Preparation method of hydrogel material for culturing small intestine organoids of mice
The recombinant protein hydrogel material prepared through genetic engineering combined with mouse small intestinal crypts has solved the problem of uncertain composition and insufficient mechanical properties of existing matrix materials, achieved efficient development and stability of mouse small intestinal organoids, and improved the accuracy of the experiment.
Patent Information
- Application Number
- CN202411615821.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-06-20
AI Technical Summary
Existing matrix materials, such as Matrigel, have problems with uncertain composition, potentially triggering immune responses, insufficient mechanical properties, and the presence of a large number of growth factors affecting quantitative cell experiments.
Recombinant protein molecules FIB-A and FIB-B were prepared by genetic engineering methods, bound to the mouse small intestinal crypts to form a hydrogel, and recombinant protein A-LIF was added thereto to simulate the in vivo microenvironment.
A hydrogel material with tunable composition and adjustable biological activity can better simulate the structure and environment of extracellular tissues in vivo, promote the development and stability of mouse small intestinal organoids, and improve the accuracy of the experiment.
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Figure CN120173858A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of hydrogel material preparation, and specifically relates to a preparation method of a hydrogel material for culturing mouse intestinal organoids. Background Art
[0002] Organoids refer to three-dimensional cell structures cultured in vitro that can mimic the morphology and function of real organs. Organoids reduce the need for animal experiments, provide an ideal model for studying cell differentiation, development processes, and tissue functions, and deepen the scientific community's understanding of the basic principles of biology. Organoids can also be used to study the pathogenesis of various cancers and genetic diseases, and the established disease models can be used to evaluate the efficacy and safety of drugs, significantly improving the accuracy of drug action in the human body compared to traditional two-dimensional cell culture. By using patient-derived cells to construct organoids, researchers can better understand the characteristics and progression of diseases, providing a basis for personalized medicine.
[0003] As an emerging research tool, organoids are driving the progress of biomedical research and have broad application potential. Compared with other organoids with more complex construction and culture maintenance processes, such as brain organoids, liver organoids, and lung organoids, mouse intestinal organoids are one of the earliest established organoid models and an important milestone in organoid research. The culture technology of this type of organoid is very mature and has been applied to various biomedical research, disease models, drug screening, and other applications.
[0004] In addition to requiring a medium supplemented with specific growth factors for in vitro culture of organoids, a matrix material that can mimic the native extracellular matrix environment is usually needed as a three-dimensional support environment to ensure the normal development of organoids. The most widely used matrix material at present is Matrigel, a gelatinous substance extracted from the mouse EHS tumor matrix, whose main components are collagen, laminin, fibronectin, glycosaminoglycans, and various growth factors. Matrigel has good biocompatibility and can support the attachment, proliferation, and differentiation of various types of cells. Matrigel is liquid at 4°C and rapidly solidifies into a gel at 37°C, providing a three-dimensional extracellular matrix environment to promote cell self-organization and organoid formation, mimicking the structure of real tissues. For the above reasons, Matrigel is widely used in organoid-related applications.
[0005] However, Matrigel also faces significant limitations: 1. The composition is uncertain. Since Matrigel is extracted from animal tumors, the specific composition is complex, which may lead to poor experimental reproducibility and batch-to-batch differences; 2. Mouse-derived materials may cause immune reactions and affect further clinical trials; 3. Mechanical property limitations. The mechanical properties of Matrigel cannot fully simulate the mechanical characteristics of real tissues, which may affect the structural stability of organoids; 4. It contains a large amount of growth factors, which has a great impact on quantitative cell experiments. In addition to Matrigel, some researchers also use other natural-source or chemically synthesized hydrogels such as collagen, alginate, fibrin, and chemically self-assembled materials (such as PVA-based materials) as alternative materials to Matrigel for culturing organoids. However, overall, the effect of culturing organoids is not ideal.
[0006] In view of this, the present invention is specifically proposed. Summary of the Invention
[0007] To solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:
[0008] A preparation method of a hydrogel material for culturing mouse small intestine organoids, comprising the following steps:
[0009] Step S1: Prepare recombinant protein molecules, wherein the recombinant protein includes FIB-A and FIB-B, and the two can be mixed to form a hydrogel;
[0010] Step S2: Pre-mix FIB-B with mouse small intestine crypts, and then add the resulting mixture of FIB-A dropwise to the center of the small holes in a 24-well plate, and incubate in a 37°C carbon dioxide incubator for 30 minutes to assemble and solidify to form hydrogel droplets;
[0011] Step S3: Add recombinant protein A-LIF to the recombinant protein hydrogel to better simulate the in vivo microenvironment, wherein A-EGF is a bioactive protein.
