Multi-enzyme digestion system and digestion method for construction of pig lung organoid

Abstract

The invention relates to the technical field of organoids, and discloses a multi-enzyme digestion system for construction of pig lung organoids, the digestion system comprises collagenase I, collagenase II, collagenase IV, DNaseI, neutral protease and an Advanced DMEM / F12 culture medium; wherein the working concentrations of the components are as follows: the collagenase I is 400 to 500 U / mL, the collagenase II is 100 to 200 U / mL, the collagenase IV is 40 to 60 U / mL, the DNaseI is 8 to 12 U / mL, and the neutral protease is 4 to 6 U / mL. The invention provides a multi-enzyme digestion system, the system is composed of collagenase I, collagenase II, collagenase IV, DNaseI and neutral protease, the lung tissue is efficiently dissociated through the synergistic effect, and high-activity single cells or cell groups are obtained. Under the synergistic effect of the multi-enzyme digestion composition, the cell release rate and stem / progenitor cell survival rate of the pig lung tissue can be remarkably improved, a stable cell source can be provided for efficient formation of subsequent organoid, and a solid foundation is laid for construction of the pig lung organoid with complete functions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organoids, and particularly relates to a multi-enzyme digestion system and a digestion method for constructing a pig lung organoid. BACKGROUND

[0002] Organoid technology has become a key tool for biomedical research due to its ability to highly simulate the structure and function of native organs, and has shown great potential in disease modeling, drug screening, developmental biology and regenerative medicine. At present, organoid models of liver, intestine and lung and other organs have been successfully constructed and widely applied.

[0003] The respiratory system of pigs has high similarity in physiological structure and function to humans, making pig lungs an ideal object for studying human lung diseases, conducting drug screening and constructing respiratory system models. Therefore, it is of great scientific and application value to establish a high-quality pig lung organoid model.

[0004] However, the current pig lung organoid construction technology still has significant bottlenecks, and the core problem lies in the tissue digestion link. The current mainstream method mainly relies on a single trypsin digestion system. Although this system can obtain a single-cell suspension, due to the single type of enzyme and the rough action mechanism, it is easy to cause damage to the cell membrane, resulting in a significant reduction in cell activity. The consequences are twofold: on the one hand, the cell extraction efficiency is low; on the other hand, the self-renewal and self-organization ability of damaged cells are destroyed, which seriously affects the formation efficiency, structural integrity and functional stability of the subsequent organoids.

[0005] Therefore, it has become a key technical challenge in the field to develop a digestion method that can efficiently and gently dissociate pig lung tissue while maximizing the preservation of cell activity and stemness. SUMMARY

[0006] To solve the technical problems proposed in the background art, the present application provides a multi-enzyme digestion system and a digestion method for constructing a pig lung organoid.

[0007] One of the purposes of the present application is to propose a multi-enzyme digestion system for constructing a pig lung organoid, which comprises collagenase I, collagenase II, collagenase IV, DNase I, neutral protease and Advanced DMEM / F12 medium.

[0008] Among them, the working concentration of each component is: collagenase I 400-500 U / mL, collagenase II 100-200 U / mL, collagenase IV 40-60 U / mL, DNase I 8-12 U / mL, and neutral protease 4-6 U / mL.

[0009] Preferably, the concentration of collagenase I is 450 U / mL, the concentration of collagenase II is 150 U / mL, the concentration of collagenase IV is 50 U / mL, the concentration of DNase I is 10 U / mL, and the concentration of neutral protease is 5 U / mL.

[0010] Preferably, the collagenase I is from Gibco, the collagenase II, collagenase IV and neutral protease are from Solarbio, and the DNase I is from Sigma.

[0011] The second object of the present application is to provide a method for digesting pig lung tissue, which uses the above-mentioned multi-enzyme digestion system to digest the pig lung tissue, comprising the following steps:

[0012] Step 1, cutting the pig lung tissue into a homogenate;

[0013] Step 2, mixing the tissue homogenate with the multi-enzyme digestion system and incubating at 37°C for 25-35 minutes;

[0014] Step 3, after terminating the digestion, centrifuging to collect the cells and treating with a red blood cell lysis solution;

[0015] Step 4, resuspending the cells to obtain a single cell suspension.

[0016] Preferably, during the incubation in step 2, a microscope examination is performed to determine the degree of digestion and determine the timing of terminating the digestion.

