Method for producing snake oil by culturing snake adipocytes

Abstract

The present invention relates to a method for producing snake oil by culturing snake adipocytes. The method comprises digesting an adipose sample of a living snake body to obtain a single cell mixture; culturing the single cell mixture and carrying out purification by means of differential adherent culture to obtain preadipocytes; carrying out continuous subculture on the preadipocytes to obtain immortalized preadipocytes; carrying out adipogenic differentiation on the immortalized preadipocytes to obtain a large quantity of mature adipocytes; and centrifuging to collect the mature adipocytes and obtaining a snake oil component. The immortalized preadipocytes and the large quantity of mature adipocytes can be obtained by means of culturing by using only a small quantity of snake adipose tissue. The present invention shows high safety and good stability, is convenient for industrial production, can partially replace snake oil produced by traditional technologies, and is applicable to the fields of skin care product production, medical and scientific research, etc.

Description

A method for producing snake oil by culturing snake fat cells Technical Field

[0001] The present invention relates to the field of stem cells, and in particular to a method for producing snake oil by culturing snake fat cells. Background Art

[0002] Snake oil's main chemical components are various fatty acids. They can promote skin metabolism, enhance cell vitality, provide antioxidant and anti-aging benefits, and enhance radiance. They also possess strong affinity for the skin and mucous membranes and good permeability, making them commonly used in the production of high-end cosmetics for beauty and skincare. Furthermore, snake oil has the potential to reduce vascular permeability in burns, provide anti-inflammatory and analgesic effects, lower blood lipids and blood sugar, and inhibit bacteria. Consequently, it is often combined with other medications to create compound topical preparations for the treatment of various skin conditions.

[0003] Snakes have a long history of use as medicinal materials in traditional Chinese medicine and folk medicine. Following the 2020 ban on eating wild animals, the use of snakes has been limited to medicinal and ornamental purposes. However, current methods for preparing snake oil directly from the snakes often involve extracting fatty tissue and refining the oil from the snake fat using acid hydrolysis or heating. While this method can directly extract the desired medicinal materials, it is complex, inefficient, and costly.

[0004] Furthermore, due to the numerous issues surrounding snake oil extraction, including animal ethics, hygiene, and infectious diseases, research capabilities and treatment levels for snake oil treatments have been limited. However, there is currently a growing body of research on the use of cell culture to produce various animal tissues, such as cultured meat, and the technology is relatively mature. Therefore, establishing a cell culture-based snake oil extraction technology to replace direct extraction is urgent and has the potential to provide a new direction and contribution to scientific research.

[0005] There is currently no literature reporting on the culture and utilization of snake cells. How to culture snake cells and whether snake oil can be produced by culturing snake fat cells need further exploration.

[0006] Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a method for producing snake oil by culturing snake fat cells. The method not only provides a feasible method for large-scale cultivation of snake fat cells, but also successfully obtains snake oil through the cultivation of snake fat cells.

[0008] A first aspect of the present invention provides a method for obtaining immortalized preadipocytes of snakes, comprising the following steps:

[0009] S1: Digestion and processing of fat samples from living snakes to obtain single-cell mixtures;

[0010] S2: Cultivate the single cell mixture and purify preadipocytes through differential adherence culture;

[0011] S3: Continuously subculture the preadipocytes to obtain immortalized preadipocytes.

[0012] In some embodiments, before the digestion treatment in step S1, the step further includes: obtaining a small amount of fat sample from the living snake, and performing disinfection and / or cleaning.

[0013] In some embodiments, the disinfectant used for disinfection is an ethanol solution, such as a 75% ethanol solution, and the cleaning solution used for rinsing is a saline solution containing antibiotics. The disinfection and cleaning times are each 3 to 5 minutes. Disinfection of fat samples helps remove bacteria and viruses, preventing infection that can lead to loss of proliferation and differentiation, or even death. Cleaning the samples also helps remove impurities, such as blood and non-fat tissues like mucous membranes.

