WO2020246781A1 - Method for micronizing soft living tissue, and soft living tissue particulate graft produced thereby - Google Patents

Abstract

The present invention provides a method for micronizing soft living tissue, the method comprising: a step in which soft living tissue isolated from a human or animal is sliced using a cutting tool; and a step for grinding the sliced soft living tissue together with a solid biocompatible material in a rotor mill equipped with a sieve.

Description

Method for micronizing soft living tissue and soft living tissue microscopic graft prepared by the method

The present invention crushes soft biological tissues such as placenta, anionic membrane, chorionic membrane, umbilical cord, and cartilage separated from humans or animals It relates to a method of converting, and the pulverized soft biological tissue fine particles are usefully used as a material for a biological tissue graft.

Various biological tissues can be isolated from humans or animals. The separated biological tissues can be used as a medical material or a pharmaceutical material through a series of processes.

The separated biological tissue may be used as an allograft, preferably an allograft, for tissue repair and wound healing. The living tissue graft can be used as an injectable graft or a patchable graft.

Biological tissues such as bone and cartilage can generally be obtained from a carcass. On the other hand, biological tissues such as placenta, amniotic membrane, chorion, and umbilical cord can be obtained after natural delivery or cesarean section of a pregnant woman.

The placenta is an organ that connects the fetus with the mother's uterus, and supplies oxygen and various nutrients to the fetus. The placenta also produces hormones necessary for the fetus and is known to synthesize various factors necessary for immunity.

The placenta has long been used as a graft for repairing human tissues or healing wounds.

The placenta is composed of an umbilical cord and an amniotic sac, and the amniotic sac is composed of an aminiotic membrane, a chorionic membrane, and a maternal decidua. Between the amnion and the chorionic membrane is an intermediate layer, called a "spongy layer".

The amniotic membrane is a thin membrane surrounding the fetus at the innermost part of the placenta, and the amniotic fluid is filled inside the amniotic membrane. The amniotic membrane consists of a single epithelium layer and extracellular matrix (ECM), and the extracellular matrix is again reticular fibers (or basement membrane), a thick compact layer and fibroblasts. It consists of a fibroblast layer.

The chorionic membrane is composed of reticular fibers, a basement membrane, and a trophoblast.

The amniotic and chorionic membranes contain various growth factors, cytokines, collagen, and cell-adhesive proteins, as well as stem cells.

Growth factors include, for example, epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor Beta (TGF-b), nerve growth factor (nerve growth factor). factor, NGF), hepatic growth factor (HGF), and vascular endothelial growth factor (VEGF).

Cytokines are, for example, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, metalloproteinase tissue inhibitors (TIMP)-1, TIMP-2, TIMP-3, TIMP- 4th place.

Collagen is, for example, collagen type I, type III, type IV, type V, and type VII.

Cell-adhesive proteins are, for example, fibronectin, laminin, fibrillin, nidogen, proteoglycan, glycosaminoglycan, and the like.

Mesenchymal stem cells are known to exist in large amounts in the epithelial cell layer of the amnion and the feeder cell layer of the chorion.

The tissue or membrane derived from the placenta has been used as a substrate for wound recovery such as diabetic foot ulcers. A variety of placenta products are commercially available for wound treatment. For example, there are products such as:

AmnioBand ^® Membrane (MTF Biologics, USA) is a dehydrated (dried) allograft derived from the human placenta and consists of the amniotic membrane and chorionic membrane.

Biovance ^® (Alliqua BioMedical, USA) is a decellularized and dehydrated human amniotic membrane allograft.

Epifix ^® (MiMedx Group, Inc., USA) is a dehydrated human amniotic/chorionic allograft (dHACM) used to treat diabetic foot ulcers.

EpiFix ^® Micronized Amniotic Membrane is a micronized dHACM that can be applied to deep wounds of cartilage, bones and muscles.

Grafix ^® (Osiris Therapeutics, Inc., USA) is a cryopreserved placenta-derived membrane composed of an extracellular matrix.

Grafts for these wound repairs generally do not need to be micronized.

On the other hand, the placenta-derived tissue graft may be administered into the human body by surgical incision or non-incision. In the surgical incision, an incision is made at the site of the procedure and the graft is inserted and then sutured. This surgical incision causes another scar.

Non-incisional surgery can be performed using an injection-type graft. In other words, a tissue graft is injected into the human body, such as muscles, cartilage, and ligaments through a syringe. In such injections, the micronization of the tissue graft is essential, and the size of the microparticles is very important. The fine particles of the graft should be sufficiently smaller than the inner diameter of the needle, and the larger the diameter of the needle, the more painful the patient will be, and may cause side effects such as skin injury or tissue destruction. The size of the graft microparticles suitable for injection is at least 1000 µm or less, preferably 500 µm or less, more preferably 300 µm or less.

