KR101288587B1 - A method of mass production of growth factors secreted stem cells by circulating filtration system - Google Patents

Abstract

The present invention relates to a method for mass separation and concentration of growth factors secreted from stem cells, and more particularly, growth factors to be separated from a culture of human mesenchymal stem cells using a plurality of circulating filter systems. To a method for rapidly separating and concentrating a large quantity. According to the method for isolating and enriching growth factors secreted from stem cells of the present invention, there is an effect that can rapidly separate and concentrate a large amount of a desired growth factor derived from stem cells without using a genetic recombination method.

Description

Mass separation and concentration method of growth factor secreted from stem cells {A METHOD OF MASS PRODUCTION OF GROWTH FACTORS SECRETED STEM CELLS BY CIRCULATING FILTRATION SYSTEM}

The present invention relates to a method for mass separation and concentration of growth factors secreted from stem cells, and more particularly, growth factors to be separated from a culture of human mesenchymal stem cells using a plurality of circulating filter systems. To a method for rapidly separating and concentrating a large quantity.

Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage.

There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, embryonic stem cells and adult stem cells are characterized by differentiation into various cell types.

Embryonic stem cells have very good differentiation ability and long telomers, but they have ethical problems and difficult to obtain large amounts of cells, while adult stem cells can obtain a large number of cells but can be transplanted to others. When the risk of infection or differentiation capacity is relatively low.

Despite the above disadvantages, adult stem cells are characterized by a very safe medical application. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration, and since the immune rejection reaction does not occur because it is in an adult body, autologous transplantation using its own cells is possible.

In addition, there is a site-specific differentiation ability to differentiate itself according to the characteristics of the surrounding tissue, and since injection does not cause cancer even when injected in the undifferentiated state, in addition to producing the cells needed immediately after transplantation The advantage is the ability to self-renewal to regenerate and store the necessary undifferentiated stem cells.

According to the above advantages, adult stem cells have been recently highlighted in importance, and various studies are being conducted to obtain adult stem cells in vivo.

In addition, based on recent advances in biochemistry and molecular biology, small amounts of signal substances (growth factors) present in our living organisms have been discovered, and the biological aging theory based on them has been redefined (Stanley Cohen, Nobel Lecture 1986, Dec, 8). In addition, this signal substance (growth factor) decreases in vivo with increasing age, and it is revealed that this growth factor is closely related to our aging (Sporn M and Roberts A, Handbook of Experimental Pharmacology). , Vol. 1, Vol. 95/1, 1990, Springer-Verlag, DE, Berlin., Pp. 667-698).

Therefore, it has been studied that the supply of growth factors of the living body can inhibit the aging of the living body, and the specific effects of the material are being studied (GE Pierce and TA Mustoe, Annu Rev Med, 46. 467-481 (1995).

Specifically, studies on the structure and synthesis of this signaling material (growth factor) have been conducted, but most growth factors have a protein structure, and the structure is three-dimensionally complex, causing various problems in chemical synthesis. In reality, the cost of such synthesis is also very expensive.

The present inventors came to pay attention to the fact that mesenchymal stem cells secrete growth factors while studying how to obtain an active human growth factor at a low price while maintaining activity in the human body (Rehman, J.). et al., Circulation, 109: 1292-1298 (2004)).

However, most of the studies on mesenchymal stem cells use the cells themselves, but there are few studies on differentiation, and there is almost no research on the method of synthesizing growth factors using them.

As such, studies on the synthesis of growth factors using mesenchymal stem cells have been insufficient. The method of using adult stem cells is focused on the differentiation of cells into different cells. We believe this was because of some incompleteness.

In addition, as described above, the method for synthesizing the growth factor is described in Korean Patent Registration No. 101436, "Method of Manufacturing Recombinant Human Endothelial Growth Factor," and Korean Patent Registration No. 6625, "Generation of Human Epithelium by Genetic Recombination Technology." Method for the production of growth factors by genetic recombination technology, such as the method for preparing the factor ", Korean Patent Publication No. 2003-45032" The method for producing biologically active human acid fibroblast growth factor and its use for promoting angiogenesis " Has been studied.

