TWI776368B - Cell differentiation medium composition, high secretory insulin producing cell and preparation method thereof - Google Patents

Abstract

The present invention relates to a cell differentiation medium composition, a high-secretion insulin-producing cell and a preparation method thereof. The high-secretion insulin-producing cells obtained by using the cell differentiation medium composition to induce stem cell differentiation under specific conditions can secrete A large amount of insulin, and when the high-secretion insulin-producing cells are transplanted into the human body, they are not easily phagocytosed by macrophages, which can prolong the time of insulin secretion and avoid being phagocytosed by macrophages in the body when transplanted into the human body. , with excellent in vivo survival rate.

Description

Cell differentiation medium composition, high secretory insulin producing cell and preparation method thereof

The present invention relates to a cell differentiation medium, and a preparation method for differentiating stem cells by using the cell differentiation medium, in particular, to a high-secretion insulin-producing cell and a preparation method thereof.

Diabetes is an endocrine disease caused by the pancreas not producing enough insulin or the patient's body not using insulin effectively. It disrupts blood sugar regulation, making the blood sugar in the patient's body too high. Based on pathogenesis, the main types of diabetes are type 1 diabetes, type 2 diabetes, and gestational diabetes. Left untreated, high blood sugar causes problems such as cardiovascular disease, stroke, chronic kidney disease, foot ulcers, nerve damage, eye damage, and cognitive impairment.

Prevention and treatment of diabetes, especially for type 2 diabetes, involves maintaining a healthy diet, regular exercise, a normal weight, and avoiding tobacco use. Type 1 diabetes must be managed with insulin injections. Type 2 is largely preventable by maintaining a normal body weight, exercising regularly, and eating right. However, many patients may eventually require insulin injections as well. Also, gestational diabetes usually resolves after the baby is born.

In recent years, some researchers have differentiated mesenchymal stem cells into insulin-producing cells based on the ability of mesenchymal stem cells to differentiate into various cell types, and injected insulin-producing cells into animals for diabetes treatment. In addition, in order to make the characteristics of insulin-producing cells similar to those of pancreatic islet β cells, the differentiated appearance of mesenchymal stem cells is usually aggregated into spheres or clumps.

However, the current techniques for culturing insulin-producing cells in three-dimensional forms such as spheroids or clumps still have some challenges that must be overcome, including different sizes of spheroids or clumps, difficult quantification, multiple steps in the purification process, and the necessity of There are special scaffolds or materials, which will increase the manufacturing cost. In addition, the size of the cell spheroid will affect the oxygen content of the inner central cells, which may easily lead to the death of the inner central cells. These problems are urgently needed to be clarified and overcome.

Therefore, the applicant provides a cell differentiation medium composition and a novel differentiation method to solve the above-mentioned problems and further improve the insulin secretion amount of insulin-producing cells and the survival rate in vivo.

That is, the present invention can provide a cell differentiation medium composition comprising at least glucose, nicotinamide, activin-A, glucagon-like peptide-4, hepatocyte growth factor, pentagastrin, B-27 serum-free and N-2-supplemented serum-free DMEM/F12 medium, and the cell differentiation medium composition does not contain antibiotics.

According to an embodiment of the present invention, the cell differentiation medium composition comprises at least 5-25 mM glucose, 5-15 mM nicotinamide, 1-10 pM activin-A, and 5-20 nM glucagon-like Peptide-4, 80~120fM hepatocyte growth factor (HGF), 5~20nM pentagastrin, 0.1~5% B-27 serum-free supplement and 0.1~5% N-2 supplement Serum-free DMEM/F12 medium.

In addition, the present invention can also provide a method for preparing high-secretion insulin-producing cells, comprising: (a) a cell attachment step: pouring a solution containing stem cells into a culture vessel, and leaving it for at least 24 hours to allow the cells to attach. The number of the stem cells on the peripheral wall of the culture vessel is between 6,000 and 15,000 cells/cm ² , preferably between 6,000 and 12,000 cells/cm ² , more preferably between 6,000 and 10,000 cells/cm ² between 6,000 and 8,000 cells/cm ² optimally. (b) Cell differentiation step: After removing the liquid from the culture vessel, add the aforementioned cell differentiation medium composition, and culture at an ambient temperature of 35.5-39.5°C and a _CO concentration of 5%, so that the The stem cells are induced to differentiate into a high-secretion insulin-producing cell, and the high-secretion insulin-producing cell is collected after being cultured for at least 2 days or more.

