Rana sauteri hind limb / toe tissue cells and rapid procurement method and uses thereof

Abstract

The application discloses a kind of Vibration Mountain beard toad hind leg / toe tissue cells and its quick acquisition method and application.The method comprises: the body surface disinfection of Vibration Mountain beard individual, cut and take hind leg or toe tissue, and put back after wound disinfection;After tissue is soaked in 75% alcohol and washed with PBS containing penicillin, streptomycin and amphotericin B, it is temporarily stored in 4 ℃ temporary storage solution;After tissue is cut, it is sequentially digested with trypsin and type I collagenase;In the optimized low permeability medium, primary culture is carried out at 25-27 ℃;When cell confluence reaches 60%-70%, it is passaged using trypsin-EDTA digestion;Freezing and recovery.The application realizes the non-lethal cell of Vibration Mountain beard for the first time Quick acquisition, cell first time migration time is only 3 days, grow into monolayer only 12-17 days, pollution rate is reduced to 5.6%, recovery survival rate reaches 76.19%, and provides efficient and reliable technical support for endangered amphibian germplasm resource preservation and biological research.

Description

Technical Field

[0001] This application relates to the field of animal cell culture technology, specifically to a hind limb / toe tissue cell of the Leishan bullfrog and its rapid acquisition method and application. Background Technology

[0002] Animal cell culture is a fundamental technology in bioengineering. Currently, the most mature culture systems are concentrated in the fields of humans and mammals. The research and application of amphibian cell lines are still seriously lagging behind. The number of amphibian species for which cell lines have been established accounts for less than 0.3% of all amphibians, and is highly concentrated in a very small number of species such as the African clawed frog. Cell line resources for endangered amphibians are almost non-existent.

[0003] Compared to mammalian cell lines, the construction of amphibian cell lines faces significant challenges, primarily in cell source, cell culture conditions, and infection by bacteria, mycoplasma, and viruses. As poikilothermic animals, amphibians have an optimal culture temperature of 19-30 °C; proliferation is inhibited above 30 °C, and death occurs at 37 °C. Their body fluid osmotic pressure is low, requiring culture medium osmotic pressure equivalent to 60%-70% of standard mammalian culture medium. Furthermore, the physiological requirements of cells from different tissue sources vary significantly. In addition, this field has long been constrained by insufficient literature review, immature cryopreservation systems, and a lack of technical standards.

[0004] Preliminary explorations have been made in cell culture for rare amphibians in existing technologies. For example, Chinese patent application number CN202610250028.3 discloses "Construction Method and Application of Emei Bearded Toad Ovarian Cell Line," which successfully established the Emei Bearded Toad ovarian epithelial cell line for the first time. Through steps such as selecting metamorphosing larvae, optimizing low-osmotic culture medium, and adding cell conditioned medium, stable passage and cryopreservation of ovarian cells were achieved. However, this method and current mainstream technologies still have significant shortcomings: First, the tissue source is the ovary, which inevitably requires a lethal donor. For rare and endangered species such as the Leishan Bearded Toad, lethal sampling directly damages wild resources; second, ovarian tissue is limited to female individuals and specific developmental stages, resulting in unstable sources and poor reproducibility; third, it focuses on constructing long-term passaged cell lines, with cumbersome procedures and long cycles, failing to meet the need for rapid acquisition of viable cells, and lacking solutions for short-term temporary storage of field samples.

[0005] The Leishan bearded toad (Leptobrachium leishanense) is a Class II protected animal in China and is listed as Endangered (EN) by the IUCN. It is found only in Leigong Mountain, Guizhou Province, and its population is declining, posing a serious conservation challenge. Currently, there are no publicly available reports of successful primary cell lines constructed from Leishan bearded toad tissues.

[0006] Therefore, there is an urgent need to develop a new method that breaks through lethal sampling, achieves rapid cell acquisition and efficient amplification, and also takes into account the temporary storage of field samples, so as to provide technical support for the preservation of germplasm resources of Leymus chinensis and similar endangered amphibians. Summary of the Invention

[0007] To address the problems of lethal sampling, limited tissue sources, and long cell acquisition cycles in existing technologies, this application provides a rapid method for obtaining hind limb / toe tissue cells from the Leishan bearded toad and its applications. Through systematic condition optimization and extensive experimental exploration, the optimal culture protocol for cell growth was determined, including: a specific combination and reaction time of digestive enzymes (0.25% trypsin + type I collagenase), an optimized basal culture medium system (79% DMEM / F12 diluted medium + 20% FBS + 1% penicillin-streptomycin-amphoteric acid triple antibiotic solution), and low-temperature culture conditions (25-27 ℃). Through this technical solution, this application achieves rapid cell acquisition, reducing the initial migration time to 3 days and the time to form a monolayer to only 12-17 days, while simultaneously enabling non-lethal sampling. This provides efficient and reliable technical support for the preservation of germplasm resources of rare and endangered amphibians.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] In a first aspect, this application provides a method for rapidly obtaining hind limb / toe tissue cells from the Leishan bullfrog, comprising the following steps:

[0010] S1. Disinfect the body surface of individual Leishan bearded toads, and under aseptic conditions, cut hind limb or toe tissues. Disinfect and clean the wounds, and place the cut tissues in a temporary storage solution containing antibiotics. The temporary storage solution is prepared by DMEM / F12 basal culture medium, sterile water, and penicillin-streptomycin-amphoteric B triple antibiotic solution, with a final antibiotic concentration of 500 U / ml penicillin, 0.5 mg / ml streptomycin, and 1-2 μg / ml amphotericin B.

[0011] S2. Cut the tissue obtained in step S1 into small pieces, digest it with trypsin first, then digest it with type I collagenase, and wash it with primary culture medium to remove residual enzymes after digestion.

