A 3D cell culture scaffold, its preparation method, and a method for culturing cells.

Abstract

This invention discloses a 3D cell culture scaffold, its preparation method, and a method for culturing cells. By adjusting the temperature and humidity during the silkworm cocooning process, multi-layered thin cocoons are produced. The size of the thin cocoon layer is cut according to the needs of the cultured cells, and after high-temperature and high-pressure sterilization, it can be used for adherent cell culture, increasing cell culture efficiency by more than three times. Based on the physiological characteristics of silkworms' environmental adaptability, this invention produces a 3D culture scaffold suitable for adherent cell culture by precisely controlling the temperature and humidity conditions. It features readily available materials, convenient operation, low cost, and easy application.

Description

Technical Field

[0001] This invention relates to a method for fabricating a 3D cell culture scaffold from multilayer silkworm cocoons, specifically to the fabrication of multilayer silkworm cocoons, the preparation of the 3D cell culture scaffold, and its application in cell culture. Background Technology

[0002] The silkworm's cocooning behavior serves two purposes: firstly, it protects the pupa from predators during pupation and metamorphosis; secondly, it helps eliminate excess amino acids, preventing amino acid poisoning. Typically, the silkworm's cocooning process is continuous, taking approximately 60 hours at a temperature of 23–25°C and humidity of 70% ± 5%, after which it enters the pupal stage. The silkworm's spinning behavior is influenced by ambient temperature and humidity. Low temperatures reduce the silkworm's metabolism, slowing down the spinning process, while low humidity promotes moisture loss from the silk, increasing the cocoon's unwinding rate.

[0003] Cell culture is a crucial experimental technique supporting cell engineering in the field of bioengineering. For adhering cell culture, various methods exist: trypsin digestion to suspend cells; repeated rinsing with pipettes; and scraping with a specialized cell scraper. All three methods cause some degree of cell damage and are technically challenging. Furthermore, adhering cells utilize only the bottom surface of the culture dish, reducing culture efficiency. To address this technical bottleneck, 3D cell culture scaffolds are commercially available, but their high price (approximately 45 yuan per scaffold, more expensive than cell culture flasks) limits their widespread application. Cell culture platforms urgently need to develop an affordable and user-friendly 3D cell culture scaffold. Summary of the Invention

[0004] The purpose of this invention is to provide a method for fabricating 3D cell culture scaffolds from multilayer silkworm cocoons, providing a practical 3D scaffold for cell culture and promoting the application of silk products in the field of biomedical materials.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for preparing a 3D cell culture scaffold includes the following steps: cutting silkworm cocoons to obtain thin cocoon layers, and then sterilizing the thin cocoon layers under high temperature and high pressure to obtain a 3D cell culture scaffold. The silkworm cocoons are elliptical; after removing both ends, the abdomen of the cocoon is cut open, and the molted skin of the silkworm pupa and larva is removed to obtain multiple layers of thin cocoon layers, which are then further layered to obtain thin cocoon layers. Preferably, the number of thin cocoon layers in the multiple layers is 5 to 15.

[0006] In this invention, silkworms are spun into cocoons to obtain cocoons; the temperature of the silkworm spun into cocoons is alternated between 23℃±1℃ / 5 hours and 12℃±2℃ / 1 hour; the spun into cocoons lasts for 48 to 72 hours, that is, the temperature is alternated 8 to 12 times; the humidity of the silkworm spun into cocoons is 60%±5%.

[0007] In this invention, the temperature for high-temperature and high-pressure sterilization is 121±1℃ and the pressure is 0.12±0.01MPa.

[0008] This invention discloses the application of the above-mentioned 3D cell culture scaffold in culturing cells or preparing cell culture scaffolds; preferably, the cell culture is the subculture of adherent cells.

[0009] This invention discloses a method for culturing cells using the aforementioned 3D cell culture scaffold. The method involves placing the 3D cell culture scaffold in a culture flask, inoculating cells, adding culture medium, and then culturing. Preferably, the 3D cell culture scaffold has 1 to 30 layers.

[0010] The present invention directly uses conventionally sterilized silkworm cocoon layers to make 3D cell culture scaffolds, including: (1) making multi-layer silkworm cocoons; (2) preparing 3D cell culture scaffolds; (3) cell culture.

[0011] The silkworm variety of this invention adopts a conventional first-generation hybrid, with oval cocoon shape being preferred, avoiding undesirable cocoons such as spherical cocoons, deformed cocoons, and double cocoons.

