Hypothalamic stem cell culture medium and preparation method thereof

Abstract

According to the hypothalamic stem cell culture medium and the preparation method thereof provided by the invention, the human platelet lysate is introduced into the basal culture medium for extracting and culturing the hypothalamic stem cells, and the human platelet lysate is long in activity maintenance time and has a remarkable effect on proliferation of the hypothalamic stem cells; the human-derived platelet lysate reduces the possibility of influence of exogenous protein, is rich in a large amount of growth factors, is simple in extraction process and small in loss, and can play a good role in primary extraction culture of cells as a culture medium additive.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a hypothalamic stem cell culture medium and a preparation method thereof. Background Art

[0002] There is a type of stem cell in the hypothalamus that controls neurogenesis, called hypothalamic neural stem cell (htNSC). In addition to its role in adult neurogenesis, it can also sense the body's metabolic state and respond, manifested as changing the differentiation direction and exosome secretion function, inhibiting the activation of inflammatory pathways, etc., thereby regulating the body's energy metabolism balance. There is great potential for the clinical application of hypothalamic neural stem cells in the treatment of various diseases. At present, there is a great demand for the number of hypothalamic neural stem cells in preclinical studies and clinical trials. Therefore, how to extract and expand hypothalamic neural stem cells more safely and qualified in vitro has become particularly crucial.

[0003] Existing conventional primary cell extraction culture media require a high proportion of fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). Fetal bovine serum is a commonly used natural medium additive in cell culture, providing various nutrients required for growth (such as proteins, hormones, growth factors, etc.). However, since it is derived from animals, there is a risk of carrying infectious sources (including viruses and toxic substances), and any infectious source may pose a hazard to the growing cell line or product; bFGF is a polypeptide growth factor and a conventional active additive in existing primary nerve cell extraction culture media, which can specifically stimulate the proliferation and differentiation of cells (such as fibroblasts, stem cells, endothelial cells, etc.). However, bFGF has been proven to be unstable under physiological conditions, and its half-life is about 8 hours under standard mammalian cell culture conditions (37 °C, 5% CO2). At this decay rate, after culturing at 37 °C for 72 hours, the activity level of bFGF decreases significantly. This instability poses a major challenge to the use of bFGF in cell culture. Usually, it is necessary to increase the concentration of growth factors. In some cases, it is necessary to change the culture medium or supplement bFGF every day, which is not conducive to the realization of economic goals. Therefore, there is an urgent need for a safe, stable, low-loss and high-efficiency hypothalamic neural stem cell extraction culture medium.

[0004] Existing studies have shown that some factors constituting platelet lysate include platelet-derived growth factor (PDGF-AA, AB, and BB subtypes), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-b or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and bone morphogenetic proteins -2, -4, and -6 (BMP-2, -4, -6). PL can provide essential nutrients and growth factors, but due to its complex composition, there is currently no report on the effect of platelet lysate on the culture of hypothalamic stem cells. Summary of the Invention

[0005] The purpose of the present invention is to overcome the disadvantages and deficiencies of the existing technology and provide a hypothalamic stem cell culture medium and its preparation method.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a hypothalamic stem cell culture medium, including a basal medium, and human platelet lysate is added to the basal medium.

[0008] Preferably, the basal medium is Neurobasal-A medium added with 0.24% GlutaMAX, 2% B27, and 1% antibiotic. The percentages are volume percentages, and VitA is not contained in the B27.

[0009] Preferably, the addition amount of the human platelet lysate in the basal medium is 0.05%. The percentage is volume percentage.

[0010] Preferably, the antibiotic includes 100 U / mL penicillin and 100 μg / mL streptomycin.

[0011] The second aspect of the present invention provides a preparation method of the hypothalamic stem cell culture medium as described above, including the following steps:

[0012] S1. Pretreat the human platelet lysate: Add calcium chloride after thawing, fully dissolve and freeze-thaw 3 times repeatedly, centrifuge the human platelet lysate, and filter the human platelet lysate with a 0.22 μm filter.

[0013] S2. Add the human platelet lysate to the basal medium in proportion and then filter and sterilize again to obtain the hypothalamic stem cell culture medium.

[0014] Preferably, the pretreatment of human platelet lysate in step S1 is specifically as follows: after thawing, add 0.025 mol / L calcium chloride, fully dissolve and freeze-thaw 3 times repeatedly, centrifuge the human platelet lysate at 300G for 5 min to remove excess calcium chloride, fat and impurities precipitated from fibrinogen in the human platelet lysate, and filter the human platelet lysate with a 0.22 μm filter to remove bacteria and impurities.