[0012] As a preferred embodiment of the present invention, in the step S1, the recombinant protein preparation steps are as follows:
[0015] S1: Plasmid construction: Select a suitable expression plasmid pQE80l, which usually contains a T7 promoter, a His tag, and an antibiotic resistance gene; clone the coding sequences of FIB-A, FIB-B, and A-EGF into the multiple cloning site of pQE80l;
[0016] S2: Plasmid amplification and extraction: Transform the constructed plasmids into Escherichia coli BL21, DE3 or other appropriate host cells respectively; Screen and culture monoclonal cells containing the target plasmid using a medium containing an appropriate antibiotic; Amplify the cell culture containing the target plasmid, usually by culturing at 37°C.
[0017] S3: Protein expression: Pre-culture: Pre-culture Escherichia coli in LB medium containing an appropriate antibiotic, usually at 37°C, until the bacterial concentration reaches an appropriate OD600 (generally about 0.6 - 0.8); Initiate expression: Transfer the pre-cultured Escherichia coli to a medium containing an appropriate amount of IPTG (isopropyl thiogalactoside), usually induce at 30°C to induce protein expression; The IPTG concentration can be optimized as needed; Culture: The duration of the induced culture depends on the stability and expression level of the target protein; Sample at different induction time points (such as 2h, 4h, 6h) to monitor the protein expression level.
[0018] As a preferred embodiment of the present invention, the steps for purifying the recombinant protein are as follows:
[0019] S1: Cell recovery: Harvest cells: Usually within 4 - 6 hours after induction, harvest Escherichia coli cells according to the expression of the target protein; Centrifuge to precipitate the cells.
[0020] S2: Protein lysis and affinity chromatography: Cell lysis: Use an appropriate cell lysis method, such as ultrasonic wave or high-pressure homogenization, to release the protein; Affinity chromatography: Use a metal chelating resin such as Ni-NTA agarose column for affinity chromatography to capture the target protein containing a His tag; Use gradient elution during the elution process, usually using a buffer containing 0.5M Imidazole to purify the protein.
[0021] As a preferred embodiment of the present invention, the steps further include dialysis to remove salt ions: Use an appropriate dialysis bag or column for buffer exchange to remove Imidazole and other salt ions.
[0022] As a preferred embodiment of the present invention, the steps further include lyophilization: Lyophilize the purified protein to remove moisture and store it for subsequent experiments or applications; Lyophilization is usually carried out at a low temperature, -80°C or lower.
[0023] The present invention has the following beneficial effects compared with the prior art:
[0024] The technology of the present invention enables a specific protein solution encapsulating mouse small intestinal crypts to self-assemble in vitro, simulating the structure and environment of the extracellular tissue in vivo, and promoting the development of crypts into mouse small intestinal organoids. The composition of this hydrogel is based on protein molecules obtained by fermentation of genetically engineered bacteria, and has excellent properties such as definite composition and adjustable biological activity, which can improve the experimental accuracy compared with ordinary animal-derived hydrogels. At the same time, this hydrogel can also carry bioactive proteins, such as extracellular proteins, growth factors, and hormones, etc., which enables this hydrogel system to better simulate in vivo conditions, mimic the environment for the development of mouse organs in vivo, and has broad potential in biological research and medical applications.
[0025] The following further describes in detail the specific implementation manners of the present invention in conjunction with the accompanying drawings. Description of the Drawings
[0026] In the accompanying drawings:
[0027] Figure 1 It is the protein sequence diagram of FIB-A, FIB-B, and A-EGF of the present invention;
[0028] Figure 2 It is the diagram of culturing mouse small intestinal organoids using the recombinant protein hydrogel of the present invention. Specific Implementation Manners
[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments in conjunction with the accompanying drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention.
[0030] Embodiment:
[0031] As Figure 1 shown, a preparation method of a hydrogel material for culturing mouse small intestinal organoids includes the following steps:
[0032] Step S1: Prepare recombinant protein molecules, where the recombinant protein includes FIB-A and FIB-B, and the two can form a hydrogel when mixed;
[0033] Step S2: Pre-mix FIB-B with mouse small intestinal crypts, and then add the obtained mixture of FIB-A dropwise to the center of the small holes in a 24-well plate, and incubate in a 37°C carbon dioxide incubator for 30 minutes to assemble and solidify to form hydrogel droplets;
[0034] Step S3: Add recombinant protein A-LIF to the recombinant protein hydrogel to better simulate the in vivo microenvironment, where A-EGF is a bioactive protein.