[0012] In this invention, the silkworm cocooning environment is set at 23℃±1℃ for 5 hours and 12℃±2℃ for 1 hour, with multiple sets alternating. Under environmental conditions of 60%±5% humidity, a thin cocoon layer suitable for 3D cell scaffold applications can be completed in 5 hours. The thin cocoon layers do not stick together and are easy to separate.

[0013] In this invention, approximately 10% of the length of each end of the elliptical silkworm cocoon is removed, and the abdomen of the cocoon is cut open to remove the molted skin of the pupa and larva, thus obtaining a multi-layered, thin cocoon. The reason for removing the top of the cocoon is that its curvature is too large, which is not conducive to the preparation of 3D cell culture scaffolds.

[0014] According to the above conditions, a multi-layered silkworm cocoon with about 10 layers can be prepared. According to the needs of cell culture, the size of the thin cocoon layer is cut. Preferably, 6 pieces are cut along the long axis of one silkworm cocoon. One silkworm cocoon can prepare 60 pieces of 3D cell culture scaffold. The natural curvature of the silkworm cocoon layer can be used to form a three-dimensional 3D structure. There is no difference between the front and back of the concave and convex curved surface in the cultured cells, and the culture effect is the same.

[0015] In this invention, the thin cocoon layer is sterilized under high pressure at a conventional temperature (121°C, 0.12 MPa) for 30 minutes to obtain a scaffold for 3D cell culture. Before sterilization, the thin cocoon layer needs to be placed in a stainless steel container, and after sterilization, it needs to be sealed and aseptically stored.

[0016] The 3D cell culture scaffold prepared in this invention can be used for the culture of adherent cells, but is not suitable for the culture of suspension cells. Multiple 3D cell culture scaffolds can be placed in cell culture flasks with larger areas, allowing cells to colonize the scaffolds. It is suitable for cell line subculture. Using the 3D cell culture scaffold prepared in this invention, the 3D cell culture scaffold can be directly removed for cell replacement, reducing cell damage during subculture and facilitating the collection of cell samples during experiments.

[0017] Due to the application of the above technical solution, the present invention has the following advantages: 1. This invention achieves the goal of preparing multi-layered silkworm cocoons for the first time by controlling the temperature and humidity conditions during the cocooning process.

[0018] 2. This invention can produce 3D cell culture scaffolds by cutting and sterilizing thin cocoon layers. It adopts a different technical approach from existing silk fibroin scaffolds, and in particular, it solves the problem that existing technologies require cumbersome steps such as degumming, dissolving, gelling, and solidification in addition to sterilization.

[0019] 3. The raw material cost of producing the 3D cell culture scaffold of this invention is about RMB 0.0013 per piece, which is far lower than the price of similar products on the market.

[0020] The method disclosed in this invention for preparing 3D cell culture scaffolds has the advantages of easy material sourcing, convenient operation, low cost, and easy promotion and application. Attached Figure Description

[0021] Figure 1 An inverted microscope image of the 3D cell culture scaffold (thin cocoon layer) fabricated for Example 1.

[0022] Figure 2 This is a SEM image of existing silkworm cocoons.

[0023] Figure 3 The cell morphology after 9 days of culture in Example 2. Detailed Implementation

[0024] In this invention, a conventional first-generation hybrid silkworm variety is used. The cocoon shape is preferably elliptical. Approximately 10% of the top portion of each end of the long axis of the elliptical cocoon is removed. The abdomen of the cocoon is cut open to remove the molted skin of the pupa and larva, resulting in a multi-layered thin cocoon. Based on the above conditions, approximately 10 layers of cocoon can be produced. The size of the thin cocoon layers is cut according to the needs of cell culture, preferably 6 or more pieces along the long axis of a single cocoon, utilizing the natural curvature of the cocoon layers to form a three-dimensional 3D structure. The thin cocoon layers are then sterilized under high pressure at a conventional temperature (121℃, 0.12MPa) for 30 minutes to obtain a 3D cell culture scaffold. This scaffold is used for adhering cell culture but not for suspension cell culture. Multiple 3D cell culture scaffolds can be placed in cell culture flasks, allowing cells to colonize the scaffold for cell line subculture, reducing cell damage during subculture and facilitating cell sample collection during experiments.

[0025] The present invention will be further described below with reference to embodiments. The silkworms and their rearing, cocooning tools, harvesting, and specific silkworm cocoon cutting involved in the present invention are all conventional techniques.