[0014] In some embodiments, in step S1, the digestion treatment uses digestive enzymes, which include dispase and pancreatic enzyme substitutes. The dispase is a mixed enzyme of type I collagenase and neutral protease II.

[0015] In some embodiments, in step S1, the final concentration of type I collagenase in the digestive enzymes is 0.1% to 0.5% (w / v), the final concentration of neutral proteinase II is 0.2% to 0.5% (w / v), and the final concentration of pancreatic enzyme substitute is 1% to 5% (w / v). Higher concentrations than these may significantly damage the activity of stem cells and be detrimental to maintaining the integrity of preadipocytes; lower concentrations than these may hinder tissue digestion.

[0016] In some embodiments, in step S1, the digestion time is 0.5 to 2 hours, and the digestion temperature is 30 to 40°C.

[0017] In some embodiments, in step S2, the culture medium for culturing the single-cell mixture comprises a low-glucose medium, FBS, glutamine, and recombinant epidermal growth factor. Using a low-glucose medium in the early stages of cell culture helps limit premature adipocyte differentiation and maintain stable cell quality. Recombinant epidermal growth factor helps rapidly establish a cell line.

[0018] In some embodiments, in step S2, the low-glucose culture medium is selected from the basal culture medium DMEM containing 1 g / L glucose.

[0019] In some embodiments, the culture medium for culturing the single-cell mixture comprises: 75-85% (v / v) DMEM, 15-20% (v / v) fetal bovine serum (FBS), 0.5-2% (v / v) 100× glutamine, and 0.01-0.02% (w / v) human recombinant epidermal growth factor. The addition of glutamine and human recombinant epidermal growth factor enhances the self-renewal and potential differentiation capacity of snake preadipocytes in the culture medium.

[0020] In some embodiments, in step S2, the single cell mixture is cultured at a temperature of 31-34°C, and the adherent culture time is 1-6 hours. A culture temperature below 37°C (e.g., 31-34°C) is closer to the natural body temperature of snakes in non-hibernating conditions and is more suitable for the growth and differentiation of snake cells. Selecting an adherent culture time of 1-6 hours can quickly and effectively eliminate fat cells and other non-fat cells in a differentiated state that are slow to adhere.

[0021] In some embodiments, in step S3, the immortalized preadipocytes are spontaneously generated through subculture, and 0.5-2% CEE (chicken embryo extract) may be added to induce and improve immortalization efficiency, for example, by subculture for more than 25 generations. This avoids the high costs and risks of impacting final product quality associated with traditional methods such as the use of viral vectors to introduce SV40 and TERT.

[0022] In some embodiments, in step S3, the culture medium for continuous subculture comprises: 75-85% (v / v) DMEM, 15-20% (v / v) fetal bovine serum (FBS), 0.5-2% (v / v) 100× glutamine, 0.01-0.02% (w / w) human recombinant epidermal growth factor, and 0.5-2% (v / v) CEE.

[0023] A second aspect of the present invention provides a method for obtaining a large number of snake fat cells, comprising the following steps:

[0024] S1: Digestion and processing of fat samples from living snakes to obtain single-cell mixtures;

[0025] S2: Cultivate the single cell mixture and purify preadipocytes through differential adherence culture;

[0026] S3: Continuously subculture the preadipocytes to obtain immortalized preadipocytes;

[0027] S4: Immortalized fat precursor cells are cultured for adipogenic differentiation to obtain a large number of mature fat cells.

[0028] The steps S1-S3 are the same as steps S1-S3 in any embodiment of the first aspect of the present invention.

[0029] In some embodiments, in step S4, the adipogenic differentiation culture medium contains additives such as glucose, NMN (β-nicotinamide mononucleotide), Y-27632 (RhoA / ROCK inhibitor), insulin, and phosphatidylcholine. The addition of glucose, NMN, Y-27632, insulin, and phosphatidylcholine helps promote the differentiation of preadipocytes into mature adipocytes containing a large amount of lipid components. Y-27632 also helps prevent cell senescence and fibrogenic differentiation.