[0017] Preferably, in step 3, the centrifugation condition is 300g for 5 minutes.

[0018] The third object of the present application is to provide a method for constructing a pig lung organoid, which comprises three-dimensional culturing the cells obtained after digesting the pig lung tissue using the above-mentioned digestion method, and using Matrigel and a special induction medium during the culturing process, and the culturing time is at least 7 days.

[0019] Preferably, the pig lung tissue is derived from healthy piglets, preferably 3-day-old Huoshou black pig lungs.

[0020] The fourth object of the present application is to provide an application of the multi-enzyme digestion system in preparing a pig lung organoid, constructing a disease model or a drug screening reagent.

[0021] The fifth object of the present application is to provide a pig lung organoid, which is obtained by the above-mentioned method for constructing a pig lung organoid, and the organoid has alveolus-like structure and high expression of stemness markers.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1. Digestion efficiency and cell activity significantly improved: The innovative multi-enzyme combination (collagenase I, II, IV + DNase I + neutral protease) of the present application, through synergistic effect, efficiently and gently dissociates different components in pig lung tissue (such as type I / II / IV collagen, cell junction protein, free DNA). Compared with the traditional single trypsin method, this system can significantly improve the cell yield and greatly protect the cell membrane integrity, thereby obtaining higher activity of single cells or cell clusters.

[0024] 2. Stem / progenitor cells are effectively preserved: This system is particularly beneficial to the release and survival of stem / progenitor cells. Through flow cytometry verification, the cells obtained after digestion by this system have a higher positive rate of stemness markers (such as EpCAM, CD44, etc.), providing a high-quality seed cell source for organoid formation.

[0025] 3. Enhanced organoid formation ability: The cells obtained by this system digestion in three-dimensional culture have significantly improved organoid formation efficiency, structural stability (presenting a typical alveolar-like structure), and passage stability, solving the problems of difficult organoid formation and poor morphology caused by single enzyme system.

[0026] 4. Provides a reliable platform for downstream applications: The present application successfully solves the key bottleneck in the construction of pig lung organoids, providing a stable, efficient, and repeatable technical foundation for the widespread application of this technology in lung disease mechanism research, drug toxicity and effectiveness screening, and regenerative medicine.

[0027] In summary, the present application improves the efficiency and quality of the whole process from tissue digestion to organoid construction through an optimized multi-enzyme digestion scheme, with outstanding technical progress and practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A flowchart of the digestion method proposed by the present application is provided;

[0029] Figure 2 A comparison of pig lung tissue before and after digestion and a cell suspension preparation flowchart are provided for the present application;

[0030] Figure 3 A comparison chart of cell activity and stemness marker expression of multi-enzyme and single-enzyme digestion systems for the present application is provided;

[0031] Figure 4 A comparison chart of organoid formation number and morphology structure in the embodiments of the present application is provided. DETAILED DESCRIPTION

[0032] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0033] Embodiment 1

[0034] The present embodiment proposes a multi-enzyme digestion system digestion method based on the construction of pig lung organoids, and the specific operation steps are as follows:

[0035] 1. Construction method of pig lung organoids

[0036] Healthy piglets with smaller age (such as 3-day-old Huoshou black pigs) are preferably used as donor animals. After tissue collection, the lung tissue is first subjected to disinfection treatment, and then the lung tissue block is transferred to a sterile operating environment through cutting treatment. Sterile forceps or scissors are used to remove the residual connective tissue and mucus on the surface. Then the tissue block is repeatedly washed with PBS buffer to remove impurities and free cell fragments. The washed tissue block is placed in a sterile 1.5 mL centrifuge tube (EP tube), and the tissue is cut into small fragments with an ophthalmic scissors to make it into a homogenate.

[0037] 2. Multi-enzyme digestion system

[0038] The above homogenate is evenly divided into 4 15 mL centrifuge tubes, and the multi-enzyme combined digestion system used in the present application and the control collagenase I++, collagenase II++, and collagenase IV++ are added, and incubated at 37°C water bath for 30 minutes. The specific enzyme combination and concentration are shown in the following table:

[0039] Tissue buffer components Concentration Collagenase I (Gibco) 450 U / ml Collagenase II (Sigma) 150 U / ml Collagenase IV (Sigma) 50 U / ml Neutral protease (Sigma) 5 U / ml DNase I (Sigma) 10 U / ml Advanced DMEM / F12 (Gibco) 1X

[0040] Note: Collagenase I++, collagenase II++, and collagenase IV++ are respectively a mixture of collagenase I, II, and IV, neutral protease, DNase I, and Advanced DMEM / F12, used for control experiments.

