KR102180733B1 - A Composition for Isolating Stem Cells Comprising Skim Milk as an Active Ingredient - Google Patents

Abstract

The present invention provides a method for obtaining stem cells from a biological specimen isolated from a human body and a method for proliferating urine-derived stem cells. The present invention improves the efficiency of both a separation step of stem cells and a cultivation step after separation of stem cells through a simple process of adding skim milk to a biological sample or a cell culture component, thereby being able to be usefully used as an efficient process for remarkably increasing a yield of the ultimately desired urine stem cells.

Description

Composition for Isolating Stem Cells Containing Skim Milk as an Active Ingredient {A Composition for Isolating Stem Cells Comprising Skim Milk as an Active Ingredient}

The present invention relates to a composition for separating stem cells containing skim milk as an active ingredient, and to a method for separating stem cells from a biological sample using the same, specifically, a urine sample.

Stem cells are cells that have pluripotency or multipotency that can differentiate into various cells that make up a tissue, and are undifferentiated at the pre-differentiation stage that can be separated from each tissue of the embryo, fetus, and adult. Cells are referred to collectively. Stem cells differentiate into specific cells by stimulation of differentiation, and unlike differentiated cells in which cell division is stopped, they have self-renewal ability, and are converted to other cells by different environments or different differentiation stimulation. It can also be differentiated, so it has plasticity in differentiation.

Stem cells are largely obtained from embryos and are pluripotency embryonic stem cells (ES cells) that have the potential to differentiate into all cells, and multipotency obtained from each tissue. ) Of adult stem cells (adult stem cells). Embryonic stem cells are undifferentiated cells capable of unlimited proliferation and can differentiate into all cells, and unlike adult stem cells, germ cells can also be made, so they can be inherited to the next generation. However, despite these advantages, the use of embryonic stem cells as a cell therapy is difficult to commercialize, such as tumor formation, immune rejection, and ethical legal restrictions. As an alternative to overcome these problems, mesenchymal stem cells are in the spotlight, which is capable of differentiating into adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, cardiomyocytes, as well as the ability to regulate immune responses. Also have.

Urine stem cells can be separated from human urine and, like other somatic stem cells (adipose stem cells, umbilical cord stem cells, bone marrow stem cells, etc.), have the ability to differentiate into various tissues and organs. In addition, urine stem cells can be obtained regardless of the donor's age, sex, or health status, and has the advantage of being able to isolate cells through a safe and low-cost, non-invasive procedure. In addition, it is easy to separate pure stem cells without enzymatic treatment, and exhibits high telomerase activity, which has the advantage of obtaining more cells than teratomas or tumors, and podocytes, Differentiation efficiency into smooth muscle, endothelial, and urinary tract cells is higher than that of other stem cells.

The ease of separation of urine stem cells is the greatest advantage compared to other stem cell separation methods, and urine collection does not require any special procedures or techniques, and sedimentation of cells using only centrifugation allows for separation at a simple procedure and low cost. However, the minimum required number of cells in cell therapy or regenerative medicine is about 1 to 6 X 10 ⁹ , and the amount increases further when the necessary cells are considered in the step of establishing culture conditions and standards. In order to supply this amount to stem cells of various origins, at least 10 passages in vitro are required, and in this process, the cells are aged and deformed, making them no longer suitable for the concept of treatment. In the case of a normal person, although it is possible to secure urine repeatedly, the amount of urine and the number of stem cells separated from it are limited depending on the patient's condition, age, and environment. Therefore, in order to utilize the various advantages of urine stem cells, it is required to develop an efficient technology for securing a therapeutically effective amount of cells.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Patent Document 1. U.S. Patent Publication No. 5,866,418

The present inventors have made intensive research efforts to develop an efficient process for separating and obtaining stem cells having high value for therapeutic and research purposes from biological samples isolated from the human body with high yield. As a result, the present invention was completed by finding that the efficiency of separation of stem cells can be significantly increased by adding a simple process of adding skim milk to the sample before separation from biological samples to stem cells by a conventional method. Was done.

Accordingly, an object of the present invention is to provide a method for obtaining stem cells from a biological specimen isolated from a human body.

Another object of the present invention is to provide a method for proliferating urine-derived stem cells.

Another object of the present invention is to provide a composition for separating stem cells from a biological sample.

Another object of the present invention is to provide a composition for proliferating urine-derived stem cells.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for obtaining stem cells from a biological specimen isolated from a human body comprising the following steps:

(a) adding skim milk to the biological sample;

(b) separating stem cells from the biological sample to which the skim milk is added.