[0030] In some embodiments, in step S4, the culture medium for adipogenic differentiation culture contains: 75-85% (v / v) DMEM medium, 15-20% (v / v) fetal bovine serum, 0.5-2% (v / v) 100X glutamine, 35-60 mg / mL glucose, 200-400 mg / L NMN, 5-10 μM Y-27632, 1.5-3 μg / mL insulin and 1.5-3 mg / mL phosphatidylcholine.

[0031] In some embodiments, before the adipogenic differentiation culture in step S4, the step further includes a step of large-scale culture of the immortalized preadipocytes.

[0032] In some embodiments, the medium for large-scale culture comprises a high-glucose medium, fetal bovine serum, glutamine, recombinant epidermal growth factor, glucose, NMN, and Y-27632 (RhoA / ROCK inhibitor) medium. Fetal bovine serum, high glucose (glucose), and NMN help provide the energy and NAD+ required for the rapid growth and proliferation of adipocytes; Y-27632 helps prevent cell aging and fibrogenic differentiation.

[0033] In some embodiments, the high-glucose culture medium is selected from the basal medium DMEM containing 4.5 g / L glucose.

[0034] In some embodiments, the culture medium for large-scale culture comprises: 75-85% (v / v) DMEM medium, 15-20% (v / v) fetal bovine serum, 0.5-2% (v / v) glutamine, 0.01-0.02% (w / w) recombinant epidermal growth factor, 35-60 mg / mL glucose, 200-400 mg / L NMN and 5-10 μM Y-27632 (RhoA / ROCK inhibitor).

[0035] A third aspect of the present invention provides a method for producing snake oil by culturing snake fat cells, comprising the following steps:

[0036] S1: Digestion and processing of fat samples from living snakes to obtain single-cell mixtures;

[0037] S2: Cultivate the single cell mixture and purify preadipocytes through differential adherence culture;

[0038] S3: Continuously subculture the preadipocytes to obtain immortalized preadipocytes;

[0039] S4: Immortalized preadipocytes are cultured for adipogenic differentiation to obtain a large number of mature adipocytes;

[0040] S5: collecting mature fat cells by centrifugation, and obtaining snake oil components from the collected fat cells.

[0041] The steps S1-S4 are the same as steps S1-S4 in any embodiment of the second aspect of the present invention.

[0042] In some embodiments, in step S5, the mature adipocytes are collected by low-speed centrifugation; the low-speed centrifugation method can quickly collect the cells and avoid rupture of the mature adipocyte membrane.

[0043] In some embodiments, in step S5, the collected mature adipocytes are then subjected to low-temperature ultracentrifugation to obtain the snake oil component. Ultracentrifugation ruptures the cells and separates the oil components from the aqueous phase, thereby avoiding the damage to certain active components of the adipocytes caused by traditional high-temperature heating of snake oil, thereby effectively protecting all active components in the snake oil.

[0044] The fourth aspect of the present invention provides a culture medium for continuous subculture of snake preadipocytes: 75-85 (v / v)% DMEM, 15-20% (v / v) fetal bovine serum (FBS), 0.5-2% (w / w) 100× glutamine and 0.01-0.02% (w / w) human recombinant epidermal growth factor, and 0.5-2% (v / v) CEE.

[0045] The fifth aspect of the present invention provides a culture medium for adipogenic differentiation of immortalized preadipocytes of snakes: 75-85% (v / v) DMEM medium, 15-20% (v / v) fetal bovine serum (FBS), 0.5-2% (v / v) 100× glutamine, 35-60 mg / mL glucose, 200-400 mg / L NMN, 5-10 μM Y-27632 (RhoA / ROCK inhibitor), 1.5-3 μg / mL insulin and 1.5-3 mg / mL phosphatidylcholine.

[0046] The sixth aspect of the present invention provides a culture medium for large-scale culture of immortalized preadipocytes of snakes: 75-85% (v / v) DMEM medium, 15-20% (v / v) fetal bovine serum (FBS), 0.5-2% (v / v) 100× glutamine, 0.01-0.02% (w / w) recombinant epidermal growth factor, 35-60 mg / mL glucose, 200-400 mg / L NMN and 5-10 μM Y-27632 (RhoA / ROCK inhibitor).

