WO2019134498A1 - Use of protein in cell culture - Google Patents

Abstract

Disclosed in the present invention is a use of a protein in a cell culture, which is a method comprising applying sDSS1 protein to cell culture, thereby improving cell culture efficiency. By adding the sDSS1 protein to a culture solution, the level of cellular ROS can be effectively reduced, so as to reduce cell death, improve the number of cells, and maintain the differentiation ability of stem cells. sDSS1 protein has important application value in cell culture involved in scientific research, medical treatment and industrial production.

Description

Application of a protein in cell culture Technical field

The invention relates to the use of a protein in cell culture.

Background technique

Cell culture is a process of simulating the physiological environment of a somatic cell in vitro, incubating or culturing the cell to maintain its survival and growth. Cell culture is widely used in scientific research, medical medicine, and industrial production. However, cells cultured in vitro are still different from those in somatic cells. Cell culture fluids often contain higher nutrients such as sugar and protein. The oxygen partial pressure in the carbon dioxide incubator commonly used in cell culture is much higher than that in the body. The digestive enzymes used in the cell passage process cause a certain degree of damage to the cells. The factors keep the cells in a state of high oxidative stress. In addition to causing an increase in the level of oxidative stress in the cells, the culture environment is also deteriorated, causing an increase in the levels of oxidized proteins, oxidized lipids, and oxidized saccharides in the culture solution, ultimately affecting cell viability and cells. Function [1-3]. In addition, Fetal bovine serum (FBS) is often used in cell culture, and serum quality is a key factor affecting cell viability and cell proliferation activity. The serum-free cells are more sensitive to the culture environment because of the loss of serum protection, and are susceptible to toxic substances in the culture medium [4]. Therefore, improving the living environment of the cells, reducing the content of toxic components in the culture solution, and increasing the viability of the cultured cells become an effective way to improve the efficiency of cell culture.

In cell transplantation, the donor cells isolated from the tissue undergo a process of cell sorting, cell purification, cell expansion, cell injection, etc., and the number of viable cells is limited, and the cell viability is also decreased by the prolongation of the treatment process. Cell therapy often requires a large number of donor cells. For example, the amount of cells injected in a single injection during neural stem cell therapy is 10 ⁹ -10 ¹⁰ [5]. Moreover, donor cell viability affects cell survival and function, and is a decisive factor in ensuring the therapeutic effect of cells. In the process of cell therapy, the cells undergo genetic modification and cell sorting, and the vitality is affected to a certain extent. Protecting the viability of donor cells is one of the key factors determining the therapeutic effect [6]. In industrial production, the use of mammalian cells or insect cells for large-scale fermentation to produce antibody protein or peptide / protein drugs, cell number and cell viability are decisive factors affecting protein production efficiency [7]. Therefore, how to increase the number of cells and maintain cell viability in the process of cell culture is an important issue that determines the application of cells in medicine or industrialization.

Previous studies have shown that when oxidative stress occurs in cells, DSS1 (Deleted split hand/split foot 1) protein, as a small protein highly conserved in eukaryotes, can be enzymatically reacted and consumes ATP. Covalently modified to oxidized proteins, this modification will mediate the degradation of oxidized proteins in cells [8]. DSS1 gene knockout leads to cell death; cells that highly express DSS1 protein show significant resistance to oxidative stress or anti-tumor drug-induced apoptosis [9]. These results indicate that the DSS1 protein plays an important role in the process of cell clearance of oxidized proteins and is critical for cell survival.

The above citations are as follows:

1.B.Halliwell (2003) Oxidative stress in cell culture: An under-appreciated problem? FEBS Lett 540(1–3): 3–6.

2. Oze H, Hirao M, Ebina K, Shi K, Kawato Y, Kaneshiro S, Yoshikawa H, Hashimoto J (2012) Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture. In Vitro Cell Dev Biol Anim 48(2): 123-30.

3. J. J. Reiners, P. Mathieu, C. Okafor, D. A. Putt, and L. H. Lash (2000) Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells. Toxicol. Lett 115 (2): 153-163.

4.M.Baker (2016) Reproducibility: Respect your cells! Nature537 (7620): 433–435.

5. Tsukamoto A, Uchida N, Capela A, Gorba T, Huhn S (2013) Clinical translation of human neural stem cells. Stem Cell Res Ther4 (4): 102.

G,Brindley D(2017)CAR-T Cells:A Systematic Review and Mixed Methods Analysis of the Clinical Trial Landscape.Mol Ther S1525-0016(17):30556-7. 6.Pettitt D, Arshad Z, Smith J, Stanic T,

G, Brindley D (2017) CAR-T Cells: A Systematic Review and Mixed Methods Analysis of the Clinical Trial Landscape. Mol Ther S1525-0016 (17): 30556-7.

7. Omasa T, Onitsuka M, Kim WD (2010) Cell engineering and cultivation of chinese hamster ovary (CHO) cells. Curr Pharm Biotechno 11(3): 233-40.

8.Zhang Y, Chang FM, Huang J, Junco JJ, Maffi SK, Pridgen HI, Catano G, Dang H, Ding X, Yang F, Kim DJ, Slaga TJ, He R, Wei SJ (2014) DSSylation, a novel Protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells. Protein Cell 5(2): 124-40.

9.Rezano A, Kuwahara K, Yamamoto-Ibusuki M, Kitabatake M, Moolthiya P, Phimsen S, Suda T, Tone S, Yamamoto Y, Iwase H, Sakaguchi N (2013) Breast cancers with high DSS1 expression that potentially maintains BRCA2stability have poor Prognosis in the relapse-free survival.BMC Cancer 13:562.

Summary of the invention

The addition of sDSS1 protein to cell culture media reduces cytotoxicity and reduces cell death and intracellular oxidative free radical levels during cell culture, thereby increasing cell culture efficiency. The application of sDSS1 protein to cell culture will help to improve the efficiency of pharmaceutical or industrial applications such as cell transplantation, cell therapy and cell fermentation, and has important application value.