Placenta-derived grafts for injection that are commercially available include, for example, the following products.

AmnioFix ^® Injectable (MiMedx Group, Inc., USA) is a micronized dHACM graft used to treat knee arthritis, orthopedic soft tissue, tendon injuries and tendonitis. The product is a dehydrated powder formulation of an allogeneic tissue graft, and the shelf life is at least 5 years at room temperature.

ReNu ^® and NuCel ^® (NuTech Medical, Inc., USA) are cryopreserved bioactive suspension grafts containing amniotic membrane and amniotic fluid. The product is used to treat degenerative diseases such as knee osteoarthritis, joint and tendon injuries.

Clarix ^® Flo and Clarix ^® Injectable (Amniox Medical, Inc., USA) are lyophilized micro human placenta allografts consisting of an amniotic membrane and umbilical cord.

Soft biological tissues such as placenta, amnion, chorion, umbilical cord, and cartilage must be micronized in order to be used as an injection.

U.S. Patent No. 9,943,551 discloses a method of pulverizing placental tissue, amnion tissue, and chorionic tissue to a microscopic size in order to make an injection composition, and the pulverizer discloses a Retsch ^TM MM400 ball mill and a Retsch ^TM Cryomill.

1 is a photograph of Retsch ^TM MM400, a representative model of a laboratory ball mill (vibration mill).

Referring to FIG. 1, in a general laboratory ball mill, a sample and a milling ball are placed in a milling cup, the cover of the milling cup is closed, and the milling cup is inserted into a cup holder. When the ball mill is operated, the milling cup vibrates (reciprocates) at high speed, and the sample is effectively crushed by a strong impact due to the inertia of the ball. Soft biological tissues such as amnion and chorion must be almost completely dehydrated (dried) before performing a ball mill. Otherwise, it will be barked rather than crushed and the desired shape of the fine particles cannot be obtained. Since dehydration refers to a non-viable environment, cells in living tissues are destroyed or killed.

Figure 2 shows a representative model of cryomill Retsch ^TM Cryomill. As shown in Fig. 2, the cryomyl is a structure in which a liquid nitrogen circulation system is installed in a conventional ball mill (vibration mill). Liquid nitrogen is supplied to the outside of the milling cup, and the sample is rapidly frozen and hardened for grinding. Cryomyl quickly cools the sample to -196°C for effective pulverization, so cells in living tissues, particularly stem cells, are almost killed.

Currently, micronization of soft biological tissues such as placenta, amnion, and chorion is all performed using cryomyl or ball mill. Such cryomyl or ball mill is useful for micronizing soft biological tissues, but since it is crushed like a large mortar by ball strike, there is a fatal problem in cell survival of micronized biological tissues.

An object of the present invention is to provide a micro-pulverization method that minimizes cell destruction of living tissue.

Another object of the present invention is to provide a method for preparing a biological tissue graft having a high content of active ingredients per unit volume.

Another object of the present invention is to provide a method for pulverizing biological tissues, which has a simpler pulverization method, high production time efficiency, and low production cost compared to the prior art.

The present invention comprises the steps of slicing soft living tissue isolated from humans or animals using a cutting tool; And it provides a method for micronizing a soft biological tissue comprising the step of pulverizing the shredded soft biological tissue together with a solid biocompatible material in a rotor mill equipped with a sieve (sieve).

In one aspect, the biocompatible material may be a biocompatible polymer or sugar.

The biocompatible polymer is not limited, but serum albumin, hyaluronic acid, starch, gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), Polylysine, carboxymethyl titine, fibrin, agarose, pullulan, cellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), Hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, alginate, cyclodextrin, dextrin, palatinite, polylactic acid, polyglycolic acid (polyglycolic acid), polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, and may include one or more polymer materials selected from the group consisting of polymaleic acid.

The saccharide is not limited, but the group consisting of glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melesitos, dextran, sorbitol, xylitol It may contain one or more saccharides selected from.

In one aspect, the biocompatible material may be serum albumin.

In one aspect, the biocompatible material may further include an active ingredient required for survival or growth of cells.

In one embodiment, the active ingredients required for survival or growth of the cells are DMEM (Dulbecco's Modified Eagle Media), MEM (Minimum Essential Media), RPMI 1640, Iscove's Modified Dulbecco's Media, Defined Keratinocyte-SFM (without BPE), Keratinocyte-D. -MEM, AmnioMAX-II Complete Culture media, AmnioMAX-C100 Complete Culture media, Plasmalyate-148 and Plasmalyte-A may be selected from the group consisting of.

In one aspect, the soft biological tissue of the present invention may be selected from the group consisting of amniotic membrane, chorionic membrane, placenta, umbilical cord, and cartilage.

In one aspect, the average diameter of the pulverized soft biological tissue may be 500 μm or less.

