KR102283848B1 - Porous scaffold for use in tissue engineering and disease therapy - Google Patents

Abstract

To a porous cell support comprising a serum-derived protein and a method for producing the same, according to one embodiment of the porous cell support, cells can be stably and continuously cultured, as well as a culture pattern suitable for the characteristics of each cell It can simulate actual tissue, has stable culture and high in vivo engraftment rate, and can be usefully used for evaluation of drug activity and toxicity using organoids, cell therapy agents, or production of target proteins.

Description

Porous cell scaffold for tissue engineering use or disease therapeutic use {POROUS SCAFFOLD FOR USE IN TISSUE ENGINEERING AND DISEASE THERAPY}

It relates to a porous cell support comprising a serum-derived protein and a method for preparing the same.

Porous scaffolds are widely applied as scaffolds for wound healing, burn healing, drug delivery, and regeneration of damaged tissues. In particular, the scaffold for the regeneration of damaged tissue requires a structure and stability suitable for culturing cells extracted and separated from a small amount of tissue in a sufficient amount, and must have biocompatibility and biodegradability that does not show side effects after transplantation into the body. Therefore, a biodegradable scaffold to effectively realize cell regeneration and growth should have a porous structure in which tissue cells adhere to the surface of the support to form a three-dimensional structure, and the movement of moisture and cell growth factors, etc. is easy, and It should be suitable for cell adhesion and growth. In addition, the porous scaffold should be well compatible with the surrounding tissues after being implanted in the body, and there should be no side effects such as an inflammatory reaction, and after the tissue is regenerated, it should have high biocompatibility because it does not biodegrade itself and does not remain as a foreign substance. Therefore, a porous scaffold with excellent biocompatibility, biodegradability, and mechanical properties is essential for tissue regeneration and restoration.

The cell culture-related biopharmaceutical industry market has been showing double-digit growth every year since 2011. In particular, in 2014, a market of more than $ 120 billion was formed, but in Korea, there is a shortage of specialized manpower related to 3D cell culture, so it is dependent on foreign institutions. 3D cell culture technology is a cutting-edge technology with bright future prospects that is used not only for drug efficacy and toxicity screening but also for the development of artificial organs. It is expected to grow to $8 billion by 2019, maintaining a growth rate of more than 10% annually. In addition, the tissue engineering and regeneration-related market (including cell-based artificial organs) is expected to reach USD 60.8 billion in 2021 from USD 13.6 billion in 2016, and is a promising market with a high annual average growth rate of 34.9%. is the field

Therefore, there is a need for research on porous cell scaffolds for producing a cell aggregate that has a high engraftment rate and storability and is implemented similar to an actual human tissue.

One aspect is to provide a porous cell support comprising a serum-derived protein obtained by treating the separated serum or plasma protein aqueous solution with a crosslinking agent to cross-link the protein in the serum plasma protein aqueous solution, and then reducing it with a reducing agent.

Another aspect, the porous cell support; And to provide a composition for tissue repair or regeneration comprising the cells dispensed in the porous cell support.

In another aspect, dispensing cells on a porous cell support and three-dimensional culture to form a three-dimensional cell aggregate; And to provide a method for evaluating the pharmacological activity or toxicity of the candidate substance comprising the step of treating the candidate substance to the three-dimensional cell aggregate.

In another aspect, dispensing cells on a porous cell support and three-dimensional culture to form a three-dimensional cell aggregate; And to provide a method for producing a target material comprising the step of isolating the target material from the culture or culture medium of the three-dimensional cell aggregate.

Another aspect includes the steps of treating the separated serum or plasma protein aqueous solution with a crosslinking agent to cross-link the protein in the serum or plasma protein aqueous solution; reacting the protein in the cross-linked serum or plasma protein aqueous solution with a reducing agent to obtain a reactant; and homogenizing the reaction to obtain a serum-derived protein.

In order to achieve the above object, one aspect is a porous cell support comprising a serum-derived protein obtained by treating the separated serum or plasma protein aqueous solution with a crosslinking agent to cross-link the protein in the serum or plasma protein aqueous solution, and then reducing it with a reducing agent. provides

As used herein, the term "serum-derived protein" refers to fetal bovine serum (FBS), bovine calf serum (BCS) or other animals (eg, mammals or humans) used for tissue culture. It may refer to a protein obtained by treating the derived serum with a crosslinking agent to cross-link the protein in the serum. In addition, the proteins in serum: serum albumin, lipoprotein, haptoglobin, transferrin, ceruloplasmin, immunoglobulin, various complements, fibrinogen, prothrombin, plasminogen, kininogen, prekallikrein, fibronectin, α2-HS- It may refer to a protein obtained by cross-linking a protein in an aqueous solution of a protein temporarily present in blood with glycoprotein, angiotensinogen, hormone, or the like.