Therefore, this researcher has studied the mass production method of growth factors through mesenchymal stem cells for many years, and developed the method of applying them to the multi-filter system by paying attention to the size of the desired growth factors in the stem cell culture. .

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is to isolate and concentrate the growth factors to be separated in the stem cell culture using a plurality of circulation filter system To provide a way.

In order to solve the above problems, the present invention is to check the molecular weight of the growth factor (growth factor) to be separated through a database, subcultured stem cells to obtain a stem cell culture, and the stem cell culture data A growth factor having a molecular weight identified from the database is passed through a first filter system having a filter diameter through which a growth factor having a molecular weight identified from the base can be passed through, and the stem cell culture passed through the first filter system. Passed through a second filter system having a filter diameter that cannot be passed, and collecting growth factors having a molecular weight to be separated that remain unfiltered on the filters of the second filter system. Provided are methods for separating and concentrating growth factors.

The growth factors are acidic FGF, basic FGF, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2). , Keratinocyte growth factor (KGF), platelet derived growth factor (PDGF), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF) , Epidermal growth factor (EGF), neuronal growth factor (NGF) and interleukin-1 family 9 (IL-1-f9).

Preferably, the database is GenBank and ExPASy.

The stem cells may be mesenchymal stem cells.

The mesenchymal stem cells are preferably derived from bone marrow, cord blood, placental tissue isolated from the individual.

The stem cells may be derived from humans.

The first filter system applies a filter system having a filter diameter through which stem cells cannot pass, or a filter system having a filter diameter through which a growth factor having a molecular weight identified from a database can be passed, in order of decreasing filter diameter. There may be several filter systems.

The several filter systems preferably consist of three to four filter systems.

The several filter systems are preferably circulating filter systems.

The second filter system preferably has a filter diameter through which particles of size 40 to 60% of the growth factor molecular weight to be separated can pass.

Preferably, the first filter system and the second filter system are circulating filter systems.

According to the method for isolating and enriching growth factors secreted from stem cells of the present invention, there is an effect that can rapidly separate and concentrate a large amount of a desired growth factor derived from stem cells without using a genetic recombination method.

Figure 1 shows a flow chart of the method for isolation and concentration of growth factors derived from stem cells of the present invention. 1 to 3 represent a first filter system and 4 to a second filter system.
2 is a photograph showing a filter system of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method of simply concentrating a growth factor (growth factor) that is produced externally while being naturally differentiated in stem cell culture. That is, the present invention relates to a method for rapidly separating a large amount of a naturally produced material from stem cells, rather than an artificially produced candidate material.

Therefore, the present invention is to check the molecular weight of the growth factor to be separated through a database, subcultured stem cells to obtain a stem cell culture, the stem cell culture growth factor having a molecular weight identified from the database Is passed through a first filter system having a filter diameter through which the stem cell culture passed through the first filter system cannot pass through a growth factor having a molecular weight identified from the database. 2 passing through a filter system and collecting growth factors having a molecular weight to be separated that remain unfiltered on the filter of the second filter system, wherein the growth factor secreted from the stem cells is provided. do.

The growth factor to be separated of the present invention is secreted from stem cells, and if it is applicable to the medical field, there is no limitation in its kind. Examples of preferred growth factors include acidic FGF, basic FGF, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-) 2), keratinocyte growth factor (KGF), platelet derived growth factor (PDGF), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), endothelial cell growth factor ( VEGF), epidermal growth factor (EGF), neuronal growth factor (NGF) or interleukin-1 family 9 (IL-1-f9).

After selecting the growth factor to be separated, first to investigate the physical properties of the growth factor to be separated, for example, molecular weight (Mw), isoelectric point (pI) and the like. At this time, it is preferable that the databases used are, for example, GenBank and ExPASy. Zenbank is accessible from http://www.ncbi.nlm.nih.gov/ and Expasi can be accessed from http://www.expasy.ch/.

The stem cells may be mesenchymal stem cells. The mesenchymal stem cells are preferably derived from bone marrow, cord blood, placental tissue isolated from the individual. The stem cells may be derived from humans.

Mesenchymal stem cells according to the present invention can be obtained through the purification process among the cells present in human bone marrow, fat, umbilical cord blood, placenta. The process of extracting stem cells according to the characteristics of each tissue is already well known to those skilled in the art through domestic and foreign patents and papers. Mesenchymal stem cells can be obtained.