[0047] Compared with the prior art, the beneficial effects of the present invention include at least:

[0048] (1) The present invention provides for the first time a method for producing snake oil by culturing snake fat cells, including obtaining immortalized snake fat precursor cells, culturing them through adipogenic differentiation to obtain a large number of mature fat cells, and obtaining snake oil from the mature fat cells, providing a complete solution.

[0049] (2) The present invention only requires obtaining a small amount of snake adipose tissue. Through subculture, high-purity, high-activity, and strong proliferation and self-renewal ability of fat precursor cells can be obtained. The cells can be subcultured for more than 40 generations, and a large number of mature fat cells can be obtained through differentiation.

[0050] (3) The present invention provides culture media for each stage of cell culture, which are highly safe and stable. The cell culture media for the early and middle stages of culture are beneficial for maintaining the adipocyte precursor cells in an undifferentiated state with high growth rate, while the differentiation culture media for the later stages can effectively promote the differentiation and maturation of adipocytes.

[0051] (4) The snake oil obtained by the present invention comes from the fat cells of snakes, and its components are consistent with those obtained from the fat cells of snake bodies. A large amount of snake body fat cells and snake oil components can be obtained, which is convenient for industrial production. It can partially replace the snake oil produced by traditional technology and be used in the production of skin care products and medical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is a photograph of the snake used in Example 1;

[0053] FIG2 is a light microscopic image of preadipocytes obtained by the differential cell attachment method in Example 1;

[0054] FIG3 is a light microscopic image of preadipocytes during the massive growth and expansion process in Example 1;

[0055] FIG4 is a light microscopic image of mature adipocytes after differentiation;

[0056] FIG5 shows adipocytes harvested by low-speed centrifugation in Example 1, with the bottom of the tube being white;

[0057] Figure 6 shows the snake oil obtained by ultracentrifugation in Example 1;

[0058] FIG7 is a light microscopic image of preadipocytes during the massive growth and expansion process in Example 2;

[0059] FIG8 is a light microscopic image of differentiated mature adipocytes in Example 2. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, intended to be used for explaining the present invention, and do not constitute a limitation of the present invention.

[0061] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0062] Example 1 Isolation and Culture of Yellow-chain Snake Fat Cells and Production of Snake Oil

[0063] 1. Reagent formula and preparation method

[0064] 1.1 Preparation of the digestive enzyme system: Dissolve 75 mg of Neutral Protease II powder (Solarbio, China) in 25 mL of PBS to a final concentration of 3 mg / mL of Neutral Protease II solution. Add 50 mg of type I collagenase powder (Gibco, USA) to a final concentration of 0.2% (w / v). Place the mixed enzyme system in a 4°C refrigerator until ready for use. Dissolve 0.5 g of pancreatic enzyme substitute (Gibco, USA) in 25 mL of PBS to a final concentration of 2% (w / v) and place in a 4°C refrigerator until ready for use.

[0065] 1.2 Cell culture medium preparation: To 390 mL of DMEM (Gibco, USA), add 100 mL of fetal bovine serum (Gibco, USA) to a final concentration of 20% (v / v), 5 mL of 100× glutamine (Gibco, USA) to a final concentration of 1% (v / v), 50 mg of human recombinant epidermal growth factor (Gibco-BRL, USA) to a final concentration of 100 ng / mL to form 0.1% (w / v), and 5 mL of CEE to a final concentration of 1%. Mix thoroughly, filter through a sterile filter, and store the filtrate at 4°C.