According to an embodiment of the present invention, before the cell attaching step, it further comprises: culturing the stem cells with a proliferation medium for at least 3 days to expand the number of cells; the proliferation medium comprises at least fetal bovine serum, N-acetyl-L - Serum-free medium for keratinocytes with cysteine, L2 ascorbic acid and phosphate.

According to an embodiment of the present invention, in the cell differentiation step, the cell culture time is 3 to 30 days, preferably 3 to 21 days; more preferably 3 to 14 days; and most preferably, 3 to 7 days.

According to an embodiment of the present invention, the stem cell line is selected from any one of adipose stem cells, bone marrow stem cells, peripheral blood stem cells, and umbilical cord blood stem cells.

According to an embodiment of the present invention, the daily insulin secretion per 100,000 of the high-secretion insulin-producing cells is above 1,000 (mIU/L).

In addition, the present invention can also provide a high-secretion insulin-producing cell, the insulin-producing cell having a spiny appearance and having an insulin gene (insulin gene), IPF-1 gene, ISL-1 gene, and the The same surface markers of stem cells, which are CD73 (positive), CD90 (positive) and CD45 (negative).

According to an embodiment of the present invention, the expression level of CD47 mRNA of the high-secretion insulin-producing cells is more than 3 times the expression level of CD47 mRNA of the stem cells.

In order to make the purpose, technical features and advantages of the present invention more understandable to those in the relevant technical field, and to implement the present invention, the technical features and embodiments of the present invention are described in detail here in conjunction with the accompanying drawings, and are listed here. Description of the preferred embodiment progress. The drawings contrasted in the following text are schematic representations related to the features of the present invention, and do not and do not need to be completely drawn according to the actual situation.

Herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, unless otherwise clearly contradicted by context, singular terms used herein shall include the plural and plural terms shall include the singular.

Notwithstanding that the numerical ranges and parameters setting forth the broader scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains the standard deviation resulting from individual testing methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value lies within an acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. In addition to the examples, or unless expressly stated otherwise, all ranges, amounts, values and percentages used herein (eg, to describe material amounts, lengths of time, temperatures, operating conditions, quantitative ratios, and the like) are understood to be ) are modified by "covenant". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying claims are approximate numerical values and may be changed as required. At a minimum, these numerical parameters should be construed as indicating the number of significant digits and values obtained by applying ordinary rounding.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the embodiments and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The features of various specific embodiments as well as method steps and sequences for constructing and operating these specific embodiments are encompassed in the detailed description. However, other embodiments may also be utilized to achieve the same or equivalent function and sequence of steps. "Proliferation of Human Adipose Stem Cells (hADSC)"

The cell line used in this example is Human Adipose-derived Stem Cells (hADSC).

By performing liposuction from healthy donors during abdominal surgery, 2-5 g of adipose tissue is collected from the subcutaneous fat of the abdominal wall. All donors provided written consent. Human adipose tissue was placed in Ca2 ⁺ / ^Mg2+ free phosphate buffered saline (PBS) and immediately transferred to the laboratory.

Human adipose tissue was removed from the transport medium and placed in petri dishes and cut into small pieces (approximately 1-2 ^mm3 in volume) in the presence of Ca2 ⁺ / ^Mg2+ in PBS. Tissues were dissociated with 0.1% collagenase and incubated at 37°C for 60 minutes. After collagenase digestion, the resulting cells are harvested and cultured with a proliferation medium, which is a serum-free medium, typically comprising at least 10% fetal bovine serum, N-acetyl-L-cysteine, L2 ascorbic acid, and Phosphate keratinocyte serum-free media. After culturing for 2 days, the supernatant and debris were removed from the petri dish to obtain primary cultured adipose stem cells.

