Mouse tooth embryo organoid culture method
By constructing tooth germ organoids from SD rats at 18.5 days of embryonic development using mechanical separation and enzymatic digestion methods, this approach solves the problems of long research cycles, high costs, and incomplete structures in existing tooth germ research techniques. It enables efficient and low-cost simulation and differentiation of tooth germ development, providing a reliable tool for tooth regeneration research.
Patent Information
- Application Number
- CN202511097620.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-11-11
AI Technical Summary
Existing technologies for tooth germ research mostly rely on animal models or two-dimensional cell culture, which have drawbacks such as long cycles, high costs, and difficulty in simulating the in vivo microenvironment. Furthermore, organoid structures are incomplete and have weak differentiation capabilities, making it impossible to accurately simulate the tooth germ development process.
The tooth germs of the first mandibular molars of SD rats at 18.5 days of embryonic development were dissociated into single cells using mechanical separation and enzymatic digestion methods. These cells were then embedded in Matrigel matrix gel, cultured in a medium containing specific growth factors, and the tooth germ organoids were harvested after 7 days of culture for immunofluorescence staining and gene expression verification.
Successfully constructed a histological structure and cellular composition similar to natural tooth germs, with strong differentiation ability, forming a layered structure similar to the bell-shaped tooth germ, solving the problems of low number and uneven morphology of organoids, reducing costs by 60%, shortening the cycle to 7 days, and avoiding the influence of species differences.
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Figure CN120924487A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organoid culture technology, specifically a method for culturing mouse tooth germ organoids. Background Technology
[0002] Teeth are one of the most important organs in the human body. Besides their functions of chewing and pronunciation, they are crucial for facial aesthetics, interpersonal communication, and mental health. However, tooth loss or malformation, often caused by various congenital or acquired factors, severely affects chewing, pronunciation, and facial appearance. These factors include primary / permanent tooth decay, periodontal disease, trauma, congenital tooth absence, and surgical removal of various tumors. With the improvement of living standards, people are paying significantly more attention to oral health, leading to higher demands for the treatment and restoration of dental diseases.
[0003] Researchers have long studied tooth regeneration. Wang Songling et al. cultured single-celled tooth germ epithelial cells from the budding and capping stages combined with mesenchyme. The epithelial cells reconstructed the complete tooth germ development process, including the budding, capping, and bell stages. Tooth germ epithelial cells possess high plasticity and can reconstruct the tooth development process under the induction of tooth germ mesenchyme. However, this recombinant method requires epithelial cells and complete dental papillae, and is limited by the availability of tissue sources and the number of recombined tooth germs. In contrast, organoid technology utilizes the self-aggregation and multi-directional differentiation capabilities of stem cells to form micro-organoids similar to the source tissue in vitro, and can increase the number of organoids through long-term stable passage culture.
[0004] Compared to traditional two-dimensional cell culture techniques, 3D organoid culture utilizes the characteristics of cell self-aggregation, assembly, proliferation, and differentiation, and maintains stable gene inheritance by simulating cell-cell and cell-extracellular matrix interactions. Compared to animal models, organoid culture is less time-consuming, lower in cost, and more operable, while avoiding species-specific differences. Organoid culture technology serves as a crucial bridge connecting two-dimensional cell culture and animal models, enabling the complementary advantages of both systems. Organoids can stably express the physiological characteristics and differentiation levels of their original organs and tissues in vitro, forming "micro-organs" with structures and functions more closely resembling those in vivo. This allows for better simulation of organogenesis and physiological and pathological states, thus holding broad application prospects in basic and clinical research. Significant progress has been made in studying organ differentiation and development, disease models, drug screening, tissue damage repair, organ transplantation, and even tumorigenesis mechanisms. Currently, successfully constructed oral organoids include salivary glands, lingual epithelium, taste buds, and palate organoids. However, tooth germ tissues have complex structures, and a systematic method for culturing tooth germ organoids is currently lacking.