[0035] In the step S1, the preparation steps of the recombinant protein are as follows:
[0036] S1: Plasmid construction: Select a suitable expression plasmid pQE80l, which usually contains a T7 promoter, a His tag, and an antibiotic resistance gene; clone the coding sequences of FIB-A, FIB-B, and A-EGF into the multiple cloning site of pQE80l;
[0037] S2: Plasmid amplification and extraction: Transform the constructed plasmid into Escherichia coli BL21, DE3, or other appropriate host cells respectively; use a medium containing an appropriate antibiotic to screen and culture monoclonal cells containing the target plasmid; amplify the cell culture containing the target plasmid, usually cultured at 37 °C;
[0038] S3: Protein expression: Pre-culture: Pre-culture Escherichia coli in an LB medium containing an appropriate antibiotic, usually at 37 °C, until the bacterial concentration reaches an appropriate OD600 (generally about 0.6 - 0.8); Initiate expression: Transfer the pre-cultured Escherichia coli to a medium containing an appropriate amount of IPTG (isopropylthio-β-D-galactoside), usually induced at 30 °C to induce protein expression; The IPTG concentration can be optimized as needed; Culture: The duration of the induced culture depends on the stability and expression level of the target protein; Samples are taken at different induction time points (such as 2 h, 4 h, 6 h) to monitor the protein expression level
[0039] As Figure 1 shown, in the specific implementation manner, the recombinant protein purification steps are as follows:
[0040] S1: Cell recovery: Harvest cells: Usually within 4 - 6 hours after induction, harvest Escherichia coli cells according to the expression of the target protein; Centrifuge to precipitate the cells;
[0041] S2: Protein lysis and affinity chromatography: Cell lysis: Use an appropriate cell lysis method, such as ultrasonic wave or high-pressure homogenization, to release the protein; Affinity chromatography: Use a metal chelating resin such as Ni-NTA agarose column for affinity chromatography to capture the target protein containing the His tag; Gradient elution is used during the elution process, usually using a buffer containing 0.5 M Imidazole to purify the protein.
[0042] As Figure 1 shown, further, the recombinant protein preparation steps also include dialysis to remove salt ions: Use an appropriate dialysis bag or column for buffer exchange to remove Imidazole and other salt ions, and the steps also include lyophilization: Lyophilize the purified protein to remove moisture and store it for subsequent experiments or applications; Lyophilization is usually carried out at a low temperature, -80 °C or lower.
[0043] Hydrogel preparation
[0044] Protein dissolution: 1. Select a common buffer solution, ensuring that its pH value is within the range of 7.5 ± 0.5, including but not limited to water, PBS, DMEM medium, etc. 2. Under low-temperature conditions below 10°C, dissolve FIB-A and FIB-B proteins separately in the selected buffer solution. Usually, it takes about 10 hours to complete the dissolution process. Depending on the required gel strength, the dissolution concentration can be between 8% and 12%.
[0045] Hydrogel formation: 1. Mix the dissolved FIB-A and FIB-B proteins in a molar ratio of 1:1 and place them in an environment at 37°C for 30 minutes to obtain a hydrogel material. Ensure thorough mixing to obtain a uniform gel material. 2. In this patent, mouse intestinal crypts will be pre-mixed with the FIB-B protein solution and then the FIB-A protein solution will be added to form a 3D culture system for mouse intestinal organoids.
[0046] Selective addition of bioactive proteins: Bioactive protein A-EGF can be selectively added to the FIB-A protein solution according to needs. This can be used to improve the efficiency of mouse intestinal organoid development and maintain the active state of stem cells.