[0012] S3. Seed the tissue blocks treated in step S2 into a culture container, add primary culture medium, and culture them at 25-27 ℃ to obtain primary cells;

[0013] S4. When the confluence of primary cells reaches 60%-70%, digest with trypsin-EDTA digestion solution, add passage culture medium to neutralize, and passage culture in proportion.

[0014] S5. After passage, mix the cells with the cryopreservation solution and freeze them under programmed cooling. During thawing, thaw the cryopreservation tubes in a 37°C water bath, centrifuge to remove the cryopreservation solution, and then resuspend them in complete culture medium.

[0015] In some preferred embodiments, in step S1, the individual Leishan bearded toad is a tadpole at stage 40-45 or an adult; for tadpoles at stage 40-45, hind limb tissue is cut from 2 / 3 away from the end of the trunk; for adults, toe tissue is cut; after cutting, the wound is treated with 10-30 mg / L potassium permanganate solution and then released back into the wild.

[0016] In some embodiments, in step S1, the disinfection and cleaning process is as follows: before cutting the tissue, the individual Leishan bearded toad is immersed in a 10-30 mg / L potassium permanganate solution for 10-15 min to clean the body surface, and then 75% alcohol is applied to the hind limbs or toes for secondary disinfection; after the cut tissue is immersed in 75% alcohol for 20-30 s, it is washed three times with a PBS solution containing 500 U / ml penicillin, 0.5 mg / ml streptomycin, and 1-2 μg / ml amphotericin B to remove residual alcohol from the surface.

[0017] In some embodiments, in step S1, the composition of the temporary storage solution is: 60% DMEM / F12 basal culture medium, 35% sterile water, and 5% penicillin-streptomycin-amphoteric B triple antibody solution; the temporary storage temperature is 4 ℃, and the temporary storage time does not exceed 4 days.

[0018] In some embodiments, in step S2, the digestion containing the antitrypsin is performed by digesting with 0.25% trypsin for 20 min; the type I collagenase digestion is performed by digesting with 1 mg / ml type I collagenase for 40 min; and the primary culture medium used for washing after digestion contains 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B.

[0019] In some preferred embodiments, in steps S2 and S3, the primary culture medium is composed as follows: first, DMEM / F12 basal medium is mixed with sterile water at a volume ratio of 6:4 to obtain a 60% diluted DMEM / F12 basal medium; then, it is prepared by mixing the diluted medium at a volume percentage of 79%, 20% fetal bovine serum, and 1% penicillin-streptomycin-amphotericin B triple antibody solution; the osmotic pressure of the primary culture medium is 155-190 mOsm / kg; wherein, the addition of the triple antibody solution makes the final working concentrations of penicillin, streptomycin, and amphotericin B in the primary culture medium reach 100 U / ml, 0.1 mg / ml, and 0.25 μg / ml, respectively; the composition of the passage medium in step S4 and the complete medium in step S5 is the same as that of the primary culture medium.

[0020] In some embodiments, step S3 specifically involves inoculating the tissue block onto a 12.5cm substrate. 2 In the cell culture flask, the bottom of the flask is moistened with primary culture medium before inoculation. After inoculation, the culture flask is inverted and incubated overnight in an incubator at 25-27 ℃. The next day, primary culture medium is added again. The culture medium is replaced by half every three days. Cells begin to migrate out on days 3-6 and grow into a cell monolayer on days 12-17.

[0021] In some embodiments, in step S4, the mass-volume concentration of the trypsin-EDTA digestion solution is 0.25%; during subculturing, the initial inoculation is at a 1:1 ratio, followed by subculturing at a 1:2 ratio, with subculturing every 5-7 days.

[0022] In some embodiments, in step S5, the cryopreservation solution is prepared by mixing DMEM / F12 basal culture medium, sterile water, fetal bovine serum, and DMSO in a volume ratio of 3:2:3:2.

[0023] Secondly, this application provides a hind limb / toe tissue cell of the Leishan bearded toad, which is obtained by the method described in the first aspect. The karyotype of the cell is 2n=26, containing 12 pairs of autosomes and 1 pair of sex chromosomes.

[0024] Thirdly, this application provides for the use of the Leishan bearded toad hind limb / toe tissue cells described in the second aspect in at least one of the following (a)-(c):

[0025] (a) Preparation of in vitro models for amphibian biological research;

[0026] (b) Screening or evaluating compounds that act on amphibian cell function;

[0027] (c) In vitro preservation of amphibian germplasm resources.

[0028] Compared with the prior art, this application has at least the following advantages:

[0029] 1. This application establishes for the first time a non-lethal cell acquisition protocol for the Leishan bearded toad. Only a small amount of tissue needs to be collected from the hind limbs or toes to obtain viable material without endangering the individual's survival. This fills a gap in the cell resources of this endangered species and provides a replicable case study for the protective utilization of rare amphibian genetic resources.

[0030] 2. The method described in this application significantly shortens the cell acquisition cycle. The optimized culture protocol (stepwise digestion with trypsin and collagenase, low osmotic medium, and culture at 25-27 °C) reduces the time for the first cell migration to 3 days and the time to grow into a monolayer to only 12-17 days, which is much faster than other amphibians reported in this application.

[0031] 3. This application establishes a standardized aseptic operation and field sample storage system. The use of a combination of potassium permanganate and alcohol for disinfection reduces the primary culture contamination rate to 5.6%; 4°C low-temperature storage allows tissues to be effectively preserved for 8 days (optimal ≤4 days), overcoming the time difference bottleneck between field sampling and laboratory work.

[0032] 4. The cell types obtained in this application are diverse and genetically stable. The obtained cells include epithelial-like, fibroblast-like, and dendritic cells, with a recovery survival rate of 76.19%. The chromosome karyotype is 2n=26 (12 pairs of autosomes + 1 pair of sex chromosomes), which can be used for the construction of amphibian in vitro models, compound screening, and in vitro preservation of germplasm resources. Attached Figure Description

[0033] Figure 1 A flowchart illustrating the rapid acquisition of hind limb / toe tissue cells from the Leishan bullfrog, as provided in this application embodiment.