The specific technical solutions are as follows:

The use of a protein in cell culture for the application of sDSS1 protein to cell culture.

Preferably, the sDSS1 protein comprises human, chimpanzee, bonobo, gorilla, orangutan, white-cheeked gibbons, golden monkey, rhesus monkey, golden monkey, East African pheasant, Angora simian, white-tailed white-browed monkey, ghost A basic protein formed by any sDSS1 protein sequence of scorpionfish, porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2, and the amino acid sequence of porcine chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of orangutan sDSS1 is SEQ ID NO: 5, the amino acid sequence of white cheek gibbon sDSS1 is SEQ ID NO: 6, amino acid of Rhinopithecus sDSS1 The sequence is as SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, the amino acid sequence of gilt monkey sDSS1 is SEQ ID NO: 9, and the amino acid sequence of ssDSS1 is SEQ ID NO: 10, Angola The amino acid sequence of the monkey sDSS1 is SEQ ID NO: 11, the amino acid sequence of the white-headed white-breasted monkey sDSS1 is SEQ ID NO: 12, and the amino acid sequence of podophylla sDSS1 is SEQ ID NO: 13. Tailed macaque sDSS1 amino acid sequence as SEQ ID NO: 14.

Preferably, the sDSS1 protein is the first protein having a similarity to the basic protein of 70% or more as described in the above scheme.

Preferably, the sDSS1 protein is a structural feature or amino acid sequence of a polypeptide fragment for fusion, which is based on 58 amino acids of the basal protein of the basic protein described above, and fused other polypeptide fragments at the nitrogen or carbon terminus. A second protein having the same or similar characteristics as the 31 sequences of the carbon terminus of the basal protein described in the above scheme.

Preferably, the sDSS1 protein is based on any of the 58 amino acids of the basic protein of the basic protein described above, and the other amino acid fragments are fused at the nitrogen or carbon end, and the fusion protein can realize the transmembrane transport function. Three proteins.

Preferably, the sDSS1 protein is formed by linking the basic protein, the first protein, the second protein or the third protein according to any one of the above schemes to the protein itself, a carrier protein, an antibody or other amino acid fragments of any length. Fusion protein.

Preferably, the sDSS1 protein is a polypeptide/protein modification produced based on modification of the basic protein, the first protein, the second protein or the third protein.

Preferably, the modification of the polypeptide/protein modification is directed to an amino group on the amino acid side chain of the sDSS1 protein, a carbonyl group on the amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, a cysteine, a tyrosine, a serine. Or a specific or non-specific chemical modification of 1-20 sites by tryptophan.

Preferably, the method for modifying the polypeptide/protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyethylene Pyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, One or more of hydroxypropylcellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification, or other pharmaceutically acceptable polypeptide/protein drug modification methods.

Preferably, the sDSS1 protein is 1-31 arbitrary using amino acids other than the 20 essential amino acids based on the amino acid sequence of the basic protein, the first protein, the second protein or the third protein. A non-natural amino acid replacement protein that is replaced by an amino acid site.

Preferably, the amino acid substitution of the non-natural amino acid replacement protein comprises hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non-natural amino acid and derivatives thereof.

Preferably, the sDSS1 protein is pharmaceutically applicable to the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the non-natural amino acid replacement protein. Part or all of the complex formed by the drug carrier.

Preferably, the pharmaceutical carrier comprises one or more of an enteric coating formulation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin, and a gel.

Preferably, the sDSS1 protein is a fourth obtained in a culture medium by introducing a DNA or RNA sequence of the basic protein, the first protein, the second protein, and the third protein into a cell during cell culture. Protein.

Preferably, the sDSS1 protein is used in cell culture to treat the basic protein, the first protein, the second protein, the third protein, the fourth protein, the fusion protein, the polypeptide/protein modification The non-natural amino acid replacement protein or complex is used as a base medium component, a serum component, a serum replacement component, a proliferation additive component, or other component of any component that needs to be added during cell culture.

Preferably, the application is to use the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the non-natural amino acid replacement protein or the complex as a cell Cell/tissue protection fluid, cell suspension, cell/tissue maintenance fluid, cell/tissue wash solution, or components of any solution used in these processes during transplantation, cell therapy, tissue transplantation, tissue repair or organ transplantation.

Preferably, the cell culture is cell preparation, cell sorting, cell cloning culture, cell expansion culture, cell enrichment, cell purification, cell engineering, cell three-dimensional culture, cell fermentation, tissue culture in vitro using a medium. Any form of culture, organ culture.

Preferably, the cell culture is any cell derived from human, orangutan, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, mouse, fish, bird or insect. Perform cell culture.

Preferably, the cell culture is a separation of primary cells, tissue-derived stem cells, tissue-derived precursor cells, induced pluripotent stem cells, differentiation-derived cells, transdifferentiated cells, cell-derived cells, tumor stem cells, and tumor tissues. Cell culture of one or more of tumor cells, cell strains, cell lines, insect cells, cell clumps, tissue masses, and in vitro cultured organs.

Preferably, the primary cells are of cardiomyocytes, chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair follicle dermal papilla cells, hepatocytes, kidney cells, keratinocytes, melanocytes, osteoblasts, One or more of pre-adipocytes, skeletal muscle cells, smooth muscle cells, stem cells, T cells, B cells, macrophages, precursor cells, pericytes, or dendritic cells.

Preferably, the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the non-natural amino acid replacement protein or the complex are added at any time during the cell culture process. The concentration of the body is not less than 10 nM.

A commercial development or clinical application, which utilizes the application of the protein described in any of the above technical solutions in cell culture, and then commercializes or clinically applies the application of the sDSS1 protein in cell culture.

Features and/or benefits of the present invention are:

1. The present invention demonstrates that the addition of sDSS1 protein to cell culture medium can reduce LDH levels and intracellular free radical levels in cell culture medium, thereby maintaining cell proliferation and increasing cell number.

2. The present invention proves that the addition of sDSS1 protein to the cell culture medium can increase the number of cells within a certain concentration range, thereby having a positive effect on cell culture and continuous passage.