[0005] Current technologies for tooth germ research largely rely on animal models or two-dimensional cell culture, which suffer from drawbacks such as long cycles, high costs, and difficulty in simulating the in vivo microenvironment. Although there have been a few attempts at organoid culture, the lack of clarity regarding the timing of tooth germ sampling, digestion conditions, and the combination of growth factors has resulted in incomplete organoid structures, weak differentiation capabilities, and an inability to accurately simulate the tooth germ development process. Therefore, there is an urgent need for an organoid culture system that can stably construct histological structures, cellular compositions, and functions similar to natural tooth germs, providing a reliable tool for research on tooth development mechanisms, disease model construction, and regenerative therapy. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a method for culturing mouse tooth germ organoids. The method involves harvesting tooth germs from the first mandibular molars of SD rats at 18.5 days of embryonic development. The tooth germ tissue is dissociated into single cells using mechanical separation and enzymatic digestion. These cells are then embedded in Matrigel at a concentration of 150,000 cells / 50 μL and cultured in a medium containing growth factors. The medium is changed every 2-3 days. Tooth germ organoids are harvested on day 7 of culture. Paraffin sections are prepared for immunofluorescence staining to detect tooth germ markers, and RNA is extracted for verification of tooth germ development gene expression.
[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for culturing mouse tooth germ organoids, comprising the following steps:
[0008] Step 1: Obtain the mandibular first molar tooth germ from SD rats at 18.5 days of embryonic development;
[0009] Step 2: The tooth germ tissue is dissociated into single cells by mechanical shearing and compound enzyme digestion. The compound enzyme is a mixture of collagenase I, neutral protease II, and deoxyribonuclease I in a mass ratio of 2:2:1.
[0010] Step 3: Embed the single-cell suspension in matrix gel at a concentration of 150,000 cells / 50 μL;
[0011] Step 4: Add DMEM / F-12 medium containing growth factors and culture at 37℃ and 5% CO2 for 7 days;
[0012] Step 5: Harvest organoids and perform parallel histological and gene expression verification.
[0013] Preferably, the complex enzyme in step two includes: 90-110 mg of collagenase I, 90-110 mg of neutral protease II, and 90-110 mg of deoxyribonuclease I.
[0014] Preferably, the method for preparing the complex enzyme in step two is as follows:
[0015] Collagenase I: 90-110 mg was dissolved in 33.33 mL of PBS to obtain 3 mg / mL solution, and then aliquoted and stored at -20°C.
[0016] Neutral proteinase II: Dissolve 90-110 mg in 25 mL of PBS to obtain a concentration of 4 mg / mL, then aliquot and store at 4°C.
[0017] Deoxyribonuclease I: 90-110 mg was dissolved in 20 mL of PBS to a concentration of 5 mg / mL, and then aliquoted and stored at -20°C.
[0018] Preferably, the dissociation in step two occurs within a single cell:
[0019] Mechanical separation, used to cut tooth germ tissue into 1mm pieces using ophthalmic scissors. 3 size;
[0020] Enzymatic digestion was performed by adding collagenase I, neutral protease II, and deoxyribonuclease I in a 2:2:1 ratio and incubating at 37°C until the tissue dispersed into single cells.
[0021] Preferably, the single-cell embedding concentration in step two is 150,000 cells / 50 μL of Matrigel matrix gel.
[0022] Preferably, the combination and concentration of growth factors in step four are as follows:
[0023] 20 ng / mL of recombinant mouse basic fibroblast growth factor, 10 μmol / mL of Y-27632, 20 ng / mL of recombinant mouse epidermal growth factor, and 10 μg / mL of bovine insulin.
[0024] Preferably, the growth factor preparation method in step four includes:
[0025] Recombinant mouse basic fiber growth factor: Prepare a 0.5 mg / mL stock solution with pure water and store at -20℃ for a long time;
[0026] Y-27632: DMSO is prepared as a 0.5mM stock solution;
[0027] Recombinant mouse epidermal growth factor: Prepare a 1 mg / mL stock solution with pure water and store at -20℃ for a long time;
[0028] Bovine insulin: Prepare a 0.2 mg / mL stock solution with pH 2-3 HCl and store at -20℃ for a long period of time.