[0034] Figure 2 Results showing the effect of different disinfection methods provided in the embodiments of this application on the contamination rate of primary cell cultures of the hind limbs of Leymus chinensis.

[0035] Figure 3 The effects of different digestion methods provided in the embodiments of this application on the migration of hind limb / toe tissue cells of Leysian toad.

[0036] Figure 4 The image shows the morphological characteristics of primary cells in the hind limbs of the Leishan bearded toad, as provided in the embodiments of this application.

[0037] Figure 5 Morphological diagram of passaged cells of the hind limb of *Toadus leishanensis* provided in the embodiments of this application.

[0038] Figure 6 A morphological diagram of the hind limb cells of the revived Leishan bullfrog provided in an embodiment of this application.

[0039] Figure 7 The chromosome division phase of the Leishan bearded toad provided in the embodiments of this application is shown, where A is male and B is female.

[0040] Figure 8 The chromosome karyotype results of *Leishan musculus* provided in the embodiments of this application.

[0041] Figure 9 HE staining results of hind limb cells of Leymus chinensis provided in the embodiments of this application. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0043] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0044] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0045] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0046] In this application, when “%” is used to represent concentration or proportion, unless otherwise specified, it represents volume fraction.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following explanations of terminology are provided to better understand this application.

[0048] Stages 1.40-45: These refer to the tadpole developmental stages defined by Gosner's (1960) universal amphibian developmental stage system, corresponding to stages 40 to 45 in the Gosner classification, belonging to the late metamorphosis to the peak metamorphosis period. The external morphological characteristics of this stage are: hind limbs are fully extended and well-developed; forelimbs begin to extend or are fully extended; the tail exists but begins to be absorbed; and the tadpole gradually transitions from an aquatic to a terrestrial lifestyle. The cells and tissues at this stage exhibit vigorous proliferation and remodeling activity, making it the optimal time to obtain primary cultured tissues.

[0049] 2. Non-lethal sampling: In this application, it refers to a sampling method that collects a small amount of tissue (such as the hind limb tip or toes) for cell culture without endangering the survival of the donor individual or affecting its normal activities and growth and development. After sampling, the wound is disinfected (e.g., by applying potassium permanganate solution), and the individual is released back into the wild.

[0050] 3. Penicillin-Streptomycin-Amphotericin B Triple Antibiotic Solution: In this application, it refers to a mixed antibiotic solution containing penicillin, streptomycin, and amphotericin B. Different working concentrations are used in different steps of this application.

[0051] 4. Primary culture medium / passaged culture medium / complete culture medium: In this application, the primary culture medium, passaged culture medium and complete culture medium have the same composition. Specifically, the composition is as follows: First, DMEM / F12 basal culture medium is mixed with sterile water at a volume ratio of 6:4 to obtain 60% diluted DMEM / F12 basal culture medium; then, it is prepared by mixing 79% of this diluted culture medium, 20% fetal bovine serum and 1% penicillin-streptomycin-amphoteric B triple antibody solution, with an osmotic pressure of 155-190 mOsm / kg.

[0052] 5. First cell migration time: refers to the number of days from the start of culture after tissue block inoculation to the observation of the first cell migrating from the edge of the tissue block under a microscope. In this application, it is 3 days.

[0053] 6.0.25% Trypsin-EDTA Digestion Solution: This refers to a sterile solution containing 0.25% trypsin and 0.02% EDTA by mass / volume, and is a commercially available standard reagent in this field.

[0054] 7. Passage Ratio: This refers to the volume distribution ratio when aliquoting the digested cell suspension into new culture flasks. For example, a 1:1 passage ratio for the first passage means that one portion of the cell suspension is aliquoted into one new culture flask (removing tissue fragments from the original culture environment and removing dead cells by centrifugation; higher cell density also improves the passage success rate). A 1:2 passage ratio means that one portion of the cell suspension is evenly aliquoted into two new culture flasks (each new flask is inoculated with an equal volume of cell suspension; typically, the total volume inoculated into each new flask is equivalent to half the volume of the original flask). Those skilled in the art can adjust the actual aliquot volume within the stated ratio range according to the bottom area of ​​the culture flask, the cell growth state, and experimental requirements.

[0055] 8. Karyotype formula: In this application, the karyotype of the hind limb / toe tissue cells of the Leishan bearded toad is diploid, with a chromosome number of 2n=26, including 12 pairs of autosomes and 1 pair of sex chromosomes.

[0056] 9. Programmed cooling: This refers to a method of controlling the freezing rate, usually using a programmed freezing box or a controlled rate freezer to slowly cool the cells to below -80 °C at a rate of about -1 °C / min, and then transfer them to liquid nitrogen for long-term storage.

[0057] This application addresses the technological gap in germplasm resource conservation and cell acquisition for the rare and endangered amphibian, the Leishan bearded toad, by proposing a non-lethal, rapid, and efficient method for obtaining hind limb / toe tissue cells. This application integrates a series of key technologies, including non-invasive sampling (collecting only a small amount of tissue from the hind limbs or toes, without affecting individual survival and activity), combined disinfection (potassium permanganate + alcohol for synergistic sterilization, reducing contamination rates), cryogenic storage (4°C storage can maintain tissue viability for up to 8 days, resolving the time difference between field sampling and experimentation), stepwise enzymatic digestion (stepwise treatment with trypsin + collagenase to accelerate cell migration and maintain viability), and optimized low-osmotic culture medium (60% DMEM / F12 + 40% sterile water, osmotic pressure 155-190 mOsm / kg, adapted to amphibian physiological needs). This achieves highly efficient cell acquisition, with initial migration within 3 days and cell monolayer formation within 12-15 days, and establishes a complete technical system from tissue collection, storage, culture, passage to cryopreservation and thawing. The procedure for rapidly obtaining hind limb / toe cells from *Toad leptostroboides* provided in this application embodiment is as follows: Figure 1 As shown, the specific operations and condition optimizations are detailed in the following embodiments.