In one preferred embodiment, the medium used in the culture in vitro using mesenchymal stem cells is as follows.

Prior to the start of cell culture, biopsies extracted from the source in a known manner, ie, bone marrow, placenta, etc., can be minimized through repeated washing procedures with wash media containing common antibiotics to minimize the potential for contamination in subsequent cultures.

In the present invention, the culture medium is optimized to induce maximum synthesis and secretion of growth factors in mesenchymal stem cells.

Specifically, in vitro culturing of mesenchymal stem cells is carried out by culturing in serum medium and then subcultured in serum-free medium to maximize the synthesis of growth factors.

The medium for the early stage cell culture, including serum, is preferably a medium for the purpose of maintaining and storing cell types such as mesenchymal stem cells.

The present invention is based on Dulbecco's Modified Eagle's Medium (DMEM), which is generally used in cell culture in the art, and includes serum commonly used in cell culture.

In this case, when 7-10% of dimethyl sulfoxide (DMSO) is added to the initial medium, the medium may be a freezing medium. Therefore, the stem cells may be frozen and then thawed if necessary.

Serum is preferably added 0.1-20% fetal bovine serum (FBS), more preferably antibiotics, antifungals and reagents that prevent the growth of mycoplasma causing contamination.

As antibiotics, all antibiotics commonly used in cell culture such as penicillin-streptomycin can be used, and antifungal agents include amphotericin B and tyrosine as a mycoplasma inhibitor, and gentamicin, ciprofloxacin, azithromycin, etc. It is possible to prevent mycoplasma contamination, and if necessary, oxidative nutrients such as glutamine and energy metabolites such as sodium pyruvate can be added.

More preferred medium contains 1-2 mM glutamine, 0.5-1 mM sodium pyruvate, 0.1-10% FBS, glucose supplemented with 1% antibiotic (100 IU / ml) and is called complete serum medium. At this time, the concentration range of glucose is approximately 1 g / L to 4.5 g / L. The complete serum medium provides storage and maintenance of adipose derived stem cells and stable basic culture conditions in vitro and shows effective cell stabilization.

In general culture conditions, the initial culture is the applying conditions suitable to 90-95% humidity, temperature 35 ~ 39 ℃, 5 ~ cultured in 10% CO ₂ incubator, and 5 ~ 10% CO _2, the final concentration during culture in cell culture Add carbon control sources such as sodium bicarbonate such that is 0.17 to 0.22% by weight.

During the initial culture step, the tissue fragments are preferably attached to the bottom of the culture vessel and can promote growth with short stimulation due to trypsin-EDTA treatment according to standard techniques of cell culture.

Cumulative population doubling time (doubling time) is maintained until 75-85% confluence of the cells in culture in the flask and preferably passaged by collecting the cells at 80% confluence.

In another aspect, the present invention provides a serum-free medium for differentiation of growth factors to promote differentiation of growth factors in mesenchymal stem cells.

Passage culture using serum-free medium for differentiation of growth factors is performed by washing the flask from which the culture medium has been removed with phosphorylation buffer solution, dropping the cells with trypsin-EDTA, and centrifuging the cell suspension with the phosphorylation buffer solution. It is preferable to repeat 2 or 3 times. The washed pellet is suspended by serum-free medium developed in this invention and passaged approximately three times in cell culture flasks.

Serum-free medium of the present invention is based on DMEM without a pH indicator such as phenol red and Ham's F-12 nutrient mixture (SIGMA, Cancer Research Vol 47, Issue 1 275-280) at approximately 1: 0.5-2. Add in proportions. At this time, it is possible to add an oxidative nutrient such as L-glutamine, an energy metabolite such as sodium pyruvate, and a carbon regulator such as sodium bicarbonate, and other growth factors other than the growth factor to be aimed at in the present invention. Growth hormones can also be added.

