Method for culturing parathyroid cells and cultured parathyroid cells

Abstract

To overcome the impossibility of culturing parathyroid cells derived from humans as well as animals.SOLUTION: Parathyroid cells can be cultured using a serum-free medium supplemented with B-27(R) Supplement and polyvinylpyrrolidone (PVP) as substitutes for serum. It is considered that no use of serum minimizes serum-derived effects (side effects), while the addition of PVP promotes construction of the extracellular matrix and molecular reception and alleviates stress on the cells.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to a method for culturing parathyroid cells and the parathyroid cells cultured thereby. [Background technology]

[0002] Parathyroid dysfunction is a designated intractable disease by the Ministry of Health, Labor and Welfare. When the action of parathyroid hormone is impaired, blood calcium levels fall, causing muscle spasms in the limbs and numbness in the hands, feet and mouth. In severe cases, this can lead to serious illnesses such as epilepsy-like seizures and impaired cardiac function. It is also a chronic disease that can lead to a decline in quality of life, including increased risk of dental development and cataracts throughout life.

[0003] On the other hand, regarding the treatment of the parathyroid gland, there is no fundamental therapy because there are no cells (parathyroid cells) available for use in drug discovery research.

[0004] Patent Document 1 discloses a method for obtaining parathyroid cells by culturing iPS cells and differentiating them into parathyroid cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-69345 Summary of the Invention [Problem to be solved by the invention]

[0006] It has not been possible to directly culture and grow parathyroid cells, whether in humans or animals. [Means for solving the problem]

[0007] The present invention was conceived in light of the above-mentioned title change, and relates to a hitherto unseen optimal method for culturing parathyroid cells and the parathyroid cells obtained by said method. This technology is not intended to be used directly in medical treatment, but is expected to be used in the life science industry, such as in drug discovery and experimental research.

[0008] More specifically, the parathyroid cell culture medium of the present invention is characterized by using a basal medium prepared by adding 50x B-27 Supplement manufactured by Life Technologies to KBM Neural Stem Cell Serum-Free Medium manufactured by Kohjin Bio, to which PVP 360kDa was added to make the concentration 11.34mg / ml (FVO 54%). [Effects of the Invention]

[0009] The medium of the present invention suppresses the side effects of serum by adding a serum-free culture supplement to the serum-free medium, and promotes the construction of extracellular matrix and molecular reception by adding polyvinylpyrrolidone (PVP), and also relieves stress. This is thought to be why the culture and proliferation of parathyroid cells is possible. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a photograph showing parathyroid tissue in a mouse body. [Figure 2] This figure compares the expression state of each gene with that of thyroid cells. [Figure 3] Serum calcium levels in parathyroidectomized mice were compared with serum calcium levels in intact mice. [Figure 4] The photographs are taken after culturing in each medium for two weeks. [Figure 5] The expression state of parathyroid cell-specific marker genes in each culture medium was examined. [Figure 6] This is a photograph of the culture results. [Figure 7]1 is a graph showing the expression state of parathyroid cell-specific marker genes depending on the culture medium. [Figure 8] 1 is a graph showing the results of a CCK8 assay. [Figure 9] This is a staining image showing the expression of CaSR and Pth. DETAILED DESCRIPTION OF THE INVENTION

[0011] The method for culturing parathyroid cells according to the present invention will be described below with reference to the drawings and examples. The following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included within the technical scope of the present invention. Furthermore, all documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," this means "greater than or equal to A and less than or equal to B."

[0012] The method for culturing parathyroid cells of the present invention involves culturing parathyroid cells in a serum-free medium. This is because parathyroid cells have not been easily cultured in conventional serum-containing media. Instead of the previously reported low-calcium, serum-containing medium, the addition of supplements and PVP to a low-calcium or calcium-containing serum-free medium has made it possible to culture parathyroid cells.

[0013] The parathyroid cells to be cultured are not particularly limited, and may be cells obtained directly from a living body, cells transdifferentiated from other cells, or cells differentiated and produced from pluripotent stem cells such as iPS cells or ES cells.