[0058] The main materials and reagents used in the following examples are shown in Table 1 below:

[0059] Table 1. Information on the main materials and reagents used in the examples.

[0060]

[0061] The following are specific examples:

[0062] Example 1: Disinfection of Body Surface and Tissue Acquisition

[0063] This embodiment illustrates the methods for surface disinfection, aseptic sampling, and temporary storage of the hind limb / toe tissues of the Leishan bullfrog.

[0064] 1.1 Donor selection and sampling site

[0065] Tadpoles (stages 40-45) or adults of the Leishan bearded toad (Leptobrachium leishanense) were used as donors. For tadpoles (stages 40-45), hind limb tissue was harvested from the distal two-thirds of the body; for adults, toe tissue was harvested. After harvesting, the wounds were treated with a 10-30 mg / L potassium permanganate solution, and the individuals were released back into the wild.

[0066] 1.2 Body Surface Disinfection Procedure

[0067] To screen the optimal sterilization protocol for primary cell culture of the hind limbs / toes of *Toadus leishanensis*, this example used different sterilization methods and statistically analyzed the contamination rate during primary culture. After 15 days of culture, contamination was observed and recorded regularly. The results are shown in Table 2. Figure 2 As shown.

[0068] Table 2. Effects of different disinfection methods on the contamination rate of primary cell cultures of the hind limbs of *Toad leptospira*.

[0069]

[0070] From Table 2 and Figure 2 The results showed that contamination mostly occurred 3-9 days after the start of cultivation, after which the degree of contamination slowed down. Specifically:

[0071] (1) The blank control group, which had not undergone any cleaning treatment, was the most contaminated. By day 3 of culture, the contamination rate had reached 70.8%; by day 6, the contamination rate had reached 100%. Without disinfection intervention, it was virtually impossible to obtain the target cells.

[0072] (2) Soaking tissues in 75% ethanol for 15-20 seconds still resulted in approximately 1 / 8 of the tissues remaining contaminated. This indicates that simply relying on short-term alcohol soaking may not completely remove latent microorganisms from the inner layers of the tissue. Therefore, potassium permanganate was introduced as a combined sterilization agent. As a strong oxidizing agent, it has bactericidal and disinfecting properties.

[0073] (3) First, soak the tissue surface in a 10-30 mg / ml potassium permanganate solution for 10-15 min, then soak the tissue in 75% ethanol for 15-20 s to achieve deep disinfection from the surface to the interior. The effect of this combined disinfection method is extremely significant. In 16 parallel groups, no contamination occurred on the third day. The final contamination rate remained stable at 5.6%. This indicates that the combined disinfection of potassium permanganate and ethanol can greatly reduce the probability of contamination during primary culture.

[0074] (4) The control group that only used potassium permanganate solution to soak the body surface for 15 min (without alcohol treatment) showed that although potassium permanganate alone could reduce the contamination rate, the contamination rate of the combined alcohol treatment group was always maintained at a lower level (5.6%).

[0075] Based on the above optimization results, the optimal disinfection method adopted is:

[0076] Tadpole hind limb disinfection: Soak 40-45 stage tadpoles in a 10-30 mg / L potassium permanganate solution for 10-15 minutes to clean their body surface, and then apply 75% alcohol to the hind limbs for secondary disinfection.

[0077] Disinfecting the toes of adult toads: Disinfect the toes of wild Leishan toads with 75% alcohol.

[0078] 1.3 Initial Organizational Processing

[0079] Immerse the excised hind limb or toe tissue in 75% alcohol for 15-20 seconds. After removal, wash the tissue three times with PBS solution containing 500 U / ml penicillin, 0.5 mg / ml streptomycin, and 1-2 μg / ml amphotericin B to remove residual alcohol from the surface.

[0080] 1.4 Organization Temporary Storage

[0081] The cleaned tissue was placed in a temporary storage solution. The temporary storage solution consisted of 60% DMEM / F12 basal medium, 35% sterile water, and 5% penicillin-streptomycin-amphoteric B triple antibody solution (the final working concentration of the triple antibody solution in the temporary storage solution was: penicillin 500 U / ml, streptomycin 0.5 mg / ml, and amphotericin B 1-2 μg / ml).

[0082] 1.4.1 Temporary storage temperature screening

[0083] To determine a suitable temporary storage temperature, this study compared the preservation effects of 4 ℃ low-temperature storage and room-temperature storage on the hind limb tissue of *Toadus leishanensis*, based on the aforementioned temporary storage solution. The results of continuous observation for 10 days on the clarity of the temporary storage solution, the time of turbidity appearance, and the tissue condition are shown in Table 3.

[0084] Table 3. Effects of different temporary storage temperatures on the preservation of hind limb tissues of the Leishan bullfrog.

[0085]

[0086] As shown in Table 3, under 4 ℃ low-temperature conditions, the temporary storage solution remained clear until day 8, became cloudy on day 9, and failed to be stored on day 10. Under normal temperature conditions, the temporary storage solution only remained clear until day 5, gradually becoming cloudy on days 6-7, resulting in storage failure. This indicates that 4 ℃ low-temperature storage can significantly extend the effective preservation time of tissues. Therefore, 4 ℃ was selected as the optimal temperature for tissue storage.

[0087] 1.4.2 Optimization of temporary storage time

[0088] After determining 4 °C as the optimal storage temperature, this embodiment further optimized the storage time. Tissues were stored at 4 °C for 1 to 8 days, then minced and digested with a combination of enzymes according to step S2 of this application, and then cultured in primary form according to step S3 (see steps S2 and S3 for specific operations). The effect of different storage times on tissue viability was evaluated by observing the tissue block migration rate and cell migration rate. The results are shown in Table 4.

