Construction method and application of ovary cell line of vijaynathella emeiensis

Abstract

The application discloses a construction method and application of an ovary cell line of Quasipaa spinosa. The method comprises the following steps: selecting metamorphosis period larva ovary tissue, disinfecting and obtaining the ovary tissue, and then using collagenase I digestion solution to digest the ovary tissue at 26 DEG C for 1 hour; after the tissue block adheres to the wall, using a low-osmotic-pressure complete culture medium to carry out primary culture at 25-27 DEG C, wherein the culture medium is 60% diluted DMEM / F12 culture medium containing 10%-15% fetal bovine serum; when the cell confluence degree is higher than 70%, using trypsin-EDTA to digest the cells for 1-2 minutes to subculture, and the subculture culture medium contains 30% cell conditional culture medium; and finally, the cells are cryopreserved through programmed cooling. The ovary epithelial cell line of Quasipaa spinosa is successfully established for the first time, the survival rate of the cells after recovery is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and in-vitro conservation of germplasm resources.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, specifically to the construction method and application of the Emei bearded toad ovarian cell line. Background Technology

[0002] Animal cell culture refers to the process of growing and proliferating discrete, living animal cells under artificial conditions in vitro. During this process, the cells do not form tissues. The earliest animal cell culture can be traced back to the late 19th century, when Wilhelm Roux in 1885 kept chicken embryo medullary plates alive for several days in warm saline solution. In 1907, Ross Harrison implanted a piece of tissue from the neural tube region of a frog embryo into a clot of frog lymph fluid, allowing the tissue to survive for several weeks and even grow axons from the cultured cells, proving that culturing animal tissues (cells) under in vitro conditions is entirely feasible. Building on this, in 1912, Carre introduced aseptic techniques into animal cell culture and discovered that animal body fluids contain growth factors that strongly promote animal cell growth. Cell culture has since developed rapidly and become a crucial fundamental technology in bioengineering, particularly cell engineering.

[0003] However, compared to mammals, birds, and fish, the research and application of amphibian cell lines are still severely lagging behind. Currently, among established cell lines, the proportion derived from amphibians is extremely low, less than 0.3% of all amphibian species. This lag is mainly reflected in the following aspects:

[0004] 1. Severe lack of species coverage: The established cell lines are highly concentrated on a very few model species such as the African clawed frog (Xenopus laevis), and the vast majority of amphibians, especially rare and endangered species, lack any available cell line resources.

[0005] 2. Highly Biased Tissue Sources: Existing limited amphibian cell lines are mainly derived from tissues such as the kidney, heart, liver, and embryos. Successful primary cell cultures and cell line constructions involving gonadal tissues (especially the ovary) are extremely rare. This results in a lack of effective in vitro models for research on amphibian reproductive development, reproductive toxicology, and gonadal-related functions.

[0006] 3. Specific Bottlenecks in the Technological System: As poikilothermic animals, amphibians face a series of unique challenges in the in vitro culture of their cells: ① Sensitive to Culture Temperature: The optimal culture temperature for amphibian cells is typically 19-30℃, far lower than the 37℃ of mammals. Temperatures above 30℃ significantly inhibit cell proliferation, while 37℃ leads to cell death. Therefore, precise temperature optimization based on the specific habitat of each species is essential. ② Customizable Culture Medium Osmolarity: Due to the low osmolarity of amphibian body fluids, the suitable osmolarity of their cell culture medium is typically only 60%-70% of that of standard mammalian culture media (approximately 185 mOsm / kgm / kg). Commercially available standard culture media must undergo precise dilution and composition adjustment, increasing the complexity and uncertainty of culture system construction. ③ High Tissue Specificity: The physiological needs of cells differ significantly among different tissues. As a reproductive tissue, the ovary has more stringent requirements for the culture conditions of digestive enzymes, growth factors, etc., making the establishment of stable primary cell lines far more difficult than for other tissues. ④ Lack of standardized technology: This field has long faced problems such as a lack of publicly available literature, immature cryopreservation and thawing protocols, and a lack of unified technical standards, which have restricted the promotion and application of related technologies.

[0007] The Emei bearded toad (Leptobrachium boringii) is a rare and endangered amphibian endemic to my country, and its biological research and genetic resource conservation are of great significance. However, after extensive searching, there are currently no publicly available reports, either domestically or internationally, of successfully constructing primary cell lines from Emei bearded toad ovarian tissue, or even any stable cell lines. Attempts to apply cell culture methods from other amphibians (such as the Chinese toad and the black-spotted side-flipped frog) or other tissue sources to Emei bearded toad ovarian cells have all failed to obtain effective, passageable cells.

