Method for constructing tracheal cartilage microtissue and application thereof

Abstract

The application discloses a tracheal cartilage microtissue construction method and application thereof, and the method is to culture silk gum microspheres and rat chondrocytes in DMEM-F12 culture medium to obtain tracheal cartilage microtissue. The application uses silkworm cocoons as raw materials, extracts silk gum by high-temperature alkali solution method, and then prepares silk gum microspheres by emulsification and crosslinking, and prepares silk gum microsphere powder for long-term preservation by freeze-drying. The application first uses silk gum microspheres as cell microcarriers, and first constructs tracheal cartilage microtissue by co-incubation of the silk gum microspheres and rat chondrocytes. The silk gum microspheres provided by the application have good biocompatibility, can promote the adhesion, proliferation and growth of various cells, and can be used as cell microcarriers to construct various artificial organ microtissues. The artificial tracheal microtissue constructed based on the silk gum microspheres can monitor the growth of the microtissue in the body through the fluorescence characteristics of the silk gum.

Description

Technical Field

[0001] This invention relates to the field of biomedical composite materials, specifically to a method for constructing tracheal cartilage microtissue and its application. Background Technology

[0002] Tracheal defects caused by congenital malformations, trauma, and tumors are among the most common and life-threatening conditions in clinical practice. Studies indicate that the mortality rate for patients with long-segment tracheal stenosis is over 70%. Due to its unique structure, tracheal defects can often only be repaired by directly closing the anastomosis. However, when the defect exceeds 2 cm in length, the anastomosis requires extensive dissection at both ends before suturing due to increased tension. Furthermore, when the tracheal defect exceeds 6 cm in length, replacement materials are necessary for repair.

[0003] Autologous and allogeneic transplantation cannot be widely used in clinical practice due to limited donors and the risk of immune rejection. While traditional tissue engineering techniques offer a new approach to the treatment of tracheal defects, current research can only repair small, localized defects in a patchy manner, failing to repair larger segmental defects. Furthermore, traditional tissue engineering is limited by the spatial resolution of scaffold technology, resulting in slow penetration of seed cells into the scaffold material, uneven cell distribution, and restricted oxygen, blood supply, and nutrient transport. Ultimately, this leads to decreased cell viability within the 3D scaffold and a higher likelihood of necrosis.

[0004] Microscale tissue engineering is an emerging technology that assembles large, fully functional tissues using the smallest functional tissue units. Compared to traditional tissue-engineered tracheas, tissue-engineered tracheas constructed using microscale tissue engineering technology have more uniform cell distribution, more efficient oxygen, blood supply, and nutrient delivery, and a microenvironment more conducive to the growth and proliferation of seed cells. Theoretically, artificial tracheas constructed in a house-building manner are more individualized and can adapt to the future trend of precision medicine.

[0005] Tracheal cartilage is an important component of the trachea, primarily serving to support its structural structure. Due to the slow healing process of damaged cartilage, tracheal cartilage is mainly repaired through artificial cartilage.

[0006] Currently, most artificial cartilage is constructed using traditional tissue engineering, but due to the aforementioned limitations, it is difficult to meet clinical needs. Micro-tissue engineering technology should overcome the technical barriers of traditional tissue engineering, enabling the construction of artificial tracheal cartilage suitable for the repair of clinical tracheal injuries. However, there is currently no publicly available method for constructing artificial tracheal cartilage using micro-tissue engineering technology. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for constructing tracheal cartilage microtissue and its application. This invention uses a simple and easy method to construct tracheal cartilage microtissue, which forms cartilage rings. These cartilage rings are then stacked in a specific order to form the trachea, thus adapting to the future trend of precision medicine.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0009] This invention provides a method for constructing tracheal cartilage microtissue, characterized in that: the method involves co-culturing sericin microspheres with rat chondrocytes in DMEM-F12 medium to construct tracheal cartilage microtissue.

[0010] Furthermore, the concentration of the rat chondrocytes is 1×10⁻⁶. 6 -1×10 7 cells / mL.

[0011] Furthermore, the co-culture time is 72–240 hours.