[0089] Table 4. Effects of different storage times on cell migration from the hind limb tissues of *Toad leptospira leishanensis*.

[0090]

[0091] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 20%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0092] As shown in Table 4, the risk of contamination of tissue blocks gradually increases with the extension of storage time, while the cell migration ability decreases. It is advisable to keep the storage time to no more than 4 days.

[0093] Example 2: Tissue Processing and Enzymatic Digestion

[0094] This embodiment illustrates the processing of tissue blocks and the screening and determination of combined enzyme digestion methods.

[0095] 2.1 Tissue mincing

[0096] The temporarily stored tissue was removed from the storage solution and washed with an antibiotic-containing washing solution. The washing solution was a PBS solution containing 300 U / ml penicillin, 0.3 mg / ml streptomycin, and 0.5-1 μg / ml amphotericin B. After washing three times, the tissue was cut into pieces approximately 1 mm in size using sterile ophthalmic scissors. 3 A tissue block of a certain size.

[0097] 2.2 Combined Enzyme Digestion

[0098] 2.2.1 Digestion Method Screening Experiment

[0099] To optimize the efficiency of obtaining primary cells from the hind limbs / toes of *Toadus leishanensis*, this study compared the initial migration time, migration duration, and relative migration amount of cells from tissue blocks under different digestion conditions. Hind limb tissues, after disinfection and cleaning as described in Example 1, were divided into the following four treatment groups: ① No enzyme digestion treatment (blank control group); ② Digestion with type I collagenase alone (1 mg / ml, 1 h); ③ Digestion with 0.25% trypsin alone (1 h); ④ Digestion with a combination of 0.25% trypsin and type I collagenase (0.25% trypsin digestion for 20 min + type I collagenase digestion for 40 min). After the corresponding digestion treatment, the tissues from each group were cultured in primary culture according to the method described in Example 3. Cell migration was observed and recorded, and the results are shown in Table 5. Figure 3 As shown.

[0100] Table 5. Effects of different digestion methods on the migration of hind limb / toe tissue cells from *Toad leptostroboides*.

[0101]

[0102] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 20%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0103] The results in Table 5 show that:

[0104] (1) As a blank control, no cell migration was observed in the hind limb tissue without any enzyme digestion treatment during the entire culture period (culture cycle of 30 days). This indicates that without the aid of enzyme digestion to destroy the extracellular matrix, fibroblasts and other cells are unlikely to spontaneously detach from the dense tissue.

[0105] (2) Using type I collagenase alone to digest tissues cannot achieve the result of obtaining cells.

[0106] (3) Digested with 0.25% trypsin alone for 1 hour, a small number of cells were observed to migrate out on the third day of culture. However, as the culture time was extended, these cells gradually detached from the cell wall and became suspended, failing to achieve stable proliferation.

[0107] (4) The combination of 0.25% trypsin and type I collagenase showed the best performance in terms of cell migration speed, migration duration, and final cell yield. The digestion time of the two enzymes was optimized and determined based on the tissue density and the results of previous tests. Cell migration was observed on the 3rd day of culture, and a cell monolayer (70% or more) was formed after 15 days, reaching the scale for passage culture.

[0108] The above results indicate that different digestion methods significantly affect cell migration. The superior performance of the combined enzyme digestion method is due to the following: firstly, trypsin rapidly digests intercellular connection proteins, initially loosening the tissue mass; then, type I collagenase further degrades collagen fibers in the extracellular matrix, accelerating cell migration. This step-by-step combination ensures digestion efficiency while avoiding excessive cell damage caused by prolonged treatment with a single enzyme (especially trypsin), thus maximizing the preservation of the viability and proliferative capacity of migrating cells.

[0109] Based on the above screening results, the optimal digestion plan is determined as follows:

[0110] Add 0.25% trypsin to the chopped tissue pieces and digest at 25-27℃ for 20 min. Discard the trypsin digest and add 1 mg / ml type I collagenase, then digest at 25-27℃ for 40 min.

[0111] 2.3 Post-digestion cleaning

[0112] After digestion, the tissue was washed three times with primary culture medium to remove residual enzymes from the surface. The primary culture medium was prepared by first mixing DMEM / F12 basal medium with sterile water at a volume ratio of 6:4 to obtain a 60% diluted DMEM / F12 basal medium; then, it was prepared by mixing this diluted medium at 79% by volume, 20% fetal bovine serum, and 1% penicillin-streptomycin-amphoteric B triple antibiotic solution. The final working concentrations of the antibiotics in this primary culture medium were: penicillin 100 U / ml, streptomycin 0.1 mg / ml, and amphotericin B 0.25 μg / ml, with an osmotic pressure of 155-190 mOsm / kg.

[0113] Example 3 Primary Culture

[0114] This embodiment illustrates the culture medium selection and adherence culture method for primary culture of hind limb / toe tissues of Leymus chinensis.

[0115] 3.1 Screening of basic culture medium types

[0116] To optimize the nutritional conditions for primary cell culture of the hind limbs / toes of *Toadus leishanensis*, this study compared the effects of three different basal culture media (DMEM, DMEM / F12, and L-15) on cell migration and monolayer formation time, based on a fixed digestion method (0.25% trypsin + type I collagenase digestion). All three media were prepared using the same method: first, the basal culture medium was mixed with sterile water at a volume ratio of 6:4 to obtain a 60% diluted basal culture medium; then, this diluted medium was mixed with 79% (v / v) of the diluted medium, 20% fetal bovine serum, and 1% penicillin-streptomycin-amphoteric B triple antibody solution. The results are shown in Table 6.

[0117] Table 6. Effects of different culture media types on the migration and cell growth of hind limb tissue blocks from *Toad leptospira*.