Furthermore, in order to proliferate the adipose stem cells, the primary cultured adipose stem cells can be continuously cultured in a proliferation medium to expand the number of adipose stem cells to a desired number. "Differentiation of Human Adipose Stem Cells (hADSC)"

First, a differentiation medium composition is prepared. The differentiation medium composition is a serum-free DMEM/F12 medium containing 5-25 mM glucose, 5-15 mM nicotinamide, 1-10 pM activin-A, 5-20 nM glucagon-like peptide- 4. 80~120fM hepatocyte growth factor (HGF), 5~20nM pentagastrin, 0.1~5% B-27 serum-free supplement and 0.1~5% N-2 supplement. In addition, the differentiation medium composition of the present invention does not contain antibiotics (eg penicillin/streptomycin solution), which can avoid affecting cell growth and changing cell surface markers.

In this example, the concentration of each component in the differentiation medium composition is shown in Table 1.

Table 1 component concentration Serum-free DMEM/F12 medium - glucose 5~25mM Nicotinamide 5~15mM Activin-A (activin-A) 1~10pM Glucagon-like peptide-4 (exendin-4) 5~20nM hepatocyte growth factor 80~120fM Pentagastrin (pentagastrin) 5~20nM B-27 serum-free supplement 0.1~5% N-2 Supplement 0.1~5%

After the proliferation was completed, the number of cells required for the experiment was counted with ADAM-MC (cell counting instrument), and hADSCs with a cell number of ^{5×10 5} and 1×10 ⁶ were inoculated in T75 culture flasks containing the aforementioned proliferation medium for 24 hours to allow Cells are attached to the walls of the flask. The hADSCs attached to the flask wall were then washed twice with 10 ml of serum-free DMEM/F12 medium (the supernatant removed was used to analyze the insulin content of the undifferentiated stem cells), and then 10 ml of differentiation medium was added to the flask. in adherent culture.

Place the culture flask inoculated with hADSCs in a 5% CO ₂ and CO ₂ incubator at 35.5~39.5°C for differentiation culture to obtain high secretory insulin-producing cells. The differentiation medium was changed every 7 days, and the replaced differentiation medium liquid was collected for insulin content analysis.

Insulin content analysis was performed based on chemiluminescence (model: ADVIA CentaurXPT, SIEMENS) method and the results are reported in Table 2.

Table 2 Control group* Example 1 Example 2 Number of cells in T75 flask (units) 0 5x10 ⁵ 1x10 ⁶ Cell density (cells/cm ² ) 0 6666.6 13333.3 Before differentiation 716.4 87 Insulin concentration (mIU/L) Day 0-7 65.8 48104.7 9034.2 Days 7-14 - 18539.6 3296.1 Days 14-21 - 11533.5 1791.2 Days 21-28 - 10696.4 1891.7 *Note: The control group is the unused differentiation medium.

From the results of the analysis of the insulin content listed in Table 2 and Fig. 1 above, it is clear that the insulin secretion of the high-secretion insulin-producing cells can reach a maximum after 7 days of culture, and the insulin obtained in Example 1 The concentration is significantly higher than the insulin concentration obtained in Example 2, indicating that the density of stem cells used is not proportional to the amount of insulin secreted by high-secretion insulin-producing cells. The higher the density of stem cells, the lower the insulin secretion.

Therefore, in the following examples, hADSCs with a cell number of 5× ^{10 5} were placed in T75 culture flasks for differentiation. "Analysis of Insulin Secretion"

The above differentiation test (Example 1 ) was repeated 3 times, and the mean value of insulin secretion per 100,000 hypersecreting insulin-producing cells every 7 days was calculated and recorded in Table 3.

table 3 control group Insulin secretion per 100,000 insulin-producing cells unit (mIU/L) (mIU/L) Day 0-7 57.63±51.15 10569.66±4686.63 Days 7-14 - 2526.02±934.01 Days 14-21 - 2290.09±631.54 Days 21-28 - 2409.42±694.69 *Note: The control group is the unused differentiation medium.

As shown in Table 3, the insulin secretion of the high-secretion insulin-producing cells reached a maximum on the 7th day of culture, and the insulin secretion per 10 ⁵ high-secretion insulin-producing cells was 10569.66 mIU/L. In other words, the average daily insulin secretion per 10 ⁵ high-secretion insulin-producing cells is about 1510 mIU/L. "The Morphology of Hypersecreting Insulin-Producing Cells"

On the 0th day and the 7th day in the culture process, the cells in the culture flask were photographed and observed by an inverted fluorescence microscope (model: OLYMPUS IX71-1LL100).