[0012] Furthermore, the sericin microspheres are prepared by the following steps:

[0013] (1) Preparation of sericin:

[0014] Weigh out silkworm cocoons, cut them into pieces, add the cocoon fragments to a Na2CO3 aqueous solution and soak and stir; obtain a sericin solution, centrifuge to remove insoluble substances, then dialyze to remove residual Na2CO3, and finally freeze-dry to obtain sericin powder;

[0015] (2) Preparation of sericin microspheres:

[0016] Sericin powder was dissolved in deionized water to obtain a sericin solution. The sericin solution was added dropwise to an emulsifying and crosslinking solvent and stirred for 20-60 minutes for emulsification and crosslinking. Then, the emulsion was added dropwise to acetone, dehydrated and cured for 1-2 hours, filtered and freeze-dried to obtain sericin microspheres.

[0017] Furthermore, in step (1), the amount of Na2CO3 aqueous solution required per gram of silkworm cocoon fragment is 10-100 mL, and the concentration of the Na2CO3 aqueous solution is 0.01-0.5 mol / L;

[0018] Furthermore, in step (1), the soaking and stirring temperature is 50-100℃, and the soaking and stirring time is 0.5-2h.

[0019] Furthermore, in step (1), the dialysis liquid used is purified water, and the dialysis time is 24-48 hours.

[0020] Furthermore, in step (2), the emulsifying crosslinking solvent is corn oil containing glutaraldehyde and Span 80, wherein 2.0 to 5.0 mL of glutaraldehyde (25% (w / v) and 4.0 to 8.0 mL of Span 80 are added to every 100 mL of corn oil.

[0021] Furthermore, the concentration of the sericin microspheres is 1–4 mg / mL; the particle size of the sericin microspheres is 80–140 μm.

[0022] This invention also provides an application of the tracheal cartilage microtissue prepared by the above method in the individualized construction of artificial trachea.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention uses silkworm cocoons as raw materials, extracts sericin from them using a high-temperature alkaline solution method, prepares sericin microspheres using an emulsification crosslinking method, and prepares sericin microsphere powder for long-term storage by freeze-drying.

[0025] 2. This invention is the first to use sericin microspheres as cell microcarriers and construct tracheal cartilage microtissue by co-incubating them with rat chondrocytes.

[0026] 3. The sericin microspheres provided by this invention have good biocompatibility and can promote the adhesion, proliferation and growth of various cells. They can be used as cell microcarriers to construct various artificial organ micro-tissues.

[0027] 4. The artificial tracheal microtissue constructed based on sericin microspheres in this invention can be monitored in vivo by the fluorescence properties of sericin to monitor the growth of the microtissue. Attached Figure Description

[0028] Figure 1 This is an observation image of sericin microspheres;

[0029] In the figure, A is an optical microscope image of the sericin microspheres, and B is a statistical chart of the particle size of the sericin microspheres.

[0030] C is a scanning electron microscope image of the sericin microspheres;

[0031] Figure 2 Statistical graph of cell viability values ​​for different cell types co-cultured with sericin microspheres;

[0032] In the figure, A is a statistical chart of the relative cell viability values ​​of rat chondrocytes co-cultured with sericin microspheres for 24-72 h;

[0033] B is a statistical chart of relative cell viability values ​​after 24-72 h of co-culture of rat tracheal epithelial cells and sericin microspheres.

[0034] Figure 3An observational image of the micro-tissue of artificial tracheal cartilage;

[0035] In the figure, A shows the staining of live and dead cells in the micro-tissue of artificial tracheal cartilage after 1, 5, 7, and 14 days of culture.

[0036] B is a scanning electron microscope image of the microtissue of artificial tracheal cartilage after 14 days of culture.

[0037] Figure 4 This is a diagram showing the mRNA expression levels of Sox 9, Aggrecan, and Col II in artificial cartilage microtissue.

[0038] In the figure, A represents the expression level of sox 9 mRNA in artificial cartilage microtissue after 7, 14, and 21 days of culture.