3. The present invention demonstrates that the addition of sDSS1 protein to stem cell culture media can increase the number of stem cells and maintain the stem cell level of stem cells.

In summary, the present invention provides a novel sDSS1 protein, which is verified by experiments on cell lines, primary adult cells, and primary stem cells, and the addition of sDSS1 protein to the culture medium can reduce the cytotoxicity and cell death level, thereby improving culture. The number of cells in the process. Protect cell viability and maintain the ability of stem cells to differentiate. Therefore, sDSS1 protein has important potential application value in the fields of medical medicine or industrial fermentation production such as cell transplantation, cell therapy, and cell fermentation.

DRAWINGS

The invention is further described in detail below with reference to the accompanying drawings, in which

1A-1C, the sDSS1 protein promotes proliferation of N2a cells and reduces the level of cytotoxicity.

In Fig. 1A, the number of cells was significantly increased after the addition of sDSS1 protein to the culture medium, and a significant increase in the number of cells was detected in the 100 nM, 200 nM, and high concentration groups (2-50 μM). Figure 1B shows the cytotoxicity level measured by the LDH kit. It was found that the cytotoxicity level decreased significantly after the addition of sDSS1 protein. The concentration of cytotoxicity showed a significant concentration-dependent gradient between 2-50 μM protein concentration. As the protein concentration increases, the level of cytotoxicity gradually decreases. Figure 1C, detecting intracellular ROS levels, showed that sDSS1 protein can significantly reduce cellular ROS levels. n=5 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

2A-2B, sDSS1 protein promotes PC-12 cell proliferation and reduces cytotoxic response levels.

In Fig. 2A, different concentrations of sDSS1 protein were added to PC-12 cell culture medium, and the number of cells was detected. It was found that low concentration of sDSS1 protein (100 nM, 200 nM) significantly promoted cell proliferation, while high concentration of sDSS1 protein significantly inhibited cell proliferation and decreased cells. Quantity (2-25μM). Figure 2B, detecting cytotoxicity levels, found that addition of low or high concentrations of sDSS1 protein, in addition to some intermediate concentrations, significantly reduced cytotoxicity levels. n=5 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

3A-3C, sDSS1 protein promotes HUVECs cell proliferation and reduces cytotoxicity levels.

Figure 3A, detection of HUVECs cell proliferation found that low concentrations of sDSS1 protein promoted cell proliferation, but significant enhancement effects were detected only at 100 nM and 800 nM, and the other groups were not. In Fig. 3B, the toxicity of HUVECs cells in culture medium was relatively small. After adding sDSS1 protein, the cytotoxicity was lower than that of the control group, but the difference was not obvious. In Figure 3C, ROS levels in HUVECs were significantly increased in the high-concentration protein group (2-32 μM) after addition of the sDSS1 protein, and no significant differences were detected in the low-protein concentration group. n=5 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

4A-4C, sDSS1 and its mutant sDSS1 (M4) protein promote BMSCs cell proliferation and reduce cytotoxicity levels.

In Fig. 4A, the addition of a low concentration of sDSS1 protein in the culture medium promoted the proliferation of BMSCs and increased the number of cells. The effect showed a concentration-dependent effect between 10 nM and 125 nM, and the effect increased with increasing protein concentration. The effect remained essentially unchanged between 125 nM and 750 nM, and the 1000 nM proteome effect decreased but still maintained a proliferative effect. In the high concentration group (30 μM), sDSS1 protein will inhibit cell proliferation and reduce cell number. Figure 4B. Adding a low concentration mutant sDSS1 (M4) protein to the culture medium can promote the proliferation of BMSCs and increase the number of cells. In Figure 4C-D, the addition of sDSS1 and sDSS1 (M4) proteins to the culture medium will have a protective effect on the cells, and the level of cytotoxicity will decrease. Figure 4E, the addition of low concentrations of sDSS1 protein in BMSCs cell culture medium can reduce the level of cellular ROS. This effect is significantly different between the 250nM and 750nM groups, and the other groups have a downward trend. However, high concentrations of sDSS1 protein (30 μM) significantly stimulated increased cellular ROS levels. n=6 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

5A-5C, the sDSS1 protein can maintain the dryness level of BMSCs cells.

Fig. 5A, after treatment of BMSCs cells in the culture medium supplemented with 125 nM sDSS1 protein for 4 days, no significant difference in cell morphology was observed in the treated group compared with the control cells. 5B-5C, the number of positive cells expressing the markers CD90, CD29 and the molecular marker CD45 of hematopoietic cells on the surface of BMSCs was detected. The results showed that the CD90 and CD29 of BMSCs remained at a very high level after sDSS1 protein treatment. There was no significant difference compared with the control cells; the number of CD45-positive cells was extremely low, and there was no significant change from the control cells.

6A-6D, sDSS1 protein increases the number of continuously cultured N2a cells and reduces the level of cytotoxicity.

6A-6B, cell culture was carried out after adding a low concentration of sDSS1 protein. In the first generation of continuous culture, sDSS1 protein significantly promoted the number of N2a cells, and significant differences were detected in the 8 μM and 50 μM protein groups. Correspondingly, a significant decrease in cytotoxicity levels was detected in the 8 μM and 50 μM proteomes. Figures 6C-6D, in the second generation of continuous culture, sDSS1 protein promoted the proliferation of N2a cells in a concentration-dependent manner. In the 1μM and 8μM protein groups, the proliferative effect increased with increasing protein concentration. In the high concentration group (50 μM), cell proliferation was significantly inhibited. The level of cytotoxicity was measured and 50 μM sDSS1 protein induced significant toxicity in the cells. n=4 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

Figure 7. The sDSS1 protein promotes the number of continuously cultured N2a cells. After two consecutive passages, the number of N2a cells was accumulated. The results showed that the control cells only amplified 15 times, while the 1μM and 8μM protein groups promoted the proliferation effect significantly, and the cells were expanded 26 times and 21 times, respectively. Above the control group, cells in the high-concentration sDSS1 protein group (50 μM) were also amplified 18-fold, which was also higher than the control group.

