (which is more time-consuming, more expensive, and yields are not comparable). Also, the antibodies used are not comparable to the antibodies mentioned in this invention (e.g., CD90…).

[0005] Patent Document 4 describes a novel collection container optimized for liposuction, which includes a filter system capable of concentrating cell suspensions. It relates to the "concentration" of cells and their application to cell culture plates for further purification and proliferation of the cells obtained from the sample.

[0006] Patent Document 5 describes the mechanical purification of liposuction products using a mesh filter. However, the procedure according to that invention determines a cell suspension as an input material that can be prepared in several ways. The procedure according to the present invention is not limited to filtration or purification using a mesh filter. Furthermore, the above patent application does not refer to a procedure of "affinity-based cell separation." It refers to the pre-preparation of liposuction products for further use.

[0007] Patent Document 6 describes a generally known cell separation procedure using centrifugation, enzymatic digestion, etc. This application refers to "cell concentration" and not to cell isolation or separation, and therefore does not preclude the present invention. This clearly indicates that this application is not about an affinity-based separation method. Furthermore, the yield is considerably lower than the yield of the present invention, especially since a "mixture of cells" is obtained even after centrifugation. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 794266 [Patent Document 2] U.S. Patent No. 7592431 [Patent Document 3] International Publication No. 2017 / 075389 [Patent Document 4] U.S. Patent No. 7390484 [Patent Document 5] U.S. Patent Application Publication No. 2013 / 130371 [Patent Document 6] U.S. Patent Application Publication No. 2013 / 034524 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The above problems are solved by the membrane and separation process according to the present invention. [Means for solving the problem]

[0010] The present invention relates to a membrane for isolating target stem cells from biological samples, more specifically from single-cell suspensions prepared from biological samples, to obtaining sterile target stem cells in a physiological buffer of known cell population size (number of isolated cells) and viability (live / dead cell ratio). The thus obtained target stem cells can be used for therapeutic purposes immediately after isolation or thereafter, for the development of tissue fillers and new solutions in regenerative medicine related to various tissues such as dentistry, orthopedics, and plastic surgery, or for research in stem cell biology and the testing of new therapeutic drugs. The membrane is designed as a 3D support structure consisting of at least one layer of a biocompatible polymer having a predetermined pore size, such as a carrier material. The membrane features target molecules, preferably target antibodies, covalently bound to its surface and / or pores, recognizing characteristic antigens bound to the surface of target stem cells and binding the target stem cells to the membrane. The target molecules are either directly bound to the surface and / or pores of the support structure, or bound to or incorporated into the 3D membrane structure by specific functionalized nanoparticles.

[0011] Furthermore, the present invention includes a membrane manufacturing process, an isolation process and apparatus for isolating target stem cells from a biological sample, the membrane being a component of the process. The use of membranes and the process of the present invention enable the specific and effective active separation of target stem cells from a cell mixture in a biological sample.

[0012] In this application, the term “stem cells” defines cells that possess a superior (theoretically unlimited) capacity for population self-regeneration (meaning they divide to form more cells of the same kind) and have the ability to regrow or regenerate (various) tissues after transplantation by differentiating into at least one other cell type. At the molecular level, these cells express so-called stem cell markers, such as surface antigens characteristic of stem cells. Stem cells contained in biological samples express characteristic surface antigens. For example, hematopoietic stem cells isolated from peripheral blood or umbilical cord blood particularly express the CD34 surface antigen, among others, and mesenchymal stem cells isolated from adipose tissue express the CD90 surface antigen.

[0013] The term "targeted stem cells" refers to stem cells that express characteristic surface antigens that bind to selected target molecules. The term "target molecule" refers to a molecule capable of recognizing and binding to a characteristic antigen on the surface of a target stem cell. In this process, the target molecule should not affect the target stem cell itself; that is, it should not force its differentiation, i.e., division. Furthermore, when used, it should not affect the secretion of any substance that could have instantaneous (acute) or long-term (chronic) adverse effects on the patient, nor should it affect the characteristics of the target stem cell (genotype, epigenetics, or phenotype). Preferably, the target molecule is a characteristic antigen on the surface of the target stem cell (e.g., region F c , area F ab This is an antibody, either whole or in part, that recognizes an aptamer and enables specific binding to a characteristic antigen. Preferably, it is an antibody that recognizes the following antigens: CD90, CD146, CD44, CD73, CD105, CD34, STRO-1, STRO-3, etc.

[0014] Biological samples for isolating target stem cells are human and animal organs, tissues, and body fluids that contain such cells and are taken from living or dead (donors) organisms. These include, among others, subcutaneous adipose tissue obtained by liposuction or surgical resection, bone marrow obtained by puncture, non-induced peripheral blood and peripheral blood after induction of bone marrow obtained by venipuncture or apheresis, endometrium obtained by uterine biopsy, menstrual blood, umbilical cord tissue, Wharton's jelly, and umbilical cord blood obtained during or after birth, amniotic fluid obtained at birth by amniocentesis or cesarean section, amnion obtained after birth, and dental pulp obtained from teeth, but are not limited thereto.