[0015] In the present invention, the GlutaMAX is an advanced cell culture additive, a substitute for L-glutamine, with better stability, which can improve cell health, is suitable for adherent and suspension culture of mammalian cells, and does not require adaptation. Compared with L-glutamine, it can significantly reduce the accumulation of toxic ammonia, improve cell viability and growth, remain stable in a wide temperature range, and support cell growth and metabolism; B27 is an optimized serum-free additive used to support the growth of embryonic, postnatal and adult hippocampal and other central nervous system neurons; human platelet lysate (PL) contains platelet-derived growth factors (PDGF-AA, AB and BB subtypes), transforming growth factor β (TGF-β), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-b or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and bone morphogenetic proteins-2, -4 and -6 (BMP-2, -4, -6) growth factors and other factors required for cell growth, which helps to promote cell proliferation and growth. Penicillin and streptomycin are antibiotics, and adding them to the medium can prevent bacterial infection and maintain the purity of the culture. The combination of these components can provide appropriate nutrition and protection for specific types of cells and promote their growth and proliferation under culture conditions.

[0016] The beneficial effects of the present invention are as follows:

[0017] 1. In the present invention, human platelet lysate is introduced into the basal medium for hypothalamic stem cell extraction and culture. The human platelet lysate has a long active maintenance time and has a significant effect on the proliferation of hypothalamic stem cells; the human-derived platelet lysate reduces the possibility of exogenous protein influence. The human platelet lysate is rich in a large amount of growth factors, and the extraction process is simple with small loss. As a medium additive, it can play a very good role in primary cell extraction and culture.

[0018] 2. In an embodiment of the present invention, a formulation of a hypothalamic stem cell culture medium is provided. The culture medium is Neurobasal-A medium supplemented with 0.24% GlutaMAX, 2% B27, 0.05% PL, and 1% antibiotic. Experiments have shown that adding a relatively low content of human platelet lysate can have a significant effect on the proliferation of hypothalamic stem cells. Compared with the existing culture medium, the culture time of hypothalamic stem cells is greatly shortened, the culture cost is reduced, it is more convenient for industrial production, and it is conducive to the popularization of hypothalamic stem cells in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, obtaining other drawings based on these drawings still belongs to the scope of the present invention.

[0020] Figure 1 It is a schematic diagram of the immunofluorescence detection results ((a) in the figure is the culture medium described in Comparative Example 1, and (b) is the culture medium of Example 2 of the present invention);

[0021] Figure 2 It is a light microscope image under a microscope when the cells are cultured for 5 days ((a) in the figure is the culture medium described in Comparative Example 1, and (b) is the culture medium of Example 2 of the present invention). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Experimental Example 1. Obtaining Hypothalamic Neural Stem Cells

[0024] 1) Newborn mice less than 6 hours old were sacrificed, and then the hypothalamus was quickly dissected and stored temporarily in phosphate buffer solution (PBS).

[0025] 2) The hypothalamus of the mouse was taken out from PBS and placed in a 6-well plate containing 1 ml of sterile PBS. After carefully removing the outer membrane, it was washed three times with PBS.

[0026] 3) The hypothalamus after removing the outer membrane was transferred into an EP tube, and a special enzyme (TrypLE TM Express) was added. After the tissue was minced with a sterilized ophthalmic scissors, the EP tube was placed in an incubator at 37 °C for digestion for 50 minutes, and it was inverted up and down every 10 minutes for mixing. After the digestion was completed, all the tissue blocks and liquid in the EP tube were added to a 15 ml centrifuge tube for balancing, at room temperature, 1000 r, for 5 minutes, and the supernatant was removed. The obtained precipitate was hypothalamic neural stem cells.

[0027] Example 1

[0028] This example is a method for preparing a hypothalamic stem cell extraction medium of the present invention, including the following steps:

[0029] 1) Pretreat human platelet lysate: After thawing, add 0.025 mol / L calcium chloride to remove fibrinogen in human platelet lysate as much as possible. Fully dissolve and freeze-thaw 3 times repeatedly. Centrifuge the human platelet lysate at 300G for 5 minutes to remove excess calcium chloride, fat and impurities precipitated from fibrinogen in the human platelet lysate. Filter the human platelet lysate through a 0.22 μm filter to sterilize and remove impurities;

[0030] 2) Add the human platelet lysate to the basal medium in proportion, and filter and sterilize again after preparation to obtain the hypothalamic stem cell medium.

[0031] Example 2

[0032] This example provides a hypothalamic stem cell medium, which is a Neurobasal-A medium supplemented with 0.24% (v / v) GlutaMAX, 2% (v / v) B27, 0.05% (v / v) human platelet lysate, and 1% (v / v) antibiotic. Among them, the added B27 does not contain VitA, and the added antibiotic is a mixture of 100 U / mL penicillin and 100 μg / mL streptomycin in a volume ratio of 1:1.