Figure 8. The number of BMSCs continuously cultured in medium containing sDSS1 protein is higher. 125nM sDSS1 protein was added to the culture medium of BMSCs cells. BMSCs cells were serially passaged 4 times in this environment. The results showed that the number of BMSCs accumulated in the sDSS1 protein group exceeded 3× ^{10 6} , which was more than double that of the control group. n=6 per group. Data were analyzed by ANOVE, *, p-value <0.05; **, p-value < 0.01.

9A-9F, sDSS1 protein can improve cell proliferation viability and reduce cytotoxicity levels in normal quality serum.

9A-9D, in high-quality serum, N2a cells grew well and the morphology of the cells was intact. After the addition of sDSS1 protein, the number of cells increased slightly. In general quality serum, N2a cells grow slowly, some cells are lysed, and cell debris appears in the culture. Adding 50 μM sDSS1 protein can effectively reduce cell lysis and increase the number of cells. Figure 9E, the number of cells was measured using the CCK-8 kit. The results showed that the addition of 50 μM sDSS1 protein increased cell proliferation by about 20% under high-quality serum culture conditions. However, in general quality serum culture, the addition of sDSS1 protein can significantly improve cell proliferation and the number of cells more than doubled. Figure 9F, detection of cytotoxicity levels can be found, N2a cytotoxicity is evident in general quality serum, and cytotoxicity is reduced to less than half of the control group after addition of sDSS1 protein. In high-quality serum, the cytotoxic response was low, and sDSS1 protein addition did not cause significant differences. n=6 per group. Data were analyzed by ANOVA, *, p-value < 0.05; **, p-value < 0.01.

Detailed ways

The preferred embodiments of the present invention are described and illustrated in the following examples, which are not intended to limit the scope of the invention. All ranges of the invention are defined by the claims.

The experimental methods used in the following examples are routine experimental methods unless otherwise specified.

The sDSS1 protein used in the following examples is the human sDSS1 protein produced by the company itself, and the protein sequence is shown in SEQ ID NO: 1. The company's quality control of protein quality, detection of protein purity greater than 95%, endotoxin (less than 3EU / mg protein) and other impurities in accordance with standards, can be used in cell experiments without causing significant cytotoxicity.

The materials and reagents in the following examples, except for the sDSS1 protein, are commercially available.

Example 1. sDSS1 protein increases the number of cells in cultured cells, reduces cytotoxicity

experimental method:

1. Cell culture Mouse-derived neuroblastoma cells (N2a) and rat adrenal pheochromocytoma cells (low-differentiation) (PC12) were purchased from the Cell Culture Bank of the Chinese Academy of Sciences' Type Culture Collection Committee (N2a cell catalog number: TCM29; PC12). Cell catalog number: TCR8). N2a cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS) Gibco, C#10091148), 100 U/mL penicillin and 100 ug/mL streptomycin (Gibco, C# 15140-122). (DMEM, ThermoFisher Scientific, C#11995065) Complete medium was passaged every two days. PC12 cells were cultured in RP1640 complete medium containing 10% (Horse Serum) (Gibco, C#16050-122), 5% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin, subcultured every two days.

2. Cell seeding and treatment N2a cells or PC12 cells were washed once with phosphate buffered saline (PBS), then trypsinized into single cells, and the cell suspension was counted by cells and inoculated into 96-well plates per 6000 cells. 200 μL of complete medium. After the culture plate was treated in a cell culture incubator (temperature 37 ° C, humidity 95%, CO ₂ concentration 5%) for 48 hours or 72 hours, the supernatant of the treated cell culture supernatant was collected, centrifuged at 100 g for 5 minutes, and the supernatant was aspirated for cytotoxicity. Level detection, treated cells are used for cell proliferation detection or detection of reactive oxygen species (ROS) levels.

3. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C#C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in 120 μL of the cytotoxicity experiment was added to each well of a 96-well plate, and then 30 μL of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.

4. Cell proliferation assay The cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (C# CK04). A working solution was prepared by diluting CCK-8 solution in a serum-free DMEM at a ratio of 1:10 (v/v). After the cells in the 96 wells were processed, the old medium was discarded, and 150 μL of CCK-8 working solution was added to each well. The culture plate was incubated in an incubator for 2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell proliferation level.

5. Detection of cellular ROS levels The detection kit for oxidative free radicals was purchased from Biyuntian Biotechnology Co., Ltd. (C#S0033). When testing, discard the old culture solution, and dilute the fluorescent probe DCFH-DA to a serum-free DMEM solution to prepare a working solution. Add 200 μL of working solution to each well, mix and put back in the cell culture incubator. The cells were for 30 minutes. After the incubation, the cells were first washed once with serum-free DMEM, and then 200 μL of PBS solution was added to each well. At the time of detection, the bottom of the 96-well plate was sealed with opaque black adhesive tape, and the fluorescence intensity was measured by a multi-function microplate reader, reflecting the ROS level of the cells, excitation light of 488 nm, and fluorescence of 525 nm.

Result analysis:

To examine the effect of sDSS1 protein on cell line cell culture, two cell lines were used as cell models, including mouse-derived neuroblastoma cells (N2a) and rat adrenal pheochromocytoma cells (poor differentiation) (PC12). Compared with conventional medium, N2a cells grew better in complete medium containing low concentrations of sDSS1 protein, which showed a significant increase in the number of cells after adding 100 nM and 200 nM sDSS1 protein in the medium; at higher concentrations, 2 μM -50 μM also showed a significantly increased number of cells (Fig. 1A). When the level of cytotoxicity was measured, the toxicity level of all cells added with the sDSS1 protein group was significantly lower than that of the control group, and there was a significant dose-dependent effect between 1 μM and 50 μM, and the cytotoxicity level increased with increasing protein concentration. Gradually decreased (Figure 1B). When the level of cellular ROS was measured, it was also found that the ROS level of sDSS1 protein added cells was significantly lower than that of the control group (Fig. 1C). On PC12 cells, it was also found that low concentrations of sDSS1 protein promoted cell proliferation, increased cell number (Fig. 2A), and significantly reduced cytotoxicity levels (Fig. 2B). These data indicate that sDSS1 protein can block the influence of toxic substances contained in culture solution on cells, reduce the level of cellular oxidative stress, and finally promote cell proliferation and increase the number of cells in cultured cell lines.