[0015] The term "functionalized nanoparticles" refers to nanoparticles that have surface functional groups (e.g., NH2, OH, COOH, SH, etc.) on their surface and enable binding to the 3D support structure of the membrane and / or binding of target molecules onto the surface of the functionalized nanoparticles. <​​​​​​Functionalized nanoparticles are inorganic, organic, hybrid, composite, magnetic, or combinations thereof, and consist of any combination of the above basic materials characterized by metals and / or their alloys and / or metal oxides and / or polymers, or surface functional groups (e.g., NH2, OH, COOH, SH, etc.). In one embodiment, the functionalized nanoparticles are hybrid inorganic-organic nanoparticles. Preferably, the functionalized nanoparticles are, for example, nanoparticles of a metal alloy (e.g., NiCu-nickel / copper) surrounded by a layer of silica (SiO2) having surface functional groups on its surface. The functionalized nanoparticles are metal oxides (e.g., Fe2O3 or Fe3O4) uniformly surrounded by a layer of silica having surface functional groups on its surface. Preferably, the thickness of the silica layer is in the range of several nanometers to tens of nanometers. In another embodiment, the functionalized nanoparticles are nanoparticles based on silica (chemical SiO2) prepared from various siloxane-based precursors (e.g., (3-aminopropyl)triethoxysilane-APTES, vinyltriethoxysilane-VTES, (3-mercaptopropyl)triethoxysilane-MPTES…), where the presence of desired functional groups on the surface of the nanoparticles can be ensured in situ during the synthesis of these nanoparticles. In yet another embodiment, the functionalized nanoparticles are polysaccharide-based nanoparticles (e.g., chitosan, carboxymethylcellulose, alginates, etc.). Their basic structures already contain desirable and preferred functional groups (NH2, OH, COOH, etc.) that ensure similar functionality to other exposed functionalized nanoparticle examples. Another embodiment refers to the synthesis of nanoparticles based on other synthetic polymers (e.g., dendrimers, methacrylate derivatives, polyethyleneimines, etc.), which also satisfy the initial definition of functionalized nanoparticles because their basic structures also contain desired functional groups (e.g., NH2, OH, COOH, SH, etc.). For the synthesis of nanoparticles, sol-gel methods, emulsification techniques, or any other synthetic procedure capable of preparing nanoparticles that meet the definition of functionalized nanoparticles consisting of the above and other basic materials can be used.

[0018] The single-cell suspension is a suspension of individual cells and small cell clusters, prepared from a biological sample in physiological buffers, i.e., buffers that allow for the preservation of cells under physiological conditions. Such buffers include, for example, physiological saline, culture medium, 1×PBS (phosphate-buffered saline), and other similar solutions. As a result, the cell suspension contains, in addition to target stem cells, other non-target cells, i.e., remaining tissue cells, non-target stem cells, cell debris, and other components present in the biological sample (e.g., plasma, intercellular fluid, extracellular matrix, etc.).

[0019] The term "membrane activation" refers to the binding of a target molecule, which recognizes and binds to a characteristic antigen on the surface of a target stem cell, to the membrane's 3D supporting structure. Membrane activation is achieved by incorporating the target molecule using chemical, physicochemical, or physical methods. This includes, but is not limited to, the incorporation of functionalized nanoparticles into the membrane's supporting structure, subsequent binding of the target molecule to the nanoparticles, the incorporation of biofunctionalized nanoparticles into the membrane's supporting structure, or the direct binding of the target molecule to the supporting structure.

[0020] The present invention is described below and illustrated in the embodiments and figures. [Brief explanation of the drawing]

[0021] [Figure 1] A diagram showing a replaceable cassette, which is a component of an apparatus for separating stem cells from a biological sample, including a membrane according to the present invention. [Modes for carrying out the invention]

[0022] The membrane according to the present invention consists of a 3D support structure containing target molecules on its surface and / or within its pores. The support structure consists of at least one layer of a biocompatible polymer, either structured or unstructured, having pores with a diameter of 50 to 500 μm, and containing covalently bound target molecules on its surface and / or within its pores. The target molecules recognize and bind to characteristic antigens on the surface of target stem cells. As a result, non-target cells pass through the membrane or are washed away from the membrane surface, while target stem cells are trapped by the membrane.

[0023] The structured shape of each layer of a support structure means that the pore shape, size, and distribution are uniform throughout each layer. The layers themselves are defined during a controllable manufacturing process. In an unstructured shape, the pore shape, size, and distribution in each layer occur randomly and cannot be influenced during the manufacturing process. The shape of each layer of a support structure varies depending on the film manufacturing process used. For example, when using electrospinning, the shape of each layer is largely unstructured or occurs randomly, which is characteristic of this method, whereas when using 3D printing, the shape of each layer of the support structure is structured.