[0041] 3. Cell separation and activity evaluation

[0042] The digestion degree is observed under a microscope, and the digestion is terminated when the predetermined digestion state is reached. Centrifugation at 300g for 5 minutes, and discard the supernatant. Add red blood cell lysis solution (Solabio) for treatment, and centrifuge again at 300g for 5 minutes. Resuspend the cells with Advanced DMEM / F12, and remove the lysis solution.

[0043] The cells after multi-enzyme digestion system and three single collagenase digestion were respectively resuspended with equal amount of Advanced DMEM / F12 medium. Equal amount of cell suspension was taken, and trypan blue was added for viable cell counting to calculate the rate of viable cells. Flow cytometry was used to analyze the expression of surface markers to verify the cell stemness retention. The experimental results showed that the survival rate and positive rate of stemness markers of the cells obtained by multi-enzyme digestion system were significantly higher than those by single enzyme digestion method.

[0044] 4. Organoid construction and functional identification

[0045] The cells after multi-enzyme digestion system and single enzyme digestion were mixed with Matrigel and inoculated in culture plates, and special induction medium was used for three-dimensional culture. After 7 days of culture, the morphological structure of organoids was observed, and the number of organoids was counted. The results showed that the organoids constructed by multi-enzyme digestion system were superior to those constructed by single enzyme digestion system in terms of number, morphological stability and structural integrity.

[0046] Example 2:

[0047] This example is used to illustrate the specific composition of the multi-enzyme digestion system for constructing pig lung organoids.

[0048] The multi-enzyme digestion system comprises the following components and concentrations:

[0049] Collagenase I (Gibco): 450 U / mL

[0050] Collagenase II (Solep): 150 U / mL

[0051] Collagenase IV (Solep): 50 U / mL

[0052] DNase I (Sigma): 10 U / mL

[0053] Neutral protease (Solep): 5 U / mL

[0054] Advanced DMEM / F12 (Gibco): 1X

[0055] The enzyme combination system realizes efficient and mild digestion of pig lung tissue through the synergistic effect of each enzyme. Among them, collagenase I and II target type I / II collagen to relax the interstitial structure; collagenase IV acts on the basement membrane type IV collagen to promote the release of epithelial cells; DNase I degrades free DNA to prevent cell aggregation; neutral protease mildly hydrolyzes cell junction proteins to protect cell membrane integrity. Each component in the above concentration range (such as collagenase I 400-500 U / mL, collagenase II 100-200 U / mL, etc.) can achieve better digestion efficiency and cell activity than the traditional single enzyme method. Refer to Figure 3The differences between the multi-enzyme digestion system and three single collagenase digestion systems (Collagenase I++, II++, IV++) in cell survival rate (a) and expression rate of stemness markers (such as EpCAM, CD44, etc.) (b) were compared by flow cytometry and trypan blue staining results. The results showed that the multi-enzyme system significantly improved the cell activity and stemness retention ability, providing a better cell source for organoid construction.

[0056] Example 3:

[0057] Referring to Figure 1 This example illustrates the specific method steps for digesting pig lung tissue using the multi-enzyme digestion system:

[0058] Healthy 3-day-old Houshou black piglet lung tissue was taken after disinfection and washing, and was cut into a homogenate using ophthalmic scissors;

[0059] The tissue homogenate was divided into 15 mL centrifuge tubes, and the multi-enzyme digestion system described in Example 2 was added, and incubated at 37°C water bath for 30 minutes. The digestion degree was monitored by microscopy during incubation to determine the optimal termination time;

[0060] After digestion, centrifuge at 300g for 5 minutes, discard the supernatant, add red blood cell lysis solution (Solabio) to treat residual red blood cells, and centrifuge again at 300g for 5 minutes;

[0061] Advanced DMEM / F12 was used to resuspend the cells to obtain a high-activity single-cell suspension.

[0062] This method significantly improves the cell extraction efficiency and activity through multi-enzyme synergistic digestion and microscopy control.