In one aspect, the rotor mill may be provided with a plurality of blades (multi-tooth, multi-blade), and may include a rotating circular rotor and a sieve mounted on the outside thereof.

On the other hand, the present invention provides a biological tissue fine particle graft comprising the prepared biological tissue fine particles.

In one aspect, the biological tissue microparticle graft may be obtained by freezing micronized soft biological tissue.

In one aspect, the biological tissue particulate graft may be used as an injection after freezing and thawing.

The biological tissue pulverized by the method of the present invention has significantly higher cell viability than conventional ball mill and cryomill pulverization, so that a biological tissue graft can be provided.

In addition, the pulverization method according to the present invention has a low cost and very high production efficiency compared to the prior art.

1 is a photograph of Retsch ^TM MM400, a representative model of a laboratory ball mill (vibration mill).

2 is a photograph of Retsch ^TM cryomill, a representative model of laboratory cryomill.

3A is a diagram showing a general configuration of a laboratory rotor mill, and FIG. 3B is a diagram showing the configuration of a rotor mill separated.

In Figure 4 (a) is a phase diagram showing the gelation area according to the concentration and temperature of serum albumin used as a grinding aid, (b) is a gelation form of 20% (w/v) bovine serum-derived albumin and human serum albumin It is a picture showing.

Figure 5 shows the gelled form of albumin and cell culture medium mixture, A (Figure 5A) is essential amino acids (AA), B (Figure 5B) is vitamins (VITA), C (Figure 5C) is non-essential Amino acids (NAA) are added.

6 shows a gelled form of albumin, cell culture medium and glutathione mixture

A (Fig. 6A) is essential amino acids (AA), B (Fig. 6B) is vitamins (VITA), C (Fig. 6C) is a non-essential amino acid (NAA) is added, and glutathione in these mixtures The concentration is 10mM.

In FIG. 7, A (FIG. 7A) is a grinding aid prepared with only 20% (w/v) albumin, B (FIG. 7B) is an essential amino acid (5×), a non-essential amino acid (5×), and a vitamin (1×). It shows a grinding aid prepared with 20% (w/v) albumin contained.

In FIG. 8, A (FIG. 8A) is a micrograph of an amniotic membrane tissue pulverized with a conventional cryomill, and B (FIG. 8B) is a microscopic picture of amnion tissue pulverized according to Example 4.

9 is another micrograph of the amniotic membrane tissue pulverized according to Example 4.

10 is a scanning electron microscope image showing the appearance of the extracellular matrix (ECM) of the amniotic membrane tissue pulverized according to Example 4 (magnification: 10000 times).

11 is a photograph showing the results of treatment with the Alamar Blue reagent of the amnion graft according to the present invention.

12 is a graph showing the results of measuring cell metabolic activity of the amniotic membrane graft according to the present invention using a fluorescence photometer.

13 is a graph showing the effect of promoting the growth of human fibroblasts of the amniotic membrane graft prepared according to the present invention.

As used in the present invention, "living tissue" refers to a viable tissue including living cells or viable cells.

The term "soft" in "soft biological tissue" used in the present invention is a soft tissue or material isolated from humans or animals, and includes, for example, placenta, amniotic membrane, chorion, umbilical cord, cartilage, skin, and ligaments.

As used in the present invention, "fine-particleization" means pulverizing a soft biological tissue to a size of 1000 microns or less.

The "biocompatible material" used in the present invention means a material that is not harmful to cells or tissues constituting a cartilage tissue graft.

In the "solid form of a biocompatible material" used in the present invention, "solid form" is used as a meaning including a hard form as well as a gel form.

The "pulverization agent or grinding agent" used in the present invention is a material added to effectively grind soft biological tissues in a rotor mill.

The "purging agent" used in the present invention is a material that pushes the soft biological tissue pulverized in the rotor mill to effectively exit the pores of the ring sieve.

The present inventors have made various attempts to effectively micronize soft biological tissues such as amniotic membrane, chorionic membrane, placenta, and umbilical cord while improving the cell viability of micronized soft biological tissue.

The inventors of the present invention confirmed that when crushing conventional ball mills, vibration mills, cryo mills used for crushing the amniotic membrane and chorionic membranes, cells are destroyed as these living tissues are previously dehydrated (dried) or rapidly frozen, and almost all cells are killed after fine particles I did. In order to solve the problems of these ball mills, vibration mills, and cryo mills, an attempt has been made to crush soft biological tissues such as amniotic membranes using a rotor mill without dehydration or quick freezing of the tissues. However, these biological tissues were not effectively pulverized with a rotor mill due to their soft and tough properties, and even if pulverized, they could not easily pass through the micro-pores of the desired size (micro size) of the sieve of the rotor mill.