[0026] The silkworm variety used in this invention is Suxiu × Chunfeng, an existing product, with one feeding sheet (25,000 silkworms). The specific feeding method is conventional, during the spring silkworm season. The mulberry variety is Yu 711. The cocooning frame is a cardboard grid frame (12×13=156 squares). Temperature and humidity control during cocooning is achieved using a temperature and humidity control room designed and built by Nanjing Tederic Environmental Engineering Co., Ltd. (temperature accuracy 0.1℃, humidity accuracy ±3%).

[0027] Example 1

[0028] By adjusting the ambient temperature conditions at different stages of silk spinning, the silkworm's spinning behavior was controlled. The cocooning process involved alternating between 23℃±1℃ for 5 hours and 12℃±2℃ for 1 hour, with 10 groups rotating throughout. The humidity was maintained at 60%±5% throughout the process. Each group worked for 6 hours, completing one layer, for a total of 60 hours, resulting in ten thin cocoon layers. Cocoons were harvested on the 7th day after cocooning was completed.

[0029] Compare with Example 1 Based on the commonly used cocooning temperature of 24±1℃ and humidity of 70%±5%, conventionally raised silkworms were subjected to cocooning treatment for 50 hours, and the cocoons were harvested on the 7th day after cocooning was completed.

[0030] Compare with Example 2 The cocoons were harvested on the 7th day after the cocooning process was completed. The cocoons were harvested in 10 alternating groups at 23℃±1℃ for 5 hours and 12℃±2℃ for 1 hour. The humidity was maintained at 70%±5% throughout the process.

[0031] Compare with Example 3 Based on the commonly used cocooning temperature of 24±1℃ and the humidity of 60%±5%, conventionally raised silkworms were treated with cocooning for 50 hours. The cocoons were harvested on the 7th day after cocooning and then processed in layers.

[0032] Take the oval-shaped silkworm cocoons prepared according to the above method, cut off about 10% of the top of each end of the long diameter of the oval silkworm cocoon with a craft knife, cut open the abdomen of the cocoon to remove the silkworm pupa and larvae's molted skin, and observe the results as shown in Table 1.

[0033] The results showed that the method of the present invention can achieve layering, while the control silkworm cocoons are not layered or layering is not easy. The cocoon silks are intertwined and dense, unable to form a 3D structure, and cells cannot attach, so they cannot be used for cell culture. Figure 1 An inverted microscope image of the 3D cell culture scaffold (thin cocoon layer) fabricated in Example 1; Figure 2 The image shows an SEM image of an existing silkworm cocoon (comparison example 1).

[0034] Example 2

[0035] Take the oval-shaped multi-layered silkworm cocoon prepared in Example 1, and use a craft knife to cut off about 10% of the top of each end of the long axis of the oval cocoon. Cut open the abdomen of the cocoon to remove the molted skin of the silkworm pupa and larva, thus obtaining a multi-layered thin cocoon with 10 layers. Cut the cocoon into 6 equal pieces along its long axis and tear it open as usual. One cocoon can be used to prepare 60 pieces of 3D cell culture scaffolds. The natural curvature of the cocoon layers forms a three-dimensional 3D structure. The thin cocoon layers are sterilized under high pressure at a conventional temperature (temperature 121℃, pressure 0.12MPa) for 30 minutes to obtain the 3D cell culture scaffold.

[0036] The obtained 3D cell culture scaffolds were used to culture the silkworm ovary cell line (BmN) in 50 mL slant-top culture flasks. Each flask contained 25 pieces of the 3D cell culture scaffold (placed in an overlapping tile pattern), at a ratio of 2 × 10⁻⁶. 4 pcs / cm 2 BmN cells were seeded at a density of 5 × 10⁶ cells / year and cultured in TC-100 insect medium with 10% fetal bovine serum at 27°C. 5 BmN cells were cultured with 50% fresh culture medium every 3 days. All cells were suspended with trypsin and counted using a hemocytometer. The cell counts were recorded after 3, 6 and 9 days of culture. Figure 3 The cell morphology after 9 days of culture.

[0037] Comparative Example 1 The silkworm ovary cell line (BmN) was cultured in 50 mL slant-mouth culture flasks using a commercially available 3D cell culture scaffold (JET Biotechnology, catalog number: TDD032035), at a rate of 2 × 10⁻⁶. 4 pcs / cm2 BmN cells were seeded at a density of 5 × 10⁶ cells / year and cultured in TC-100 insect medium with 10% fetal bovine serum at 27°C. 5 BmN cells were cultured with 50% fresh culture medium every 3 days. All cells were suspended with trypsin and counted using a hemocytometer. The cell counts were recorded after 3, 6 and 9 days of culture.