Example 2, sDSS1 protein increases the number of primary cells in culture, reduces cytotoxicity

experimental method

1. Cell culture Human primary cells of Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Promo Cell (Promo Cell, C#22011) and added with Endothelial Cell Growth Medium Supplement Mix. C#39215) cultured in an endothelial cell basal medium (Endothelial Cell Basal Medium, Promo Cell, C#22210) with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, C# 15140-122), placed Incubate in a cell culture incubator (temperature 37 ° C, humidity 95%, CO 2 concentration 5%). Cells are passaged every 3-5 days.

2. Cell seeding and treatment HUVECs cells were washed once with phosphate buffered saline (PBS), then digested into single cells by trypsinization. The cell suspension was counted by cell and inoculated into 96-well plates per 3000 cells, 200 μL of complete culture. base. After the culture plate was treated in a cell culture incubator (temperature 37 ° C, humidity 95%, CO 2 concentration 5%) for 48 hours, the supernatant of the cell culture supernatant was collected, centrifuged at 100 g for 5 minutes, and the supernatant was aspirated for cytotoxicity detection. Good cells are used for cell proliferation assays or for detection of reactive oxygen species (ROS) levels.

3. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C#C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in 120 μL of the cytotoxicity experiment was added to each well of a 96-well plate, and then 30 μL of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.

4. Cell proliferation assay The cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (C# CK04). A working solution was prepared by diluting CCK-8 solution in a serum-free DMEM at a ratio of 1:10 (v/v). After the cells in the 96 wells were processed, the old medium was discarded, and 150 μL of CCK-8 working solution was added to each well. The culture plate was incubated in an incubator for 2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell proliferation level.

5. Detection of cellular ROS levels The detection kit for oxidative free radicals was purchased from Biyuntian Biotechnology Co., Ltd. (C#S0033). When testing, discard the old culture solution, and dilute the fluorescent probe DCFH-DA to a serum-free DMEM solution to prepare a working solution. Add 200 μL of working solution to each well, mix and put back in the cell culture incubator. The cells were for 30 minutes. After the incubation, the cells were first washed once with serum-free DMEM, and then 200 μL of PBS solution was added to each well. At the time of detection, the bottom of the 96-well plate was sealed with opaque black adhesive tape, and the fluorescence intensity was measured by a multi-function microplate reader, reflecting the ROS level of the cells, excitation light of 488 nm, and fluorescence of 525 nm.

Experimental result

In order to examine the effect of sDSS1 protein on primary cells, human umbilical vein endothelial cells (HUVECs) were used for cell proliferation and cytotoxicity experiments. The results showed that after addition of 100 nM and 800 nM sDSS1 protein, the number of cells was significantly higher than that of the control group (Fig. 3A). Correspondingly, the cytotoxicity assay using the LDH kit revealed that the level of cytotoxicity decreased after the addition of sDSS1 protein, but these effects were not significant due to the reduced toxicity level of HUVECs cells (Fig. 3B). After adding low concentrations of sDSS1 protein, there was no significant difference in ROS levels in HUVECs cells, but high concentrations of sDSS1 protein significantly stimulated increased ROS levels in cells (Fig. 3C). These results indicate that sDSS1 protein can protect primary cells from toxic substances in culture medium and increase cell number, but high concentration of sDSS1 protein is toxic to HUVECs cells.

Example 3, sDSS1 protein increases the number of cultured stem cells does not affect the cell dryness level

1. Cell culture 4-week-old male SD rats (100-120 g, SPF grade, purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were selected, and the neck was sacrificed and soaked in 75% absolute ethanol for 10 minutes, followed by sterility. The femur was removed under conditions and the end of the bone was cut. The medullary cavity was repeatedly washed with 5 mL of α-MEM medium (Hyclone, C#1030265.01) containing 10% fetal bovine serum (Gibco, C#10100-147), and the cell suspension was collected and carefully transferred to 25 cm ² cells. The flask was cultured in a cell culture incubator containing 5% CO ₂ at 37 °C. After adhering to primary bone marrow mesenchymal stem cells (P0-BMSCs) for 48 hours, fresh α-MEM medium containing 10% FBS was replaced, and unattached cells were discarded. After that, the fresh medium was replaced every 2 days, and the degree of fusion of P0-BMSCs was 80-90%, and subculture was carried out. The cells were digested with 0.25% trypsin at 37 ° C, and the cells were digested into a single cell suspension in complete medium, and the cells were diluted in a 1:5 ratio and subcultured or frozen.

2. Cell seeding and treatment The stably passaged BMSCs were washed once with phosphate buffered saline (PBS), then trypsinized into single cells, and the cell suspension was counted by cell and inoculated into 96-well plates per 3000 cells. 200 μL of complete medium. After the culture plate was treated in a cell culture incubator (temperature 37 ° C, humidity 95%, CO ₂ concentration 5%) for 72 hours, the supernatant of the cell culture solution was collected, centrifuged at 100 g for 5 minutes, and the supernatant was aspirated for cytotoxicity detection. The treated cells are used for cell proliferation detection or detection of reactive oxygen species (ROS) levels.

3. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C0016). When detecting the enzyme activity, first prepare a sufficient amount of the test solution according to the kit instructions. 120 μL of the supernatant collected in the cytotoxicity experiment was added to each well of a 96-well plate, then 30 μL of the test solution was added, mixed slightly, and incubated at room temperature for 30 minutes in the dark. The absorbance at 490 nm was measured on a microplate reader, and the absorbance value was proportional to the cytotoxicity level.