The crosslinking agent used herein is dextran dialdehyde, 1-ethyl 3- [3-dimethylminopropyl] carboimide hydrochloride (1-ethyl-3- [3-dimethylaminopropyl] carboimide hydrochloride), vinylamine (vinylamine), 2-aminoethyl methacrylate, 3-aminopropyl methacrylamide, ethylene diamine, ethylene glycol dimethacrylate , methyl methacrylate, N,N'-methylene-bisacrylamide (N,N'-methylene-bisacrylamide), N,N'-methylenebis methacrylamide (N,N'-methylenebis-methacrylamide) ), diallyltartardiamide, allyl (meth)acrylate, lower alkylene glycol di(meth)acrylate, lower poly alkylene glycol di (meth) acrylate (poly lower alkylene glycol di (meth) acrylate), lower alkylene di (meth) acrylate (lower alkylene di (meth) acrylate), divinyl ether, divinyl sulfone, divinylbenzene, trivinylbenzene, trimethylolpropane tri(meth)acrylate, pentaderithritol tetra(meth)acrylate (pentaerythritol tetra(meth)acrylate), bisphenol A di(meth)acrylat e), methylenebis(meth)acrylamide, triallyl phthalate, diallyl phthalate, allyl glycidyl ether, alkyl choloride ),　transglutaminase, lysyl oxidase, protein disulfide-isomerase, protein-disulfide reductase, sulfhydryl oxidase (sulfhydryl oxidase), lipoxygenase, polyphenol oxidase, tyrosinase, peroxidase, laccase, horseradish peroxidase ( Horseradish peroxidase) and combinations thereof, and any other material that binds proteins is applicable.

Among the crosslinking agents, the enzyme crosslinking agent may be an enzyme derived from any microorganism (eg, Streptoverticillium mobaraense).

As used herein, the term “reducing agent” refers to a substance that reduces disulfide bonds of proteins in serum. The reducing agent used herein is beta-mercaptoethanol (b-mercaptoethanol), dithiolthreitol (ditiolthreitol: DTT), tris (2-carboxyethyl) phosphine (tris (2-carboxyethyl) phosphine: TCEP)), It includes cysteine, glutathione, and trishydroxypropyl phosphine (Tris(3-hydroxypropyl)phosphine: THPP).

The pore size of the porous cell support according to one embodiment is 50 to 600 μm, 100 to 550 μm, 150 to 500 μm, 200 to 450 μm, 250 to 400 μm, 300 to 350 μm, 50 to 300 μm, 100 to 250 μm, 150 to 200 μm, 300 to 600 μm, 350 to 550 μm, and 400 to 500 μm.

The elastic modulus of the porous cell support according to one embodiment is 1 to 10 kPa, 2 to 8 kPa, 3 to 8 kPa, 1 to 5 kPa, 4 to 10 kPa, 6 to 10 kPa, 1 to 3 kPa, or 4 to 6 kP can be

The porosity of the porous cell support according to one embodiment is 10 to 80%, 20 to 80%, 30 to 70%, 40 to 60%, 10 to 40%, 10 to 30%, 50 to 80%, or 60 to 80 It can be %.

In another embodiment, the porous cell support may undergo a reversible phase transition to a solid or semi-solid. In addition, the porous cell support may be insoluble.

The porous cell scaffold according to one embodiment is particularly suitable for tissue engineering, repair or regeneration. The difference in porosity can enable the migration of different cell types to the appropriate site of the porous cell scaffold. In another embodiment, the difference in porosity may enable the development of appropriate cell-to-cell neuronal connections among cell types comprising porous cellular scaffolds, which are required for proper structuring of developing/repairing/regenerating tissue. For example, the expansion of cell treatment can be more appropriately controlled through various porosity of the porous cell scaffold material. Therefore, the porous cell scaffold may include any tissue and cells.

The use of the porous cell scaffold according to one embodiment may include tissue engineering, 3D cell culture, or cell transport for therapeutic use.

In one embodiment, the porous cell support may be one in which cells are dispensed. The cells dispersed in the porous cell support according to one embodiment may include a group consisting of bacterial cells, yeast cells, mammalian cells, insect cells, and plant cells.

The cells, e.g., eukaryotic cells, are cells of yeast, fungi, protozoa, plants, higher plants and insects, or amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1. For example, cultured cells (in vitro), transplanted cells (graft cells) and primary cell cultures (in vitro and ex vivo), and in vivo (in vitro and ex vivo), and cells in vivo), and also mammalian cells, including humans. The organism may also be a yeast, a fungus, a protozoa, a plant, a higher plant and insect, an amphibian, or a mammal.

The cells dispensed in the porous cell support according to one embodiment include chondrocytes; fibrochondrocytes; osteocytes; osteoblasts; osteoclast; synovial membrane cells; bone marrow cells; nerve cells; adipocytes; mesenchymal cells; epithelial cells, hepatocytes, muscle cells; stromal cells; vascular cells; Stem Cells; embryonic stem cells; mesenchymal stem cells; progenitor cells derived from adipose tissue; peripheral blood progenitor cells; stem cells isolated from adult tissues; It may include any one selected from induced pluripotent stem cells (iPS cells), tumor cells derived therefrom, and combinations thereof.

The porous cell support may further include a growth factor, a growth stimulator, a physiologically active substance, or a cell-binding mediator such as peptides or polysaccharides for inducing differentiation or proliferation of the divided cells.