[0008] Therefore, given the lack of cell models for *Toadus emblica*, particularly the challenge of constructing its ovarian epithelial cell line, there is an urgent need to develop a standardized, optimized, and highly reproducible method for constructing primary ovarian cell lines for *Toadus emblica*. This will not only provide valuable in vitro tools for physiological, reproductive biological, genetic, and toxicological research on this species, but also offer crucial technical references for the construction of germplasm resource cell banks for other endangered amphibians. Summary of the Invention

[0009] In view of this, this application provides a method for constructing and applying an ovarian cell line of *Toadoptera exigua*. This method, through a systematic technical solution including donor selection at specific developmental stages, an optimized low-osmotic-pressure culture medium formulation (60% DMEM / F12), targeted culture temperature (25-27 °C) and digestion conditions, and the addition of cell conditioned medium (CCM) to the passage medium, successfully achieves the efficient acquisition, purification, stable passage, and cryopreservation and thawing of primary epithelial ovarian cells from *Toadoptera exigua*. This establishes, for the first time, an ovarian cell line of this species, providing a standardized cell model and technical platform for related scientific research and germplasm resource preservation.

[0010] To achieve the above objectives, this application provides the following technical solution:

[0011] Firstly, this application provides a method for constructing an ovarian cell line from *Toadus emeiensis*, such as... Figure 1 As shown, it includes the following steps:

[0012] S1. Donor processing: Take the larvae of Emei Bearded Toad in the metamorphosis stage 42-45, disinfect the body surface, and dissect and remove the ovarian tissue under aseptic conditions;

[0013] S2. Tissue processing: The ovarian tissue is washed in a cleaning solution containing antibiotics and then cut into small pieces to obtain tissue blocks;

[0014] S3. Primary culture: The tissue blocks are placed in a complete culture medium and cultured adherently at 25 °C to 27 °C; wherein, the complete culture medium is prepared by diluting DMEM / F12 basal medium with sterile water at a dilution ratio of 55%-65% (i.e., 55%-65% DMEM / F12 basal medium + 35%-45% sterile water) to form a DMEM / F12 diluted culture medium solution; then, 10% to 15% of fetal bovine serum and 1% penicillin-streptomycin are added to the DMEM / F12 diluted culture medium solution to prepare the complete culture medium. The osmotic pressure of the complete culture medium is 155-190 mOsm / kgm / kg.

[0015] S4. Subculture: When the confluence of primary cultured cells reaches 70% or more, digest with trypsin-EDTA digestion solution for 1 to 2 minutes, collect cells by centrifugation, resuspend in subculture medium and inoculate; wherein, the subculture medium, by volume, consists of 70% of the complete medium and 30% of the cell conditioned medium.

[0016] S5. Cryopreservation and thawing: The cells obtained in step S4 are mixed with the cryopreservation solution at a volume ratio of 1:1, and then cryopreserved after programmed cooling; during thawing, the cryopreservation tubes are thawed in a 30 °C water bath, centrifuged to remove the cryopreservation solution, and then resuspended and cultured in the complete culture medium.

[0017] The aforementioned construction method selects highly viable tissues at specific developmental stages as starting materials and tailors a complete set of in vitro survival and proliferation conditions, from osmotic pressure and temperature to growth factors, supplemented by strict aseptic operation. This systematically meets all the core requirements for the in vitro growth of Emei bearded toad ovarian epithelial cells, ultimately breaking through the bottleneck of constructing cell lines for this species "from 0 to 1." Each technical feature works synergistically and is indispensable, jointly achieving the complete technical effect from obtaining viable cells to establishing a stable cell line.

[0018] In some preferred embodiments, in step S2, the antibiotic-containing cleaning solution is prepared by mixing L-15 culture medium and sterile water at a ratio of 60%:40% by volume, and then adding 10% of penicillin-streptomycin-amphoteric B antibiotic to the total volume of the mixture.

[0019] In some preferred embodiments, in step S3, the dilution ratio in the complete culture medium is 60%.

[0020] In some preferred embodiments, prior to the primary culture in step S3, an enzyme digestion step is included: the tissue block obtained in step S2 is placed in a collagenase I digestion solution with a concentration of 1.0 mg / mL and digested at 26 °C for 1 hour.

[0021] In some preferred embodiments, in step S4, the cell conditioned medium is prepared by collecting the supernatant of Emei bearded toad ovarian epithelial cell culture with a confluence of more than 70%, and then sterilizing it by centrifugation and filtration through a 0.22 μm filter membrane.

[0022] Secondly, this application provides an ovarian epithelial cell line of *Toadus edulis*, which is obtained by the construction method described in the first aspect.

[0023] The chromosome karyotype of the above-mentioned Emei bearded toad ovarian epithelial cell line is 2n=26, including 11 pairs of mesocentric chromosomes and 2 pairs of near-mesocentric chromosomes, of which chromosomes 3 and 13 are near-mesocentric chromosomes.