The present inventors have made intensive research efforts to develop an efficient process for separating and obtaining stem cells having high value for therapeutic and research purposes from biological samples isolated from the human body with high yield. As a result, it was found that the efficiency of separation of stem cells can be remarkably increased by adding a simple process of adding skim milk to the sample before separating it from a biological sample to stem cells by a conventional method.

As used herein, the term “stem cell” refers to a cell capable of differentiating into various cells constituting a biological tissue, and has a pluripotent or pluripotent regeneration capable of, but not limited to, forming specialized cells of tissues and organs. Refers to undifferentiated cells.

In the present specification, the term "biological sample" is any sample including stem cells obtained from mammals including humans, and includes, but is not limited to, tissues, organs, cells, or cell culture.

More specifically, the biological sample of the present invention is a liquid specimen. Liquid samples are not only various body fluids that are in a liquid state at the time they are separated from the human body, but they are in a solid state at the time of separation, but after separation or during separation, by adding a solvent or adding a culture solution, all the samples become liquid. Includes without limitation biological samples.

According to a specific embodiment of the present invention, the biological sample of the present invention is a body fluid. More specifically, the bodily fluid is urine, saliva, semen, tears, amniotic fluid, cerebrospinal fluid, synovial fluid, pericardial fluid, ascites (peritoneal fluid), and blood. It is one or more body fluids selected from the group consisting of. Most specifically, the biological sample is urine.

Urine stem cells, like other adult stem cells, not only have pluripotency, but can be obtained regardless of the donor's age, sex, and health status, and exhibit high telomerase activity, resulting in cancer or cancer. It is possible to obtain safe cells without the risk of teratoma. The present invention provides an efficient separation method for obtaining a therapeutically effective amount of urine-derived stem cells from a limited amount of a urine sample.

In the present specification, the term "therapeutically effective amount" refers to the number of stem cells sufficient to provide a therapeutic (for example, regenerative treatment of a degenerative disease or treatment of an immune disease) or a prophylactic effect to a subject to which the therapeutic stem cells are administered. It means the amount, and it means including "prophylactically effective amount". As used herein, the term “subject” includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

In the present specification, the term "isolation" refers to a process of selectively obtaining a target material (eg, stem cells) from a biological sample (positive isolation) as well as a process of selectively removing impurities other than the target material ( negative isolation). Therefore, the term "separation" is used in the same meaning as "obtain", "extract", and "purify". The separation process of step (b) includes, for example, separation using an antibody specific to the surface antigen of the target cell (eg, FACS) and separation according to the difference in specific gravity of the target cell (eg, a layer separation method, a centrifugation method). ), including, but not limited to, all methods commonly used in the art as a method for isolating specific cells in a heterogeneous sample.

According to a specific embodiment of the present invention, the step (b) is performed by centrifugation.

According to a specific embodiment of the present invention, the skim milk used in the present invention is added in a final concentration of 2-7% (v/v) to the biological sample. More specifically, it is added at a final concentration of 2.2-6% (v/v), more specifically, it is added at a final concentration of 2.3-5.5 (v/v), and most specifically, 2.5-5% (v /v) is added at a final concentration.

According to another aspect of the present invention, the present invention provides a method for proliferating urine-derived stem cells comprising culturing urine-derived stem cells in a medium to which skim milk is added.

The present inventors found that the addition of skim milk to a urine sample not only increases the separation efficiency of stem cells in the sample, but also increases the proliferation rate significantly when included in a medium for culturing stem cells isolated from urine. Therefore, the skim milk of the present invention improves the efficiency of both the separation step of the stem cells and the culture step after the separation, thereby significantly increasing the yield of the desired urine stem cells.

The term "medium cell culture" herein in, in vitro, including the essential element in the growth and proliferation of cells, such as sugar, amino acids, and various nutrients, minerals refers to a mixture for cell growth and proliferation.

Components that may be additionally included in the cell culture medium include, for example, glycerin, L-alanine, L-arginine hydrochloride, L-cysteine hydrochloride-monohydrate, L-glutamine, L-histidine hydrochloride-monohydrate, L- Lysine hydrochloride, L-methionine, L-proline, L-serine, L-threonine, L-valine, L-asparagine-monohydrate, L-aspartic acid, L-cystine 2HCl, L-glutamic acid, L-isoleucine, L -Leucine, L-phenylalanine, L-tryptophan, L-tyrosine disodium salt dihydrate, i-inositol, thiamine hydrochloride, niacinamide, pyridoxine hydrochloride, biotin, D-calcium pantothenate, folic acid, riboflavin, vitamin B ₁₂ , Sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl ₂ ), sodium hydrogen phosphate monohydrate (NaH2PO4-H2O), copper sulfate pentahydrate (CuSO4-5H2O), ferric sulfate heptahydrate (FeSO4- 7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4), disodium hydrogen phosphate (Na2HPO4), zinc sulfate heptahydrate (ZnSO ₄ -7H ₂ O), D-glucose (dextrose), sodium pyruvate, hippoc Santin Na, linolenic acid, lipoic acid, putrescine 2HCl and thymidine are included, but are not limited thereto.