[0014] As shown in the following examples, the preferred medium is KBM Neural Stem Cell Serum-Free Medium (Kohjin Bio Co., Ltd.) supplemented with B-27 Supplement and polyvinylpyrrolidone (PVP) (Life Technologies, Inc.). B-27 should be added at approximately 1% to 5% (volume ratio) of the medium, and PVP should be added at least 40% or more, with a maximum FVO (Fractional Volume Occupancy) of 54%. The Ca concentration in the medium should be adjusted to 0.5 mM to 1.2 mM.

[0015] In addition to KBM, serum-free media such as DMEM / F12 (Dulbecco's Modified Eagle Medium F12) can also be used. Specifically, 0.5 mM DMEM / F12 is supplemented with 2% B-27 (volume ratio) and 54% PVP (volume ratio of FVO).

[0016] The culture method is the same as the usual culture method, in which the collected cells are dispersed, washed, and seeded in a medium using contact culture. The environment can be 37°C and 5% CO2. In addition to monolayer culture, 3D culture can also be used. [Example]

[0017] Figure 1 is a photograph showing parathyroid tissue in a mouse. This parathyroid tissue was collected. The mouse parathyroid tissue is shown in Figure 1(b). Parathyroid tissue is present in parts "a" and "b". Figure 1(a) is an enlarged view of part "a" in Figure 1(b), and Figure 1(c) is an enlarged view of part "b" in Figure 1(b).

[0018] The collected tissues strongly expressed the genes Gcm2, Casr, and Pth, which are specifically expressed in the parathyroid gland. Figure 2 compares the expression status of each gene with that of thyroid cells. Figure 2(a) shows the gene Gcm2, Figure 2(b) shows the gene Casr, and Figure 2(c) shows the gene Pth. The horizontal axis represents the type of cell (thyroid or parathyroid), and the vertical axis shows the relative expression level, with the thyroid gland set at 1. It can be seen that all genes are specifically expressed in parathyroid cells.

[0019] Figure 3 compares serum calcium concentrations in mice with and without parathyroidectomy. Referring to Figure 3, the horizontal axis represents mice with and without parathyroidectomy (labeled "untreated") and mice with and without parathyroidectomy (labeled "parathyroidectomized"). The vertical axis represents serum calcium levels (mg / dL). Since serum calcium levels in mice with parathyroidectomy were lower than in mice without parathyroidectomy, it was determined that the parathyroidectomy was successful.

[0020] Next, we prepared low-Ca culture medium and normal-Ca culture medium. Low-Ca culture medium contains 10% FBS and 0.5 mM CaCl2. Normal-Ca culture medium contains 1.2 mM CaCl2. We prepared both with and without FBS.

[0021] The collected cells were dispersed in 0.1% Collagenase Type II solution at 37°C for 30 minutes with stirring, debris was removed using a cell strainer (35 μm), and adherent culture was performed. Figure 4 shows photographs of cells cultured in each medium for two weeks. In serum-containing culture medium (Figures 4(a) and (c)), fibroblast-like cells appeared regardless of calcium concentration. On the other hand, epithelial colonies were observed in serum-free culture medium (Figure 4(b)).

[0022] Figure 5 shows the expression state of parathyroid cell-specific marker genes in each culture medium. Figure 5(a) shows the gene Gcm2, Figure 5(b) shows the gene Casr, and Figure 5(c) shows the gene Pth. The horizontal axis shows the type of culture medium. "Low Ca" is low-Ca culture medium, "Normal Ca" is normal-Ca culture medium without FBS, and "Normal Ca+FBS" is normal-Ca culture medium containing FBS.

[0023] The vertical axis shows the relative expression level when the gene expression level in the low-Ca culture medium is set to 1. Figure 5 shows that the expression of parathyroid cell-specific marker genes was maintained in the FBS-free culture medium (corresponding to Figure 4(b)).