Accordingly, the present inventors pulverized the soft biological tissue by putting a solid biocompatible material together as a pulverization agent or a purging agent, and surprisingly, the pulverization was smoothly performed, and the crushed tissue was smooth. It was confirmed that it passed through a sieve. In addition, it was found that the cell viability of the pulverized tissue is increased when a pulverization aid is used by adding components capable of inducing cell protection and activity to such a biocompatible material.

The biocompatible material used as a grinding aid or purging agent in the present invention is not limited, but, for example, a biocompatible low molecule, a biocompatible polymer may be used, and a natural polymer, a synthetic polymer, or a recombinant polymer may be used.

Hereinafter, various methods of manufacturing micro-sized biological tissue grafts will be described in detail.

Preparation of the placenta

The placenta is obtained after spontaneous delivery or cesarean section surgery with the informed consent of the pregnant woman. To prevent potentially blood-transmitting diseases to the placenta, pregnant women are pre-tested for HIV-1, HIV-2, HTLV-1, hepatitis B virus and syphilis virus using conventional serological tests. . The above test items are exemplary, and more serum tests may be performed if necessary. The placenta can be used for the graft of the present invention only if the pregnant woman's blood is serologically negative.

Placenta meeting the above criteria is placed in a sterile plastic bag, stored in an ice bucket, and transferred to the laboratory as soon as possible. The following procedure is performed in aseptic conditions routinely used for tissue culture. The sterile plastic bag containing the placenta is checked and stored in the refrigerator until ready for further processing.

Blood and clots remaining in the placenta can be removed using saline. The amniotic and chorionic membranes from the placenta can be carefully separated by hand. The separated amniotic membrane and chorionic membrane may be pulverized individually or in combination with a rotor mill to be described later.

In one aspect, the layer of epithelial cells of the amnion may be micronized without being removed. In other embodiments, the epithelial cell layer of the amnion can be physically or chemically removed by known techniques. At this time, the epithelial cell layer may be removed using a cell scraper, or nonionic surfactants such as Triton X-100, Triton X-114, NP-40, Brij-40, and Tween-80, ionic surfactants, or nucleases. Can be removed.

The amniotic membrane is washed with physiological saline or PBS added with antibiotics. Streptomycin (Streptomycin), gentamicin (Gentamicin), penicillin, polymycin B (Polymixin B), bacitracin (Bacitracin), or a mixture thereof, etc. are used as antibiotics, but all antibiotics known in the art can be used. have.

The above-described process of obtaining the placenta, separation of the amnion from the chorion membrane, and washing may be performed by a method known in the art, for example, a method described in US Patent Nos. 8,357,403 and 9,943,551.

Cutting of soft living tissue

Before pulverizing the soft biological tissue using a rotor mill to be described later, the soft biological tissue is cut (minced) into small size using a cutting tool. The cutting tool is irrelevant as long as it can cut living tissue, but a knife or scissors can be used mainly.

As a knife for slicing living tissue, for example, a rotating knife (rolling knife), a round knife, a straight knife, a mincer knife, etc. can be used, and these knives are arranged at regular intervals. A knife may be used. In addition, the living tissue may be shredded by an electric grinder equipped with a knife blade such as a household electric mixer.

In one embodiment, the soft biological tissue can be shredded in a grid pattern. In another aspect, the soft living tissue can be shredded into amorphous shape using a cutting tool.

The size of the fragmented biological tissue is not limited, but may be 1 to 10 mm in diameter, 2 to 9 mm, 3 to 8 mm, 4 to 7 mm, and 5 to 6 mm.

Micronization of soft living tissues using a rotor mill

The rotor mill grinder is a device that grinds materials with centrifugal force and shear force generated by the rotation of the rotor.

3A is a diagram showing a general configuration of a laboratory rotor mill, and FIG. 3B is a diagram showing the configuration of a rotor mill separated.

3A and 3B, the rotor mill includes a body 1 and a cover 2. A rotor 3 is provided at a central position of the main body 2, and a circular body 4 surrounds the rotor 3. There is a shear gap between the rotor 3 and the circular body 4. The rotor 3 is rotated at high speed by a motor, and the circular body 4 is fixed to the outside of the rotor 3.

The rotor 3 may be a rotor having a multi-tooth 5. The blades 5 of the rotor may be arranged vertically on the circumference of the rotor at regular intervals from each other. In one aspect, the blade 5 of the rotor may have a triangular shape of a stainless steel bar or a titanium bar. The number of blades 5 of the rotor may be 4, 6, 8, 12, or 24, but is not limited thereto. In general, as the number of rotor blades 5 increases, the size of the pulverized fine particles decreases.

A number of micropores are formed on the surface of the circular body 4. The shape of the micropores may be a round hole or a trapezoidal hole, but is not limited thereto. The size of the micropores is not limited, but may be less than 1.00 mm, less than 0.75 mm, less than 0.50 mm, or less than 0.25 mm. The size of the micropores of the prototype may be appropriately selected according to the size of the fine particles of the soft living tissue.