[0024] A suitable biocompatible polymer is either hydrophobic or hydrophilic. Preferably, it is hydrophobic and should not bind target stem cells. It should be inactive to target stem cells (i.e., should not affect their essential properties, such as differentiation state and potential, proliferation state and potential, and surface antigen expression). During use, it should not promote the secretion and / or development of substances that could have short-term or long-term adverse effects on patients being treated with such cells. It should be sterilizable without altering the polymer's properties. Preferably, it should not bind platelets or red blood cells. It should exhibit appropriate physicochemical and mechanical properties important for film production, such as viscosity, pKa value, printability, and surface tension. Suitable biocompatible polymers include, but are not limited to, natural materials for woven and nonwoven fabrics such as polysaccharide derivatives including alginates (ALG), carboxymethylcellulose (CMC), viscose (VIS), silk, collagen, nanofibrillated cellulose (NFC), and combinations thereof; semi-synthetic materials such as derivatives and chitosan (CHI), cellulose and other derivatives, and combinations thereof; synthetic materials such as polycaprolactone (PCL), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polypropylene (PP), polyhydroxyethyl methacrylate (PHPMA), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyvinyl alcohol (PVA), polyethylene oxide (PEOX); and various dendrimers such as polyamidoamines (PAMAM), polyethyleneimines (PEI), and combinations thereof.

[0025] Preferably, the biocompatible polymer is selected from PCL, CMC, CHI, ALG, PET, PEOX, and PHEMA / PHPMA. This does not exclude other biocompatible polymers or combinations thereof.

[0026] The selection of a biocompatible polymer for fabricating the support structure itself ensures that the support structure characterizes its surface and / or pores (e.g., NH2, OH, COOH, SH, etc.) with functional groups.

[0027] If the support structure is formed of several layers of structured and / or unstructured shapes, each layer can be prepared from the same or different biocompatible polymers, and the shape of each layer, i.e., the shape, size, and distribution of the pores in each layer, may be the same or different.

[0028] The shape of each layer is determined so that as many non-target cells as possible can pass through the membrane while target stem cells bind to the membrane surface and / or pores. Preferably, the pore diameter is in the range of 100 μm to 200 μm.

[0029] Optionally, functionalized nanoparticles can be covalently bonded "in situ" (using any chemical, physicochemical, or physical method) onto and / or into a support structure composed of a biocompatible polymer, i.e., on the surface of the support structure or within its pores. Functionalized nanoparticles can also be incorporated into the support structure simply "mechanically," i.e., without using any special bonding or interaction with the support structure (or simply being captured by it). Target molecules are covalently bonded to the functionalized nanoparticles via surface functional groups through the active site of the functionalized nanoparticle.

[0030] During the film fabrication process, or "in-situ," the biofunctionalized nanoparticles—that is, the functionalized nanoparticles to which the target molecule is bound—are already incorporated into the support structure. According to the present invention, various biomedical procedures can be used to manufacture the film, including 3D printing (e.g., extrusion, laser, etc., and combinations thereof), casting, electrospinning, weaving from treated or untreated infinite fibers, other techniques, and combinations thereof. Other techniques, such as polymer blending, can also be used. The resulting selected form is ensured by selectively removing the desired polymer component(s) by utilizing different melting points. Among other possible film manufacturing techniques is the sintering of beads (round particles) of various sizes (e.g., polystyrene beads or silica microspheres) followed by polymer coating. After removing the sintered component, what remains is a film structure consisting entirely of polymer and having the desired porosity (one or more levels). However, other techniques or combinations thereof that ensure the above-mentioned film properties can be used for the preparation of the film.

[0031] Preferably, 3D printing technology is used to manufacture membranes with structured or "computationally" unstructured shapes. For this purpose, it is best to use an extrusion-based 3D printer that extrudes polymers or hydrogels by heating, melting, and mechanical extrusion. A combination of all technologies is possible. 3D printers allow for the simultaneous precise (up to picoliters) pipetting of solutions or suspensions of selected active molecules to predetermined spots on the membrane structure. This represents another method for ensuring the activation of the desired membrane. Furthermore, the internal structure of each extruded filament is executed in the form of tunnels or two or more filaments (e.g., core, shell printing). This allows for control of membrane properties in active or inactive states, in addition to chemical, physicochemical, and mechanical membrane properties.

[0032] A second preferred method is electrospinning, which produces unstructured or partially structured membranes (the resulting macromaterials are always similar, if necessary). Using this method, thin layers consisting of multiple sublayers can be prepared, which can then be assembled into membranes of selective thickness. Simultaneously, electrospinning can be used to modify the surface properties of printed 3D membranes (increasing the specific surface area), alter the micro and nano properties of the membranes (such as the localized addition of functional groups that characterize the electrospinned fibers), or modify the activation of the membranes (if the electrospinning formulation contains biofunctionalized nanoparticles or other target molecules incorporated in other ways).