As shown in Figure 2, during differentiation, the appearance of the high secretory insulin-producing cells was spiny-like, and all the high-secretion insulin-producing cells were dispersed rather than aggregated into spheroids or clumps. "Representation of surface markers in hypersecreting insulin-producing cells"

Cells were collected on days 0 and 7 in culture, then fixed and stained with specific surface marker antibodies (negative markers: CD45; positive markers: CD73 and CD90). The surface antibody was labeled with FITC composite secondary antibody, and the expression of cell surface proteins was measured by flow cytometry (model: BD Accuri C6), and then the percentage of cells with positive expression was obtained. The analysis was repeated at least 2 times and the average numerical results were recorded in Table 4.

Table 4 Day 0 Day 7 Percentage of positive cells (normalized) CD73 99.775 99.935 CD90 99.985 99.985 CD45 0.095 0.095

As shown in Table 4, Figure 3A and Figure 3B, after 7 days of culture, the expression distribution of insulin-producing cells was shown to be 0.095% for the negative marker CD45, while the expression distribution of CD73 and CD90 (stem cell positive markers) were mainly found 99% or higher. In other words, the culture medium and differentiation method of the present invention do not alter the expression of specific surface markers of high secretory insulin producing cells, which remain the same as hADSCs (stem cells). "Gene expression of hypersecreting insulin-producing cells"

Cells were harvested on days 0 and 7 of the culture. Cellular mRNA was isolated using an mRNA isolation kit (Quick-RNA™ MiniPrep, ZYMO RESEARCH), and then the isolated mRNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScript™ RT reagent Kit, Takara). A 100 ng cDNA sample was mixed using SYBR® Premix Ex Taq™ II (Takara) and appropriate primers were selected for the insulin gene, IPF-1 gene, ISL-1 gene, and CD47 for Real time PCR.

Primers for specific genes are shown in Table 5 below.

table 5 Gene forward primer reverse primer Ipf-1 5'-TGATACTGGATTGGCGTTGTTT-3' 5'-TCCCAAGGTGGAGTGCTGTAG-3' Isl-1 5'-CAACTGGTCAATTTTTCAGAAGGA-3' 5'-TTGAGAGGACATTGATGCTACTTCAC-3' insulin 5'-GCAGCCTTTTGTGAACCAACA-3' 5'-TTCCCCGCACACTAGGTAGAGA-3' CD47 5'-GGCAATGACGAAGGAGGTTA-3' 5'-ATCCGGTGGTATGGATGAGA-3' HPRT (reference gene) 5'-TCAGGCAGTATAATCCAAAGATGGT-3' 5'-AGTCTGGCTTATATCCAACACTTCG-3'

After mRNA isolation, reverse transcription into cDNA and Real time PCR were performed, and the expression level of each gene was analyzed and recorded in Table 6.

Table 6 Day 0 Day 7 mRNA relative expression insulin 1 11.47 Ipf-1 1 5.89 Isl-1 1 6.13 CD47 1 3.64

As shown in Table 6 and Figure 4, after 7 days of culture, the expression of insulin gene, Ipf-1 gene, and Isl-1 gene in high-secretion insulin-producing cells was compared with that of human adipose-derived stem cells that had not yet started to differentiate on day 0. Significant improvement of at least 5 times.

In addition, the expression level of CD47 in high secretory insulin-producing cells was also significantly increased by at least 3 times compared with that in human adipose stem cells. Since CD47 is a signaling molecule that can prevent macrophage phagocytosis, this means that the high level of the present invention The secreted insulin-producing cells can avoid being phagocytosed by macrophages in the body when transplanted into the human body, and have excellent in vivo survival rate.

It can be seen from the above examples that the present invention provides a cell differentiation medium composition and a method for preparing high-secretion insulin-producing cells, which are cultured in an adherent manner to avoid cells from agglomerating into spheroids or clumps, which not only facilitates collection and measurement, but also It can secrete a large amount of insulin in a short period of time, which is helpful for industrial utilization; and when the high-secretion insulin-producing cells are transplanted into the human body, they are not easily phagocytosed by macrophages, which can prolong the time of insulin secretion.