[0047] Experimental examples:
[0048] During the experiment, the culture medium was prepared according to the standard culture medium formula of ISCO9001001 - mouse intestinal organoids. The culture medium formula is as follows: 1. Basal medium DMEM / F12: glycine 18.75 mg / L, L - alanine 4.45 mg / L, L - arginine hydrochloride 147.5 mg / L, L - aspartic acid monohydrate 7.5 mg / L, L - aspartic acid 6.65 mg / L, L - cysteine hydrochloride monohydrate 17.56 mg / L, L - cystine dihydrochloride 31.29 mg / L, L - glutamic acid 7.35 mg / L, L - glutamine 365 mg / L, L - histidine hydrochloride monohydrate 31.48 mg / L, L - isoleucine 54.47 mg / L, L - leucine 59.05 mg / L, L - lysine hydrochloride 91.25 mg / L, L - methionine 17.24 mg / L, L - phenylalanine 35.48 mg / L, L - proline 17.25 mg / L, L - serine 26.25 mg / L, L - threonine 53.45 mg / L, L - tryptophan 9.02 mg / L, L - tyrosine disodium salt 55.79 mg / L, L - valine 52.85 mg / L, biotin 0.0035 mg / L, choline chloride 8.98 mg / L, calcium pantothenate 2.24 mg / L, folic acid 2.65 mg / L, nicotinamide 2.02 mg / L, pyridoxine hydrochloride 2 mg / L, riboflavin 0.219 mg / L, thiamine hydrochloride 2.17 mg / L, vitamin B12 0.68 mg / L, inositol 12.6 mg / L, anhydrous calcium chloride 116.6 mg / L, copper sulfate pentahydrate 0.0013 mg / L, iron(III) nitrate nonahydrate 0.05 mg / L, ferrous sulfate 0.417 mg / L, magnesium chloride (anhydrous) 28.64 mg / L, magnesium sulfate 48.84 mg / L, potassium chloride 311.8 mg / L, sodium chloride 6995.5 mg / L, disodium hydrogen phosphate anhydrous 71.02 mg / L, sodium dihydrogen phosphate 62.5 mg / L, zinc sulfate heptahydrate 0.432 mg / L, D - glucose anhydrous 3151 mg / L, hypoxanthine 2.05 mg / L, linoleic acid 0.03894 mg / L, lipoic acid 0.105 mg / L, phenol red 8.1 mg / L, putrescine dihydrochloride 0.081 mg / L, sodium pyruvate 55 mg / L, thymidine 0.365 mg / L, HEPES 2383 mg / L, sodium bicarbonate 2438 mg / L, L - alanyl - L - glutamine dipeptide 2 mM / L, penicillin 100,000 U / L, streptomycin 100 mg / L; 2. Additives:
[0049] 5 mL of N2 supplement, 10 mL of B27 supplement, 50 mM / L of N-Acetylcysteine, 10 mM / L of Nicotinamide; 3. Growth factors and inhibitors: 50 μg / L of Epidermal Growth Factor (EGF), 100 μg / L of Noggin, 500
[0050] μg / L of R-spondin, 100 μg / L of Wnt3a, 500 nM / L of A83-01, 10 μM / L of SB202190, 10
[0051] μM / L.
[0052] One night before the experiment, dissolve 10% FIB-B and FIB-A protein powders in mouse intestinal organoid medium and fully dissolve the proteins in a 4°C refrigerator overnight.
[0053] On the day of the experiment, the mice were euthanized, and 3-10 cm of small intestinal tissue near the stomach of the mice was sampled aseptically at 4°C. It was immersed in PBS supplemented with antibiotics such as streptomycin, penicillin, and gentamicin. The intestinal mucosa, fat, intestinal villi, etc. were removed using ophthalmic forceps. After washing three times with PBS in a petri dish (changing the petri dish each time), it was minced and cut into wide segments approximately 2 mm in length, transferred to a new petri dish, and washed twice with PBS. The washed intestinal segments were transferred to pre-cooled PBS containing 5 mmol / L EDTA for digestion and incubated at 4°C for 30 minutes. After washing twice with PBS to remove EDTA, the tissue fragments were pipetted and resuspended in pre-cooled PBS containing 0.1% BSA using a 5 mL pipette. The mechanical shear force generated during this process would separate the crypts from the basal layer. A portion of the suspension was examined under a microscope. When a large number of crypt-like structures could be seen, pipetting was stopped, and the tissue suspension after pipetting was filtered through a 70 μm filter. The filtered tissue suspension was collected and centrifuged at 300 g for 3 minutes at 4°C. The supernatant was discarded, and the crypts were resuspended for microscopic examination and counting. Every 30 μL of FBI-B solution could resuspend 400 crypts. The suspension of crypts required was calculated according to the number of hydrogels needed, centrifuged at 300 g for 3 minutes at 4°C, the supernatant was discarded, and it was placed on ice. The crypts were resuspended using the corresponding amount of FIB-B protein solution. 30 μL of the FIB-B solution containing crypts was pipetted into the center of the bottom of the wells of a 24-well plate, and then 30 μL of FIB-A protein solution was added. It was pipetted 30-50 times with a pipette gun to mix well, and care was taken to avoid generating bubbles during pipetting, otherwise it would affect the gel formation effect. The inoculated culture plate was placed in a 37°C carbon dioxide incubator (5% carbon dioxide) and incubated for 30 minutes to wait for the hydrogel to solidify. After the hydrogel had completely solidified, the pre-prepared mouse small intestinal organoid medium was slowly added along the wall, 500 μL per well, avoiding damaging the solidified gel structure. The well plate was placed in a carbon dioxide incubator for culture. The medium was changed every 3 days, and care was taken to avoid damaging the hydrogel structure. The growth status of the organoids was closely monitored. Mouse small intestinal organoids should be established within 5-7 days (as Figure 2 shown)