In one aspect, the shredded soft biological tissue may be pre-positioned on the center of the rotor. In another aspect, the shredded soft biological tissue may be supplied to the center of the rotor through the inlet 7 of the rotor mill. When the rotor mill starts to operate, the soft biological tissue shredded by the centrifugal force generated by the high-speed rotation of the rotor is moved toward the circular body (4), and is torn by the shear force between the blade (5) and the circular body (4) of the rotor. It is ground, ground and micronized. The micronized soft biological tissue passes through the micropores formed in the circular body 4 and is collected in an external collection container.

Various rotor mills can be used in the present invention. Rotor mills are for example Retsch ^® ZM 200 (Retsch GmbH, Germany), Retsch ^® ZM 100 (Retsch GmbH, Germany), Retsch ^® ZM 1000 (Retsch GmbH, Germany), Retsch ^® TWISTER (Retsch GmbH, Germany), Retsch ^® SR300 (Retsch GmbH, Germany), Fritsch ^® Pulverisette14 (Fritsch GmbH, Germany), Glatt Rotor Sieve GSE may be used, but are not limited thereto.

Grinding aid or purging agent

In the present invention, the grinding aid or purging agent may be a solid or gel biocompatible material. In the present invention, the solid form may be a hard formulation, or may be a soft formulation like jelly. In one aspect, the biocompatible material may be prepared by itself or in a solid form or a gel form by mixing a solution such as water or a medium. In addition, the biocompatible material may be manufactured by cutting with a knife or making a tablet in a mold. If necessary, a biocompatible adhesive may be added to the biocompatible material.

In one aspect, the biocompatible material may include ingredients that are not harmful in the human body.

In one aspect, the biocompatible material is not limited, but may be a component used for medical purposes, a component used as an excipient for a drug, or a component used for a medical device.

In one aspect, the biocompatible material may be a low-molecular material or a high-molecular material as long as it is maintained in a solid form or a gel form. As the polymer material, natural polymers, recombinant polymers, and synthetic polymers can be used.

In one embodiment, the biocompatible material is not limited, but serum albumin, hyaluronic acid, starch, gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (Sulfate), polylysine, carboxymethyl titine, fibrin, agarose, pullulan, cellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, alginate, cyclodextrin, dextrin, palatinite, polylactic acid , Polyglycolic acid, polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, and polymeric materials such as polymaleic acid may be used.

In another embodiment, the biocompatible material is not limited, but glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melesitos, dextran, sorbitol, It may be a sugar such as xylitol.

In one aspect, the starch used in the grinding aid may be pharmaceutical starch or pregelatinized starch, and may be included in whole or in part. For example. PGS grade pregelatinized starch such as Cargill's C☆PharmGel™ can be used.

In one aspect, the biocompatible material may be water-soluble. At this time, after grinding with a rotor mill, the biocompatible material is dissolved in an aqueous solution, so it can be easily removed through a filter.

In one aspect, the biocompatible polymer may be a biodegradable polymer.

On the other hand, the grinding aid or purging agent may contain an active ingredient required for survival or growth of cells. In one aspect, the active ingredient may be a culture medium for cell growth. The culture medium is not limited, but DMEM (Dulbecco's Modified Eagle Media), MEM (Minimum Essential Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE), Keratinocyte-SFN (with BPE) ), KnockOut D-MEM, AmnioMAX-II Complete Medium and AmnioMAX-C100 Complete Medium, Plasmalyate-148, Plasmalyte-A, and the like may be used.

On the other hand, the active ingredient may be amniotic fluid or amniotic fluid-derived ingredients.

In one aspect, the biocompatible material may be in a solid form or a gel form, and the size is not limited, but the average diameter is 1 to 8 mm, preferably 2 to 7 mm, more preferably 3 to 6 mm. It may be, even more preferably 4 ~ 5mm.

In one aspect, the solid biocompatible material may be prepared by using a pill tablet. If necessary, purified water may be added.

In one aspect, when the biocompatible material is a sugar such as glucose or sucrose, it may be mixed with purified water and then put in a mold and dried.

In another embodiment, the solid biocompatible material may be prepared by crosslinking by adding a crosslinking agent in a liquid phase. As the crosslinking agent, glutaraldehyde may be used, but the present invention is not limited thereto, and a known crosslinking agent may be used.

In another embodiment, the solid biocompatible material may be dissolved in an aqueous solution, mixed with an excipient such as starch, and dried.

In the present invention, the grinding aid or purging agent is divided into roles, and the grinding aid and purging agent may be the same. That is, the grinding aid can be a purging agent, and the purging agent can be a grinding aid. In one aspect, the purging agent may be a biocompatible material described in the aforementioned grinding aid.