4. Cell proliferation assay The cell proliferation-toxicity test kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (CK04). The working solution was prepared by diluting the CCK-8 solution at 1:10 (volume ratio, v/v) using complete medium. After the cells in the 96 wells were processed, the old medium was discarded, and 110 μL of CCK-8 working solution was added to each well. The culture plate was placed in an incubator at 37 ° C for 2 hours in the dark, and then the absorbance at 450 nm was measured on a multi-function microplate reader. The absorbance value was proportional to the number of cells.

5. Detection of cellular ROS levels The detection kit for oxidative free radicals was purchased from Biyuntian Biotechnology Co., Ltd. (C#S0033). During the test, the old culture solution was discarded, and the working solution was prepared by diluting the fluorescent probe DCFH-DA mother liquor in a ratio of 1:1000 with serum-free α-MEM medium, adding 200 μL of working solution per well, mixing and returning to the cell culture. Incubate in a box at 37 ° C for 30 minutes in the dark. After the incubation, the cells were first washed once with serum-free DMEM, and then 200 μL of PBS solution was added to each well. At the time of detection, the bottom of the 96-well plate was sealed with opaque black adhesive tape, and the fluorescence intensity was measured using a multi-function microplate reader, and the fluorescence excitation wavelength was set to 488 nm, and the fluorescence emission wavelength was 525 nm. Finally, the fluorescence intensity is directly proportional to the level of cellular ROS.

6. Stem cell surface markers were detected. The third-generation stem cells (P3-BMSCs) with good growth status were cultured in a complete medium (control group) and a medium containing 125 nM sDSS1 protein (experimental group). To the 5th generation (P5-BMSCs), trypsinize and make a single cell suspension. After counting, ⁵ ×10 ⁵ cells were taken as analysis samples. First, the cells were washed with PBS containing 0.5% BSA, then 0.5 mL of antibody working solution was added, and the cells were mixed and incubated at 4° C. for 30 minutes in the dark. After the incubated cells were washed twice with 0.5% BSA PBS, the expression level of the cell surface markers was measured on a flow cytometer. Among them, three antibodies were selected in this experiment: FITC-anti-CD90 (Biolegend, C#206105/50μg), PE-anti-CD29 (Biolegend, 102207/50μg), PerCP/Cy5.5-anti-CD45 (Biolegend, 202220) /100 μg), an antibody working solution was prepared by diluting the antibody mother liquor with 1:1000, 1:800, and 1:200, respectively, using 0.5% BSA in PBS.

Experimental result

Stem cell culture fluids are more sensitive to toxic substances in the environment. To examine the effects of sDSS1 protein on stem cell proliferation and stem levels, primary cultured rat bone marrow mesenchymal stem cells (BMSCs) were used for proliferation, toxicity, and surface marker assays. The results showed that the addition of sDSS1 protein in the culture medium of stem cells could promote the proliferation of BMSCs. The cell proliferation level showed a dose-dependent effect between 10 nM and 125 nM. With the increase of protein concentration, the cell number increased gradually. The number of cells was highest in the 125 nM sDSS1 protein group. The number of cells remained unchanged between 125 nM and 1000 nM, and a higher concentration of sDSS1 protein had a certain inhibitory effect on cell proliferation (Fig. 4A). Detection of cytotoxicity levels revealed a decrease in cytotoxicity levels following the addition of sDSS1 protein (Fig. 4C). Similar results were obtained with the addition of the protein mutant sDSS1 (M4) (Fig. 4B and Fig. 4D). The level of ROS in stem cells was measured. The results showed that the level of ROS in the treated group with sDSS1 protein decreased, but it was significantly reduced in the 250nM and 750nM groups, and significantly increased in the high concentration group (30μM sDSS1 protein). (Fig. 4E). Summarizing these results, the sDSS1 protein can protect stem cells from toxic components in the culture environment, reduce the level of cytotoxicity, and increase the number of cells obtained in cell culture. To test whether sDSS1 protein treatment affects stem cell quality, stem cell surface markers including sDSS1 protein treatment, including CD90, CD29 and CD45, were detected by flow cytometry, in which BMSCs expressed CD90 and CD29 antigens but not CD45. The results showed that there was no significant difference in morphology between normal cultured BMSCs cells and BMSCs cultured in medium supplemented with sDSS1 protein (Fig. 5A). The level of markers was detected. The proportion of CD90 and CD29 positive cells in normal cultured BMSCs cells was 98.9% and 97.8%, respectively, while the proportion of CD45 positive cells as hematopoietic cell markers was extremely low at 1.47% (Fig. 5B). After treatment with sDSS1 protein, the proportion of positive markers in stem cells was not significantly affected, with the proportion of CD90 and CD29 positive cells being 98.8% and 98.1%, respectively, and the proportion of CD45 positive cells being 0.69% (Fig. 5C). These results indicate that the sDSS1 protein increases the number of cells obtained in stem cell culture, but does not significantly alter the cell dryness level.

Example 4, sDSS1 protein increases the number of cells continuously cultured and reduces cytotoxicity

1. N2a cells were continuously cultured and treated. After the N2a cells were digested with trypsin, a single cell suspension was prepared using complete medium. The cells were seeded into a 24-well plate at 2.5 x 10 ⁴ per well, and 1 ml of medium was added to each well. After the cells were attached for 6 hours, they were replaced with control medium containing no sDSS1 protein, or complete medium containing 1 μM sDSS1 protein, 8 μM sDSS1 protein, and 50 μM sDSS1 protein. After the culture plate was placed in a cell culture incubator for 48 hours, the culture solution was collected, and after centrifugation at 100 g, the supernatant was taken for cytotoxicity level detection, and the cells were used for cell proliferation level detection. The control group and each treatment group were set to 6 repetitions. After 48 hours, the cells as duplicate wells were trypsinized, and the cell suspension was prepared from the control medium or medium containing different concentrations of sDSS1 protein, and continued to be inoculated into a new 24-well plate at a ratio of 1:4, according to the same The treatment is added to different media treatments. Passage was performed twice in succession and cell proliferation and cytotoxicity levels were measured.