In one embodiment, the growth factor or growth stimulator is a cell adhesion mediator; biologically active ligands; integrin binding sequence; ligand; Various growth and/or differentiation agents (eg, epithelial cell growth factor, IGF-I, IGF-II, TGF-[beta], growth and differentiation factor, matrix-derived factor SDF-1; vascular endothelial growth factor, fibroblast growth factor , platelet-derived growth factor, insulin-derived growth factor and transforming growth factor, parathyroid hormone, parathyroid hormone-related peptide, bFGF; nerve growth factor (NGF) or muscle-forming factor (MMF); heparin-binding growth factor (HBGF), transforming growth factor alpha or beta, alpha fibroblast growth factor (FGF), epithelial cell growth factor (TGF), vascular endothelial growth factor (VEGF), SDF-1;TGF[beta] superfamily factor;BMP-2;BMP-4; BMP-6; BMP-12; Sonic Hedgehog; GDF5; GDF6; GDF8; PDGF); small molecules that affect the upregulation of specific growth factors; tenacin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboplastin; thrombin-derived peptide; TNF alpha/beta; matrix metalloproteinase (MMP); heparin-binding domain; heparin; heparan sulfate; DNA fragments, DNA plasmids, short interfering RNA (siRNA), transfectants, or any mixture thereof may be included.

Another aspect includes the steps of treating the separated serum or plasma protein aqueous solution with a crosslinking agent to cross-link the protein in the serum or plasma protein aqueous solution; reacting the protein in the cross-linked serum or plasma protein aqueous solution with a reducing agent to obtain a reactant; and homogenizing the reaction product to obtain a serum-derived protein.

In the step of treating the separated serum or plasma protein aqueous solution with a crosslinking agent, the specific activity of the crosslinking agent is approximately 2 to 100 unit/mg, 7 to 95 unit/mg, 12 to 90 unit/mg, 17 to 85 unit/mg , 22 to 80 unit / mg, 27 to 75 unit / mg, 32 to 70 unit / mg, 37 to 65 unit / mg, 42 to 60 unit / mg, 47 to 55 unit / mg, 2 to 50 unit / mg, 7 to 45 unit/mg, 12 to 40 unit/mg, 17 to 35 unit/mg, 22 to 30 unit/mg, 50 to 100 unit/mg, 55 to 95 unit/mg, 60 to 90 unit/mg, 65 to 85 unit/mg, 70 to 80 unit/mg, 2 to 30 unit/mg, 7 to 25 unit/mg, 12 to 20 unit/mg, 30 to 60 unit/mg, 35 to 55 unit/mg, 40 to 50 unit/mg, 60 to 100 unit/mg, 65 to 95 unit/mg, 70 to 90 unit/mg, 75 to 85 unit/mg.

Specific activity herein may refer to a unit unit per matrix protein (herein, serum, or protein in serum).

In the step of reacting the protein in the serum or plasma protein aqueous solution with the reducing agent, the concentration of the reducing agent is approximately 5 to 50 mM, 10 to 45 mM, 15 to 40 mM, 20 to 35 mM, 25 to 30 mM, 5 to 25 mM , 10 to 20 mM, 25 to 50 mM, 30 to 45 mM, 35 to 40 mM. The reaction with the reducing agent may be carried out at 20 to 40 °C.

In the step of homogenizing the reactant to obtain a serum-derived protein, the homogenization may be performed in a tissue homogenizer. The step may also include homogenizing and then centrifuging to recover a suspension of serum-derived protein.

In another embodiment, the method may further include aging the serum-derived protein. The aging may include washing serum-derived proteins; And it may include the step of aging the washed serum-derived protein at 1 to 15 ℃.

In another embodiment, the method may further comprise molding the aged serum-derived protein and freeze-drying. The molding may be performed in a casting mold having a desired shape and size. The freeze-drying may include freezing at -40°C to -240°C for 1 minute to 3 hours, followed by drying.

In addition, the method includes washing the dried serum-derived protein with an aqueous solution or an organic solvent to wash impurities other than the protein; Alternatively, it may include immersing the washed serum-derived protein in a preservative solution.

The porous cell support according to one embodiment has the effect of not only stably and continuously culturing cells, but also exhibiting a culture pattern suitable for the characteristics of each cell. In addition, the porous cell support according to one embodiment has the effect of enabling long-term culture even with the conventional 2D culture method of culturing in cell culture dishes and plates.

One aspect, the porous cell support; And it relates to a composition for tissue repair or regeneration comprising the cells dispensed in the porous cell support.

The cells dispensed in the porous cell support may be a three-dimensional cultured three-dimensional cell aggregate.

As used herein, the term "cellular therapeutic agent" refers to cells and tissues produced through isolation, culture, and special manipulation from humans, and as pharmaceuticals (U.S. FTA regulations) used for the purpose of treatment, diagnosis and prevention. Or, through a series of actions, such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells extracellularly or changing the biological properties of cells in other ways to restore the function of the tissue, these cells can be used in the treatment, diagnosis and prevention of diseases. Drugs used for that purpose.

The term “treatment” refers to or includes the alleviation, inhibition of progression or prevention of a disease, disorder or condition, or one or more symptoms thereof, and “a pharmaceutically effective amount” refers to alleviation of a disease, disorder or condition, or one or more symptoms thereof. , may mean any amount of the composition used in the practice of the invention provided herein sufficient to inhibit or prevent progression.