The cell culture medium according to the present invention may be artificially prepared and used, or commercially available ones may be purchased and used. Examples of commercially available culture media are IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12), and DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture). F-12), but is not limited thereto.

According to a specific embodiment of the present invention, the medium is added in a concentration of 1-8% (v/v) of the skim milk. More specifically, it is added at a final concentration of 1.2 to 7.5% (v/v), more specifically, it is added at a final concentration of 1.2 to 6% (v/v), and even more specifically, 1.2 to 5% It is added at a final concentration of (v/v), and most specifically, it is added at a final concentration of 1.25-4% (v/v).

According to another aspect of the present invention, the present invention provides a composition for separating stem cells from a biological sample containing skim milk as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for proliferating urine-derived stem cells comprising skim milk as an active ingredient.

Since the concentrations of the biological sample and skim milk used in the present invention have already been described above, the description thereof will be omitted to avoid excessive duplication.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method of obtaining stem cells from a biological specimen isolated from a human body.

(b) The present invention provides a method for proliferating urine-derived stem cells.

(c) The present invention significantly increases the yield of the desired urine stem cells by improving the efficiency of both the separation step of stem cells and the culture step after separation through a simple process of adding skim milk to a biological sample or cell culture component. It can be usefully used as an efficient process to make.

1 is a schematic diagram summarizing the process of separation and cultivation of urine stem cells using skim milk.
2 is a diagram showing the separation efficiency of urine stem cells according to the concentration of skim milk through the number of separated cells.
3 is a diagram showing the results of measuring the change in the proliferation rate of urine stem cells by skim milk by concentration of skim milk added to the culture medium.
4 is a picture showing a photograph of a urine stem cell culture plate to which 0-15% of skim milk was added.
5 is a schematic diagram summarizing the process of producing induced pluripotent stem cells derived from urine stem cells.
6 is a diagram showing urine stem cell-derived induced pluripotent stem cell colonies and AP staining results. Scale bar: 200 μm.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

Preparation of skim milk

Skim milk was put into a 100 mL glass bottle and then autoclaved in an autoclave. Sterile skim milk powder was added to the urine stem cell culture medium [renal epithelial (RE) media, PromoCell, Heidelberg, Germany].

Separation of urine stem cells using skim milk Experiment

Urine stem cells were collected using a sterile medical urine sampling container (SPL, Pocheon, Republic of Korea) from the urine of 34, 28, 50, 23, and 24 year old males and 21 year old females. Separation was performed within 2 hours at and within 4 hours under ice storage (4°C). The collected urine was dispensed into 15 mL conical tubes (SPL) by 10 mL, and skim milk prepared in each tube was added by concentration. After stirring the tube slowly, centrifugation was performed at 400 g for 10 minutes. Thereafter, the supernatant of the 15 mL conical tube was slowly discarded, and 10 mL of PBS containing 1× Anti-Anti (Thermo Fisher Scientific, Massachusetts, USA) was added to each tube. After centrifugation was performed again at 400 g for 10 minutes, RE medium was added to the gelatin-coated cell culture dish, and each was seeded. The culture dish was not shocked for 3 days, and fresh RE medium was added on the 4th day. After that, the medium was replaced by half every two days, and the number of cells was measured through trypan blue staining after 14 days.

Comparison of Urine Stem Cell Proliferation Rate by Skim Milk

In order to test the proliferation rate of urine stem cells according to the concentration of skim milk, the number of urine stem cells was first measured through trypan blue staining. The measured urine stem cells were seeded in a 96-well plate in 3 times of 5×10 ³ each, and skim milk was added for each concentration. Incubated in a cell culture incubator at 5% CO ² and 37°C for a total of 3 days, and after washing the cultured urine stem cells 5 times with PBS, EZ-CYTOX (WST-based cell proliferation assay kit, DAEILLAB, Chungwon, Republic of Korea) was added. After 2 hours, the cell culture dish was measured for absorbance at a wavelength of 450 nm and the measured value was analyzed.

Urine stem cell reprogramming experiment

Urine stem cells isolated and cultured using skim milk were used to produce induced pluripotent stem cells. Urine stem cells were reprogrammed according to the manufacturer's manual using the CytoTune-iPS Sendai Reprogramming Kit (Thermo Fisher Scientific). Induced pluripotent stem cell colonies formed by reprogramming are mTeSR1 (STEMCELL technologies, Vancouver, Canada) stem cell culture medium and 10 μM Y-27632 (STEMCELL technologies) on a culture plate coated with Matrigel (Becton Dickinson, New Jersey, USA). ) Was added to culture the induced pluripotent stem cells.