[0054] The protein sequences of FIB-A, FIB-B, and A-EGF are as follows:
[0055] >FIB-A
[0056] MKGSSHHHHHHVDTVYAVTGRGDSPASSAAGGSAHIVMVDAYKPTKLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELTVYAVTGRGDSPASSAAGGSAHIVMVDAYKPTKTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLDTVYAVTGRGDSPASSAAGGSAHIVMVDAYKPTKLEWKK
[0057] >FIB-B
[0058] MKGSSHHHHHHVDIPTTENLYFQTVYAVTGRGDSPASSAAGGSGAMVDTLSGLSSEQGQSGDMTI EEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDGPQGIWGQLEGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELTVYAVTGRGDSPASSAAGGSTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLVDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDTVYAVTGRGDSPASSAAGGSGPQGIWGQLEWKK
[0059] >A-EGF
[0060] MKGSSHHHHHHVDTVYAVTGRGDSPASSAAGGSAHIVMVDAYKPTKLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGENSYPGCPSSYDGYCLNGGVCMHIESLDSYTCNCVIGYSGDRCQTRDLRWWELRTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLDTVYAVTGRGDSPASSAAGGSAHIVMVDAYKPTKLEWKK。
Claims
1. A method for preparing a hydrogel material for culturing mouse small intestinal organoids, characterized in that: The steps include: Step S1: preparing a recombinant protein molecule, wherein the recombinant protein comprises FIB-A and FIB-B, and the two can be mixed to form a hydrogel; Step S2: pre-mix FIB-B with mouse small intestinal crypts, then add FIB-A to obtain a mixed solution, drop it into the center of a small hole in a 24-well plate, and incubate it in a carbon dioxide incubator at 37°C for 30 minutes to allow it to assemble and solidify to form hydrogel droplets; Step S3: adding recombinant protein A-LIF to the recombinant protein hydrogel to better simulate the in vivo microenvironment, wherein A-EGF is a biologically active protein.
2. The method for preparing a hydrogel material for culturing mouse small intestinal organoids according to claim 1, characterized in that: In step S1, the steps of preparing the recombinant protein are as follows: S1: Plasmid construction: Select an appropriate expression plasmid pQE801, which usually contains a T7 promoter, a His tag, and an antibiotic resistance gene; clone the coding sequences of FIB-A, FIB-B, and A-EGF into the multiple cloning site of pQE801; S2: Plasmid amplification and extraction: Transform the constructed plasmids into E. coli BL21, DE3 or other appropriate host cells; Use a culture medium containing appropriate antibiotics to screen and culture monoclonal cells containing the target plasmid; Amplify the cell culture containing the target plasmid, usually at 37°C; S3: Protein expression: Preculture: Preculture E. coli in LB medium containing appropriate antibiotics, usually at 37°C, until the bacterial concentration reaches an appropriate OD600; Start expression: Transfer the precultured E. coli to a medium containing an appropriate amount of IPTG, usually at 30°C to induce protein expression; IPTG concentration can be optimized as needed; Cultivation: The duration of the culture after induction depends on the stability and expression level of the target protein; samples are taken at different induction time points, such as 2h, 4h, and 6h to monitor protein expression levels.
3. The method for preparing a hydrogel material for culturing mouse small intestinal organoids according to claim 1, characterized in that: The recombinant protein purification steps are as follows: S1: Cell recovery: Harvest cells: Usually within 4-6 hours after induction, depending on the expression of the target protein, harvest the E. coli cells; centrifuge to precipitate the cells; S2: Protein disruption and affinity chromatography: Cell disruption: Use appropriate cell disruption methods, such as ultrasound or electric high-pressure disruption, to release proteins; Affinity chromatography: Use metal chelating resins such as Ni-NTA agarose columns for affinity chromatography to capture target proteins containing His tags; Use gradient elution during elution, usually using a buffer containing 0.5MImidazole to purify the protein.
4. The method for preparing a hydrogel material for culturing mouse small intestinal organoids according to claim 2, characterized in that: The step also includes dialysis to remove salt ions: Use appropriate dialysis bags or columns to perform buffer exchange to remove Imidazole and other salt ions.
5. The method for preparing a hydrogel material for culturing mouse small intestinal organoids according to claim 4, characterized in that: The steps also include freeze drying: freeze drying the purified protein to remove water and store it for subsequent experiments or applications; freeze drying is usually performed at low temperatures, -80°C or lower.