JP2007228873A - Method for measuring differentiation of mesenchymal stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and nondestructively measuring the degree of differentiation in the culture and in vitro differentiation induction of mesenchymal stem cells and solving the problem of the contamination in the preparation of a specimen. <P>SOLUTION: The degree of differentiation is nondestructively measured by culturing a mesenchymal stem cell, carrying out differentiation induction in vitro and measuring the quantity of adiponectin released in the medium. As an alternative, the degree of differentiation to a fat in the culture of mesenchymal stem cell on a three-dimensional culture base is nondestructively measured by measuring the quantity of adiponectin released in the medium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

It is expected that mesenchymal stem cells that exist in adult tissues and have multipotency capable of regenerating various tissues are used for tissue regeneration in the field of regenerative medicine.
The present invention provides a novel method capable of measuring the differentiation of such cells nondestructively and easily.

Regarding the method for culturing mesenchymal stem cells, mesenchymal stem cells and cultured tissues, for example, JP 2005-34030 (Patent Document 1) or JP 2005-117939 (Patent Document 2) by the present applicant, As a method for selectively proliferating mesenchymal stem cells from a group of adipose-derived cells, JP-A No. 2004-129549 (Patent Document 3) and the like have been proposed.

JP 2005-34030 A JP 2005-117939 A JP 2004-129549 A

In order to use this in clinical practice, it is necessary to collect tissue from a living body, isolate mesenchymal stem cells from the tissue, and then culture in vitro for a certain period to increase the number of cells.
Since the mesenchymal stem cells have pluripotency, it is necessary to induce differentiation in vitro in order to construct the target tissue. It is necessary to confirm.
In addition, in order to regenerate a tissue by transplanting it into a living body, it is necessary to perform three-dimensional culture in vitro after seeding cells on a three-dimensional substrate.

Conventionally, if a portion of the cultured tissue is not destroyed and inspected, such as performing tissue staining using a part of the reconstructed tissue and performing immunostaining, the target tissue is constructed. I couldn't judge.
In addition, when collecting a sample for preparing a tissue section, there is a possibility that the cultured tissue may be contaminated, and it may be necessary to waste a valuable sample. Met.

The present invention provides a novel method for non-destructively measuring the differentiation of cells that have been subjected to three-dimensional culture in this respect, and provides any tissue-specific factor that cells of the target tissue release extracellularly. If it can be detected outside the cell, it will be possible to measure the degree of differentiation without destroying the cultured sample, and the risk of contamination during culture will be reduced. Specifically, it is characterized by the following method.
Item 1. To measure the amount of adiponectin released into the medium after in vitro differentiation induction by culturing mesenchymal stem cells.
Item 2. To measure the amount of adiponectin released into the culture medium when the mesenchymal stem cells are cultured on a three-dimensional culture substrate.
Item 3. To automatically measure the amount of adiponectin in the medium over time.

The present invention detects a substance specifically produced by a target tissue cell, for example, a protein or polysaccharide, as an index, and if it is possible to evaluate the degree of differentiation by capturing the quantitative change thereof, Compared to a destructive method in which a tissue is fixed and a section is prepared and a target substance is detected from the tissue section, such as a staining method, a conventional defect can be eliminated, and a change in culture environment such as a medium Differentiating serum concentration and culture substrate may affect the differentiation from mesenchymal stem cells, but if it becomes possible to detect such changes in the culture environment, it will be a technique for in vitro evaluation. In the hypothesis that the adiponectin will specifically produce a protein called adiponectin and release it outside the cell, we will quantify the amount of adiponectin released from the cell into the medium. Ri, has been made to obtain a demonstration that it is possible to verify the differentiation into adipose mesenchymal stem cells.

That is, in the present invention, production of adiponectin was not observed when cultured in a growth medium, but it was confirmed that the amount of adiponectin increased over time by culturing in a fat differentiation medium. As a result of observing the cells under a load by culturing at a low serum concentration in the medium, the amount of adiponectin may correlate with the amount of lipid droplets even if the culture environment changes. As a result, the amount of adiponectin indicates the amount of adipocytes, which is a sufficient index for evaluating the degree of fat differentiation.

As a method for inducing differentiation into mesenchymal stem cells and in vitro differentiation into fat, in addition to using a commercially available differentiation medium, as a supplement to a growth medium (1 μM dexamethasone, 0.5 mM 3-isobutyl-1- methylxanthine, 200 μM indomethacin, 10 μg / mL insulin) can be exemplified, and the amount of adiponectin released into the medium after differentiation induction is measured.
On the other hand, as a method for three-dimensional culture of mesenchymal stem cells, cells are seeded on a substrate such as collagen sponge, P (LA / CL) sponge, chitin nonwoven, alginate nonwoven, PGA nonwoven, etc., and then three-dimensionally cultured in vitro. In the initial stage of culture, the cells are cultured in a cell growth medium, and after a certain period, the cells are cultured in a fat differentiation medium. In this process, the medium is periodically collected and the amount of adiponectin contained in the medium is measured.
Adiponectin released into the medium can be quantified, for example, by ELISA (Enzyme-linked immunosorbent assay: “enzyme immunoassay”) using an antigen-antibody reaction.
An example of a method for automatically measuring the amount of adiponectin over time is an automated apparatus using an enzyme immunoassay method.