Experiment result

Effect of increasing urine stem cell separation efficiency by skim milk

In order to evaluate whether the addition of skim milk to the urine sample changes the separation efficiency of urine stem cells, the skim milk was mixed with the urine sample, followed by separation (FIG. 1). After separating the urine stem cells, they were cultured in a cell culture incubator for a total of 3 days, and then a new culture medium was added on the 4th day. After that, each half was replaced with a new medium every two days, and finally the number of colonies of urine stem cells attached to the plate was counted after 5 days.

Skim milk was added to the urine at final concentrations of 0% (control), 1%, 2.5%, 3.5%, 4%, 5%, 10%, and 20%, respectively. Urine stem cells were observed in all cell culture plates, and all plates to which skim milk was added showed superior stem cell separation efficiency compared to the control. In particular, it was confirmed that the number of colonies was significantly increased from 40% to 60% compared to the control in the sample added with skim milk of 2.5% to 5% (FIG. 2).

Effect of increasing urine stem cell proliferation rate by skim milk

Urine stem cells were cultured in a 96-well plate supplemented with skim milk and RE (renal epithelial) medium (PromoCell, Heidelberg, Germany), and the concentration of skim milk of 15%, 10%, 7.5%, 5%, 3.75% 2.5% And, the concentration lower than 2.5% was tested by lowering the concentration to 0.02% while sequentially diluting 2.5% skim milk to half the concentration (Fig. 3). As a result, the proliferation rate of urine stem cells was significantly increased in the culture medium to which 1.25% of skim milk was added, a higher proliferation rate was shown at 2.5%, and the proliferation rate was maximized in the group to which 3.75% of skim milk was added. On the other hand, even in the group containing skim milk having a high concentration of 5% or more, the proliferation rate improved significantly compared to the control group, but the skim milk did not completely dissolve and partially precipitated in the plate (FIG. 4).

Urine stem cell-derived induced pluripotent stem cell production

Urine stem cells isolated from skim milk were used to produce induced pluripotent stem cells (Fig. 5). In order to produce induced pluripotent stem cells, urine stem cells were first seeded on a cell culture plate and then cultured for two days until the cells were filled to 70-80%. Then, Sendai virus (SeV) containing induced pluripotent stem cell reprogramming factor was inoculated. After 24 hours, SeV was removed through a new culture medium, and two days later, it was replaced with mTeSR1 medium, an induced pluripotent stem cell medium. After that, it was confirmed whether induced pluripotent stem cell colonies were formed by replacing the mTeSR1 medium by half every day.

As a result, it was confirmed that the urinary stem cell-derived induced pluripotent stem cell colonies were formed after the 12th day (left and center of Fig. 6), and undifferentiated induction through alkaline phosphatase (AP) staining of the produced induced pluripotent stem cells. It was confirmed that they are pluripotent stem cells (Fig. 6, right).

conclusion

In general, in order to produce induced pluripotent stem cells (iPSCs), blood or skin is collected from a cell donor to separate peripheral blood mononuclear cells (PBMC) or fibroblasts, Using this as a starting cell, induced pluripotent stem cells are produced through a reprogramming process.At this time, cells are collected even if the donor is suffering from a skin or blood-borne disease, or other blood collection is not possible, or even a healthy donor. And there are concerns about various infections that may occur with a low probability during the blood collection process. On the other hand, when cells are collected from urine, complete in vitro collection without direct contact of medical equipment to the donor body is possible, and even if not an experienced expert, cells from the donor can be collected in a short time and in a quick and simple manner. Would be said to be very high. In addition, the isolated urine stem cells can be used not only as adult stem cells, but also as raw materials for obtaining induced pluripotent stem cells, and these induced pluripotent stem cells can ultimately differentiate into immune cells or organoids. It will be said to have regenerative medicine, cell therapy, and research value.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (13)

A method of obtaining stem cells from urine comprising the following steps:
(a) adding skim milk to urine separated from the human body; And
(b) separating stem cells from the urine.
delete delete delete The method of claim 1, wherein the step (b) is performed by centrifugation.
The method of claim 1, wherein the skim milk is added at a final concentration of 2-7% (v/v) to the urine.
A method for proliferating urine-derived stem cells, comprising culturing urine-derived stem cells in a culture medium to which skim milk is added.
The method of claim 7, wherein the skim milk is added at a final concentration of 1-8% (v/v) to the culture medium.
A composition for separating stem cells from urine containing skim milk as an active ingredient.
delete delete delete A composition for proliferating urine-derived stem cells comprising skim milk as an active ingredient.

Images