[0066] 2. Isolation of Preadipocytes

[0067] Obtain snake adipose tissue and isolate and culture preadipocytes using the differential adherent culture method. The specific steps are as follows:

[0068] S1: A live yellow-banded snake (~1.1 m long) was selected as shown in Figure 1. It was anesthetized and washed with 75% ethanol for approximately 10 seconds. An abdominal incision (~1 cm long wound, sutured afterwards) was made under sterile conditions to obtain approximately 5 g of abdominal adipose tissue. The tissue was then soaked in 75% ethanol for 3 minutes. The adipose tissue was then washed twice with physiological saline containing 2% double-antibody for 3 minutes each to obtain the adipose tissue.

[0069] S2: Use sterile scissors to cut the fat tissue into 2mm pieces. 3 The tissue blocks were digested for 1 hour in a 37°C water bath using a mixture of 0.2% (w / v) type I collagenase and 0.1% (w / v) neutral proteinase II. Mix well every 10 minutes, add trypsin substitute at a final concentration of 2% after 1 hour, and continue digestion in a 37°C water bath for 0.5 hours. After centrifugation at 1000 rpm for 5 minutes, resuspend the pellet in PBS and filter it with a 70 μm cell sieve to obtain a single cell mixture. The mixture was transferred to a culture flask and cultured at 32°C. After 3 hours, the suspended cells were removed and the culture medium was changed, depending on the cell attachment rate. The results of the attached cells are shown in Figure 2.

[0070] 3. Large-scale culture and expansion of preadipocytes

[0071] The specific steps are as follows:

[0072] S1: Immortalization of adipocytes. Snake adipocytes were subcultured in vitro for approximately 25 generations. It was found that the growth of many cells slowed down while the growth of a few cells accelerated. The cells with accelerated growth were further cultured to obtain spontaneously immortalized adipocytes.

[0073] S2: Immortalized preadipocytes were cultured in a new cell culture medium containing DMEM (Gibco, USA) supplemented with fetal bovine serum (20%), glutamine (1%), recombinant epidermal growth factor (0.01%), glucose (45 mg / mL), Y-27632 (5 μM), and NMN (β-nicotinamide mononucleotide) (300 mg / L). This culture was continued for 20 days (32°C) and 0.5×10 4 Cell expansion was ~1×10 10 cells, as shown in Figure 3.

[0074] 4. Adipogenic differentiation of preadipocytes

[0075] The expanded preadipocytes were cultured in an adipogenic differentiation medium containing fetal bovine serum (20%), glutamine (1%), recombinant epidermal growth factor (0.01%), glucose (45 mg / mL), NMN (β-nicotinamide mononucleotide) (300 mg / mL), Y-27632 (5 μM), insulin (2 μg / mL), and phosphatidylcholine (2 mg / mL). The cells were cultured for 7 days (32°C), as shown in Figure 4.

[0076] 5. Separation of snake oil

[0077] The specific steps are as follows:

[0078] S1: The cultured cells were collected using trypsin and separated by low-speed centrifugation (800 rpm / min) to obtain adipocytes, as shown in FIG5 .

[0079] S2: Snake oil was obtained by low-temperature ultracentrifugation at a temperature of 4°C and a centrifugal speed of 20,000 rpm / min. The snake oil results are shown in FIG6 .

[0080] Example 2 Isolation and Culture of Red Chain Snake Fat Cells and Production of Snake Oil

[0081] Similar to Example 1, except that:

[0082] 1. Reagent formula and preparation method

[0083] 1.1 Preparation of the digestive enzyme system: Dissolve 50 mg of Neutral Protease II powder (Solarbio, China) in 25 mL of PBS to a final concentration of 0.2% (w / v) Neutral Protease II solution. Add 125 mg of type I collagenase powder (Gibco, USA) to a final concentration of 0.5% (w / v). Place the mixed enzyme system in a 4°C refrigerator until ready for use. Dissolve 1.25 g of pancreatic enzyme substitute (Gibco, USA) in 25 mL of PBS to a final concentration of 5% (w / v) and place in a 4°C refrigerator until ready for use.