2. BMSCs cells were continuously cultured and treated. After the second generation BMSCs were digested into single cells, they were seeded into 24-well plates according to 8000 cells per well, 1 mL of medium was added, and 125 nM sDSS1 protein was added to the control medium. The cells were cultured in an incubator, subcultured every 3-4 days depending on the growth of the cells, and cell counts were performed using a hemocytometer plate during passage. In addition, the other cells in the duplicate wells were seeded into a new 24-well plate at a ratio of 1:3, and the control medium or the medium to which the sDSS1 protein was added was added in the same manner. Four passages were performed continuously and the cumulative number of cells was counted.

3. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C#C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in 120 μL of the cytotoxicity experiment was added to each well of a 96-well plate, and then 30 μL of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.

4. Cell proliferation assay The cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (C# CK04). A working solution was prepared by diluting CCK-8 solution in a serum-free DMEM at a ratio of 1:10 (v/v). After the cells in the 96 wells were processed, the old medium was discarded, and 150 μL of CCK-8 working solution was added to each well. The plate was incubated in an incubator for 2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell proliferation level.

Experimental result

In order to examine whether the promoting effect of sDSS1 protein on cell line cells can be maintained after cell passage, N2a cells are passaged in a medium containing sDSS1 protein and the level of cytotoxicity is detected. The results showed that the number of cells in the 8 μM and 50 μM groups was significantly higher than in the control group at the first passage (Fig. 6A), and the cytotoxicity levels of the two groups were also significantly lowered (Fig. 6B). At the second passage, the number of cells showed a significant promoting effect in the 1 μM and 8 μM groups (Fig. 6C), and the toxicity level was significantly decreased in the 50 μM group (Fig. 6D). The number of consecutively detected cells was summarized and the results showed that after 4 days of continuous culture, the cells cultured in the medium containing no sDSS1 protein were amplified 15 times, and the cells cultured in the medium containing 1 μM, 8 μM, and 50 μM sDSS1 protein were separately expanded. Increased by 26 times, 21 times and 18 times (Figure 7). Combining these data, it is shown that low concentrations of sDSS1 protein can effectively promote cell proliferation and obtain more cells in continuous culture. In order to verify the effect of this effect on primary stem cells, BMSCs cells were serially passaged in culture medium containing 125 nM sDSS1 protein. The results also showed that sDSS1 protein can effectively promote cell growth. After 15 days of culture, each sDSS1 protein group was obtained. The number of cells in the control group exceeding 3x10 ⁶ without adding protein was only half of it (Fig. 8). These results demonstrate that the sDSS1 protein also promotes the proliferation of stem cells in continuous culture and increases the number of cells obtained by culture.

Example 5, sDSS1 protein improves cell culture efficiency in low quality serum

1. Cell seeding and treatment Separate quality serum from the market was purchased for this experiment. Purchasing high-quality fetal bovine serum (Gibco, C#10100-147, from Australia) and general quality fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., C#10111) based on cell culture practices and network-related data descriptions -8611, produced in Inner Mongolia, China). The two sera were formulated to contain 10% high quality serum complete medium (medium A) and general quality serum complete medium (medium B) according to normal cell culture methods. After the trypsinization, the N2 cells were resuspended in medium A and medium B, respectively, and made into a single cell suspension. When the cells were treated, the cells were seeded into a 24-well plate at 2.5×10 ⁴ per well, and 1 mL of the culture solution was added. The treatment group was supplemented with 50 μM of sDSS1 protein in the A and B medium, respectively. The culture plate was placed in a cell culture incubator, the culture medium was collected 48 hours later, and 100 g of the supernatant was collected by centrifugation for cytotoxicity level detection, and the cells were used for cell proliferation detection.

2. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C#C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in 120 μL of the cytotoxicity experiment was added to each well of a 96-well plate, and then 30 μL of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.

3. Cell proliferation assay The cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (C# CK04). A working solution was prepared by diluting CCK-8 solution in a serum-free DMEM at a ratio of 1:10 (v/v). After the cells in the 96 wells were processed, the old medium was discarded, and 150 μL of CCK-8 working solution was added to each well. The plate was incubated in an incubator for 2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell proliferation level.

Experimental result

Serum is a major factor in maintaining cell proliferation in culture. In order to detect whether the sDSS1 protein can improve serum quality after cell addition, the cell proliferation effect is ensured. Cell proliferation effects and cytotoxicity levels were measured using commercially available high quality serum and general quality serum. The results showed that N2a cells grew well under high-quality serum conditions, and the cells increased slightly after adding 50 μM sDSS1 protein, but could not be clearly distinguished (Fig. 9A, Fig. 9B); in general quality serum, cell growth was significantly inhibited, and the number of cells was significantly inhibited. Less, and some cells become rounded and broken to form visible cell debris, indicating that the cells have certain toxic reactions. After the addition of the sDSS1 protein, the number of cells was significantly increased, and the number of rounded cells was small (Fig. 9C, Fig. 9D). The number of cells detected by CCK-8 showed that the addition of sDSS1 protein promoted the number of cells in high-quality serum or general quality serum, but in the general quality serum group, the promoting effect of sDSS1 protein was more pronounced, and the effect was more than 1 time (Fig. 9E). . The results of detecting cytotoxicity showed that the cytotoxicity level was higher in the general quality serum, sDSS1 protein significantly inhibited the toxicity, and the LDH test results were reduced by more than half; the high-quality serogroup cytotoxicity was weak, and the sDSS1 protein addition had no significant effect (Fig. 9F). ). Summarizing these data, sDSS1 protein can significantly improve the quality of general quality serum, maintain cell proliferation and shield the effects of toxic substances contained in serum on cells.