[0029] This invention provides a method for culturing mouse tooth germ organoids. It has the following beneficial effects:
[0030] 1. The present invention enables the cultured organoids to form a layered structure similar to the bell-shaped tooth germ on day 7, which includes epithelial cells (CK14 positive) and mesenchymal cells (Vimentin positive), and can express ameloblast marker AMELX, odontoblast marker DSPP and tooth development key factor TGF-β, which are highly consistent with the characteristics of tooth germ development in vivo.
[0031] 2. This invention clearly defines the use of tooth embryos at 18.5 days of age, a specific enzymatic hydrolysis ratio (2:2:1), and a growth factor concentration, thereby increasing the organoid formation rate to over 85% and achieving a diameter of up to 150 μm. This solves the problems of low organoid quantity and uneven morphology in traditional methods.
[0032] 3. This invention provides an in vitro model for tooth germ development research, which can be used for drug screening, gene function verification, and regenerated tooth tissue engineering. Compared with animal models, the cost is reduced by 60%, the experimental cycle is shortened to 7 days, and the influence of species differences is avoided, thus promoting the translational application of tooth regeneration research. Attached Figure Description
[0033] Figure 1 This is a schematic diagram of the optimized posterior tooth germ organoids under low magnification in this invention;
[0034] Figure 2 This is a high-powered microscopic view of the optimized posterior tooth germ organoids in this invention;
[0035] Figure 3 This is a schematic diagram illustrating the optimized number and size of posterior tooth germ organoids in this invention;
[0036] Figure 4 This is a schematic diagram of HE staining results for bell-shaped secretory tooth germs and tooth germ organoids in this invention;
[0037] Figure 5 This is a schematic diagram of the immunofluorescence staining results of epithelial cell markers for tooth germ organs in this invention;
[0038] Figure 6 This is a schematic diagram of the immunofluorescence staining results of mesenchymal cell markers for tooth germ organs in this invention;
[0039] Figure 7 This is a schematic diagram of the immunofluorescence staining results of CK14 and AMELX in tooth germ organoids in this invention;
[0040] Figure 8 This is a schematic diagram of the TGF-β immunofluorescence staining results for tooth germ organoids in this invention;
[0041] Figure 9 This is a schematic diagram of the DSPP immunofluorescence staining results for tooth germ organoids in this invention. Detailed Implementation
[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] Example:
[0044] This invention provides a method for culturing mouse tooth germ organoids, comprising the following steps:
[0045] I. Experimental Methods
[0046] 1. Experimental animals: SD rats at 18.5 days of embryonic development, 1.5 days of birth, and 3.5 days of birth.
[0047] 2. Preparations before the experiment:
[0048] (1) Matrigel was stored in a -80°C freezer. The day before the experiment, Matrigel was removed from the -80°C freezer and placed on ice to thaw before being aliquoted and stored. 30 minutes before the experiment, the aliquoted Matrigel was placed on ice for later use.
[0049] (2) Place the 200μL autoclave nozzle in a 4℃ refrigerator for pre-cooling and use.
[0050] (3) The 24-well culture plate and the complete culture medium with added growth factors were placed in a 37°C incubator for preheating and preparation.
[0051] 3. Separation and extraction of tooth germs
[0052] (1) Extraction of the mandibular first molar tooth germ from SD rats at day 18.5: Pregnant rats were euthanized after isoflurane anesthesia and immersed in povidone-iodine for 5 minutes for disinfection. After the abdomen was disinfected again with povidone-iodine, the entire uterus was placed in a 100 mm culture dish. The uterus was transferred to a laminar flow hood, the amnion and placenta were removed, and the complete single fetal rat was isolated. The fetus was rinsed with PBS. The complete head was isolated, and the mandible was completely dissected from the corner of the mouth to the temporomandibular joint under a stereomicroscope using ophthalmic forceps. Soft tissue was removed, and the mandibular first molar tooth germ was completely isolated.
[0053] (2) Extraction of mandibular first molar tooth germs from P1.5 and P3.5 SD rats: SD rats that were 1.5 days and 3.5 days old were euthanized after being anesthetized with isoflurane. The head was separated with tissue scissors. The mandible was completely separated along the upper back corner of the mouth towards the temporomandibular joint with forceps. The soft tissue in the mandibular molar area was removed, the alveolar bone was removed, and the mandibular first molar tooth germ was separated.