When the rotor mill is operated, the grinding aid or purging agent is crushed between the rotor blade and the circular sieve by centrifugal and shear forces. A grinding aid or purging agent not only helps the pulverization of soft living tissues, but also serves to push the pulverized living tissues out of the prototype body (purging agent). In addition, the grinding aid serves to buffer the impact generated by the shear of the rotor mill to prevent or reduce damage or destruction of cells including tissues.

The operation sequence of the rotor mill is as follows.

3A and 3B, the cover 2 of the rotor mill is opened, and a labyrinth disc 8 is inserted on the body of the rotor mill. Then, a rotor (3) equipped with a multi-blade is mounted on the central axis of the motor, a collection container (cassette) (6) is mounted, and then a circular sieve (4) is mounted. The shredded soft living tissue is placed in the center of the rotor (3), and the grinding aid is placed in a position slightly away from the center of the rotor. Operate after closing the cover of the rotor mill.

The rotational speed and operating time of the rotor mill can be preset.

The rotor mill is not limited, but rotates at high speeds of 1,000-18,000 rpm. Meanwhile, when a separate purging agent is injected through the rotor mill inlet during operation of the rotor mill, the pulverized soft biological tissue is completely pushed out of the circular body.

Most of the pulverized biological tissue is collected in the collection container 6 and a part remains on the outer surface of the circular body 4. The pulverized biological tissue remaining in the prototype can be collected using a washing solution or a culture medium, and the pulverized biological tissue can then be obtained by filtration or centrifugation. The obtained biological tissue microparticles can be stabilized in a culture medium or a stabilizing solution. To stabilize the pulverized biological tissue, antioxidants such as N-acetyl cysteine (NAC) may be further added to the culture medium or stabilization solution.

Cryopreservation

The micronized soft biological tissue of the present invention may include living cells, for example, mesenchymal stem cells, epithelial cells, amnion-derived fibroblasts, and the like.

The micronized soft biological tissue including the living cells may be cryopreserved.

Cryoprotective agents can be added to prevent cell damage while freezing the cells. There are two types of cryoprotectants: cell-permeable cryoprotectants and non-permeable cryoprotectants. Cell-permeable cryoprotectants are low-molecular substances capable of penetrating cell membranes, and play a role of inhibiting the formation of ice cubes upon freezing by substituting water inside cells through the cell membrane. The impermeable cryoprotectant is mainly a polymer material, and serves to inhibit the formation of ice crystals outside the cell.

As the cell permeable cryoprotectant, dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (Gly), methanol (MeOH), propylene glycol (PG), and the like may be used, but are not limited thereto.

Non-permeable cryoprotectants include lactose, dextran 40, dextran 70, fructooligosaccharide, isomaltooligosaccharide, galactooligosaccharide, and penta. Isomaltose (pentaisomaltose), trehalose (trehalose), sucrose (sucrose), raffinose (raffinose), d-mannitol (mannito), etc. may be used, but is not limited thereto.

The cell permeable cryoprotectant and the impermeable cryoprotectant may be used alone or in combination. The concentration of the cryoprotectant is not limited, but may be 5 to 40% (v/v). In one aspect, a mixture of 3-5% DMSO and 9% dextran 70 may be used. The cryopreservation method of living tissue can be performed according to a cryopreservation protocol known in the art.

The soft biological tissue microparticle graft of the present invention may be used in the form of an injection after melting, and is mainly used for orthopedic treatment such as knee osteoarthritis, soft tissue damage, ligament damage, and tendon damage. I can.

Example 1: Gelation of albumin solution (preparation of grinding aid)

In order to prepare a solid grinding aid, an experiment on gelation of albumin was conducted.

Bovine serum albumin (BSA) and human serum albumin (HSA) are dissolved in ultrapure water, respectively, at pH 7.0, concentration 10%, 12%, 14%, 16%, 18%, 20% (w After preparing each solution of /v), gelation was observed according to the albumin concentration and temperature change.

In Figure 4, (a) is a phase diagram showing the gelation area according to the concentration and temperature of serum albumin used as a grinding aid, and (b) is a gelation of 20% (w/v) bovine serum-derived albumin and human serum albumin. Show the form.

Through this experiment, it was shown that the gelation temperature of albumin decreased as the concentration of albumin increased, and gelation was possible at a concentration of 10% or more.

Example 2: Gelation of albumin + cell culture medium mixture (preparation of a grinding aid)

The albumin solution and the cell culture medium were mixed to perform gelation of albumin.

Cell culture medium is MEM amino acid solution (50×: 50 times concentrate) containing essential amino acids (L-Arginine, L-Cystine, L-Histidine, L-Isoleucine), and non-essential amino acids (L-Alanine, L-Asparagine). , L-Aspartic acid) containing MEM non-essential amino acid solution (100×: 100 times concentrate), vitamins containing MEM vitamin solution (100×: 100 times concentrate) was used. Here, "x" is concentrated by the corresponding multiple based on the components that enter the cell culture medium, and is generally used after being diluted.