The terms “administering,” “applying,” “introducing,” and “implanting” are used interchangeably and refer to a method or route that results in at least partial localization to a desired site of a patch or composition according to one embodiment. may refer to the placement of a patch or composition according to one embodiment into a subject by

As used herein, the term "cell aggregate" or "three-dimensional cell aggregate" refers to a state in which two or more cells are dense, and may be in a tissue state or a multicellular state. Each cell aggregate may exist as a tissue itself or a part, or a multicellular aggregate, and may include cell-like-organisms differentiated from stromal cells.

As used herein, the term “three-dimensional” may refer to a solid having a geometric three-parameter (eg, depth, area, height, or X, Y, Z axis) model that is not two-dimensional. there is. The three-dimensional cell aggregate may refer to a cell aggregate formed in a multilayered or spherical form, rather than a cell aggregate formed of a single layer of cells like a general cell aggregate.

The tissue may be heart, joint, bone, blood vessel, stomach, liver, skin, kidney, cartilage, intestine, nerve, or pancreas. A person skilled in the art may dispense somatic cells or stem cells suitable for the desired tissue reconstruction in the porous cell support. Alternatively, the same method can be applied for the study of tumor cells derived from the tissue.

The porous cell support according to one embodiment has a high engraftment rate when transplanted in vivo, and can be used to form a three-dimensional cell aggregate or differentiate into a tissue, so that it can be usefully used for tissue repair or regeneration. In addition, since the porous cell support according to one embodiment has stable cell culture ability even in freezing and thawing, there is an effect that it can be usefully used for commercialization of cell therapeutics.

The three-dimensional cell aggregate may be a sphere or sheet of a size that can be identified with the naked eye, for example, may be a spherical three-dimensional cell aggregate having a diameter of approximately 300 to 2000 μm, and in one embodiment, 300 to 1000 μm. The diameter of the spherical three-dimensional cell aggregate can be adjusted to a size that can be visually recognized by those skilled in the art by the culture method according to one embodiment. In addition, the spherical three-dimensional cell aggregate according to one embodiment may include approximately 3.0Х10 ⁵ to 1.0Х10 ⁶ cells within a diameter of 400 μm. Also, in one embodiment, the three-dimensional cell aggregate may secrete various proteins and functional substances.

Therefore, the three-dimensional cell aggregate according to one embodiment can be usefully used as a cell source when supplying a cell therapy agent or a physiologically active material. Hereinafter, the use of the three-dimensional cell aggregate is as follows.

As described above, since the three-dimensional cell aggregate can secrete epithelial cell growth factor, various proteins and functional substances, it is transplanted into an individual in need thereof and serves as a cell supply source, thereby promoting skin regeneration or angiogenesis. can do. In addition, it can be usefully used in pharmaceutical compositions for preventing and treating diseases related to the heart, stomach, liver, skin, kidney, cartilage, intestine, nerve, or pancreas by promoting tissue regeneration or tissue regeneration.

The heart-related disease may include, for example, angina pectoris, myocardial infarction, cardiomyopathy, valvular disease, aortic disease and arrhythmia. The gastrointestinal disease may include gastritis, gastric ulcer, gastric cancer, gastric adenoma, Helicobacter pylori infection and gastric MALT lymphoma. The liver-related disease may include hepatitis, sarcoidosis, fatty liver, alcoholic liver disease, liver cancer, cirrhosis, preeclampsia, non-Hodgkin's lymphoma, Wilson's disease, Reye's syndrome, other neonatal jaundice, hepatochondrosis, liver abscess, and liver hemangioma. The skin-related diseases are eczematous skin diseases, erythema, urticaria, mild rash, papules scaly disease, insect and parasite-borne diseases, superficial dermatophytosis, bacterial infection diseases, viral diseases, adult diseases, autoimmune bullous diseases, connective tissue Diseases, dyspigmentation, acne, rosacea, and skin tumors may be included. The kidney-related diseases include acute kidney injury (acute renal failure), chronic kidney disease (chronic renal failure), end-stage chronic renal failure, chronic glomerulonephritis, rapidly progressive glomerulonephritis, acute glomerulonephritis, acute nephritis, acute interstitial nephritis, chronic interstitial nephritis. , hematuria, proteinuria, asymptomatic urinary abnormality, nephrotic syndrome, acute pyelonephritis, tubular defect, hereditary renal disease, kidney stone, hydronephrosis, renal insipidus, renal cell carcinoma, transitional epithelial cell carcinoma, angiomyolipoma, autosomal dominant polycystic kidney , simple cysts and renal tuberculosis. The cartilage-related disease may include degenerative arthritis, post-traumatic arthritis, dissociative osteochondritis, and osteomalacia. The intestinal diseases include ulcers, enteritis, bleeding, tumors, intestinal adhesions, intestinal obstruction, intestinal paralysis, colorectal cancer, colon polyps, irritable bowel syndrome, ulcerative colitis and Crohn's disease, tuberculosis enteritis, infectious diarrhea and colitis, vascular growth disease and It may include colonic diverticulosis. The nerve-related diseases may include congenital anomalies, cancer, infection, trauma, hemorrhage, and autoimmune diseases. The pancreatic-related disease may include diabetes, acute pancreatitis, chronic pancreatitis and pancreatic cancer.