[0033] A third preferred membrane manufacturing technique is the preparation of fabrics having structured microstructures and structured or unstructured micro and nanostructures. These consist of an infinite number of fibers or pre-selected polymers. The latter can be further processed using electrospinning methods, which enable additional membrane properties.

[0034] The above process allows for the generation of a membrane support structure from a biocompatible polymer to which the target molecule will later be bound. The above process allows for the generation of a membrane support structure from a biocompatible polymer having already incorporated functionalized nanoparticles to which the target molecule will later be bound. As will be explained in detail below, the above process allows for the generation of a membrane support structure from a biocompatible polymer having already incorporated functionalized nanoparticles.

[0035] Membrane activation or biofunctionalization can be performed directly on a 3D support structure. In this case, the surface of the support structure is chemically treated using known methods. For example, the so-called carbodiimide method (CDI) using the reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) generates amide bonds under "mild conditions." Thus, it is possible to obtain covalently bonded surface functional groups on the surface and within the pores of the membrane support structure to which active sites, i.e., target molecules (e.g., antibodies, parts thereof, aptamers), are covalently bonded, whether or not they have a minimal impact on their activity. EDC is a water-soluble carbodiimide that further promotes the covalent bonding of active molecules to the membrane.

[0036] A typical activation procedure involves a reaction in an aqueous medium containing, in addition to the reagent EDC, the target molecule to be bound (such as an antibody), the membrane structure, and a buffer. After approximately 30 minutes, a membrane with an active antibody bound to the supporting structure is obtained. In other words, a membrane capable of affinity binding to target stem cells expressing an antigen that recognizes the antibody (or target molecule) bound to the membrane during the above process is obtained.

[0037] Membrane activation is performed using functionalized nanoparticles that can bind (e.g., covalently) to or unbound (i.e., "capture") the membrane's supporting structure, meaning "in situ" for biocompatible polymers. As a result, the surface functional groups function as anchor points or active sites for target molecule binding, recognizing and binding characteristic antigens bound to the surface of target stem cells on the surface of the functionalized nanoparticles.

[0038] When membrane activation is performed using functionalized nanoparticles, the functionalized nanoparticles are prepared using known methods such as CDI or amine-reactive crosslinking (along with functional groups or active sites for binding to the membrane's supporting structure and to target molecules that recognize and bind to characteristic antigens bound to the stem cell surface). The CDI method is mainly used for activating carboxyl and phosphate functional groups, while the other methods mentioned are mainly used for activating amine functional groups. These methods can be used simultaneously.

[0039] Membrane activation is performed using functionalized nanoparticles if the membrane support structure into which the functionalized nanoparticles are incorporated has already been generated by one of the processes described above. In this case, the functionalized nanoparticles, which have been pre-prepared by one of the processes described above, are first incorporated into the membrane support structure by one of the processes described above during the production of the support structure. As a result, the functionalized nanoparticles are incorporated "in situ" into the surface or pores of the support structure, or are chemically bound to the support structure. The functionalized nanoparticles incorporated into the support structure are characterized on their surface by the functional groups described above (NH2, OH, COOH, SH, etc.). These functional groups are then subjected to a chemical binding method (e.g., CDI method) for target molecules to the functional groups, and therefore to the support. As a result, membrane activation, i.e., binding of target molecules that recognize and bind characteristic antigens bound to the surface of stem cells on the surface of the functionalized nanoparticles, is performed, for example, by following the membrane activation procedure described above.

[0040] To increase the number of active sites on the surface and / or within the pores of the support structure incorporated into the functionalized nanoparticles for binding target molecules, the surface of the support structure is optionally treated mechanically (e.g., by grinding, cutting, or removing the upper layer of the support structure to a thickness of ~μm) or by another method (e.g., etching). This allows for the exposure of a large number of functionalized nanoparticles on the surface or within the pores of the support structure, improving the efficiency of binding target molecules to the support structure. In other words, a greater number of target molecules per volume unit / film surface is obtained.

[0041] Optionally, the necessary active sites on the surface and / or within the pores of the membrane support structure for binding target molecules can be obtained, particularly in the case of woven or electrospun membranes, by using oxygen plasma to ensure surface hydrophilicity and the presence of OH-, COOH- groups on the surface and / or within the pores of the support structure; by using nitrogen plasma (or ammonium plasma) to ensure surface hydrophilicity and the presence of NH2 functional groups on the surface and / or within the pores of the support structure; or by using hydrogen fluoride plasma (or HF) to optimize the hydrophobicity of the support structure. Plasma-treated surfaces allow for further steps of biofunctionalization beyond the use of the above activation methods. Each plasma treatment method may be repeated or combined as additional treatment of the membrane support structure is required.