To sum up, the content of the present invention has been illustrated by the above embodiments, but the present invention is not limited to these embodiments. Those with ordinary knowledge in the technical field to which the present invention pertains can make various changes and modifications without departing from the spirit and scope of the present invention; As new embodiments, these embodiments are of course regarded as one of the contents of the present invention. Therefore, the scope of protection in this case also includes the scope of the patent application and the scope defined in the following.

none

FIG. 1 is a graph showing the analysis of insulin content obtained by differentiation and culture of hADSCs with cell numbers of ⁵ ×10 5 and 10 ⁶ , respectively. Figure 2 is a graph showing the morphological changes on day 0 and day 7 during cell differentiation (magnifications of 40X and 100X). Figure 3A is a graph showing the fluorescence signal representation of cells on day 0 and day 7 during cell differentiation. Figure 3B is a schematic diagram showing normalized results of cell surface marker expression on day 0 and day 7 during cell differentiation. Figure 4 is a schematic diagram showing the comparison of mRNA expression levels of cells on day 0 and day 7 during cell differentiation.

<110> Guoxi Stem Cell Application Technology Co., Ltd.

<120> Cell differentiation medium composition, high secretory insulin-producing cell and preparation method thereof

<130> 20PV0125TW

<140> TW 110102610

<141> 2021-01-25

<150> US 63/031,596

<151> 2020-5-29

<160> 10

<170> PatentIn version 3.5

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Claims (8)

A cell differentiation medium composition, which is used to induce and differentiate stem cells into high secretory insulin-producing cells capable of secreting insulin, the cell differentiation medium composition comprising 5-25 mM glucose, 5-15 mM nicotinamide, 1~10pM activin-A, 5~20nM glucagon-like peptide-4, 80~120fM hepatocyte growth factor, 5~20nM pentagastrin, 0.1~5% B-27 none Serum-free DMEM/F12 medium with serum supplement and 0.1-5% N-2 supplement. The cell differentiation medium composition of claim 1, wherein the cell differentiation medium composition does not contain antibiotics. A method for preparing high-secretion insulin-producing cells, comprising: (a) a cell attachment step: pouring a solution containing stem cells into a culture container, and leaving it to stand for 24 hours to make the cells attached to the peripheral wall of the culture container. The number of stem cells is between 6,000 and 15,000 cells/cm ² ; (b) cell differentiation step: after removing the liquid from the culture vessel, add the cell differentiation medium composition as described in claim 1 or 2, in Cultured at an ambient temperature of 35.5-39.5°C and a CO ₂ concentration of 5% to induce differentiation of stem cells into a high-secretion insulin-producing cell, and during the period from the 0th day to the 7th day of culture, the high secretion The insulin secretion of the high-volume insulin-producing cells reaches the maximum value; wherein the appearance of the high-secretion insulin-producing cells is spiny-like, and has the insulin gene (insulin gene), IPF-1 gene, ISL-1 gene, and the The stem cells have the same surface markers. The method for preparing high secretory insulin-producing cells according to claim 3, wherein before the cell attaching step, it further comprises: Culturing the stem cells for 3 days with a proliferation medium to expand the cell number; wherein the proliferation medium is a serum-free medium for keratinocytes comprising at least fetal bovine serum, N-acetyl-L-cysteine, L2 ascorbic acid and phosphate . The method for producing high-secretion insulin-producing cells according to claim 3, wherein the stem cell line is selected from any one of adipose stem cells, bone marrow stem cells, peripheral blood stem cells, and umbilical cord blood stem cells. The method for preparing high secretory insulin-producing cells according to claim 3, wherein the surface markers are CD73 (positive), CD90 (positive) and CD45 (negative). A high-secretion insulin-producing cell, wherein the high-secretion insulin-producing cell has a spiny-like appearance, and has an insulin gene (insulin gene), an IPF-1 gene, and an ISL-1 gene, and shows a relationship with stem cells. The same surface marker; the stem cell line is selected from any one of adipose stem cells, bone marrow stem cells, peripheral blood stem cells, and umbilical cord blood stem cells, and the surface markers are CD73 (positive), CD90 (positive) and CD45 (negative). The high-secretion insulin-producing cell according to claim 7, wherein the expression level of CD47 mRNA of the high-secretion insulin-producing cell is more than 3 times the expression level of CD47 mRNA of the stem cell.

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