[0084] 1.2 Cell culture medium preparation: To 85 mL of DMEM (Gibco, USA), add 15 mL of fetal bovine serum (Gibco, USA) to a final concentration of 15% (v / v), 100× glutamine (Gibco, USA) to a final concentration of 0.5% (v / v), human recombinant epidermal growth factor to a final concentration of 0.01%, and CEE to a final concentration of 1.5%. Mix thoroughly, filter through a sterile filter membrane, and store the filtrate at 4°C.

[0085] 2. Isolation of Preadipocytes

[0086] S1: A live red snake (~0.9 m long) was selected and anesthetized. The snake was washed with 75% ethanol for approximately 10 seconds. An abdominal incision (~1 cm long wound, sutured afterwards) was made under sterile conditions to obtain approximately 4 g of abdominal adipose tissue. The tissue was then soaked in 75% ethanol for 2 minutes. The adipose tissue was then washed twice with saline containing 2% double-antibody for 2 minutes each.

[0087] S2: Use sterile scissors to cut the fat tissue into 2mm pieces. 3 Tissue fragments were digested in a 37°C water bath with a mixture of 0.5% collagenase type I and 0.2% neutral proteinase II for 1 hour. Mix every 10 minutes, then add trypsin substitute to a final concentration of 5% and continue digestion at 37°C for 0.5 hours. After centrifugation at 1000 rpm for 5 minutes, the pellet was resuspended in PBS and filtered through a 70 μm cell sieve to obtain a single-cell mixture. The mixture was transferred to a culture flask and cultured at 32°C. After 4 hours, the suspended cells were removed and the culture medium was replaced, depending on the cell attachment rate.

[0088] The cells expanded in vitro and after adipogenic differentiation are shown in Figures 7 and 8 .

[0089] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for producing snake oil by culturing snake fat cells, characterized in that: The following steps are involved: Step S1: digesting and processing a fat sample from a living snake to obtain a single-cell mixture; Step S2: culturing the single cell mixture and purifying preadipocytes through differential adherence culture; Step S3: Continuously subculturing the preadipocytes to obtain immortalized preadipocytes; Step S4: performing adipogenic differentiation culture on the immortalized preadipocytes to obtain a large number of mature adipocytes; Step S5: collecting mature fat cells by centrifugation, and then obtaining the snake oil component from the collected fat cells.

2. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S1, the digestion treatment uses digestive enzymes, which include dispase and a pancreatic enzyme substitute. The dispase is a mixed enzyme of type I collagenase and neutral protease II. The final concentration of type I collagenase in the digestive enzyme is 0.1% to 0.5% (w / v), the final concentration of neutral protease II is 0.2% to 0.5% (w / v), and the final concentration of the pancreatic enzyme substitute is 1% to 5% (w / v).

3. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S1, the digestion time is 0.5 to 2 hours, and the digestion temperature is 30 to 40°C.

4. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S2, the culture medium for culturing the single cell mixture contains: low-glucose culture medium, FBS, glutamine and recombinant epidermal growth factor; the culture temperature for culturing the single cell mixture is 31-34° C., and the adherent culture time is 1-6 hours.

5. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S3, the immortalized preadipocytes are spontaneously generated through subculture or added with 0.5-2% (v / v) chicken embryo extract; the culture medium for continuous subculture comprises: 80-85% (v / v) DMEM, 15-20% (v / v) fetal bovine serum, 0.5-2% (w / v) 100× glutamine, 0.01-0.02% (w / v) human recombinant epidermal growth factor, and 0.5-2% (v / v) chicken embryo extract.

6. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S4, the culture medium for adipogenic differentiation contains glucose, NMN, RhoA / ROCK inhibitor, insulin and phosphatidylcholine.

7. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: Before the adipogenic differentiation culture in step S4, the step also includes a step of large-scale culture of the immortalized fat precursor cells; the medium for the large-scale culture includes a high-glucose medium and an NMN medium.

8. The method for producing snake oil by culturing snake fat cells according to claim 1, wherein: In step S5, the mature adipocytes are collected by low-speed centrifugation; and the collected mature adipocytes are subjected to low-temperature ultracentrifugation to obtain the snake oil component.

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