[0042] When activating a membrane using functionalized nanoparticles, this is performed before the membrane is fabricated using 3D printing. This involves adding pre-prepared biofunctionalized nanoparticles, already bound to target molecules on their surface functional groups, "in situ" to a solution of a selected biocompatible polymer, followed by the membrane fabrication process described above. As a result, the biofunctionalized nanoparticles, already bound to the target molecules, are incorporated into the 3D membrane structure during the fabrication process.

[0043] In a preferred embodiment, the membrane is a 3D support structure consisting of several layers having a structured shape and pore sizes in the range of 100 μm to 200 μm. Each layer of the support structure is composed of the same biocompatible polymer, and activation of the membrane is performed using functionalized nanoparticles.

[0044] Preferably, the membrane according to the present invention is manufactured using 3D printing (bioprinting). A selected biocompatible polymer is melted, and molten, pre-prepared functionalized nanoparticles having surface functional groups are added "in situ." This creates a structured 3D membrane structure by 3D printing. This incorporates the functionalized nanoparticles into the 3D support structure of the membrane. The 3D support structure of the thus-fabricated membrane is activated via the functionalized nanoparticles containing the corresponding target molecule. That is, the selected target molecule binds to the functional groups of the nanoparticles on the membrane support structure (thus, biofunctionalized nanoparticles are obtained within the membrane). The above describes the method for manufacturing the membrane of the present invention.

[0045] The membrane of the present invention utilizes a method for separating and isolating target stem cells from a biological sample. Thus, sterile target stem cells in a physiological buffer of a known cell population size are obtained. Before being introduced into the membrane, the biological sample is pre-prepared accordingly. Specifically, the single-cell suspension is prepared. This ensures that the appropriate size of each cell in the suspension and the appropriate density of the suspension are maintained.

[0046] The preparation of single-cell suspensions involves known processes of biological sample disintegration and mechanical filtration. Biological sample disintegration processes include, but are not limited to, mechanical treatment (e.g., maceration, cutting, scraping, centrifugation), chemical treatment (e.g., treatment with erythrocyte lysis buffer, addition of anticoagulants), enzymatic treatment (e.g., use of collagenase, hyaluronidase, trypsin, or a combination thereof), and / or a combination thereof.

[0047] In particular, when the suspension is obtained from blood and / or blood-rich biological samples, the step of removing red blood cells by means of, for example, erythrocyte lysis buffer, density gradient centrifugation, labeled magnetic beads, a method called "buoyant" separation, or other known techniques may be optionally added to the preparation of the single-cell suspension.

[0048] The next step of the process is mechanical filtration as a preliminary method for separating particles (cells) from the selected biological sample based on not only the pore size of the filter (through which particles smaller than the pores of the filter pass) but also the chemical composition of the filter material. For example, certain substances adhere to the material constituting the selected filter more than other substances. Mechanical filtration includes mesh filters with different porosities (e.g., 10 μm to 100 μm). These are used either as a single unit or in a cascade of successive filters with decreasing pore sizes. Under the condition that the mesh filter material does not bind to the target stem cells, examples of the chemical composition of the filter include, but are not limited to, nylon, cellulose acetate, polylactic acid, polyglycolic acid, polyethylene terephthalate, polypropylene, and polycaprolactone.

[0049] When using a cascade filter, each filter in the cascade is made of a different material. The filters consist of commercially available filters (such as the Cell Strainer (registered trademark) manufactured by Corning) and / or self-developed 3D printed filters, or filters manufactured by other techniques and their combinations for this purpose. By pre-preparing the biological sample, an input single-cell suspension is obtained, whereby the size of each cell and / or potential smaller cell clusters in the suspension does not exceed the pore size of each membrane in at least two dimensions, and the density of the input cell suspension is less than 2×10 8 cells / mL, preferably maintained at 1×10 6 ~1×10 7 cells / mL.

[0050] The appropriate density of the input single-cell suspension is achieved, if necessary, by adding a physiological buffer (to achieve dilution) or increasing the amount of cells in the suspension (by concentration). The supply of the input cell suspension to the membrane is automatically controlled. A cell counter (in biological impedance mode) detects the number of cells approaching the membrane and automatically supplies or removes the physiological buffer to maintain the input cell suspension density at 2×10 8Maintain a level below cells / mL.

[0051] The size of cells and / or small cell clusters in the suspension does not exceed the pore diameter of the membrane. The preferred size of each cell and / or small cell cluster in the suspension does not exceed 70 μm in at least two dimensions.

[0052] The isolation of target stem cells from the input material, i.e., the biological sample, is performed based on the free flow of the input single-cell suspension through at least one membrane. During this process, the target stem cells are captured on the membrane surface and membrane pores by specific recognition and binding between characteristic antigens on the surface of the target stem cells and target molecules immobilized on the membrane, i.e., antibodies or fragments of antibodies against the characteristic antigens. The passage of other non-target cells through the membrane is not hindered (or is slightly hindered, for example, because the number of bound target stem cells increases, thereby reducing the effective porosity of the membrane).