[0038] Comparative Example 2 The silkworm ovary cell line (BmN) was cultured in a standard 50 mL slant-top culture flask according to the formula, at a ratio of 2 × 10⁻⁶. 4 pcs / cm 2 BmN cells were seeded at a density of 5 × 10⁶ cells / year and cultured in TC-100 insect medium with 10% fetal bovine serum at 27°C. 5 BmN cells were cultured with 50% fresh culture medium every 3 days. All cells were suspended with trypsin and counted using a hemocytometer. The cell counts were recorded after 3, 6 and 9 days of culture.

[0039]

[0040] Referring to Table 2, the results showed that using the 3D cell culture scaffold of this invention, cells proliferated continuously for 9 days. Comparative Example 1 showed slightly lower cell proliferation compared to this invention, while in Comparative Example 2, the cells completely adhered to the bottom of the flask after 3 days, showed cell accumulation and apoptosis after 6 days, and cell increase was not significant after 9 days. This indicates that the 3D cell culture scaffold of this invention can be used for continuous 9-day culture, with a cell density more than three times that of Comparative Example 2. Furthermore, although the culture effect of the scaffold in Comparative Example 2 was acceptable, its price was 45 yuan per piece, while the price of each silkworm cocoon of the 3D cell culture scaffold of this invention is approximately 0.08 yuan, allowing for the production of 60 scaffolds. 25 scaffolds would only cost 0.033 yuan, and the price could be further reduced through mass production. Therefore, the product of this invention has significant market competitiveness.

[0041] silkworm( Bombyx moriThe silkworm (Sericultured in China) is an important economic insect and a poikilothermic insect, domesticated for over 5700 years. It is a holometabolous lepidopteran insect, undergoing a complete metamorphosis, progressing through egg, larva, pupa, and adult stages. Its nutrient intake primarily comes from mulberry leaves during the larval stage. Silkworms typically have five instars: 1st, 2nd, 3rd, 4th, and 5th instars. The 5th instar spins a cocoon, and raw silk is produced by reeling the cocoons, providing raw materials for the textile industry. The silk fibroin in silkworm cocoons has excellent biocompatibility and is widely used in biomedical materials. Current technologies often use fibroin to prepare three-dimensional scaffolds, which, in addition to conventional sterilization, require complex dissolution, gelation, and freeze-drying methods, resulting in the waste of other components of the cocoon. This invention utilizes the biological characteristics of the silkworm to produce multi-layered cocoons, obtaining thin cocoon layers, and is applied for the first time in cell culture. In particular, the surface of the 3D cell culture scaffold contains sericin, facilitating cell colonization while fully utilizing the cocoon. This invention demonstrates technical feasibility and a broad market potential for the production of 3D cell culture scaffolds.

Claims

1. A method for preparing a 3D cell culture scaffold, characterized in that, The process includes the following steps: cutting the silkworm cocoon to obtain a thin cocoon layer, and then sterilizing the thin cocoon layer under high temperature and high pressure to obtain a 3D cell culture scaffold; the silkworm cocoon is oval in shape; After removing both ends, cut open the cocoon abdomen, then remove the molted skin of the silkworm pupa and larva to obtain multiple thin cocoon layers. Then, layer them again to obtain thin cocoon layers. The silkworms then spin their cocoons to obtain cocoons. The temperature of the environment for the silkworms to spin their cocoons is alternated between 23℃±1℃ / 5 hours and 12℃±2℃ / 1 hour. Spinning lasts for 48 to 72 hours. The humidity of the environment for the silkworms to spin their cocoons is 60%±5%.

2. The method for preparing the 3D cell culture scaffold according to claim 1, characterized in that, In multi-layered thin cocoons, the number of thin cocoon layers ranges from 5 to 15.

3. The method for preparing the 3D cell culture scaffold according to claim 1, characterized in that, The temperature for high-temperature and high-pressure sterilization is 121±1℃, and the pressure is 0.12±0.01MPa.

4. The cell 3D culture scaffold prepared by the method described in claim 1.

5. The application of the 3D cell culture scaffold according to claim 4 in culturing cells or preparing cell culture scaffolds.

6. The application according to claim 5, characterized in that, Cell culture is a subculture of adherent cells.

7. The method for culturing cells using the 3D cell culture scaffold according to claim 4, characterized in that, The cell 3D culture scaffold is placed in a culture flask, cells are seeded, culture medium is added, and then the cells are cultured.

8. The method for culturing cells according to claim 7, characterized in that, The cell 3D culture scaffold has 1 to 30 layers.

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