Patent Description:
Conventional first-generation stem cell therapeutics are isolated cells obtained by treatment using proteases, such as trypsin or dispase, wherein intercellular bindings and basement membrane proteins of the cells are not maintained because all proteins exposed on cell membranes are nonselectively degraded by the treatment using proteases. In addition, animal-derived substances, such as fetal bovine serum (FBS), added to inactivate a protease is removed by several times of washing-centrifugation process, wherein <NUM>% to <NUM>% of cells are lost by one washing-centrifugation process. Therefore, a cell collection process using a protease is very inefficient.

In addition, the ratio of settled cells is low in a transplantation of isolated single cells to a disease part because transplanted cells are discharged from a target part through diffusion and absorption. Moreover, because mesenchymal stem cells are highly adhesive, the survival rate of isolated single mesenchymal stem cells is very low as it dies within <NUM> to <NUM> hours after isolation.

The patent <CIT> provides a method of de novo in vivo synthesis of soft tissues having a predetermined shape and size from adult mesenchymal stem cells in biocompatible scaffolds and a composition prepared by the same method. The patent provides a method of using hydrogel polymer, more specifically, polyethylene glycol diacrylate as a biocompatible scaffold. However, since the composition is for recovery of soft tissue binding, the abovementioned patent only presumes that there should be almost no change in the diameter of the scaffold prepared by using polyethylene glycol diacrylate, and neither provides nor implies a manufacturing method including the filling a cultured substance to a syringe for immediate administration.

The <CIT> provides a sphincter-regenerative cell therapeutic agent including amniotic fluid-derived stem cells and describes that the cell therapeutic agent may be injected to a hydrogel composite, specifically to an alginate/PF-<NUM>/hyaluronic acid to enhance the effect. However, the abovementioned patent does not provide that the process of collecting stem cells may be improved to increase the yield.

The <CIT> provides a method of differentiating mesenchymal stem cells to chondrocytes, more specifically, a method of fixing mesenchymal stem cells at a composite scaffold including fibrin/Ha for culturing, wherein fibrin/Ha is a biodegradable polymer. However, the abovementioned patent only provides that the differentiation of mesenchymal stem cells to chondrocytes may be enhanced when fibrin/Ha is used as a scaffold even without adding TGF-beta, w is added in a conventional method of differentiating cells.

In conventional methods, intercellular bindings and basement membrane proteins may be damaged by an enzyme treatment performed in a final process of preparing transplanted cells, cells may be lost in each of cell collection, washing, vial filling, and syringe filling using materials contained in a vial, and most bioactive substances synthesized during cell culture, such as collagen, are eliminated as only single cells are collected and injected after an enzyme treatment.

The present invention provides a composition including mesenchymal stem cells cultured in hydrogel, washed, and filled into a syringe, wherein the composition of the present invention may be readily administered without modification and may not need an enzyme treatment in a final process of preparing cells to be transplanted, almost no cell is lost because the composition is washed as hydrogel without an washing process using centrifugation or other methods, and the therapeutic effect is shown immediately after administration to an individual because bioactive substances are patched in pores of the hydrogel.

The purpose of the present invention is to prepare mesenchymal stem cells separated from adipose tissues and to use them for disease treatment. The present invention provides an improved method of preparing a cell composition to <NUM>) prevent loss of cells in a process of collecting cells cultured in a sufficient amount, <NUM>) prevent damage to cells by protease treatment, and <NUM>) minimize cell loss at a target part; and a composition prepared by the method. The cell composition prepared by the improved method is provided after filling the composition to a syringe to make the utilization easy. The cell-hydrogel prepared and filled by the method provided by the present invention may be applied to all therapeutic agents that may be locally administered by using a <NUM> to <NUM>-gauge needle.

The present disclosure provides a method of culturing adipose tissue-derived mesenchymal stem cells in a hydrogel and collecting stem cell-hydrogel by using a syringe.