By mixing 25% (w/v) serum albumin and the MEM solution, the albumin concentration was adjusted to a concentration of 20% (w/v) with respect to the total mixture, and the concentration of amino acids and vitamins was diluted with respect to the total mixture. For example, if 400 µl of 25% (w/v) albumin solution and 100 µl of amino acids (100×) are mixed, a total of 500 µl is obtained, while albumin is diluted to 20% (w/v) and amino acids are 20 times (20). ×).

Figure 5 shows the gelled form of albumin and cell culture medium mixture, A (Figure 5A) is essential amino acids (AA), B (Figure 5B) is vitamins (VITA), C (Figure 5C) is non-essential Amino acids (NAA) are added.

As shown in FIG. 5, both albumin and cell culture medium mixtures were gelled, and in the case of non-essential amino acids, when 10 times or more was added, a translucent or milky albumin gel was formed.

Example 3: Preparation of albumin starch paste (preparation of grinding aid)

A 20% (w/v) human serum albumin solution, pregelatinized starch, and the cell culture medium mentioned in Example 2 were mixed to prepare a partial starch paste.

Figure 6 shows the form of the albumin starch paste, A (Figure 6A) is essential amino acids (AA), B (Figure 6B) is vitamins (VITA), C (Figure 6C) is non-essential amino acids (NAA) Is added.

As shown in FIG. 6, albumin and albumin starch paste were all prepared in a milky gel form.

Example 4: Micronization of soft living tissue

After separating the amniotic membrane from the placenta obtained with the consent of the pregnant woman, Triton X was treated, and then the epithelial cell layer of the amniotic membrane was removed using a cell scraper.

The amniotic membrane was washed with PBS or physiological saline to which 1% streptomycin/penicillin antibiotic was added, and then stabilized in DMEM medium.

The amnion was shredded into a size of about 4mm×4mm using a rolling multi-blade.

The albumin gel prepared in the same manner as in Examples 1 and 2 was cut into cubes and used as a grinding aid.

In FIG. 7, A (FIG. 7A) is a grinding aid prepared with only 20% (w/v) albumin, B (FIG. 7B) is an essential amino acid (5×), a non-essential amino acid (5×), and a vitamin (1×). It shows a grinding aid prepared with 20% (w/v) albumin contained.

A 6-bladed rotor and a ring sieve having a diameter of 0.25 mm were mounted on a rotor mill (retsch zm 200).

Several grinding aids were placed around the inside of the rotor, and a shredded amnion was placed in the center of the rotor.

While rotating the rotor mill at a speed of 3000 rpm, a separate grinding aid was introduced into the inlet of the rotor mill, and the amniotic membrane crushed in the rotor was pushed out of the sieve.

The pulverized amnion adhered to the collection container and sieve of the rotor mill was washed with DMEM medium to collect the pulverized amnion, and stabilized at room temperature for 1 hour.

Experimental Example 1: Micronization of soft living tissue

Example 4 by observing the tissue with the amniotic membrane and the amniotic membrane tissue ground to a conventional keurioh mill (Retsch Cryomill ^TM) milling according to the microscope naeteotda receive the results in Figs. 8 and 9 (Scale Bar: 200 μm) .

In FIG. 8, A (FIG. 8A) is a micrograph of a conventional amnion tissue pulverized with a cryomill, and B (FIG. 8B) is a micrograph of a pulverized amnion tissue according to Example 1. In FIG. As shown in Figure 8, it can be seen that the cells in the amniotic membrane tissue crushed with conventional cryomyl are all destroyed, whereas the amniotic membrane tissue according to Example 4 survives.

9 is another micrograph of the amniotic membrane tissue pulverized according to Example 4. As shown in Figure 9, it can be seen that the size of the micronized amniotic membrane tissue according to the present invention is about 500 μm or less.

10 is a scanning electron microscope image showing the appearance of the extracellular matrix (ECM) of the amniotic membrane tissue pulverized according to Example 4 (magnification: 10000 times). As shown in FIG. 10, it can be seen that the reticular fibers are preserved as they are.

Experimental Example 2: Measurement of cell metabolic activity of micronized amniotic membrane graft

Cellular metabolic activity of micronized amniotic membrane grafts was measured according to Example 4.

Each amniotic membrane graft prepared according to the present invention was branched into a 24-well plate, incubated in 20% serum high-concentration glucose DMEM medium (1 ml) for 12 hours, and a recovery period was held for 12 hours.

After adding 100 µl of Alamar Blue reagent to the well plate, further incubation was performed for 3 hours. FIG. 11 shows Alamar Blue reagent-treated amniotic membrane grafts, where "Fresh Amniotic Tissue" represents unground amniotic membrane grafts, and "Processed Amniotic Tissues" represents amniotic membrane grafts pulverized according to the present invention.