The uniqueness of the serum-free medium of the present invention can be found in Ham's F-12 nutrient mixture. This mixed solution is a growth factor secreted from various minerals and amino acids and mesenchymal stem cells that are involved in the growth and homeostasis of the cells and in promoting the stability and maintenance of the cells in the early and post-culture of adipose derived stem cells. Vitamin-based nutrients and other factors, which can promote higher production, are formed by mixing in proportions. In the serum-free medium containing Ham's F-12 mixed solution of the present invention, the production of mesenchymal stem cells and growth factor were not significantly reduced or negative when compared to the conditions of serum medium containing animal serum. Some growth factors show higher productivity in serum-free media and, unlike serum media with unknown components by serum, can identify all component contents of the culture medium.

This suggests that various variables that can come from animal serum included in serum medium can be minimized, and the present invention can achieve the desired efficacy at a cost as low as about 50%.

One filter system of the present invention reduces the filter diameter to a filter system having a filter diameter through which stem cells cannot pass or a filter system having a filter diameter through which a growth factor having a molecular weight identified from a database can be passed. There may be several filter systems applied in the order of The several filter systems preferably consist of three to four filter systems. The several filter systems are preferably circulating filter systems. The circulation refers to passing the cell culture on the filter system of the present invention, replacing the filter with a filter having a smaller diameter, then filtering the first filtrate again, and repeating this process. As such, by sequentially decreasing the filter diameter and filtering the cell culture in a circular manner, only materials having a desired growth factor and a molecular weight of less than can be efficiently separated in a short time.

The second filter system of the filter system of the present invention preferably has a filter diameter through which particles of 40 to 60% of the growth factor molecular weight to be separated can pass. By adopting a filter having a diameter smaller than the molecular weight of the growth factor to be separated as described above, it is possible to effectively remove substances having a molecular weight smaller than the growth factor to be separated, thereby efficiently concentrating only the desired growth factor. Do.

In the filter system of the present invention, the first filter system and the second filter system are preferably circulating filter systems. As a result, several first filter systems and a second filter system enable circulation systems by using filters having different filter diameters.

In one preferred embodiment, the filter system of the present invention may be QuixStand ^™ Systems (GE helthcare, USA) (see FIG. 2). The system is capable of applying up to 10 L of cell culture at a time, with a volume of about 30-50 ml. The system has the advantage of being easily replaced with a filter of a desired size, which is suitable for the circulating filter system of the present invention.

By employing the filter system of the present invention described above, only the specific growth factor to be separated having a known molecular weight can be concentrated quickly and efficiently.

Hereinafter, the present invention will be described in more detail by way of examples.

< Example >

1. Molecular weight prediction of growth factor

In order to concentrate the EGF, the estimated isoelectric point (pI) and molecular weight (Mw) using GenBank Accession number, ExPASy were obtained. EGF was Accession number AAS83395, pi 4.78, Mw 6.2kDa.

2. Stem Cell Culture Preparation

Bone marrow-derived mesenchymal stem cells hMSCs were purchased from Human Mesenchymal stem cells (Cambrex, PT-2501) and were isolated from the bone marrow of a 19 year old healthy male.

Initial cell culture of bone marrow-derived mesenchymal stem cells was performed using DMEM and 10% FBS was added. In addition, 1% penicillin-streptomycin (100 IU / ml, GIBCO) was added as an antibiotic, amphotericin B (0.5ug / ml, Amresco) as an antifungal agent, and tyrosine (10ug / ml, Serva, as a mycoplasma inhibitor. Heidelberg), 2 mM glutamine and 1 mM sodium pyruvate were further added. Culture conditions were incubated in a 95% humidity, 37 ℃, 5% CO ₂ incubator, sodium bicarbonate was added to the final concentration of 0.17% in 5% CO ₂ culture.

Cumulative population doubling time was maintained until 80% confluence of the cells in culture in the flask and passage was performed at 80% confluence.

Subculture was washed with PBS to remove the culture medium, the cells were dropped with 0.25% Trypsin-EDTA (GIBCO), centrifuged the cell suspension, and then subcultured three times after measuring the cell number and viability. Serum-free medium used for subculture was based on DMEM without the addition of pH indicators such as phenol red and Ham's F-12 nutrient mixture (SIGMA) in a ratio of 1: 1, 2 mM L-glutamine and 1 mM sodium pyruvate. After addition of the bait, sodium bicarbonate was added to 0.1 wt%, and the process was repeated three times.