[0053] Target stem cells can be isolated using a single membrane. However, cells can also be isolated using several membranes within a cascade, where each subsequent membrane in the cascade has the same or smaller pore size.

[0054] The separation process can be further enhanced by allowing multiple filters of the cell suspension across the membrane, thereby increasing its efficiency. Depending on the end use, target stem cells can be removed from the membrane or the membrane can be used together with the target stem cells in one of the following ways: In the latter case, for example, the membrane can be used as a tissue graft to be transplanted into a patient with a major trauma. After being placed in its natural environment, the membrane-bound stem cells differentiate into the desired surrounding tissue and effectively contribute to the regeneration of one or more surrounding tissues. The membrane to which the target stem cells are bound is used as a growth substrate to proliferate these cells in order to apply them in a desired manner (e.g., in therapy). Another way to use the membrane with captured target stem cells is to differentiate the cells into the appropriate tissue by external stimuli (e.g., by adding selected growth factors to the growth medium or by other stimuli). However, the selection of tissue is limited by the type of captured target stem cells. Thus, for example, bone segments can be obtained and transplanted into a patient. These are just examples, and there are many other possibilities for directly using the membrane with captured target stem cells.

[0055] Processes for removing target stem cells from a membrane include, but are not limited to, physical and mechanical processes (e.g., pressure fluctuations, pressure increases and decreases), physicochemical processes (e.g., ionic strength fluctuations by adding salts, buffers, or ultrapure water washing), biochemical processes (e.g., the use of enzymes such as peptidases to cleave bonds between the membrane and antibodies), chemical processes (e.g., reduction of disulfide bonds to thiol groups), and affinity processes (e.g., by adding compounds that have a higher affinity for the selected active functionalized surface (e.g., antibodies) than for the cells (which are relatively "large" particles)), and other processes and combinations thereof. These procedures for removing target stem cells from a membrane may be used separately or as a combination of the above procedures, or as a cascade system of the same or different procedures with any number of further repetitions. Preferred separation procedures are those that minimize stress and reduce the impact on the isolated target stem cells being removed from the membrane, such as processes that apply appropriate pressure difference intervals. The selected removal procedure will vary depending on the characteristics of the selected membrane, functionalized (or biofunctionalized) nanoparticles, target molecules, and target stem cells. As a result, various removal procedures or combinations thereof can be applied to various selected method processes for isolating target stem cells from biological samples.

[0056] The apparatus for isolating target stem cells from biological samples, i.e., from single-cell suspensions, consists of a housing fitted with electronic and mechanical components with appropriate controls, and a replaceable cassette. The electronic components include all the electronic components necessary for the operation of the apparatus. For example, these include an uninterruptible UPS power supply system, sensors for measuring flow rate and temperature to ensure and monitor the flow rate and the optimal temperature of 37°C suitable for working with the biological material (the temperature is adjusted as needed), a cell counter, an analog-to-digital converter, an electrical converter, etc. The mechanical parts of the apparatus also include all the mechanical components necessary for the operation of the apparatus. For example, these include a valve system, a pump and / or compressor, an opening and closing track for inserting the replaceable cassette, fittings for mounting the replaceable cassette to the housing, and a fluid system connection for the replaceable cassette based on a coupling for easy cassette replacement. The control unit ensures appropriate apparatus control to ensure optimal apparatus operating conditions, such as proper temperature control and appropriate flow rate adjustment to bring the input cell suspension to the desired concentration.

[0057] Optionally, the device may include a cleaning cassette for automatic cleaning when non-replaceable parts come into contact with biological materials. Cleaning of the device is performed automatically according to the relevant protocol.

[0058] The interchangeable cassette shown in Figure 1 contains container FC for the input single-cell suspension prepared from a biological sample, and the input suspension density can be increased to 2 × 10 by adding physiological buffer from container PBS as needed. 8 The apparatus comprises a mixing chamber MIX that reduces the cell / mL to less than one foil membrane AM of the present invention, a waste container W, and a collector SC for collecting the target stem cell suspension. A VVR control valve connected to a single pump compressor unit within the apparatus ensures adequate supply of the liquid or suspension. A replaceable cassette is inserted into the apparatus from the top or front.

[0059] The raw materials to be introduced, i.e., the single-cell suspension, are inserted into a replaceable cassette within container FC, along with a needle or other sterile transport and storage techniques. If necessary, physiological buffer is supplied from a PBS container to ensure that the single-cell suspension is of appropriate density within the mixing chamber MIX.