[0063] Example 4:

[0064] This example illustrates the method of constructing pig lung organoids using the cells after digestion:

[0065] The single-cell suspension obtained by the multi-enzyme digestion system in Example 3 was mixed with Matrigel and inoculated into a culture plate, and a special induction medium was added for three-dimensional culture. After 7 days of culture, it was observed that the cells successfully self-organized to form pig lung organoids with typical alveolar-like structure. The organoids were stable in shape, complete in structure, and highly expressed stemness markers. The experimental results showed that the organoid formation efficiency and stability of the cells obtained using this digestion method were significantly better than those of the traditional single-enzyme digestion method. Referring to Figure 4, the comparison results of the quantity (a) and morphological structure (b) of the constructed pig lung organoids after different digestion systems are shown. The multi-enzyme digestion system is significantly better than the single enzyme digestion system in the number of organoid formation, structural integrity and alveolar-like morphology, which confirms its technical advantages in improving the efficiency and stability of organoid construction.

[0066] Example 5:

[0067] This example illustrates the effect of the multi-enzyme digestion system in related applications:

[0068] The multi-enzyme digestion system can be used to prepare pig lung organoids, which are suitable for lung disease model construction and drug screening. The cells obtained by digestion of the system show high activity and stemness retention ability, and can efficiently form organoids with stable structure and complete function, providing a reliable and efficient experimental platform for disease mechanism research, drug toxicity and effectiveness testing.

[0069] Example 6:

[0070] This example illustrates the characteristics of the pig lung organoids constructed by the method.

[0071] The pig lung organoids are constructed by the method proposed in Example 4, which has a typical alveolar-like three-dimensional structure, ordered cell arrangement, obvious luminal structure, and high expression of stemness markers (verified by flow cytometry). It still maintains the stability of morphology and function in long-term subculture, and is suitable for subsequent biological research and application.

[0072] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A multi-enzyme digestion system for pig lung organoid construction, characterized by, The digestion system comprises collagenase I, collagenase II, collagenase IV, DNase I, neutral protease and Advanced DMEM / F12 medium. The working concentrations of each component are as follows: collagenase I 400-500 U / mL, collagenase II 100-200 U / mL, collagenase IV 40-60 U / mL, DNase I 8-12 U / mL, and neutral protease 4-6 U / mL.

2. The multi-enzyme digestion system of claim 1, wherein, The concentration of collagenase I is 450 U / mL, the concentration of collagenase II is 150 U / mL, the concentration of collagenase IV is 50 U / mL, the concentration of DNase I is 10 U / mL, and the concentration of neutral protease is 5 U / mL.

3. A method for digesting pig lung tissue, characterized in that, The pig lung tissue is subjected to digestion treatment using the multi-enzyme digestion system according to any one of claims 1-2, comprising the following steps: Step 1: cutting the pig lung tissue into a homogenate; Step 2: mixing the tissue homogenate with the multi-enzyme digestion system and incubating at 37°C for 25-35 minutes; Step 3: after terminating the digestion, centrifuging to collect the cells and treating with red blood cell lysis solution; Step 4: resuspending the cells to obtain a single-cell suspension.

4. The method of claim 3, wherein the porcine lung tissue digestion is performed at a temperature of about 37°C. During the incubation in Step 2, microscopy is performed to determine the degree of digestion and determine the timing of terminating the digestion.

5. The method of claim 3 or 4, wherein the porcine lung tissue is digested by a combination of enzymes. The centrifugation condition in Step 3 is 300g for 5 minutes.

6. A method of constructing a pig lung organoid, characterized by, The cells obtained after digesting the pig lung tissue using the method according to any one of claims 3-5 are subjected to three-dimensional culture, and during the culture, Matrigel and a special induction medium are used, and the culture time is at least 7 days.

7. The method of claim 6, wherein the lung organoids are porcine lung organoids. The pig lung tissue is derived from healthy piglets, preferably 3-day-old Huoshou black pigs.

8. Use of the multi-enzyme digestion system according to any one of claims 1-2 in the preparation of pig lung organoids, disease model construction, or drug screening reagents.

9. A pig lung organoid, characterized in that, The organoids obtained by the construction method according to claim 6 or 7 have alveolus-like structures and high expression of stemness markers.

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