According to the present invention, the degree of differentiation induction of mesenchymal stem cells can be measured without destruction or contamination, and a difference in the production amount of adiponectin is observed depending on the culture environment. The effect on cells can also be evaluated.
Furthermore, changes in the culture environment, such as the concentration of serum in the medium and the difference in the culture substrate, also affect the differentiation from mesenchymal stem cells. This makes it possible to evaluate the culture environment in vitro.

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

Human mesenchymal stem cells (hMSC) are dispersed in a growth medium (α-MEM + 15% FCS) and seeded on a collagen sponge (12 mmφ) (initial seeding density: 5.25 × 10 ⁵ cells / medium 1 mL) 37 ° C., 5% The cells were cultured for 6 hours in a carbon dioxide incubator. Thereafter, the medium was replaced with an adipose differentiation medium (FCS 15%; 2.0 mL / well) and cultured at 37 ° C. in a 5% carbon dioxide incubator (6 to 20 days; medium exchange once every few days).
Quantification of adiponectin in the medium collected at the time of replacement was performed using AssayMax Human Adiponectin (Acrp30) ELISA Kit (Assaypro). In addition, the value which measured the fresh culture medium was used as control. In order to quantify the amount of lipid in the cells, the cultured cell / collagen sponge was washed with PBS (twice) and fixed with 10% neutral formalin (4 ° C., 1 hr). Thereafter, it was washed with a 60% aqueous isopropanol solution and stained with oil red O (15 min). After washing with water (three times), the red pigment was extracted with isopropanol (15 min), and the absorbance (520 nm) was measured. The results are shown in FIGS.
According to this example, as shown in FIG. 1, it was found that when cultured in a differentiation medium, the amount of adiponectin in the medium increased, but when cultured in a growth medium, adiponectin was not produced.
In addition, as shown in FIG. 2, when the amount of lipid in the cells was measured by the amount of oil red O, the amount of lipid increased in the cells cultured in the differentiation medium. It was found to be related to the amount of adiponectin.

Human mesenchymal stem cells (hMSC) are dispersed in a growth medium (α-MEM + 15% FCS) and seeded on a collagen sponge (12 mmφ) (initial seeding density: 5.25 × 10 ⁵ cells / medium 1 mL) 37 ° C., 5% The cells were cultured for 6 hours in a carbon dioxide incubator. Thereafter, the medium was replaced with an adipose differentiation medium (2.0 mL / well) and cultured at 37 ° C. in a 5% carbon dioxide incubator (6 to 20 days; medium exchange once every two or three days). Culturing was performed with the amount of FCS (bovine serum) added to the adipose differentiation medium being 1,5,10,15%.
Quantification of adiponectin in the medium collected at the time of replacement was performed using AssayMax Human Adiponectin (Acrp30) ELISA Kit (Assaypro). In addition, the value which measured the fresh culture medium was used as control. In order to quantify the amount of lipid in the cells, the cultured cell / collagen sponge was washed with PBS (twice) and fixed with 10% neutral formalin (4 ° C., 1 hr). Thereafter, it was washed with a 60% aqueous isopropanol solution and stained with oil red O (15 min). After washing with water (three times), the red pigment was extracted with isopropanol (15 min), and the absorbance (520 nm) was measured. The results are shown in FIGS.
According to FIG. 3, the amount of adiponectin increased as the amount of FCS increased.
According to FIG. 4, the amount of neutral fat contained in the cells also increases as the amount of FCS increases.
FIG. 5 shows the correlation between the amount of adiponectin in the medium and the amount of lipid in the cells. There was a correlation between the two, and it was possible to determine the proportion of mesenchymal stem cells differentiated into fat by measuring the amount of adiponectin released into the medium.

When culturing mesenchymal stem cells and inducing differentiation in vitro, the degree of differentiation can be measured easily and non-destructively. It can be used suitably.
To evaluate non-destructively whether mesenchymal stem cells are differentiated into cells such as cartilage, bone, and heart muscle in addition to fat by selecting the type of cytokine that cells differentiated other than adiponectin release into the medium Is possible.

The figure showing the time-dependent change of the adiponectin production amount in the culture medium from the fat-cultured hMSC. The figure showing the time-dependent change of the amount of triglycerides (the amount of oil red staining) in hMSCs subjected to adipogenic culture. The figure showing the result of having performed the time-dependent change of the adiponectin production amount in the culture medium from the fat differentiation culture hMSC, changing the amount of FCS added to the fat differentiation medium. The figure showing the result of having performed the time-dependent change of the amount of triglycerides (the amount of oil red staining) in hMSCs subjected to adipose differentiation culture by changing the amount of FCS added to the adipose differentiation medium. HMSCs subjected to adipose differentiation culture; a graph showing a comparison between the amount of intracellular neutral fat and the amount of adiponectin produced in the medium.

Claims (3)

  A method of non-destructively measuring the degree of differentiation by measuring the amount of adiponectin released into the medium after culturing mesenchymal stem cells and inducing differentiation in vitro. A method of non-destructively measuring the degree of differentiation into fat by measuring the amount of adiponectin released into the medium when mesenchymal stem cells are cultured on a three-dimensional culture substrate.   The method according to claim 1 or 2, wherein the amount of adiponectin in the medium is automatically measured over time.