[0118]

[0119] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 20%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0120] As shown in Table 6, the culture system containing DMEM / F12 performed best, with cell migration observed as early as day 3 and remaining stable until day 15, at which point cell confluence exceeded 70%. In the L-15 culture group, more cells migrated around tissue blocks on day 3, but the persistence of cell migration was not as good as in the DMEM / F12 group, and there were more apoptotic floating cells in the solution. In the DMEM culture group, cells were only observed around a few tissue blocks, and the cell size did not increase over time. Therefore, DMEM / F12 was selected as the optimal basal medium.

[0121] 3.2 Optimization of culture medium osmotic pressure

[0122] Based on the determination that DMEM / F12 is a suitable basal medium, this example further optimized the osmotic pressure conditions of the medium. Considering that amphibians are poikilothermic aquatic / semi-aquatic animals, their somatic cells have adapted to a lower osmotic pressure environment (usually lower than 280-320 mOsm / kg for mammalian cell culture), this experiment compared four different dilution ratios (basal medium: sterile water), following the same preparation principle (first dilute the basal medium, then add FBS and antibiotics at a ratio of 79%:20%:1%), to investigate the effect of osmotic pressure on the migration of primary cells from the hind limbs of *Toadoptera litura*. Cell confluence was observed on day 15, and the results are shown in Table 7.

[0123] Table 7. Effects of different osmotic pressures on the migration of primary cells from the hind limbs of *Toad leptospira*.

[0124]

[0125] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 20%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0126] As shown in Table 7, the optimal culture results were obtained when the osmotic pressure was 155-190 mOsm / kg, with cell confluence exceeding 70% by day 15. When the culture medium concentration was increased to 65% (osmotic pressure 170-210 mOsm / kg), the final cell migration decreased, and an effective cell monolayer failed to form; similarly, no monolayer formed was achieved when the osmotic pressure increased to 180-225 mOsm / kg. This indicates that cell migration is significantly inhibited by increasing osmotic pressure. These results suggest that the optimal osmotic pressure range is 155-190 mOsm / kg, corresponding to a DMEM / F12 to sterile water volume ratio of 60:40.

[0127] Based on the screening results in sections 3.1 and 3.2 above, the optimal formulation of the primary culture medium was determined as follows: First, DMEM / F12 basal medium was mixed with sterile water at a volume ratio of 6:4 to obtain a 60% diluted DMEM / F12 basal medium; then, this diluted medium was prepared by mixing 79% (v / v) of this diluted medium, 20% fetal bovine serum, and 1% penicillin-streptomycin-amphoteric B triple antibiotic solution. The final working concentrations of the antibiotics in this primary culture medium were: penicillin 100 U / ml, streptomycin 0.1 mg / ml, and amphotericin B 0.25 μg / ml, with an osmotic pressure of 155-190 mOsm / kg.

[0128] 3.3 Inoculation and Adherence

[0129] Tissue blocks digested and cleaned in Example 2 were pre-wetted to a depth of 12.5 cm with the aforementioned primary culture medium. 2 Place the tissue block evenly on the bottom of the cell culture flask, invert the flask, and incubate overnight (approximately 24 hours) at 25-27°C to allow the tissue block to adhere fully. The next day, add sufficient primary culture medium to the flask, invert the flask, and continue culturing at 25-27°C. Replace half of the primary culture medium every three days.

[0130] 3.4 Primary Culture Results

[0131] Observe under a microscope during days 3-6 of culture. Figure 4 As shown, cells begin to migrate from the edge of the tissue block. The morphology of the migrating cells includes irregular polygonal (epithelial-like cells), spindle-shaped, or triangular (fibroblast-like cells). By days 14-17 of culture, the cell confluence reaches more than 70%, forming a cell monolayer.

[0132] In this embodiment, primary culture of hind limb / toe tissues was performed using six culture flasks, and epithelial cells and fibroblasts were successfully obtained in all flasks without contamination. A total of four tubes of primary cells were cryopreserved. The migration results of cells from various tissues of *Toadoptera litura* are shown in Table 8 below.

[0133] Table 8. Statistics on cell migration from various tissues of *Toad leptospira*.

[0134]

[0135] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 20%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0136] Example 4: Subculture

[0137] This example illustrates the method for passage, purification, and amplification of primary cells.

[0138] 4.1 Timing of propagation and digestion

[0139] When the confluence of primary cultured cells reaches 60%-70%, passage them. Discard the old culture medium, add 0.25% trypsin-EDTA digestion solution, gently shake to cover the cell layer with the digestion solution, and incubate at 25-27 ℃ for 1-2 minutes. Observe under a microscope; when most cells become rounded and detach, add 1-2 mL of passage culture medium to stop the digestion.

[0140] 4.2 Subculture inoculation

[0141] Centrifuge the cell suspension at 800 rpm for 8 min, discard the supernatant, and resuspend the cells in passage medium. For the first passage, seed at a 1:1 ratio, and thereafter at a 1:2 ratio. Continue culturing in an incubator at 25-27 ℃. Passage every 5-7 days.

[0142] 4.3 Results of subculture

[0143] like Figure 5 As shown, the cells exhibit strong three-dimensional morphology after passage, displaying a peak-and-valley pattern. The cell growth rate decreases slightly with each passage, but the cell morphology remains good at passage P4, allowing for further passage and expansion.

[0144] Example 5: Cell Cryopreservation and Thawing

[0145] This example illustrates the cryopreservation protection of cells and the detection of cell viability after thawing.

[0146] 5.1 Preparation of cryopreservation solution

[0147] The cryopreservation solution is prepared by mixing DMEM / F12 basal culture medium, sterile water, fetal bovine serum, and DMSO in a volume ratio of 3:2:3:2.

[0148] 5.2 Cryopreservation Procedures

[0149] Cells in good growth condition with confluence exceeding 70% (e.g., P3 generation) were digested and centrifuged according to the method in Example 4. The cells were resuspended in an appropriate amount of primary culture medium, and the cell suspension was mixed with cryopreservation solution at a 1:1 volume ratio and aliquoted into cryovials. The cryovials were placed in a programmed cryopreservation box and stored at -80 °C overnight to achieve programmed cooling (approximately -1 °C / min). The following day, the cryovials were transferred to liquid nitrogen for long-term storage.