[0033] Comparative Example 1

[0034] This comparative example provides a hypothalamic stem cell extraction medium, which is a Neurobasal-A medium supplemented with 0.24% (v / v) GlutaMAX, 2% (v / v) B27, 0.4% (v / v) epidermal growth factor (EGF), 0.4% (v / v) bFGF, and 1% (v / v) antibiotic.

[0035] Among them, the concentrations of the added EGF and bFGF are both 10 ng / μl. The added B27 does not contain VitA, and the added antibiotic is a mixture of 100 U / mL penicillin and 100 μg / mL streptomycin in a volume ratio of 1:1.

[0036] Experimental Example 2. Immunological Identification of Nerve Cells

[0037] The hypothalamic neural stem cells in Experimental Example 1 were respectively added to the hypothalamic stem cell extraction medium described in Example 2 prepared by the method of Example 1 and the hypothalamic stem cell extraction medium described in Comparative Example 1, transferred to a six-well plate, and a small amount of medium was added to make the tissue blocks just adhere to the wall. After 3 h, 1 ml of medium was added, and then observed every day, and the medium was changed every 2 days.

[0038] Take the cells cultured for 7 days, fix them with 4% paraformaldehyde at room temperature for 30 min, and use bovine serum albumin (BSA) combined with immunostaining permeabilization solution (Triton X-100, 0.1%) to permeabilize and block for 30 min. Add glial fibrillary acidic protein (GFAP) / nestin (Nestin) / SOX2 primary antibody and incubate overnight at 4 °C. The next day, place the specimen at 37 °C for 1 h for rewarming, wash 3 times with phosphate buffered saline with Tween (PBST), incubate the secondary antibody in the dark for 1 h, wash 3 times with PBST, incubate DAPI in the dark for 5 min, observe and take pictures under a fluorescence microscope. After immunofluorescence staining identification of specific indicators GFAP / Nestin / SOX2, it is shown that the hypothalamic neuron cells extracted by the hypothalamic stem cell extraction medium of the present invention are indeed hypothalamic neuron cells, proving that the medium of the present invention can replace the existing medium for culturing hypothalamic neural stem cells (as Figure 1 shown).

[0039] Experimental Example 3

[0040] Take the cells cultured for 5 days in Experimental Example 2 and observe them under a microscope. The results show that the cell aggregation in the hypothalamic stem cell extraction medium described in Comparative Example 1 is less severe and there is no obvious morphological change, while obvious slender synapses can be seen in the cells in the hypothalamic stem cell extraction medium of the present invention, and the cell density is more dense than that in Comparative Example 1. Thus, it is proved that using the hypothalamic stem cell extraction medium of the present invention to culture hypothalamic stem cells can significantly shorten the culture time, reduce the culture cost, and is beneficial to the popularization of hypothalamic stem cells in clinical applications (as Figure 2 shown).

[0041] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. A hypothalamic stem cell culture medium, characterized in that: It includes a basal medium, and human platelet lysate is added to the basal medium.

2. The hypothalamic stem cell culture medium according to claim 1, wherein: The basal medium is Neurobasal-A medium supplemented with 0.24% GlutaMAX, 2% B27, and 1% antibiotic. The percentages are volume percentages, and VitA is not contained in the B27.

3. The hypothalamic stem cell culture medium according to claim 2, wherein: The addition amount of the human platelet lysate in the basal medium is 0.05%. The percentage is volume percentage.

4. The hypothalamic stem cell culture medium according to claim 2, characterized in that: The antibiotic includes 100 U / mL penicillin and 100 μg / mL streptomycin.

5. The preparation method of the hypothalamic stem cell culture medium according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Pretreatment of human platelet lysate: After thawing, add calcium chloride, fully dissolve and freeze-thaw 3 times repeatedly, centrifuge the human platelet lysate, and filter the human platelet lysate with a 0.22 μm filter; S2. After adding the human platelet lysate to the basal medium in proportion, filter and sterilize again to obtain the hypothalamic stem cell medium.

6. The method for preparing a hypothalamic stem cell culture medium according to claim 5, characterized in that, The specific pretreatment of the human platelet lysate in step S1 is: After thawing, add 0.025 mol / L calcium chloride, fully dissolve and freeze-thaw 3 times repeatedly, centrifuge the human platelet lysate at a speed of 300G for 5 minutes to remove excess calcium chloride, fat and impurities precipitated from fibrinogen in the human platelet lysate, and filter the human platelet lysate with a 0.22 μm filter to sterilize and remove impurities.

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