[0039] B represents the expression levels of aggrecan mRNA in artificial cartilage microtissue after 7, 14, and 21 days of culture.

[0040] C represents the expression level of col II mRNA in artificial cartilage microtissue after 7, 14, and 21 days of culture. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0042] Example 1: A method for preparing sericin microspheres, comprising the following steps:

[0043] 1. Selection of silkworm cocoons:

[0044] The main chemical components are silkworm cocoons, sericin, and fibroin.

[0045] 2. Extraction and preservation of sericin

[0046] (1) Weigh 50g of silkworm cocoons, cut them into pieces, wash them three times with deionized water and remove the water;

[0047] (2) Soak the silkworm cocoon fragments from step 1 in 1-2 L of Na2CO3 solution with a molar concentration of 0.02 mol / L, and stir at 100℃ for 1 h to obtain sericin solution;

[0048] (3) Centrifuge the solution obtained above to remove insoluble substances;

[0049] (4) Transfer the sericin solution from step (3) into a dialysis bag with a molecular weight cutoff of 3.5 kDa, place it in ddH2O, and dialyze for a total of 48 h to remove residual Na2CO3 and obtain sericin solution.

[0050] (5.) Place the sericin liquid obtained in step (4) into a freeze dryer and freeze dry it into sericin powder, and store it in a -20℃ refrigerator.

[0051] 3. Preparation of sericin microspheres

[0052] (1) Weigh 20g of sericin powder and dissolve it in 60mL of deionized water at room temperature;

[0053] (2) Add 3.0 mL of 25% (w / v) glutaraldehyde and 5.0 mL of Span 80 to 100 mL of corn oil and mix well to obtain an emulsified crosslinking solvent;

[0054] (3) The sericin solution from step 1 was added dropwise to the emulsified crosslinking solvent and stirred for 1 hour.

[0055] (4) Add the emulsion from step 3 to 500ml of acetone, dehydrate and solidify for 1h, centrifuge and freeze-dry the precipitate to obtain sericin microspheres.

[0056] Depend on Figure 1 As shown in Figure A, the sericin microspheres appear spherical and well-dispersed under an electron microscope, proving that the sericin microspheres have been successfully prepared.

[0057] Depend on Figure 1 As shown in Figure B, the sericin microspheres have a particle size of approximately 80-140 μm, which basically meets the requirements for cell microcarriers.

[0058] Depend on Figure 1 As shown in C: the sericin microspheres have regular shapes, uniform size distribution, and rough surfaces, which are suitable for cell adhesion and growth.

[0059] Example 2: Cell viability detection of sericin microspheres

[0060] 1. Experimental Procedure

[0061] (1) First, rat chondrocytes and rat tracheal epithelial cells were cultured for 24 h at 37°C, 5% CO2, and 100% humidity using DMEM-F12 high-glucose medium.

[0062] (2) Different concentrations of sericin microspheres (0, 1, 3 mg / mL) were seeded in 96-well plates containing cells, and three parallel samples were set up for each concentration of sericin microspheres.

[0063] (3) The rat chondrocytes and rat tracheal epithelial cells collected from the cell culture flasks were resuspended and dispersed using culture medium, and then mixed with the sericin microspheres from step 2, with approximately 1 × 10⁻⁶ cells per well. 4 ;

[0064] (4) After co-culturing for 48 and 96 hours, the cell viability of each well in step 3 was detected using the CCK-8 kit.

[0065] 2. Experimental Analysis

[0066] like Figure 2 As shown, sericin microspheres did not significantly affect the cell viability of rat chondrocytes and rat tracheal epithelial cells within 72 hours, and the cells could grow and proliferate normally.

[0067] Example 3: Construction of artificial tracheal cartilage microtissue and detection of cartilage-related genes

[0068] 1. Experimental Procedure

[0069] In this embodiment, the preparation method of sericin microspheres is the same as that in Example 1. The method for constructing artificial tracheal cartilage microtissue using the prepared sericin microspheres is as follows:

[0070] (1) The density is 1×10 6 Rat chondrocytes were dispersed in 10 mL of DMEM-F12 medium at a concentration of cells / mL.