[0060] The single-cell suspension is then sent to a membrane AM, and the isolation of target stem cells from the input single-cell suspension is performed based on the free flow of the input single-cell suspension passing through at least one membrane AM. Target stem cells bind to the membrane AM, while all non-target cells are removed from the membrane surface by passing through the membrane AM or by washing into a waste container W as residual suspension. Target stem cells are removed from the membrane AM using physiological buffer under pressure supplied from a PBS container via a feedback loop. As a result, a sterile suspension of target stem cells in physiological buffer is collected in an SC collector. This target stem cell suspension can be applied to a patient immediately or subsequently used for various purposes.

[0061] The delivery of single-cell suspensions to the membrane AM is automatically regulated. A cell counter detects the number of cells approaching the membrane and automatically supplies physiological buffer from the container PBS, thereby maintaining the density of the cell suspension at 2 × 10⁶. 8 Maintain a level below cells / mL.

[0062] In a preferred embodiment, the cell counter is based on two electrodes of any material (such as silver) that detect bioimpedance, i.e., changes in electrical resistance. The cell counter is installed at two different locations: before the input single-cell suspension reaches the membrane AM, and before the sterile target stem cell suspension in physiological buffering reaches the collector container SC. A key part of the cell count is the characterization of living and dead cells using bioimpedance techniques to measure membrane conductivity AM (changes in the conductivity of dead cells due to leached cytosol) or viability staining, and small samples of cells from the collector container SC are collected online to assess living cells.

[0063] All sensors in the device are connected to a main computer equipped with a touchscreen and user interface (UI). The device connects to the internet via a LAN port to the main computer. This device is installed in hospitals, private outpatient clinics, or various research institutions.

[0064] The final product of the separation process obtained by the method and apparatus according to the present invention is sterile target stem cells in a physiological buffer (as defined in this application) having a known cell population size (number of isolated cells) and a known cell viability (live / dead cell ratio).

[0065] The final product is intended for use in medical and / or research settings. In particular (but without excluding other possible uses), possible uses include direct autologous and allogeneic cell transplantation into patients, laboratory animals, or diseased animals; in vitro culture, proliferation, and cell differentiation; direct cryopreservation of cells without culture for subsequent use; and further isolation of cell (sub)populations using other markers. [Examples]

[0066] (Membrane production) The membrane is fabricated using 3D printing technology. The initial 3D support structure of the membrane consists of 10 layers of polycaprolactone with a pore size of 100 μm. Each layer of polycaprolactone incorporates functionalized NiCu nanoparticles surrounded by a silica layer, along with NH2 functional groups on its surface. Before activation (i.e., binding of the target molecule), this support structure of the membrane undergoes a grinding process. As a result, more nanoparticles with functional groups are exposed. Separately, solutions of antibodies against CD90 and EDC are prepared and obtained so that the activated antibodies bind to the support structure of the membrane. The support structure is immersed in the activated antibody solution. After about 30 minutes, the antibodies bind to the support structure of the membrane, i.e., the NH2 functional groups. Subsequently, the membrane is washed three times with deionized water. This activates the membrane, preparing it for the isolation of target stem cells, in this case, stem cells expressing the CD90 antigen.

[0067] Procedure for isolating target stem cells from a biological sample (Example 1) CD34 from healthy donor bone marrow for transplantation in leukemia patients after chemotherapy + hematopoietic stem cell preparation The biological sample used to prepare the cells is peripheral blood collected by using apheresis following bone marrow induction. The introduced single-cell suspension is prepared as a "buffy coat," which is part of the blood sample produced by density gradient centrifugation and further washed with 1×PBS to remove red blood cells and serum.

[0068] The final single-cell suspension is prepared by injection in 1×PBS buffer. Using a needle, the suspension is added to a cassette with a 70 μm mechanical filter to remove the major cell clusters and the membranes to which the target molecule that recognizes and binds to the surface antigen CD34 expressed on hematopoietic stem cells is bound.

[0069] A flow regulator on the membrane ensures the optimal dosage of physiological fluid or buffer to prevent system clogging. Once all non-target cells are collected in the waste container, an efficient feedback loop re-filters the same solution to capture any remaining cells that did not bind to the membrane.

[0070] The next step involves washing non-target or unbound cells from the membrane. This is done in each feedback loop (not connected to a waste container) using a physiological solution or a buffer under pressure (e.g., 1 bar (100 kPa) or higher). Target cells are washed and collected in a separate container at the bottom of the apparatus. The washed and selected cells are suitable for direct dispensing.

[0071] The final product is sterile CD34 suitable for patient application. + This is a hematopoietic stem cell suspension. (Example 2) CD90 for autologous transplantation + Preparation of (mesenchymal stem cells) Swelling liposuction of subcutaneous fat is used as a biological sample for cell preparation. The introduced single-cell suspension is prepared as an interstitial vascular fraction (SVF) according to a known method in the following order: washing the liposuction with 1×PBS (removal of red blood cells and serum), enzymatic digestion with collagenase Ia (breakdown of intercellular junctions), gradient centrifugation and vibration (final separation of SVF cells and adipocytes), and removal of adipocytes by pipetting. The final single-cell suspension is prepared by injection with 1×PBS buffer.