[0150] 5.3 Recovery Operations

[0151] Remove the cryovials from liquid nitrogen and quickly transfer them to a 37°C water bath, shaking rapidly to thaw completely within 1-2 minutes. Add an appropriate amount of pre-cooled (4°C) primary culture medium to a centrifuge tube, then transfer the entire thawed cell suspension to the centrifuge tube and mix well. Centrifuge at 800 rpm for 8 minutes and carefully discard the supernatant. Resuspend the cell pellet in fresh complete culture medium (with the same composition as the primary culture medium), seed it into culture flasks, and incubate at 25-27°C.

[0152] Figure 6 This is a microscopic image of the hind limb cells of the Leishan bearded toad after thawing. As can be seen from the image, the cells after cryopreservation and thawing have good morphology and strong three-dimensionality.

[0153] 5.4 Determination of resuscitation survival rate

[0154] The cell viability after resuscitation was detected using trypan blue staining, and four parallel experiments were set up. The results are shown in Table 9 below. As can be seen from the table, the average total cell density of the hind limb cells (P3) of *Toadus leishanensis* after resuscitation was 2.08 × 10⁻⁶. 5 Cells / ml, average viable cell density is 1.53 × 10⁻⁶ 5 The number of cells / ml was 76.19%, indicating good survival.

[0155] Table 9. Cell survival rate of hind limb cells after resuscitation from *Toad leptospira*.

[0156]

[0157] Example 6: Identification of Cell Biological Characteristics

[0158] In this embodiment, the karyotype and staining of the obtained Leishan bullfrog hind limb / toe tissue cells were identified.

[0159] 6.1 Chromosome karyotype analysis

[0160] (1) Take cells in good growth condition, add colchicine to the culture medium to a final concentration of 0.1 μg / mL, and treat under normal culture conditions for 2-4 hours.

[0161] (2) Add 0.25% trypsin to digest the cells into a single-cell suspension. Drop the suspension onto a glass slide and add 2-3 drops of 0.4% KCl hypotonic solution to dissolve the cells. After removing insoluble impurities, add hypotonic solution again to cover the glass slide. Transfer the glass slide to a large glass dish and support it with a glass rod to keep it horizontal. Allow it to stand under hypotonic conditions for 30 minutes.

[0162] (3) First fixation: Prepare a mixed fixative solution with the composition of ethanol:acetic acid:water = 1:2:3 (volume ratio). Add the fixative solution to the culture dish containing the glass slide, ensuring that the fixative solution fills the bottom of the culture dish but does not touch the glass slide. Cover the culture dish and perform steam fixation at 24-28 °C for 120 min;

[0163] Second fixation: After steam fixation, the first fixative solution at the bottom of the petri dish is aspirated and replaced with anhydrous ethanol for a second fixation time of 30 min.

[0164] Water curtain fixation: After the second fixation, remove the slide from the petri dish and tilt it to allow the hypotonic solution to drain away. While keeping the slide tilted, pour a fixative solution of ethanol:acetic acid = 1:2 (volume ratio) from the top of the slide, ensuring the fixative covers the entire slide and flows down. Repeat this process 3-4 times. After fixation, allow the slide to air dry naturally.

[0165] (4) Staining and observation: Stain the slide with 10% (v / v) Giemsa stain for 30 min, rinse off the excess stain with running water, and air dry.

[0166] (5) Karyotype analysis: Observe and photograph clear metaphase chromosome divisions under an oil immersion microscope.

[0167] Results: Using the above method, clear images of male and female chromosome division were successfully obtained, such as... Figure 7 As shown. Microscopic observation and photography were used to measure the chromosomes of *Leishan muscularis*. The results indicate that the chromosome number in the hind limb / toe tissue cells of *Leishan muscularis* is 2n=26, containing 12 pairs of autosomes and 1 pair of sex chromosomes, as shown... Figure 8 As shown. Karyotype analysis confirmed that the karyotype obtained according to the method of this application was stable and no abnormal chromosomes were found.

[0168] 6.2 HE staining identification

[0169] To morphologically identify the cell types of the ovarian hindlimbs of the obtained *Toadus leishanensis*, hematoxylin-eosin (HE) staining was used for observation. The specific steps are as follows:

[0170] (1) After sterilizing the cell slides with high temperature in advance, gently place them into a 24-well cell culture plate. Before placing them, you can add a small amount of sterile aqueous solution to the 24-well cell culture plate to make the cell slides contact more closely.

[0171] (2) Inoculate each well of the 24-well cell culture plate with 2.0 × 10⁻⁶ mg of the drug. 4 Each cell is cultured under suitable conditions for 2-3 days until the cell confluence reaches approximately 70%-80%.

[0172] (3) In a 24-well plate, the cell smears were rinsed three times with PBS solution and excess solution was removed.

[0173] (4) Fixation: Fix with 4% paraformaldehyde for 10 min, then remove excess solution.

[0174] (5) Hematoxylin staining: Add hematoxylin to the slide, stain for 15 min, and wash away the excess stain with distilled water.

[0175] (6) Differentiation: Differentiate with differentiation solution for 20 seconds, then add tap water or rinse twice, each time for 3-5 minutes.

[0176] (7) Eosin staining: stain with eosin for 5 min, pour off the excess staining solution and dehydrate quickly.

[0177] (8) Dehydration: Wash with gradient ethanol (75%, 85%, 95%, 100%) for 2-3 seconds each, and finally wash with 100% ethanol for 1 minute.

[0178] (9) Preparation of mounting: After adding an appropriate amount of glycerol to the slide, carefully remove the cell smear with tweezers and syringe tip, mark the smear on the slide, and carefully tilt the smear onto the glycerol, being careful not to create air bubbles.

