A urine-derived mixed cell culture method

Abstract

The present invention discloses a urine-derived mixed cell culture method, comprising the following steps: centrifuging urine, collecting a precipitate, and culturing cells in the precipitate using an improved urine-derived cell culture medium of the present invention. The urine-derived mixed cell culture method of the present invention not only provides nutrients for a variety of cells but also reduces the expression of senescence and apoptosis genes within the cells, inhibiting apoptosis and senescence of urine-derived cells, thereby promoting their proliferation and growth, maintaining cell diversity, and enabling stem cells to synergize with other cell types to enhance the therapeutic efficacy of cell therapy.

Description

Technical Field

[0001] The invention relates to a urine-derived mixed cell culture method and belongs to the field of biotechnology. Background Art

[0002] In theory, autologous cells are the ideal source for cell therapy. Mesenchymal stem cells (MSCs) possess the potential for multidirectional differentiation and self-renewal, and have been shown to differentiate into neurons, muscle cells, osteoblasts, chondrocytes, and other cells. MSCs are widely used as autologous seed cells in tissue engineering and regenerative medicine, but they are primarily derived from invasive sources such as bone marrow and adipose tissue, or from sources like the umbilical cord and amniotic fluid. In 2008, Zhang et al. first isolated stem cells from human urine, naming them urine-derived stem cells. Compared to other MSCs, urinary-derived stem cells offer advantages such as non-invasive retrieval and abundant sources, making them an ideal source of autologous stem cells with promising clinical applications. However, a major challenge with MSCs is that after multiple passages in vitro, cells gradually age and undergo apoptosis, leading to impairment of stem cell characteristics and phenotype, which in turn affects clinical efficacy. Furthermore, the lack of synergistic effects of other cell types in the treatment of MSCs alone results in limited therapeutic efficacy. The traditional culture medium for urine-derived cells is a mixture of serum-free keratinocyte culture medium and embryonic fibroblast culture medium. However, the urine-derived cells cultured in this traditional culture medium are homogeneous (over 90% are mesenchymal stem cells), and the proliferation rate decreases significantly after the sixth passage due to cell apoptosis and senescence. This is why the present invention was proposed. Summary of the Invention

[0003] To solve the above problems, the present invention provides a urine-derived mixed cell culture method, which not only provides nutrients for multiple cells, but also reduces the expression of aging and apoptosis genes in cells, inhibits the apoptosis and aging of urine-derived cells, and thus promotes their proliferation and growth.

[0004] The first object of the present invention is to provide a method for culturing urine-derived mixed cells, comprising the following steps:

[0005] S1. Centrifuge the urine and take the precipitate;

[0006] S2. Culturing the cells in the precipitate using a urine-derived cell culture medium; wherein the urine-derived cell culture medium comprises a first culture medium and a second culture medium:

[0007] The first culture medium is a serum-free keratinocyte culture medium containing epidermal growth factor, bovine pituitary extract, penicillin-streptomycin and L-glutamine.

[0008] The second culture medium is DMEM medium containing F-12, fetal bovine serum, hydrocortisone, insulin, transferrin, triiodothyronine, epidermal growth factor, adenine, penicillin-streptomycin and L-glutamine.

[0009] Furthermore, in the first culture medium, the contents of the components are: 5-10 μg / L epidermal growth factor, 50-100 μg / L bovine pituitary extract, 1-3% penicillin-streptomycin, and 1-3% L-glutamine.

[0010] Furthermore, the second culture medium contains the following components: 50-75% DMEM, 25-50% F-12 culture medium, 10% fetal bovine serum, 0.4-2 mg / L hydrocortisone, 10-50 μg / L insulin, 5-25 mg / L transferrin, 2-10×10 - 9 mol / L triiodothyronine, 10-50μg / L epidermal growth factor, 1.8-9×10 -4 mol / L adenine, 1-3% penicillin-streptomycin, 1-3% L-glutamine.

[0011] Furthermore, the volume ratio of the first culture medium to the second culture medium is 1-9:9-1.

[0012] Furthermore, the urine-derived mixed cells include mesenchymal stem cells, epithelial cells, endothelial cells and fibroblasts.

[0013] A second object of the present invention is to provide a culture medium for urine-derived mixed cell culture, the culture medium comprising a first culture medium and a second culture medium:

[0014] The first culture medium is a serum-free keratinocyte culture medium containing epidermal growth factor, bovine pituitary extract, penicillin-streptomycin and L-glutamine.

[0015] The second culture medium is DMEM medium containing F-12, fetal bovine serum, hydrocortisone, insulin, transferrin, triiodothyronine, epidermal growth factor, adenine, penicillin-streptomycin and L-glutamine.

[0016] Furthermore, in the first culture medium, the contents of the components are: 5-10 μg / L epidermal growth factor, 50-100 μg / L bovine pituitary extract, 1-3% penicillin-streptomycin, and 1-3% L-glutamine.

[0017] Furthermore, the second culture medium contains the following components: 50-75% DMEM, 25-50% F-12 culture medium, 10% fetal bovine serum, 0.4-2 mg / L hydrocortisone, 10-50 μg / L insulin, 5-25 mg / L transferrin, 2-10×10- 9 mol / L triiodothyronine, 10-50μg / L epidermal growth factor, 1.8-9×10 -4 mol / L adenine, 1-3% penicillin-streptomycin, 1-3% L-glutamine.

[0018] Furthermore, the volume ratio of the first culture medium to the second culture medium is 1-9:9-1.

[0019] The third object of the present invention is to provide the use of the above-mentioned culture medium for urine-derived mixed cell culture in the preparation of cell therapy reagents.