The dosage of the cell therapy agent or pharmaceutical composition according to one embodiment is approximately 1.0Х 10 ⁵ to 1.0Х 10 ⁸ cells/kg (body weight), or 1.0Х 10 ⁷ to 1.0, based on the three-dimensional cell aggregate constituting the active ingredient. Х 10 ⁸ cells/kg (body weight). However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be one time or two or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site. For animals other than humans, the same dose as that of humans per kg or, for example, the above dose is converted by the volume ratio (for example, the average value) of the target animal and the human organ (heart, etc.) One dose can be administered. Examples of the animal to be treated according to one embodiment include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs and others. This includes pets.

The cell therapy agent or pharmaceutical composition according to one embodiment may include a cell aggregate and a pharmaceutically acceptable carrier and/or additive as an active ingredient. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, it is also preferable to combine with organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, etc. . When the cell therapy agent or pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

The composition for tissue reconstruction or repair, cell therapy or pharmaceutical composition according to one embodiment, if necessary depending on the administration method or formulation, suspending agent, solubilizing agent, stabilizing agent, isotonic agent, preservative, adsorption inhibitor, surfactant, Diluents, excipients, pH adjusters, soothing agents, buffers, reducing agents, antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

A composition for tissue reconstruction or repair according to an embodiment, a cell therapy agent or a pharmaceutical composition is a pharmaceutically acceptable carrier and / Alternatively, it may be prepared in a unit dose form by formulating using an excipient, or it may be prepared by internalizing it in a multi-dose container. Herein, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets or capsules.

Another aspect provides a three-dimensional culture system for drug screening comprising a three-dimensional cell aggregate according to an embodiment.

In another aspect, dispensing cells on a porous cell support and three-dimensional culture to form a three-dimensional cell aggregate; And it provides a method for evaluating the pharmacological activity or toxicity of the candidate substance comprising the step of treating the candidate substance to the three-dimensional cell aggregate.

The candidate substance, for example, the test compound or test composition is a small molecule compound, an antibody, an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA ( short hairpin RNA, shRNA), nucleic acid, protein, peptide, and other extracts or natural products.

The three-dimensional cell aggregate has an artificial cell shape that mimics the in vivo environment, and can be usefully used for research on actual cell types and functions, or therapeutic agents (eg, the above skin diseases or vascular diseases). Therefore, the three-dimensional culture system for drug screening including the three-dimensional cell aggregate can replace animal experiments in tests for the efficacy of disease treatment agents such as pharmaceuticals or cosmetics, or tests for inflammation and allergy.

In another aspect, dispensing cells on a porous cell support and three-dimensional culture to form a three-dimensional cell aggregate; And it provides a method for producing a target material comprising the step of isolating the target material from the culture or culture medium of the three-dimensional cell aggregate.

The target substance may be a target protein.

The culture may be cultured in a medium containing a carbon source.

As used herein, the term "carbon source" refers to a fermentable carbon substrate desirable as an energy source for a host organism or microorganism, such as one that can be metabolized by a production cell line, usually in a purified form or raw material, such as a source carbohydrate, complex nutritional substance, etc. It means a source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, alcohols including glycerol, provided as a substance. The carbon source may be used according to the invention as a single carbon source or as a mixture of different carbon sources.

As used herein, the term “product of interest (POI)” refers to a polypeptide or protein produced by recombinant technology in a host cell. More specifically, the protein may be a polypeptide that does not naturally occur in the host cell (i.e., a heterologous protein), or may be native to the host cell (i.e., a homologous protein to the host cell), for example, By transformation with a self-replicating vector containing a nucleic acid sequence encoding the POI, or by recombinant technology integration of one or more copies of the nucleic acid sequence encoding the POI into the genome of the host cell, or, for example, a promoter produced by recombinant modification of one or more regulatory sequences that control the expression of the gene encoding the POI of the sequence. In some instances, the term POI, as used herein, refers to any product of metabolism by the host cell as mediated by a recombinantly expressed protein. Specifically, the POI is a eukaryotic protein, preferably a mammalian protein. POI is a heterologous recombinant polypeptide or protein produced as a protein secreted by a eukaryotic cell, preferably a yeast cell. Examples of proteins include immunoglobulins, immunoglobulin fragments, aprotinins, tissue factor pathway inhibitors or other protease inhibitors, and insulin or insulin precursors, insulin analogues, growth hormones, interleukins, tissue plasminogen activators, transforming growth factor a or b, glucagon, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GRPP, factor VII, factor VIII, factor XIII, platelet derived growth factor 1, serum albumin, enzyme, e.g. For example, lipases or proteases, or functional analogues, functionally equivalent variants, derivatives and biologically active fragments having functions similar to those of a native protein. A POI may be structurally similar to a native protein, and may include the addition of one or more amino acids to either or both the C- and N-terminus or to a side chain of the native protein, one or more amino acids at one or multiple different sites in the native amino acid sequence. It can be derived from a native protein by substitution of amino acids, deletion of one or more amino acids at one or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence. . Such modifications are known for several of the proteins mentioned above.