[0071] (2) Weigh 20 mg of sericin microspheres and dissolve them in the DMEM-F12 medium from step (1);

[0072] (3) Add the DMEM-F12 medium from step (2) to the low adhesion cell culture plate and replace the DMEM-F12 medium every 24 hours.

[0073] (4) After culturing for 240 hours, centrifuge to remove the culture medium in step (3) to obtain artificial tracheal cartilage microtissue;

[0074] (5) The constructed artificial tracheal microtissue was stained with live / dead cells on days 1, 5, 7 and 14 after culture and observed using a microscope.

[0075] (6) RNA was extracted from the constructed artificial tube microtissue after 7, 12 and 21 days of culture, and the expression of cartilage-related genes was detected.

[0076] 2. Experimental Analysis

[0077] (1) The results are as follows Figure 3 As shown in Figure A, the chondrocyte survival rate in the micro-tissue was high. Furthermore, on day 14 after culture, we performed electron microscopy on the micro-tissue, and the results are as follows... Figure 3 As shown in Figure B, obvious cell adhesion can be observed on the surface of the sericin microspheres. These results indicate that we have successfully constructed artificial tracheal cartilage microtissue.

[0078] (2) Results of cartilage-related gene testing, as shown in the figure Figure 4As shown in AC, after 7, 12, and 21 days of culture, the expression of cartilage-related genes (sox 9, aggrecan, col II) mRNA in the artificial tracheal cartilage microtissue was significantly higher than that in the control group, further indicating that the constructed artificial tracheal cartilage has been successfully constructed.

[0079] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing tracheal cartilage microtissue, characterized in that: This method involves mixing sericin microspheres with a concentration of 1-4 mg / mL and a particle size of 80-140 µm with a concentration of 1×10... 6 -1×10 7 Rat chondrocytes at a density of [number] cells / mL were mixed in DMEM-F12 medium, added to a low-adhesion cell culture plate, and co-cultured with the DMEM-F12 medium being replaced every 24 hours; tracheal cartilage microtissue was constructed; wherein, the sericin microspheres were prepared by the following steps: (1) Preparation of sericin: Weigh out silkworm cocoons, cut them into pieces, add the cocoon fragments to a Na2CO3 aqueous solution and soak and stir; obtain a sericin solution, centrifuge to remove insoluble substances, then dialyze to remove residual Na2CO3, and finally freeze-dry to obtain sericin powder; (2) Preparation of sericin microspheres: Sericin powder was dissolved in deionized water to obtain a sericin solution. The sericin solution was added dropwise to an emulsifying and crosslinking solvent and stirred for 20-60 minutes for emulsification and crosslinking. Then, the emulsion was added dropwise to acetone, dehydrated and cured for 1-2 hours, filtered and freeze-dried to obtain sericin microspheres.

2. The method for constructing tracheal cartilage microtissue according to claim 1, characterized in that: The co-culture time is 72-240 hours.

3. The method for constructing tracheal cartilage microtissue according to claim 1, characterized in that: In step (1), the amount of Na2CO3 aqueous solution required per gram of silkworm cocoon fragment is 10-100 mL, and the concentration of Na2CO3 aqueous solution is 0.01-0.5 mol / L.

4. The method for constructing tracheal cartilage microtissue according to claim 1, characterized in that: In step (1), the soaking and stirring temperature is 50-100℃, and the soaking and stirring time is 0.5-2h.

5. The method for constructing tracheal cartilage microtissue according to claim 1, characterized in that: In step (1), the dialysis liquid used is purified water, and the dialysis time is 24-48 hours.

6. The method for constructing tracheal cartilage microtissue according to claim 1, characterized in that: In step (2), the emulsifying crosslinking solvent is corn oil containing glutaraldehyde and Span 80, wherein 2.0-5.0 mL of glutaraldehyde and 4.0-8.0 mL of Span 80 are added to every 100 mL of corn oil.

7. The application of the tracheal cartilage microtissue prepared by the method of claim 1 in the individualized construction of artificial trachea.

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