[0072] Using a hypodermic needle, the suspension is added to a cassette with a 70 μm mechanical filter to remove the major cell clusters and the membranes to which target molecules that recognize and bind to the surface antigen CD90 expressed on mesenchymal stem cells from subcutaneous adipose tissue are bound.

[0073] A flow regulator on the membrane ensures the optimal dosage of physiological fluid or buffer to prevent system clogging. Once all non-target cells are collected in the waste container, an efficient feedback loop re-filters the same solution to capture any remaining cells that did not bind to the membrane.

[0074] The next step involves washing non-target or unbound cells from the membrane. This is done in each feedback loop (not connected to a waste container) using a physiological solution or a buffer under pressure (e.g., 1 bar (100 kPa) or higher). Target cells are washed and collected in a separate container at the bottom of the apparatus. The washed and selected cells are suitable for direct dispensing.

[0075] The final product is a sterile CD90 suitable for patients receiving treatment for various medical conditions (orthopedics, cardiology, plastic surgery, oral medicine, urology, oncology). + This is a hematopoietic stem cell suspension. When using single-cell suspensions in orthopedics, the obtained CD90 + The mesenchymal stem cell suspension is applied directly to the joint cavity (intra-articular injection) of joints with surface cartilage damage.

[0076] In oral pathology, the use of single-cell suspensions often leads to extensive bone resorption in the jaw after tooth extraction. This frequently hinders finding aesthetically appropriate prosthetic solutions for replacing the affected jaw and teeth. The optimal solution for reconstructing jaw defects is the use of mesenchymal stem cells. Existing voids need to be protected by the periosteum to prevent them from becoming too large, ensuring space for bone growth. Titanium mesh is typically used; however, 3D-printed meshes made of biocompatible materials are superior. Stem cells can then be applied to this pre-fabricated space. Through the activation of platelet-derived growth factors, the stem cells begin to differentiate into osteoblasts (young bone cells) and finally into osteocytes (adult bone cells). Over several months, the bone defect is filled with healthy, natural tissue.

Claims

1. A membrane manufacturing process, The aforementioned membrane is a membrane for separating target stem cells from a single-cell suspension containing stem cells. The single-cell suspension is obtained from a biological sample containing stem cells. The aforementioned membrane consists of a 3D support structure comprising at least one layer of a biocompatible polymer having pores. The biocompatible polymer is inactive with respect to target stem cells and does not affect the essential properties of the target stem cells. The pores have a diameter of 50 μm to 500 μm, which allows for the free flow of the introduced single-cell suspension through the support structure. The support structure has a selected target molecule covalently bonded to its surface and / or within its pores, and the target molecule is Recognizing characteristic antigens on the surface of isolated target stem cells, It binds to characteristic antigens on the surface of the isolated target stem cells. This method selectively isolates target stem cells by binding them to selected target molecules. The manufacturing process of the aforementioned film is as follows: Pre-preparing functionalized nanoparticles having a functional group on its surface that recognizes and binds to a target molecule that binds to a characteristic antigen bound to the surface of stem cells, During the film manufacturing process, pre-prepared functionalized nanoparticles are incorporated into or chemically bonded to a support structure made of a biocompatible polymer, thereby enabling the functionalized nanoparticles to be incorporated into or chemically bonded to the support structure in situ. To increase the number of active sites on the surface and / or within the pores of the support structure into which the functionalized nanoparticles are incorporated or chemically bonded, the functionalized nanoparticles are exposed on the surface or within the pores of the support structure by removing the upper layer of the support structure by mechanical, chemical, or plasma treatment. Activation of the membrane, wherein the support structure into which the functionalized nanoparticles are incorporated or chemically bonded is immersed in a solution of the target molecule, thereby causing the target molecule to bond to the functional groups of the functionalized nanoparticles. A film manufacturing process comprising the following features.

2. In the film manufacturing process described in claim 1, A film manufacturing process in which the integration of pre-prepared functionalized nanoparticles into a support structure involves the melting of a biocompatible polymer, and while the functionalized nanoparticles are being integrated into the film structure, previously prepared functionalized nanoparticles are added to the melt along with their surface functional groups, and then the support structure is fabricated.

3. In the film manufacturing process according to claim 1 or 2, The support structure is manufactured using a film manufacturing process, which involves 3D printing, electrospinning, weaving from treated or untreated infinite fibers, or a combination thereof.

4. In the film manufacturing process described in any one of claims 1 to 3, A film manufacturing process in which the activation of the film with functionalized nanoparticles is performed before film production, and pre-prepared biofunctionalized nanoparticles, along with target molecules already bound to surface functional groups, are added "in situ" to a solution of a selected biocompatible polymer, and then film production by 3D printing is performed, and the biofunctionalized nanoparticles to which the target molecules are bound are incorporated into the support structure during the manufacturing process.

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