The POI produced according to the present invention may be a multimeric protein, preferably a dimer or a tetramer. POIs include therapeutic proteins, enzymes and peptides, proteins, antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, process enzymes, growth factors, including antibodies or fragments thereof; It is a recombinant or heterologous protein selected from hormones and cytokines or metabolites of POIs. A specific POI is an antigen-binding molecule such as an antibody or a fragment thereof. Among the specific POIs are monoclonal antibodies (mAbs), immunoglobulins (Ig) or immunoglobulin class G (IgG), heavy chain antibodies (HcAb') or fragments thereof, e.g., fragment-antigen binding (Fab), Fd, single chain variable fragments (scFvs), or engineered variants thereof, such as Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers or minibodies and VH or VHH or V-NAR, etc. Antibodies, such as single-domain antibodies.

POI can be produced using a recombinant host cell line by culturing the transformant thus obtained in an appropriate medium, isolating the expressed product or metabolite from the culture, and optionally purifying it by a suitable method.

The transformant according to the present invention can be obtained by introducing such a vector DNA, for example, a plasmid DNA into a host, and selecting a transformant that expresses the POI or the host/cell/metabolite in high yield. Host cells are treated to introduce foreign DNA by methods commonly used for transformation of eukaryotic cells, such as the electric pulse method, protoplast method, lithium acetate method, calcium chloride method, and modifications thereof.

Preferred host cell lines according to the present invention retain the genetic characteristics used according to the present invention, and the production level is maintained at a high level, for example, at a level of at least μg after about 20 passages, at least 30 passages, at least 50 passages. do.

The methods disclosed herein may further comprise culturing said recombinant host cell under conditions that permit expression of the POI in a secreted form or as an intracellular product. Then, the recombinantly produced POI or host-cell metabolites can be isolated from the cell-culture medium and further purified by techniques known to those skilled in the art.

The POI produced according to the present invention may be isolated and purified using conventional state of the art, which comprises increasing the desired POI concentration and/or reducing the concentration of at least one impurity.

When the POI is secreted from the cell, it can be isolated and purified from the cell medium using state of the art. The secretion of the recombinant expression product from the host cell is generally for reasons including facilitation of the purification process, since it is recovered from the culture supernatant rather than the complex mixture of proteins that occurs when the yeast cell is disrupted to release the intracellular protein. It is advantageous.

In addition, the cultured transformant cells can be disrupted by ultrasonic waves or mechanically, enzymatically or chemically to obtain a cell extract containing the desired POI, from which the POI is isolated and purified.

As separation and purification methods for obtaining a recombinant polypeptide or protein product, methods using solubility differences (eg, salting out and solvent precipitation), methods using molecular weight differences (eg, ultrasound and gel electrophoresis) , a method using charge difference (e.g., ion exchange chromatography), a method using specific affinity (e.g. affinity chromatography), a method using hydrophobicity difference (e.g., reversed-phase high performance liquid chromatography) ) and a method using isoelectric point difference (eg, isoelectric point electrophoresis) may be used.

The highly purified product is essentially free of contaminants and proteins and preferably has a purity of at least 90%, more preferably at least 95% or at least 98%, 100% or less. A purified product can be obtained by purification of cells, culture, and supernatant from cell debris.

The following standard methods for isolation and purification are preferred: cell destruction (when POI is obtained intracellularly), cell (debris) separation and microfiltration or washing by means of a tangential flow filter (TFF) or centrifugation, sedimentation or heat POI purification by treatment, POI activation by enzymatic digestion, chromatography such as ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion (SEC) or HPLC chromatography POI purification by , POI precipitation of concentration and washing by ultrafiltration steps.

The isolated and purified POI can be identified by a conventional method, for example, Western blotting, HPLC, activity assay or ELISA.

In general, the host cell expressing the recombinant product may be any eukaryotic cell suitable for recombinant expression of a POI. Examples of mammalian cells include BHK, CHO (CHO-DG44, CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1SV, CHO-S), HeLa, HEK293, MDCK, NIH3T3, NS0, PER.C6, SP2/0 and VERO cells. Examples of yeast used as the host cell include, but are not limited to, genus Saccharomyces (eg, Saccharomyces cerevisiae), genus Pichia (eg, P. pastoris or P. metanorica). ), genus Comagataella (K. pastoris, K. pseudopastoris or K. papii), Hansenula polymorpha or Kluyveromyces lactis.

The porous cell support according to one embodiment can not only stably and continuously culture the cells, but also simulate the actual tissue by displaying a culture pattern suitable for the characteristics of each cell, and has a stable culture and a high engraftment rate in vivo. , there is an effect that can be usefully used for evaluation of drug activity and toxicity using organoids, cell therapy products, or production of target proteins.

1 is a result of culturing the SNU017 gastric cancer cell line for 9 days using a porous cell support according to an embodiment.
2 is a result of culturing a SiHA cervical cancer cell line using a porous cell support according to an embodiment for 14 days.
3 is a result of culturing hACs (chondrocytes) for 7 days using a porous cell support according to an embodiment.
4 is a result of culturing enteroendocrine cells for 13 days using a porous cell support according to an embodiment.
5 is a week after injection of a three-dimensional cell aggregate of enteroendocrine cells (EEC) using a porous cell support according to an embodiment into the renal membrane of a mouse.
6 is a result of freeze-thaw test after culturing liver cancer cells for 12 days using a porous cell support according to an embodiment.
7 is a graph comparing the expression levels of hepatocellular carcinoma cells cultured using a porous cell support according to an embodiment and albumin of hepatocellular carcinoma cells cultured in a positive control using the ELISA method.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of a porous cell scaffold

A porous cell scaffold using serum-derived protein is prepared as follows.