[0054] 4. Dental germ enzyme digestion and cell extraction
[0055] The isolated tooth germ tissue was placed in a 30 mm culture dish and washed three times with PBS supplemented with 10% penicillin and streptomycin (1000 units / mL penicillin, 1000 μg / mL streptomycin), followed by three washes with PBS without penicillin and streptomycin. The tooth germ tissue was then transferred to a new culture dish and minced into 1 mm³ pieces using ophthalmic scissors. Collagenase I, neutral protease II, and deoxyribonuclease I were added in a 2:2:1 ratio. The culture dish was placed in a 37°C incubator, and the dish was shaken every 10 minutes, with the tooth germ tissue being pipetted to ensure complete digestion. Microscopic observation was possible during digestion. Once the tooth germ tissue had been digested into single cells, digestion was terminated using complete culture medium (DMEM / F-12 medium containing 10% fetal bovine serum). After mixing, the cell suspension was filtered through a 70 μm cell sieve to remove any incompletely digested tissue.
[0056] 5. Cell counting:
[0057] (1) Preparation of cell suspension: After the tooth germ tissue was digested and filtered, it was centrifuged at 1000 rpm / min for 5 minutes, the supernatant was removed, and 1 mL of complete culture medium was added to resuspend the cells.
[0058] (2) Cell counting: Place the coverslip on a clean and dry cell counting plate, blow the cell suspension to mix it, and take 10 μL of cell suspension and pour it into the cell counting plate along the edge of the coverslip. Count all cells in the four large squares (each large square contains 16 small squares) under a microscope.
[0059] (3) Cell density calculation: Cell count in cell suspension / mL = Cell count in four large squares / 4 × 10 4 .
[0060] 6. Dental germ organoid culture
[0061] (1) After cell counting, centrifuge at 1000 rpm / min for 5 minutes, remove the supernatant, and calculate the number of organoid wells based on the cell counting results at 150,000 cells / well.
[0062] (2) Calculate the required Matrigel based on a cell concentration of 150,000 / 50 μL. Resuspend the tooth germ cells in the calculated Matrigel. During resuscitation, try to avoid the formation of air bubbles. Place on ice for later use.
[0063] (3) Take out the preheated 24-well culture plate and the pre-cooled 200μL autoclave tip. Using the pre-cooled 200μL autoclave tip, draw up 50μL of cell suspension per well and quickly drop it vertically into the center of the preheated 24-well culture plate, forming an arched droplet. After standing for 1 minute, invert the 24-well culture plate containing the cell suspension and place it in a 37°C incubator to allow the Matrigel to completely solidify.
[0064] (4) After 30 minutes, remove the culture plate and observe the solidification of the Matrigel matrix. After complete solidification, add 500 μL of complete culture medium containing growth factors to each well (the culture medium is preheated in a 37°C incubator for 30 minutes) and observe the cell distribution in the Matrigel matrix under a microscope.
[0065] (5) Observe organoid growth under an optical microscope and take pictures every day. Change the culture medium every 2-3 days (preheat the culture medium in a 37°C incubator for 30 minutes).
[0066] (6) Organoid counting: Take 10 images of each well under an inverted microscope (50×).
[0067] (7) Organoid size: Take pictures of 10 wells under an inverted microscope, with 2 organoids in each well (select the largest organoid, 400×).
[0068] 7. Organoid harvesting, fixation, and embedding
[0069] (1) Place the autoclaved PBS and Matrigel cell recovery solution in a 4°C refrigerator for pre-cooling.
[0070] (2) On the 7th day of culture, tilt the 24-well culture plate, remove the culture medium from the culture plate, take 500 μL of pre-cooled PBS and slowly inject it into the 24 wells along the well wall. Gently shake the culture plate and remove the PBS. Repeat 3 times.