Cell number measurement and viability test were performed by mixing 0.1 ml of cell suspension with the same amount of 0.2% trypan blue (SIGMA), and counting the stained and missing cells in the microscope field using a hemocytometer to determine the percentage of total cells. It was.

3. Identify growth factors

After centrifuging 1 ml of the culture medium from 2 for 5 minutes at 1200 g, filtered with a 0.22 mm filter to filter out the cell residues, and diluting the filtrate step by step to set the optimum conditions for nonspecific reactions. The concentration of EGF secreted in the culture was measured using a sandwich ELISA kit (Quantikine Human FGF basic Immunoassay, R & D systems). The measured concentration was 250 pg / ml.

4. Filter System  Enrichment of growth factors

1 L of cell culture according to the cell culture method of 2 was obtained. The resulting cell culture was filtered using Quixstand ^™ from GE. The advantage of this machine is that the material in the target solution can be separated and concentrated with high accuracy and at high speed depending on the molecular weight. Since it is possible to build a filter system that can distinguish the molecular weight of various sizes, it can be effectively used to separate and concentrate the material to be obtained in the present invention.

First, the supernatant is recovered to remove the largest stem cells, and various sizes of debris and candidates are separated together with the supernatant. Next, debris of size 100 kDa or more are removed, and debris of less than that size and candidate material are separated. Next, debris with size greater than 20 kDa are removed and candidate material less than that is isolated. Since EGF has a molecular weight of 6.2 kDa, a 3 kDa filter was mounted and filtered, and the remaining EGF candidate material in the filter was concentrated to a volume of 20 ml.

The above description is merely illustrative of the technical spirit of the present invention, and those skilled in the art to which the present invention pertains may make various modifications and changes without departing from the essential characteristics of the present invention.

In addition, the embodiments disclosed herein are not intended to limit the technical spirit of the present invention but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments.

Therefore, the protection scope of the present invention should be interpreted by the following claims, and all technical ideas within the equivalent scope will be construed as being included in the scope of the present invention.

Claims (11)

The molecular weight of the growth factor to be isolated (growth factor) is confirmed through a database, the stem cells are passaged to obtain a stem cell culture, and the stem cell cultures have a growth factor having a molecular weight identified from the database. Passing through a first filter system having a filter diameter that can be passed through, and a stem cell culture that has passed through the first filter system having a filter diameter such that a growth factor having a molecular weight identified from the database cannot be passed through Passing through a filter system and collecting the growth factors with the molecular weight to be separated which remain unfiltered on the filters of the second filter system,
The first filter system applies a filter system having a filter diameter through which stem cells cannot pass, or a filter system having a filter diameter through which a growth factor having a molecular weight identified from a database can be passed, in order of decreasing filter diameter. Method for isolation and concentration of growth factors secreted from stem cells, characterized in that several filter systems. The method of claim 1, wherein the growth factor is acidic fibroblast growth factor (acidic FGF), basic fibroblast growth factor (basic FGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor- 2 (IGF-2), keratinocyte growth factor (KGF), platelet derived growth factor (PDGF), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), endothelial vessels Secreted from stem cells, characterized in that it is selected from the group consisting of cell growth factor (VEGF), epidermal cell growth factor (EGF), neuronal growth factor (NGF) and interleukin-1 family 9 (IL-1-f9). Method of isolation and concentration of growth factors. The method of claim 1, wherein the database is GenBank and ExPASy. The method of claim 1, wherein the stem cells are mesenchymal stem cells. The method of claim 4, wherein the mesenchymal stem cells are derived from bone marrow, umbilical cord blood, and placental tissue isolated from an individual. The method of claim 4 or 5, wherein the stem cells are derived from a human, characterized in that the method for separating and enriching growth factors secreted from stem cells. delete The method of claim 1, wherein the several filter systems comprise three to four filter systems. 9. The method of claim 1 or 8, wherein said several filter systems are circulating filter systems. The method of claim 1, wherein the second filter system is characterized in that the separation of the growth factor secreted from the stem cells, characterized in that having a filter diameter that can pass through the particles of the size of 40 to 60% of the molecular weight of the growth factor to be separated Way. The method of claim 1, wherein the first filter system and the second filter system are circulating filter systems.

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