[0024] Next, parathyroid cells were cultured in a serum-free culture medium prepared by adding 50X B-27® Supplement (Life Technologies) to KBM® Neural Stem Cell Serum-Free Medium (Kojin Bio Co., Ltd.) as a substitute for serum. Note that "50X" refers to a 2% volume of the medium used, equivalent to adding 2 ml to 100 ml of medium.

[0025] Photographs of the results are shown in Figure 6. Figure 6(a) shows a photograph of the culture in B-27® Supplement, and Figure 6(b) shows a photograph of the culture in B-27® Supplement with PVP 360 kDa added to achieve an FVO of 54%. Figure 6(c) is an enlarged photograph of Figure 6(b). Hereinafter, B-27 Supplement will also be referred to as "Supplement A," and PVP will also be referred to as "Additive X."

[0026] Figure 7 is a graph showing the expression of parathyroid cell-specific marker genes in different culture media. Figure 7(a) shows the Gcm2 gene, Figure 7(b) shows the Casr gene, and Figure 7(c) shows the Pth gene. In each graph, the horizontal axis represents the type of culture media. "Low Ca" represents low-Ca culture media, "Supplement A" represents serum-free media supplemented with B-27® Supplement, "Supplement A + Additive X" represents serum-free media containing B-27® Supplement and PVP, and "Parathyroid Tissue" represents parathyroid tissue itself. The vertical axis represents the relative expression level, with the gene expression level in low-Ca culture media set at 1.

[0027] Referring to Figures 6 and 7, when cells were cultured with supplement A, expression of parathyroid cell-specific marker genes was observed (see "Supplement A" in Figure 7). However, little cell proliferation was observed (see Figure 6(a)). However, by adding additive X, it was possible to promote cell proliferation while maintaining the properties (see Figures 6(b) and (c), and Figure 7 "Supplement A + Additive X").

[0028] Figure 8 shows the results of the CCK8 (Cell COUNTING Kit-8 (registered trademark)) assay. The horizontal axis represents the type of culture medium. "Low Ca" represents a low-Ca culture medium, "Supplement A" represents a serum-free medium supplemented with B-27 (registered trademark) Supplement, and "Supplement A + Additive X" represents a serum-free medium containing B-27 (registered trademark) Supplement and PVP. The vertical axis represents the relative proliferation rate, with the cell proliferation rate in the low-Ca culture medium set at 1. The CCK8 assay also indicates that the serum-free medium containing Supplement A and Additive X has the ability to promote parathyroid cell proliferation.

[0029] Figure 9 shows staining images of CaSR and Pth expression. The dark areas represent DAPI. This staining image also confirmed that the proliferated cells expressed CaSR and Pth.

[0030] Primary parathyroid cells derived from normal tissue lose their proliferation ability and PTH secretion function, and a complete in vitro culture method has not been established. Conventionally, calcium concentration has been considered important for the culture of parathyroid tissue. In the present invention, by adding supplement A and additive X to a culture medium with a typical calcium concentration, it is possible to maintain cells expressing PTH and other hormones even after two weeks of culture, demonstrating their effectiveness as a primary culture cell model.

[0031] This invention not only establishes a model for physiological research into the parathyroid gland, but also leads to the development of a treatment aimed at regenerating parathyroid tissue. [Industrial Applicability]

[0032] The present invention makes it possible to culture parathyroid cells, which have been almost impossible to culture in the past, and can therefore be suitably used for mass production of parathyroid cells.

Claims

1. A method for culturing parathyroid cells using a medium in which B-27 Supplement and polyvinylpyrrolidone are added to KBM Neural Stem Cell serum-free medium and the Ca concentration is adjusted to 0.5 mM or more and 1.2 mM or less.

2. The serum-free medium is KBM Neural stem cell serum-free medium, 2. The method for culturing parathyroid cells according to claim 1, wherein the B-27 supplement is present at 50x the serum-free medium, and the polyvinylpyrrolidone is present at 54% FVO relative to the serum-free medium.

3. Parathyroid cells cultured by the culture method of claim 1 or 2.

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