[0071] (3) Add 500 μL of cell recovery solution to each well (the amount of cell recovery solution is 10 times that of Matrigel), place the 24-well plate on ice, and then place the 24-well plate and ice on a shaker to promote the dissolution of Matrigel and expose the organoids.
[0072] (4) Observe the lysis of Matrigel under an optical microscope every 10 minutes. When Matrigel is completely lysed and the organoids are exposed (usually 60 minutes), transfer the organoids and cell recovery solution together to a centrifuge tube and centrifuge at 1000 rpm / min for 5 minutes.
[0073] (5) After centrifugation, remove the supernatant, add 4% paraformaldehyde solution, resuspend the organoids, and fix them at 4°C for 24 hours.
[0074] (6) Preparation of low melting point agarose: 3% low melting point agarose was prepared using sterile PBS and melted by heating in a water bath at 65°C.
[0075] (7) While the agarose solution is still warm and liquid, gently resuspend the organoids using 75 μL of agarose solution, avoiding the formation of air bubbles (although a small number of air bubbles may be unavoidable). Immediately transfer the resuspended organoids and agarose solution to a mold placed on ice (remove the tail of a 200 μL LEP tube), and the agarose will solidify immediately. If using a 200 μL or smaller pipette tip, cut off the tail to prevent damage to the organoids.
[0076] (8) After the agarose solidification, observe the number and location of organoids under an inverted microscope.
[0077] (9) Place the solidified agarose in a 4°C refrigerator for 2 hours to ensure that the agarose is completely solidified.
[0078] (10) Remove the fully solidified agarose from the mold and place it in a tissue embedding cassette for storage using 70% ethanol.
[0079] II. Experimental Results
[0080] 1. Light microscopic observation of tooth germ organoids
[0081] On day 0, cells were evenly distributed in Matrigel. After 12 hours, cells began to aggregate and form organoids, which were spherical structures. As the culture time increased, the number and size of organoids gradually increased, reaching their maximum size (150 μm) on day 6. Figure 1 , Figure 2 , Figure 3 ), followed by a layered structure similar to a tooth germ ( Figure 4 Furthermore, organoid permeability is reduced.
[0082] 2. Histological structure of tooth germ organoids
[0083] On day 7, organoids were collected from Matrigel, fixed, dehydrated, and embedded, and paraffin sections were prepared for HE staining. The tooth germ organoids were observed to be round with a relatively loose outer structure and cuboidal epithelium with a high nucleus-to-cytoplasm ratio. Figure 4 (.b Black arrow), similar to the inner enamel epithelial cells in the bell-shaped secretory phase ( Figure 4 (.a black arrow), also has a loose mesenchymal cell structure similar to that in dental papillae ( Figure 4 a, b (green arrows); the inner layer consists of tightly packed, layered epithelial cells ( Figure 4(b. red arrow) is similar to the intermediate layer of cells composed of 2-3 layers of flattened cells during the bell-shaped secretory phase. Figure 4 (a red arrow); the center is a cell-free red matrix-like substance ( Figure 4 (b yellow arrow), similar to the enamel and dentin of teeth in the bell-shaped stage ( Figure 4 (a yellow arrow).
[0084] 3. Detection of tooth germ organoid cell types
[0085] The tooth germ organoids cultured for 7 days were fixed, dehydrated, and embedded to prepare paraffin sections for immunofluorescence detection of cell markers in the tooth germ organoids.
[0086] (1) Detection of epithelial cell markers
[0087] Keratin 14 (CK14) is an epithelial cell marker. Immunofluorescence assays showed that CK14 was positive (green fluorescence) in organoids cultured for 7 days, mainly distributed in the inner layer and center of the organoids, and more abundant in the nuclei of the outer layer cells (blue fluorescence). Figure 5 This suggests that the in vitro cultured tooth germ organoids contain epithelial cells, which are mainly distributed in the inner layer of the organoid.
[0088] (2) Mesenchymal cell detection
[0089] Vimentin is a marker of mesenchymal cells. Immunofluorescence assays showed that vimentin was positive (green fluorescence) in organoids cultured for 7 days, and it was scattered throughout the organoids. Figure 6 This suggests that the in vitro cultured tooth germ organoids contain mesenchymal cells.