In the composition for disease treatment of the present disclosure, the mesenchymal stem cells are positive for CD29, CD44, CD73, CD90, and etc., or may be autologous or allogenic cells positive to CD34 and CD45.

In one example of the present invention, hydrogel is fibrin glue, wherein the concentration of fibrinogen constituting the fibrin glue is from <NUM> to <NUM>/mL.

In one example of the present invention, <NUM>,<NUM> to <NUM>,<NUM>,<NUM> stem cells are mixed with <NUM> of hydrogel, <NUM> to <NUM> of the resulting mixture is added to <NUM> to <NUM>-well plate to produce a gel, and a medium is added to the gel to culture for three to six days. The medium may be a Dulbecco's Modified Eagle's Medium (DMEM) including <NUM>% FBS and basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) provided by a conventional patent (Autologous and Allogenic Adipose Tissue-Derived Stromal Stem Cells Composition for Treatment of Fistula; Patent registration number: <NUM>,<NUM>,<NUM>) or a DMEM including <NUM>% to <NUM>% human serum albumin and <NUM>% to <NUM>% of a composition prepared by a method provided by a convention patent (Method for Preparation of Mesenchymal Stem Cell Culture Comprising High Concentration of Cell Growth Factors and Composition Prepared Therefrom; Patent application number: <NUM>-<NUM>-<NUM>).

The method of preparing a composition including an adipose tissue-derived mesenchymal stem cell-hydrogel of the present patent is very useful, because no enzyme treatment is needed in a final process of preparing transplanted cells as cells are cultured in a hydrogel, washed, and immediately filled to a syringe for use, almost no cell is lost because the composition is washed as hydrogel without a washing process using centrifugation or other methods, and the therapeutic effect is shown immediately after administration to an individual because bioactive substances are patched in pores of the hydrogel.

More specifically, the method of preparing a composition including an adipose tissue-derived mesenchymal stem cell-hydrogel according to the present invention does not include cell isolation (screening) performed by using a protease, and thus cells may be efficiently obtained by preventing cell loss in a conventional cell collection process. In addition, stem cells obtained by the method provided by the present invention may be administered to a disease part in a highly active state, and the administered cells remain at a target part until all the hydrogel is degraded and absorbed completely to show a therapeutic effect continuously. In addition, extracellular matrix secreted from mesenchymal stem cells during culture, such as collagen, laminin, fibronectin, elastin, is completely present as being patched in pores of the hydrogel, and thus the therapeutic capability of the composition of the present invention is significantly higher than that of conventional therapeutic agents, and treatment period may be reduced by using the composition of the present invention.

Experimental results showed that the adipose tissue-derived mesenchymal stem cell-hydrogel of the present invention has a cell survival period significantly longer than that of conventional stem cell therapeutic agents because the stem cells survive more than one week in the hydrogel and maintain the shape of fibroblast, and may enhance healing of wounds because various growth factors and cytokines enhancing cell growth and angiogenesis are continuously secreted for one week, various kinds of extracellular matrix are secreted in a considerable amount, and the secreted extracellular matrix remains in the hydrogel to provide various kinds of matrix when transplanted to a body. In addition, the composition of the present invention does not cause an immune response but inhibits tumor necrosis factor (TNF)-alpha to mitigate inflammation to help healing of wounds, wherein TNF-alpha is an inflammatory substance secreted from activated immune cells.

The present invention provides a method of preparing a composition including a adipose tissue-derived mesenchymal stem cell-hydrogel as defined in the claims.

The present invention provides a composition including mesenchymal stem cells cultured in hydrogel, washed, and then immediately filled into a syringe, wherein the composition of the present invention does not need an enzyme treatment in a final process of preparing cells to be transplanted, almost no cell is lost because the composition is washed as hydrogel without an washing process using centrifugation or other methods, and the therapeutic effect is shown immediately after administration to an individual because bioactive substances are patched in pores of the hydrogel.

The present invention is described more specifically below.