First, serum proteins were reduced with a crosslinking and reducing agent with a crosslinking agent. Specifically, in a clean booth of Class 100 or less, the same amount of fetal bovine serum or bovine serum was treated with 1 mg/ml microvial transglutaminase (specific activity unit 20 U/mg, Sigma Aldrich). Thereafter, a reducing agent dithiothreitol (DTT, Sigma Aldrich) was added to a final concentration of 15 mM and reacted at 37° C. for 12 hours to prepare a reactant. Thereafter, a washing buffer (6M urea and 0.1M sodium acetate, pH 5.0) was added to the reaction mixture, homogenized with an electric tissue grinder (Homogenizer D1000, Benchmark Scientific), and centrifuged to recover a suspension of serum-derived protein. The suspension was washed with lactic acid and then stirred at 10,000 rpm for 20 minutes using a high-speed stirrer (MaXtir® 500S, DAIHAN-brand®), and aged at 4° C. for 8 hours. The aged serum-derived protein was transferred to a casting mold to form a desired shape, frozen at -80 °C for 1 hour, and then freeze-dried for 24 hours. Thereafter, the dried serum-derived protein was washed with 100% ethanol and sterile tertiary distilled water 5 times each at room temperature to wash impurities other than the serum-derived protein to prepare a porous cell support for serum-derived protein. In subsequent experiments, the porous cell support of the serum-derived protein was immersed in a preservative solution (1XPBS, cell culture media, DW, etc.) and used.

The characteristics of the prepared cell support are shown in Table 1 below.

pore size Average diameter about 200㎛ (100 to 400㎛) pH About 7.0 to 7.4 in PBS or tissue culture medium suspension storage temperature room temperature

Experimental Example 1. Three-dimensional culture of cancer cells using a porous cell scaffold By culturing cancer cells using the porous cell scaffold prepared in Example 1, culture stability and sustainability were confirmed.

Specifically, in order to remove the preservative solution as much as possible from the porous cell support prepared in Example 1, 1x PBS was added to sufficiently submerge the porous cell support and gently shaken and rinsed, and then 1x PBS was removed. This was repeated once more to remove the preservative. Thereafter, SNU017 (KCLB), a gastric cancer cell line, and SiHA (KCLB), a cervical cancer cell line, were dispensed to the ^{porous cell support in the number of 5 x 10 5 cells.} Thereafter, the porous cell support in which the cells were dispensed was cultured in a cell culture medium (RPMI, Gibco) at 37° C. and CO ₂ 5% in an incubator.

On the 9th day and 14th day of culture, a paraffin block was formed, and a slide was made, and H&E staining was performed to perform histological examination, and the results are shown in FIGS. 1 and 2 .

As shown in FIG. 1 , as a result of culturing the SNU017 cell line for 9 days using the porous cell support, it was confirmed that it was cultured while forming a spherical cancer tissue structure similar to that in an actual tissue in the body.

In addition, as shown in FIG. 2 , as a result of culturing the SiHA cell line for 14 days using the porous cell support, it was confirmed that it was cultured while forming a desk tissue-type cancerous tissue structure similar to that in an actual tissue in the body.

Experimental Example 2. Three-dimensional culture of chondrocytes using a porous cell scaffold

Chondrocytes are generally known to be difficult to culture in vitro. In this example, chondrocytes were cultured using the porous cell support prepared in Example 1 to confirm culture stability and sustainability.

Specifically, chondrocytes were three-dimensionally cultured in the same manner as in Experimental Example 1, except that chondrocytes (hACs) collected from actual patients were ^{aliquoted in the number of 5 x 10 5 cells.}

Thereafter, Live and Dead Cell staining was performed on the three-dimensionally cultured chondrocytes on the 7th day of culture. Thereafter, the stained cells were confirmed with a confocal laser microscope (LSM880 with Airyscan, Zeiss), and the results are shown in FIG. 3 . In FIG. 3 , green indicates live cells, and red indicates dead cells.

Z1 to Z9 of FIG. 3 mean that a cross section of every 10 μm in height is taken by fluorescence on the vertical axis.

As shown in FIG. 3 , it can be observed that the dead cells displayed in red were not observed for one week and that they were stably cultured. The patient-derived chondrocytes cultured on the porous cell support according to one embodiment showed a high survival rate.

Experimental Example 3. Three-dimensional culture of enteroendocrine cells using a porous cell scaffold

Enteroendocrine cells were cultured using the porous cell support prepared in Example 1 to confirm culture stability and sustainability.

Specifically, enteroendocrine cells were three-dimensionally cultured in the same manner as in Experimental Example 1, except that enteroendocrine cells collected from mice were ^{aliquoted in the number of 5 x 10 5 cells.}

Thereafter, DAPI and Hematoxylin & Eosin (H&E) staining was performed on the three-dimensionally cultured enteroendocrine cells on the 11th and 13th days of culture. The stained cells were confirmed with a fluorescence microscope (Axiovert 200, Zeiss), and the results are shown in FIG. 4 .