The fluorescence intensity of the well plate was measured using a fluorescence photometer to measure cell metabolic activity, and the results are shown in FIG. 12 (Ex: 560 nm; Em: 590 nm).

As shown in FIG. 12, the amniotic membrane graft according to the present invention has a lower cell metabolic activity compared to the uncrushed amnion tissue, but it means that the cell survival is possible even after pulverization. In particular, a grinding aid made of 20% (w/v) albumin containing essential amino acids (5×), non-essential amino acids (5×) and vitamins (1×) has the highest cellular metabolism activity compared to other grinding aids. Can be seen.

Experimental Example 3: Promoting growth of human fibroblasts (Detroit 562) of micronized amniotic membrane graft

300 µl of a mixed solution of the amniotic membrane graft and serum-free medium 1:1 (w/w) according to the present invention was prepared, a serum-free medium was prepared as a negative control, and a 10% FBS medium was prepared as a positive control.

Human fibroblasts (Detroit 562) were mixed with 1 ml of serum-free medium, and 3 wells were inoculated into well plates of a total of 3 groups at 5×10 ⁵ per well, and the cells were attached for 24 hours. After mounting a culture plate insert having a 3 μm pore (cell culture plate insert), the amnion graft mixture solution, the negative control group, and the positive control group according to the present invention were respectively added to the culture plate inserts and cultured for 24 hours.

The MTT reagent was dissolved in PBS (phosphate-buffered saline) at a concentration of 5 mg/mL, treated with 100 µl each in a cultured 24-well plate, and stored at 37°C for 1 hour, and the amount of MTT formazan produced at 540 nm absorbance was measured. By quantification, the cell activity of all cells was analyzed.

13 is a graph showing the effect of promoting the growth of human fibroblasts of the amniotic membrane graft prepared according to the present invention.

As shown in Figure 13, the amniotic membrane graft (Processed Amniotic Tissues) according to the present invention showed a high MTT activity (activity) compared to the negative and positive control groups, which included an active ingredient that helps cell growth in the amniotic membrane graft according to the present invention. It means being

The present invention crushes soft biological tissues such as placenta, anionic membrane, chorionic membrane, umbilical cord, and cartilage separated from humans or animals It relates to a method of converting, and the pulverized soft biological tissue fine particles are usefully used as a material for a biological tissue graft.

Claims (12)

1. Slicing the soft living tissue isolated from humans or animals using a cutting tool; And

   Comprising the step of placing the shredded soft biological tissue together with a solid biocompatible material in a rotor mill equipped with a sieve and grinding,

   A method of micronizing soft living tissue.
2. The method of claim 1,

   The biocompatible material is a biocompatible polymer or sugar, characterized in that, the method for micronizing soft living tissue.
3. The method of claim 2,

   The biocompatible polymers are serum albumin, hyaluronic acid, starch, gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), polylysine. , Carboxymethyl titine, fibrin, agarose, pullulan, cellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), hydroxyethylcellulose ( HEC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, alginate, cyclodextrin, dextrin, palatinite, polylactic acid, polyglycolic acid, Polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid and polymaleic acid, characterized in that it comprises at least one polymer material selected from the group consisting of, a method for micronizing a soft living tissue.
4. According to claim 2

   The saccharide is one selected from the group consisting of glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melesitos, dextran, sorbitol, and xylitol A method for micronizing a soft biological tissue, characterized in that it contains the above saccharides.
5. The method of claim 1,

   The biocompatible material further comprises an active ingredient required for survival or growth of cells.
6. The method of claim 5,

   Active ingredients required for the survival or growth of the cells are DMEM (Dulbecco's Modified Eagle Media), MEM (Minimum Essential Media), RPMI 1640, Iscove's Modified Dulbecco's Media, Defined Keratinocyte-SFM (without BPE), Keratinocyte-D-MEM, AmnioMAX. -II Complete Culture media, AmnioMAX-C100 Complete Culture media, Plasmalyate-148 and Plasmalyte-A, characterized in that selected from the group consisting of, a method for micronizing soft living tissue.
7. The method of claim 1,

   The biological tissue is one or more selected from the group consisting of amniotic membrane, chorionic membrane, placenta, umbilical cord, and cartilage.
8. The method of claim 1,

   The average diameter of the pulverized biological tissue is 500 μm or less, the method of micronizing soft biological tissue.
9. The method of claim 1,

   The rotor mill is provided with a plurality of blades, and includes a rotating circular rotor and a sieve mounted on the outside thereof.
10. A biological tissue fine particle graft comprising the biological tissue fine particles prepared in claim 1.
11. The method of claim 10,

    The biological tissue particulate graft is obtained by freezing the particulate soft biological tissue.
12. The method of claim 11,

    The biological tissue microparticle graft is to be used as an injection after freezing and thawing, a biological tissue graft.

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