[0020] Beneficial effects of the present invention:

[0021] The present invention maintains the characteristics and diversity of urine-derived cells by optimizing the methods and culture medium for extracting, separating and culturing a variety of mixed primary urine-derived cells, thereby significantly reducing the apoptosis and aging of urine-derived cells, laying a solid foundation for the actual effect of the clinical application of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results are analyzed by flow cytometry;

[0023] Figure 2 Results of gene expression analysis. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0025] Example 1 Isolation and culture of urine-derived mixed cells

[0026] The extraction, isolation, proliferation, and characterization of various primary mixed cells from human urine include the following steps:

[0027] (1) 100 ml of urine was collected using a sterile culture flask. The urine was centrifuged for 10 minutes at 500 RPM, the supernatant was discarded, and the cells were washed with PBS. The cells were centrifuged again for 10 minutes at 500 RPM, and the supernatant was discarded to obtain a cell pellet. The pellet was then resuspended and seeded in a six-well plate (2 ml of 0.1% gelatin was added to each well for 4 hours to improve cell attachment). The cells were then cultured in a 37°C cell culture incubator. 3 ml of culture medium was added every 3 days. The cell growth status was observed under an inverted microscope and photographed for preservation.

[0028] (2) After 10 to 14 days in the incubator, the urine-derived mixed cells proliferate and cover about 80% of the entire culture dish.

[0029] (3) Flow cytometry analysis of cell typing.

[0030] The culture medium used above is a novel urine-derived primary mixed cell culture medium prepared by mixing the first culture medium and the second culture medium in a ratio of 1:1.

[0031] The first culture medium, serum-free keratinocyte culture medium (KSFM), was supplemented with 8 μg / L epidermal growth factor, 70 μg / L bovine pituitary extract, 2% penicillin-streptomycin, and 2% L-glutamine.

[0032] The second culture medium, precursor cell culture medium, was prepared as follows: 60% DMEM, 40% Ham's F-12 culture medium, 10% fetal bovine serum, 1 mg / L hydrocortisone, 30 μg / L insulin, 15 mg / L transferrin, 5×10 -9 mol / L triiodothyronine, 20 μg / L epidermal growth factor, 3×10 -4 mol / L adenine, 2% penicillin and streptomycin, 2% L-glutamine.

[0033] like Figure 1 As shown, urine-derived cells are a heterogeneous population composed of multiple cell types. CD90 and CD105 indicate that 30-50% are mesenchymal stem cells, CD13 indicates that 35% are renal tubular epithelial cells, and an additional 1% are CD31-positive angioblasts and 6% are CD140a-positive fibroblasts.

[0034] Example 2 Senescence and apoptosis of cells in subculture

[0035] The traditional culture medium was used as the control group, and the culture medium in Example 1 was used as the experimental group. The cells were separated and cultured according to the method of Example 1, and then the cells were extracted and the expression of apoptosis and senescence genes was analyzed.

[0036] The traditional culture medium used above is a mixture of serum-free keratinocyte culture medium and embryonic fibroblast culture medium in a 1:1 ratio, wherein:

[0037] Serum-free keratinocyte culture medium: KSFM supplemented with 8 μg / L epidermal growth factor, 70 μg / L bovine pituitary extract, 2% penicillin-streptomycin, and 2% L-glutamine.

[0038] Embryonic Fibroblast Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% GlutaMAX, 1% non-essential amino acids, 1% antibiotic / antimycotic solution, 10 ng / ml basic fibroblast growth factor, 10 ng / ml platelet-derived growth factor, and 10 ng / ml epidermal growth factor.

[0039] like Figure 2 As shown, Caspase3 is a gene for cell apoptosis, and P21 is a gene for cell senescence. Genetic analysis results show that compared to a mixture of a traditional serum-free keratinocyte culture medium and an embryonic fibroblast culture medium (UM: third-generation traditional culture medium with urine-derived mixed cells), the novel mixed culture medium of the present invention (U3, U4, and U5: third-, fourth-, and fifth-generation novel culture medium with urine-derived mixed cells) can effectively reduce cell apoptosis and senescence, while maintaining cell characteristics and diversity.

[0040] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing urine-derived mixed cells and reducing cell apoptosis and delaying cell senescence, characterized in that: The following steps are involved: S1. Centrifuge the urine and take the precipitate; S2. Culturing the cells in the precipitate for 10-14 days using a urine-derived cell culture medium; wherein the urine-derived cell culture medium is obtained by mixing the first culture medium and the second culture medium in a ratio of 1:1: The first culture medium is a serum-free keratinocyte culture medium containing epidermal growth factor, bovine pituitary extract, penicillin-streptomycin, and L-glutamine. The content of each component is: 8 μg / L epidermal growth factor, 70 μg / L bovine pituitary extract, 2% penicillin-streptomycin, 2% L-glutamine, The second culture medium is DMEM medium containing F-12, fetal bovine serum, hydrocortisone, insulin, transferrin, triiodothyronine, epidermal growth factor, adenine, penicillin and streptomycin, and L-glutamine. The content of each component is: 60% DMEM, 40% F-12 culture medium, 10% fetal bovine serum, 1 mg / L hydrocortisone, 30 μg / L insulin, 15 mg / L transferrin, 5×10 -9 mol / L triiodothyronine, 20 μg / L epidermal growth factor, 3×10 -4 mol / L adenine, 2% penicillin-streptomycin, 2% L-glutamine; The urine-derived mixed cells are a mixture of mesenchymal stem cells, epithelial cells, endothelial cells and fibroblasts.

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