[0090] 4. Differentiation and functional testing of tooth germ organoids
[0091] The tooth germ organoids cultured for 7 days were fixed, dehydrated, and embedded, and paraffin sections were prepared for immunofluorescence detection of tooth germ organoid differentiation and function.
[0092] (1) Amelogenin detection
[0093] Amelogenin (AMELX) is a matrix protein of tooth enamel synthesized and secreted by ameloblasts. It is present in cell vesicles or secreted in the extracellular matrix. Immunofluorescence assay results showed that AMELX was positive (green fluorescence). CK14 was mainly distributed in the inner layer and center of the organoid, while AMELX was mainly distributed in the outer layer of the organoid. Figure 7 The presence of ameloblasts in the organoids indicates the presence of ameloblasts secreting ameloblasts. This suggests that tooth germ epithelial stem cells differentiate into ameloblasts, which have the function of secreting enamel matrix proteins.
[0094] (2) DSPP detection
[0095] Dentin sialophosphoprotein (DSPP) is mainly synthesized and secreted by odontoblasts and is currently recognized as a tooth-specific protein. DSPP has been detected as positive (red fluorescence) in cultured tooth germ organoids, mainly distributed in the outer layer of the organoid. Figure 9 This indicates that stem cells differentiate into dentin cells and synthesize and secrete DSPP.
[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for culturing mouse tooth germ organoids, characterized in that, Includes the following steps: Step 1: Obtain the mandibular first molar tooth germ from SD rats at 18.5 days of embryonic development; Step 2: The tooth germ tissue is dissociated into single cells by mechanical shearing and compound enzyme digestion. The compound enzyme is a mixture of collagenase I, neutral protease II, and deoxyribonuclease I in a mass ratio of 2:2:
1. Step 3: Embed the single-cell suspension in matrix gel at a concentration of 150,000 cells / 50 μL; Step 4: Add DMEM / F-12 medium containing growth factors and culture at 37℃ and 5% CO2 for 7 days; Step 5: Harvest organoids and perform parallel histological and gene expression verification.
2. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The complex enzyme in step two includes: 90-110 mg of collagenase I, 90-110 mg of neutral protease II, and 90-110 mg of deoxyribonuclease I.
3. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The preparation method of the complex enzyme in step two is as follows: Collagenase I: 90-110 mg was dissolved in 33.33 mL of PBS to obtain 3 mg / mL solution, and then aliquoted and stored at -20°C. Neutral proteinase II: Dissolve 90-110 mg in 25 mL of PBS to obtain a concentration of 4 mg / mL, then aliquot and store at 4°C. Deoxyribonuclease I: 90-110 mg was dissolved in 20 mL of PBS to a concentration of 5 mg / mL, and then aliquoted and stored at -20°C.
4. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The dissociation in step two occurs within a single cell: Mechanical separation, used to cut tooth germ tissue into 1mm pieces using ophthalmic scissors. 3 size; Enzymatic digestion was performed by adding collagenase I, neutral protease II, and deoxyribonuclease I in a 2:2:1 ratio and incubating at 37°C until the tissue dispersed into single cells.
5. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The single-cell embedding concentration in step two is 150,000 cells / 50 μL Matrigel matrix gel.
6. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The combination and concentration of growth factors in step four are as follows: 20 ng / mL of recombinant mouse basic fibroblast growth factor, 10 μmol / mL of Y-27632, 20 ng / mL of recombinant mouse epidermal growth factor, and 10 μg / mL of bovine insulin.
7. The method for culturing mouse tooth germ organoids according to claim 1, characterized in that, The growth factor preparation method in step four includes: Recombinant mouse basic fiber growth factor: Prepare a 0.5 mg / mL stock solution with pure water and store at -20℃ for a long time; Y-27632: DMSO is prepared as a 0.5mM stock solution; Recombinant mouse epidermal growth factor: Prepare a 1 mg / mL stock solution with pure water and store at -20℃ for a long time; Bovine insulin: Prepare a 0.2 mg / mL stock solution with pH 2-3 HCl and store at -20℃ for a long period of time.