[0179] Cells in their vigorous growth phase were seeded into 24-well plates containing cell spreaders, with 2.0 × 10⁶ cells per well. 4 Cells were cultured for 2-3 days until confluence reached 70%-80%. Cell slides were removed, washed three times with PBS, and fixed with 4% paraformaldehyde for 10 min. Hematoxylin staining was performed for 15 min, differentiation was carried out with differentiation medium for 20 s, eosin staining was performed for 5 min, dehydrated with graded ethanol, mounted with glycerol, and examined under a microscope.

[0180] Result: As Figure 9 As shown, after HE staining, the cell nuclei appeared blue-purple, and the cytoplasm appeared pale red. Under the microscope, the cells were observed to grow in a single layer adhering to the wall, mainly fibroblasts, with no signs of contamination in the background and clear staining.

[0181] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A rapid method for obtaining hind limb / toe tissue cells from the Leishan bullfrog, including the following steps: S1. Disinfect the body surface of individual Leishan bearded toads, and under aseptic conditions, cut hind limb or toe tissues. Disinfect and clean the wounds, and place the cut tissues in a temporary storage solution containing antibiotics. The temporary storage solution is prepared by DMEM / F12 basal culture medium, sterile water, and penicillin-streptomycin-amphoteric B triple antibiotic solution, with a final antibiotic concentration of 500 U / ml penicillin, 0.5 mg / ml streptomycin, and 1-2 μg / ml amphotericin B. S2. Cut the tissue obtained in step S1 into small pieces, digest it with trypsin first, then digest it with type I collagenase, and wash it with primary culture medium to remove residual enzymes after digestion. S3. Seed the tissue blocks treated in step S2 into a culture container, add primary culture medium, and culture them at 25-27 ℃ to obtain primary cells; S4. When the confluence of primary cells reaches 60%-70%, digest with trypsin-EDTA digestion solution, add passage culture medium to neutralize, and passage culture in proportion. S5. After passage, mix the cells with the cryopreservation solution and freeze them under programmed cooling. During thawing, thaw the cryopreservation tubes in a 37°C water bath, centrifuge to remove the cryopreservation solution, and then resuspend them in complete culture medium.

2. According to the method of claim 1, in step S1, the individual Leishan bearded toad is a tadpole at stage 40-45 or an adult; for tadpoles at stage 40-45, hind limb tissue is cut from 2 / 3 away from the end of the trunk; for adults, toe tissue is cut; after cutting, the wound is treated with 10-30 mg / L potassium permanganate solution and then released.

3. According to the method of claim 1, in step S1, the disinfection and cleaning process is as follows: before cutting the tissue, the individual of the Leishan bearded toad is immersed in a 10-30 mg / L potassium permanganate solution for 10-15 min to clean the body surface, and then 75% alcohol is applied to the hind limbs or toes for secondary disinfection; after the cut tissue is immersed in 75% alcohol for 15-20 s, it is washed 3 times with a PBS solution containing 500 U / ml penicillin, 0.5 mg / ml streptomycin, and 1-2 μg / ml amphotericin B to remove residual alcohol on the surface.

4. According to the method of claim 1, in step S1, the composition of the temporary storage solution is: 60% DMEM / F12 basal culture medium, 35% sterile water, and 5% penicillin-streptomycin-amphoteric B triple antibody solution; the temporary storage temperature is 4 ℃, and the temporary storage time does not exceed 4 days.

5. According to the method of claim 1, in step S2, the trypsin digestion is performed by digesting with 0.25% trypsin for 20 min; the type I collagenase digestion is performed by digesting with 1 mg / ml type I collagenase for 40 min; the primary culture medium used for washing after digestion contains 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B.

6. According to the method of claim 1, in steps S2 and S3, the primary culture medium is composed of: first, mixing DMEM / F12 basal medium with sterile water at a volume ratio of 6:4 to obtain a 60% diluted DMEM / F12 basal medium; then, mixing the diluted medium at a volume percentage of 79%, 20% fetal bovine serum, and 1% penicillin-streptomycin-amphoteric B triple antibody solution; the osmotic pressure of the primary culture medium is 155-190 mOsm / kg; wherein, The addition of the three-antibiotic solution brings the final working concentrations of penicillin, streptomycin, and amphotericin B in the primary culture medium to 100 U / ml, 0.1 mg / ml, and 0.25 μg / ml, respectively; the subculture medium in step S4 and the complete culture medium in step S5 have the same composition as the primary culture medium.

7. The method according to claim 1, wherein in step S3, the specific operation of the adherent culture is as follows: inoculating the tissue block into a 12.5 cm² culture medium. 2 In the cell culture flask, the bottom of the flask is moistened with primary culture medium before inoculation. After inoculation, the culture flask is inverted and incubated overnight in an incubator at 25-27 ℃. The next day, primary culture medium is added again. The culture medium is replaced by half every three days. Cells begin to migrate out on days 3-6 and grow into a cell monolayer on days 12-17.

8. According to the method of claim 1, in step S4, the mass-volume concentration of the trypsin-EDTA digestion solution is 0.25%; during subculturing, the initial inoculation is at a 1:1 ratio, followed by subculturing at a 1:2 ratio, with subculturing every 5-7 days.

9. A type of hind limb / toe tissue cell of *Leishan muscularis*, obtained by the method described in any one of claims 1-8, wherein the chromosomal karyotype of the cell is 2n=26, comprising 12 pairs of autosomes and 1 pair of sex chromosomes.

10. Use of the hind limb / toe tissue cells of the *Leishan bullfrog* according to claim 9 in at least one of the following (a)-(c): (a) Preparation of in vitro models for amphibian biological research; (b) Screening or evaluating compounds that act on amphibian cell function; (c) In vitro preservation of amphibian germplasm resources.

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