The present invention relates to a method of preparing a mesenchymal stem cell-hydrogel composition, wherein the method includes (a) culturing of adipose tissue-derived mesenchymal stem cells; (b) forming a gel by mixing the cultured adipose tissue-derived mesenchymal stem cells and a hydrogel solution; and (c) culturing the gel and(d) filling the gel of step (c) into an ampoule, a vial, or a syringe, wherein the hydrogel is fibrin glue, wherein the fibrin glue includes fibrinogen of a concentration from <NUM> to <NUM>/mL.

In one aspect, one or a composite of two or more selected from the hydrogel group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, a <NUM>-hydroxyethyl methacrylate derivative or a copolymer thereof, polyethylene oxide, and polyvinyl alcohol may be used as a hydrogel, more specifically, one or a composite of two or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, and pectin may be used, and more specifically, fibrin glue may be used.

In one embodiment of the present invention, the fibrin glue may include fibrinogen of a concentration from <NUM> to <NUM>/mL. An experimental result showed that cells did not grow well or cells were separated from a hydrogel when a final concentration of fibrin glue was out of the range described above.

In one aspect, the fibrin glue may include thrombin of a concentration from <NUM> to <NUM> I. /mL, specifically, from <NUM> to <NUM> I. /mL, more specifically, from <NUM> to <NUM> I. No problem occurs in cell growth and cell-hydrogel adhesiveness within the range described above.

In one embodiment of the present invention, the method of the present invention includes filling of the mesenchymal stem cell-hydrogel composition to an ampoule, a vial, or a syringe following (c). A adipose tissue-derived mesenchymal stem cell-hydrogel composition prepared by using the preparation method of the present invention may be locally administered by using a <NUM> to <NUM>-gauge syringe immediately after the preparation.

In one embodiment of the present invention, the number of cells counted in the mesenchymal stem cell-hydrogel filled into an ampoule, a vial, or a syringe in (d) may be <NUM>% or more, more specifically, <NUM>% or more of the number of counted mesenchymal stem cells collected in (c).

An experimental result showed that about <NUM>% of cells prepared by a conventional method were lost after being filled into a syringe but only <NUM>% or less of cells were lost when the preparation method of the present invention was used, indicating that the cell loss rate of the method provided by the present invention was significantly lower than that of a conventional method.

The present invention provides a cell therapeutic agent including as an active ingredient a mesenchymal stem cell-hydrogel composition prepared by the method.

The term "cell therapeutic agent" used herein refers to a medical product used for the purpose of treatment, diagnosis and prevention, which contains a cell or tissue prepared through isolation from humans, culture and specific operation. Specifically, it refers to a medical product used for the purpose of treatment, diagnosis and prevention through a series of behaviors of in vitro multiplying and sorting living autologous, allogenic and xenogenic cells or changing the biological characteristics of cells by other means for the purpose of restoring the functions of cells and tissues. Cell therapeutic agents are broadly divided into somatic cell therapeutic agents and stem cell therapeutic agents according to the differentiation level of cells. More specifically, the present invention relates to a adipose tissue-derived mesenchymal stem cell therapeutic agent.

The term "subject" used herein refers to all kinds of animals, including humans, already having or likely to have a disease that may be prevented or treated by the administration of the cell therapeutic agent of the present invention.

Hereinafter, the present invention will be described in further detail with reference to examples.

A adipose tissue may usually be obtained by liposuction, but the method of obtaining a adipose tissue is not limited thereto.