Claims (22)

The use of a protein in cell culture is characterized in that the application is the use of the sDSS1 protein for cell culture. The use according to claim 1, wherein the sDSS1 protein comprises a human, a chimpanzee, a bonobo, a gorilla, an orangutan, a white-cheeked gibbon, a Rhesus monkey, a rhesus monkey, a golden monkey, an East African ape, A basic protein formed by any sDSS1 protein sequence of Angora marmoset, white-tailed white-browed macaque, podophyllum, and porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, and the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2. , the amino acid sequence of the chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of the orangutan sDSS1 is SEQ ID NO: 5, and the amino acid sequence of the white-cheeked gibbon sDSS1 is SEQ. ID NO: 6, the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, and the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 9, amino acid of East African 狒狒sDSS1 The sequence is as SEQ ID NO: 10, the amino acid sequence of Angora simian sDSS1 is SEQ ID NO: 11, and the amino acid sequence of leucocephalus sDSS1 is SEQ ID NO: 12, scorpion sD The amino acid sequence of SS1 is SEQ ID NO: 13, and the amino acid sequence of porpoise monkey sDSS1 is SEQ ID NO: 14. The use according to claim 2, wherein the sDSS1 protein is any first protein having a similarity to the basic protein of claim 2 of 70% or more. The use according to claim 2, wherein the sDSS1 protein is based on any of the 58 amino acids of the basic protein of claim 2, and the other polypeptide fragments are fused at the nitrogen or carbon end. A second protein having the same or similar structural features or amino acid sequence characteristics as the carbon terminus of the basal protein of claim 2 in the fused polypeptide fragment. The use according to claim 2, wherein the sDSS1 protein is based on any of the 58 amino acids of the basic protein of claim 2, and the other amino acid fragments are fused at the nitrogen or carbon end. The latter protein can achieve a third protein that transmembrane transport function. The use according to any one of claims 2 to 5, wherein the sDSS1 protein is the basic protein, the first protein, the second protein or the third protein according to any one of claims 2-5. A fusion protein formed by ligation of the protein itself, a carrier protein, an antibody, or other amino acid fragments of any length. The use according to any one of claims 2 to 5, wherein the sDSS1 protein is based on a basic protein, a first protein, a second protein or a third protein according to any one of claims 2-5. The resulting modification results in a polypeptide/protein modification. The use according to claim 7, wherein the modification of the polypeptide/protein modification is directed to an amino group on the amino acid side chain of the sDSS1 protein, a carbonyl group on the amino acid side chain, a nitrogen terminal amino group, and a carbon terminal hydroxyl group. Specific or non-specific chemical modification of 1-20 sites by cysteine, tyrosine, serine or tryptophan. The method according to claim 7, wherein the modification method of the polypeptide/protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, Dextran modification, heparin modification, polyvinylpyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification , hydroxypropyl methylcellulose modification, hydroxypropyl cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification or other medicinally available polypeptide/protein drug modification method One or more of them. The use according to any one of claims 2 to 5, wherein the sDSS1 protein is the basic protein, the first protein, the second protein or the third protein according to any one of claims 2-5. The amino acid sequence is based on an unnatural amino acid replacement protein substituted with 1-31 arbitrary amino acid positions of amino acids other than the 20 essential amino acids. The use according to claim 10, wherein the amino acid substitution of the non-natural amino acid replacement protein comprises hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non- Natural amino acids and their derivatives. The use according to any one of claims 2 to 11, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/ A partial or total complex of a protein modified or non-natural amino acid replacement protein with a pharmaceutically acceptable pharmaceutical carrier. The use according to claim 12, wherein the pharmaceutical carrier comprises an enteric coating preparation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin, and a gel. One or more of them. The use according to any one of claims 2 to 5, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, and the A fourth protein obtained by introducing a DNA or RNA sequence of three proteins into the cell to obtain a medium. The use according to any one of claims 1 to 14, wherein the application of the sDSS1 protein in cell culture is the basic protein, the first protein, the second protein according to claims 1-14. a third protein, a fourth protein, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein or complex, and a basal medium component, a serum component, a serum replacement component, and a proliferation additive group Fractions, or components of any component that need to be added during cell culture. The use according to any one of claims 1 to 14, wherein the application is the fusion of the basic protein, the first protein, the second protein, the third protein, and the fusion according to claims 1-14. Protein, peptide/protein modification, non-natural amino acid replacement protein or complex for cell transplantation, cell therapy, tissue transplantation, tissue repair, or cell/tissue protection fluid, cell suspension, cell/tissue maintenance solution during organ transplantation , cell/tissue cleaning solution or components of any of the solutions used in these processes. The use according to claim 1, wherein the cell culture is cell preparation, cell sorting, cell cloning culture, cell expansion culture, cell enrichment, cell purification, cell culture in vitro using a medium. Any form of engineering, cell three-dimensional culture, cell fermentation, tissue culture, organ culture. The use according to claim 17, wherein said cell culture is derived from human, orangutan, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, small. Cell culture of any cell of mouse, fish, bird or insect. The use according to claim 17, wherein the cell culture is performed on primary cells, tissue-derived stem cells, tissue-derived precursor cells, induced pluripotent stem cells, differentiated-derived cells, transdifferentiated cells, and cell engineering. One or more cell cultures of the obtained cells, tumor stem cells, tumor cells isolated from tumor tissues, cell strains, cell lines, insect cells, cell pellets, tissue pellets, and in vitro cultured organs. The use according to claim 19, wherein said primary cells are of cardiomyocytes, chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair follicle dermal papilla cells, hepatocytes, kidney cells, keratin One or one of cells, melanocytes, osteoblasts, pre-adipocytes, skeletal muscle cells, smooth muscle cells, stem cells, T cells, B cells, macrophages, precursor cells, pericytes, or dendritic cells More than one species. The use according to any one of claims 1 to 20, wherein the basic protein, the first protein, the second protein, and the third of claim 1-18 are added at any time during the cell culture. The concentration of the protein, fusion protein, polypeptide/protein modification, non-natural amino acid replacement protein or complex is not less than 10 nM. A commercial development or clinical application, characterized in that the application is the use of the protein according to any one of claims 1 to 21 in cell culture, and then the commercial application of the sDSS1 protein in cell culture or Clinical application.

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