As shown in FIG. 4 , it is usually difficult for enteroendocrine cells to be cultured for more than a week, but the three-dimensional cell aggregate of enteroendocrine cells cultured on a porous cell support according to one embodiment was stably cultured for more than two weeks without deterioration in cell status. Could know.

As a result of the above, it was found that the porous cell support according to an embodiment can not only stably and continuously culture the cells, but also observe a culture pattern suitable for the characteristics of the cells for each cell. In addition, it was found that the conventional 2D culture method of culturing in cell culture dishes and plates has the effect of enabling long-term culture.

Experimental Example 4. Engraftment rate of three-dimensional cell aggregates using porous cell support

In this example, in order to apply the porous cell scaffold prepared in Example 1 to tissue regeneration treatment, the engraftment rate of the three-dimensional cell aggregate of enteroendocrine cells on the renal membrane was measured.

Specifically, after culturing the cells as in Example 3 in the same way, they were transplanted into the mouse kidney membrane with a Hamilton syringe. One week after transplantation, the mice were euthanized, and the kidneys were separated and dissected, and the remaining amount was directly observed. Afterwards, only the transplanted part was removed, the wet weight was measured, the engraftment rate was confirmed with the number of engrafted cells, a paraffin block was formed, a slide was made, and H&E staining was performed to perform histological examination. The results are shown in FIG. 5 .

As shown in Figure 5, the cell body obtained by culturing enteroendocrine cells in FBS-free for 2 weeks on a porous cell support according to one embodiment was transplanted into the renal membrane of a mouse, and as a result of culturing for 1 week, about 80% or more The survival rate was confirmed.

Considering that the in vivo engraftment rate is less than 5% when the stem cells are injected, it can be seen that the porous cell support according to one embodiment can be usefully used for cell therapy for tissue regeneration.

Experimental Example 5. Freeze-thaw test of three-dimensional cell aggregates using a porous cell support

In this example, in order to confirm that the porous cell scaffold prepared in Example 1 is easy to commercialize and transport for practical use in the commercialization of tissue regeneration treatment, a freeze-thaw experiment was performed.

Specifically, the lung cancer cell lines NCI-H28 and NCI-H2170 (KCLB) were ^{dispensed in the number of 5 x 10 5} cells on the porous cell support according to one embodiment, and then cultured for 28 days. For cryopreservation, deepfreezer was stored in a freezing solution (Media: FBS: DMSO, 5:4:1) for 30 days at -80 ° C, thawed and cultured for 12 days. Thereafter, H&E staining was performed on the cells cultured on the support, and the results are shown in FIG. 6 .

As shown in FIG. 6 , it was confirmed that even when the porous cell support according to one embodiment was frozen or thawed, it was possible to re-culture the cells.

These results mean that the porous cell support according to one embodiment has a beneficial effect on commercialization and delivery as a cell therapy agent.

Experimental Example 6. Protein production using porous cell support

In order to analyze whether a useful substance (target protein) can be produced using the porous cell scaffold prepared in Example 1, the protein production ability of the liver cancer cells was measured for the three-dimensional cell aggregate.

Specifically, HepG2 cells were seeded in a 24-well plate in ^{the number of 5 x 10 5} cells, and then cultured in MEM (10% FBS added) medium for 7 days, 14 days and 21 days. Thereafter, the concentration of albumin in the cell culture was measured by the method of Human ALB solid-phase sandwich ELISA (enzyme-linked immunosorbent assay). As a control, a conventional two-dimensional cell culture method was performed. Two-dimensional cell culture is performed in a 6-well plate. carried out. In addition, as a positive control, Matrigel ^® (Corning, 356231) and Alvetex™ (ReproCell, Glasgow, UK) were used. Specifically, cells were cultured in the same manner as above, except that the commercially available three-dimensional cell culture support was used. The concentration of albumin was measured in the cell cultures of the control groups, and the results are shown in FIG. 7 .

As shown in FIG. 7 , in the case of the porous cell scaffold according to one embodiment, the expression level of albumin indicating the degree of activation of liver cancer cells was higher than in the case of using ^{Matrigel ® and Alvetex™, which are positive controls.} The above results mean that the porous cell support according to an embodiment can be usefully used for the production of a target substance (protein).

Claims (1)

After treating the separated serum with transglutaminase to cross-link the proteins in the serum, the reaction product obtained by treatment with a reducing agent that reduces the disulfide bonds of the proteins in the serum is homogenized, and the suspension is recovered by centrifugation. After stirring the recovered suspension, the stirred suspension is left at 1 to 15° C. to include the obtained serum-derived protein composition,
The reducing agent beta-mercaptoethanol (b-mercaptoethanol), dithiothreitol (ditiolthreitol: DTT), triscarboxyethyl phosphine (tris (2-carboxyethyl) phosphine: TCEP)), cysteine (cysteine), glutathione ( glutathione), trishydroxypropyl phosphine (Tris(3-hydroxypropyl)phosphine: THPP)), and one selected from the group consisting of combinations thereof,
The composition pore size is 150 to 400㎛,
The elastic modulus of the composition is 1 to 10 kPa,
The porosity of the composition is 30 to 70%,
Wherein the reducing agent is treated at a concentration of 5 mM to 50 mM,
A porous cell scaffold for tissue engineering use through three-dimensional cell culture, or therapeutic use.

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