Adipose tissue-derived mesenchymal stem cells were separated from adipose tissue obtained by liposuction by the following method. The adipose tissue was washed with the same volume of PBS for three or four times to remove blood. A collagenase solution of a volume same as the adipose tissue was added to the adipose tissue and kept in a water bath at <NUM> for reaction. The resulting mixture was transferred to a centrifugation tube and underwent centrifugation at <NUM> and <NUM> rpm for ten minutes. The supernatant lipid layer was removed, and the bottom collagenase solution layer was carefully separated not to be shaken. A substrate medium was added to the separated collagenase solution layer and suspended, and the resulting mixture underwent centrifugation at <NUM> and <NUM> rpm for five minutes. The subsiding matter was a stroma-vessel fraction, from which the supernatant was removed. The stroma-vessel fraction was suspended in a substrate medium and then the suspension was inoculated to a culture vessel. Culturing was performed at <NUM> in a <NUM>% CO<NUM> incubator for <NUM> hours. After removing the culture solution, the cells were washed with PBS, and then proliferated by using a substrate medium, a growth medium prepared by adding <NUM> ng/ml bFGF to a substrate medium, or a medium prepared by adding <NUM> ng/ml EGF to a substrate medium. When the adipose tissue-derived mesenchymal stem cells grew to a volume of <NUM>% to <NUM>% of the culture vessel, the cells were treated with trypsin, separated as single cells, and then collected.

A thrombin solution was added to the adipose tissue-derived mesenchymal stem cells of a concentration of <NUM> × <NUM><NUM>/mL obtained in Example <NUM> at ratios of <NUM>:<NUM>, <NUM>:<NUM>, and <NUM>:<NUM>. Fibrinogen (<NUM> to <NUM>/mL) was prepared by diluting at dilution factors of <NUM>:<NUM>, <NUM>:<NUM>, and <NUM>:<NUM>. A Duploject syringe system was used to add <NUM> to <NUM> uL of the cell suspensions containing fibrinogen and thrombin to each well of a <NUM>-well plate. After the gel was completely solidified, a culture medium containing <NUM>% FBS and <NUM> ng/mL bFGF was added, and the mixture was cultured at <NUM> in a <NUM>% CO<NUM> incubator for five days. After washing the cells with DMEM for three times, DMEM containing <NUM> ng/mL bFGF was added, and the resulting mixture was cultured at <NUM> in a <NUM>% CO<NUM> incubator for about <NUM> hours. After removing the supernatant, a <NUM>-syringe was used to collect a cell-hydrogel. A <NUM>-gauge needle was connected to the syringe to push the cell-hydrogel to a dish, and the cell-hydrogel was observed with an optical microscope.

<FIG> is an image of the cell-hydrogel transferred to a dish after culturing at a <NUM>-well plate, indicating that the formed hydrogel was maintained during the culturing period.

<FIG> is an image of the cell-hydrogel filled into a syringe, indicating that the gel state was maintained even after being filled into the syringe.

<FIG> shows the injection of the cell-hydrogel filled into a syringe through a <NUM>-gauge needle, and <FIG> shows that the hydrogel shape of the cell-hydrogel was maintained even after being injected through the <NUM>-gauge needle.

<FIG> shows a microscopic image of the cell-hydrogels prepared at different ratios, and then filled into a syringe and injected to a plate by using a <NUM>-gauge needle. The cell growth was better, as the fibrin glue dilution factor was higher. The cell growth was good and the cells and the fibrin glue hydrogel were well mixed with each other when the final fibrin glue concentration was from <NUM> to <NUM>/mL or from <NUM> to <NUM>/mL. No significant difference depending on the thrombin concentration was found.

<FIG> is a microscopic image of a cell-hydrogel prepared at different ratios, filled into a syringe, kept at <NUM>, the human body temperature, for <NUM> hours, and then injected to a plate by using a <NUM>-gauge needle. The cells and the hydrogel were separated from each other almost completely at a final fibrinogen concentration from <NUM> to <NUM>/mL and from <NUM> to <NUM>/mL, but the cells and the hydrogel were well mixed with each other for up to <NUM> hours at a final fibrinogen concentration from <NUM> to <NUM>/mL. No significant difference depending on the thrombin concentration was found.

A thrombin solution was added to the adipose tissue-derived mesenchymal stem cells of a concentration of <NUM> × <NUM><NUM>/mL obtained in Example <NUM> at a ratio of <NUM>:<NUM>. Fibrinogen was prepared by diluting at a dilution factor of <NUM>:<NUM>. Then, the cells were cultured, washed, and filled into a syringe according to Example <NUM>.

The following method was used as a conventional cell therapeutic agent culture method.

Adipose tissue-derived mesenchymal stem cells were cultured in a culture vessel. When the cells grew to a volume of about <NUM>% of the culture vessel, the cells were separated as single cells by adding trypsin-EDTA. After deactivating enzymes by adding DMEM containing FBS, the cells were collected to a centrifugation tube and underwent centrifugation at <NUM> rpm. After removing the supernatant, DMEM was added to suspend the cells in order to remove the FMS. Then, the cells again underwent centrifugation at <NUM> rpm and the resulting supernatant was removed. The cells were washed for a total of three times, suspended in the final washing with a cell suspension agent of an appropriate volume, and then filled to a vial. The filled cells were transferred to a <NUM> syringe connected with a <NUM>-gauge needle.

A triple expression enzyme was added to the cell-hydrogel at each stage of Example <NUM> to dissolve fibrin glue, and the number of cells was measured. Similarly, the number of cells was measured at each stage of Comparative Example <NUM>.

Figure 3a is a plot showing the recovery rate of the cells at each stage of the conventional method of preparing the cell therapeutic agent (Comparative Example <NUM>) in comparison with the initial cell collection stage (trypsin-EDTA deactivation), indicating that the cell recovery rate was decreased by <NUM> to <NUM>% at each stage and only about <NUM> of the cells were recovered after the cells were finally filled to a syringe in comparison with the initial collection stage.

Figure 3b is a plot showing the recovery rate of the cells at each stage of the preparation method of the present invention (Example <NUM>) in comparison with the initial cell collection stage (after completion of cell culture), indicating that the cell recovery rate was decreased by <NUM> to <NUM>% at each stage and more than <NUM>% of the cells were recovered after the cells were finally filled to a syringe.

The survival rate of the cells cultured in a hydrogel according to Example <NUM>, filled into a <NUM> syringe, and kept at room temperature was measured after <NUM>, <NUM>, and <NUM> hours and seven days by staining the cells with acridin orange/etidium bromide. The cells prepared by a conventional method of Example <NUM> and filled to a vial were used as controls.

As shown in <FIG>, the cells prepared by the conventional method and filled to a vial maintained a high survival rate of <NUM>% or higher at Hour <NUM>, but the cells failed to maintain the shape or were aggregated with each other on Hour <NUM> as the cell membrane was destroyed due to cell death, and almost no cell was observed on Hour <NUM> as all the cells were dead. On the other hand, the cells cultured in the cell-hydrogel survived until Day <NUM>, and the shape of the fibroblast was maintained.

The cell-hydrogel prepared by culturing cells in a hydrogel and filling into a <NUM> syringe according to Example <NUM> was dissolved by treating with an enzyme, and the quantity of collagen Type I was analyzed by ELISA. A cell suspension prepared by culturing cells by a conventional method of Example <NUM> and packing into a <NUM> syringe was used as a control.

As shown in <FIG>, almost no collagen was included in the cell suspension prepared by culturing cells by the conventional preparation method of a cell therapeutic agent, but <NUM> ug/mL or more collagen was included in the cell-hydrogel.

Claim 1:
A method of preparing a mesenchymal stem cell-hydrogel composition, wherein the method includes the steps of:
(a) culturing of adipose tissue-derived mesenchymal stem cells;
(b) mixing the cultured adipose tissue-derived mesenchymal stem cells and a hydrogel solution to form a gel;
(c) culturing the gel; and
(d) filling the gel of step (c) into an ampoule, a vial, or a syringe,
wherein the hydrogel is fibrin glue, wherein the fibrin glue includes fibrinogen of a concentration from <NUM> to <NUM>/mL.