[0024] Thirdly, this application provides the use of the Emei bearded toad ovarian epithelial cell line described in the second aspect in the preparation of in vitro models for amphibian biological research.

[0025] Fourthly, this application provides the use of the Emei bearded toad ovarian epithelial cell line described in the second aspect in screening or evaluating compounds that act on ovarian function or reproductive development in amphibians.

[0026] The establishment of the cell line and the determination of its biological characteristics such as growth characteristics and karyotype stability enable it to be stably used as an in vitro model to simulate the physiological function of the ovary of the Emei bearded toad. This allows it to be used to test or evaluate the effects of various chemical substances, biological agents or environmental factors on its cell proliferation, survival or gene expression, providing a tool for reproductive toxicology research, screening of endocrine disruptors and development of reproductive-related drugs.

[0027] Fifthly, this application provides the use of the Emei bearded toad ovarian epithelial cell line described in the second aspect in the in vitro preservation of amphibian germplasm resources.

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

[0029] 1. This application provides a standardized and reproducible method for constructing ovarian epithelial cell lines from the Emei bearded toad. By systematically optimizing and integrating key steps such as surface disinfection, low-osmotic culture medium, suitable culture temperature, and tissue block adhesion and CCM-enhanced passage, the ovarian epithelial cell line of this species was successfully established for the first time. This method significantly reduces the risk and uncertainty of microbial contamination during the operation process. The constructed cell line has a high success rate in primary culture and stable cell migration, filling a gap in the cell resource bank for rare tailless animals.

[0030] 2. The culture system of this application is specifically adapted to the physiological characteristics of *Toadoptera exigua*. The optimal combination, determined through extensive experimental screening, is a passage system consisting of low-osmotic-pressure complete medium (60% DMEM / F12) and 30% conditioned medium (CCM). This system significantly promotes cell adhesion, proliferation, and passage stability, enabling stable passage to P5 and above, with a recovery survival rate of 77.48% ± 4.22%, achieving efficient cell acquisition and long-term preservation.

[0031] 3. The ovarian epithelial cells obtained by the method described in this application are of high purity, uniform in state, and exhibit stable growth characteristics. The cell growth curve follows a typical "S"-shaped pattern, and chromosome karyotype analysis shows that their genetic characteristics are stable, with a karyotype of 2n=26 (11M + 2SM), consistent with species characteristics. This method has been repeatedly verified, demonstrating good reproducibility and experimental consistency, providing a reliable technical standard for related research in different laboratories.

[0032] 4. The cell line constructed in this application provides an important in vitro model for the reproductive biology, genetics, and toxicology research of the Emei bearded toad. This system can also provide technical reference for cell culture of ovaries or other tissues from other amphibians, especially rare and endangered species, and has broad application prospects in the fields of in vitro preservation of germplasm resources, screening of environmental toxins, and reproductive endocrine research. Attached Figure Description

[0033] Figure 1 A flowchart illustrating the construction process of the Emei bearded toad ovarian cell line provided in this application.

[0034] Figure 2 This is a morphological diagram of primary ovarian cells from the Emei Bearded Toad provided in Example 1 of this application.

[0035] Figure 3 The image shows the morphology of passaged Emei bearded toad ovarian cells provided in Example 2 of this application, where A, B, C, and D are the cell morphology images of Emei bearded toad ovarian cells cultured to P2, P3, P4, and P5, respectively.

[0036] Figure 4 The P3 cells of the ovary of the Emei Bearded Toad provided in Example 3 of this application after 24 hours of resuscitation.

[0037] Figure 5 The growth curves of ovarian cells of the Emei Bearded Toad in Example 3 of this application are shown at different generations.

[0038] Figure 6 The images show chromosome splitting phases of male and female Emei bearded toads provided in Embodiment 3 of this application, where A represents male and B represents female.

[0039] Figure 7 HE staining results of the ovarian cell line of Toad hyacinthus provided in Example 3 of this application. Detailed Implementation

[0040] 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.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] 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 definitions are provided to better understand it.

[0043] 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.”

[0044] In this application, when “%” is used to represent concentration, unless otherwise specified, it represents volume fraction.

[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 vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0046] To enable those skilled in the art and the general public to more accurately understand this application, the key terms used in the specification are defined as follows:

[0047] 1. Metamorphosis (Stages 42-45): This refers to the specific developmental stage of the Emei Bearded Toad, as defined by Gosner's (1960) universal amphibian developmental stage system, from the complete extension of its forelimbs to the significant absorption of its tail, but not yet complete disappearance. This stage corresponds to stages 42 to 45 in Gosner's stage system, marking the critical period of transformation from an aquatic tadpole to a terrestrial adult. During this period, the tissue cells exhibit vigorous proliferation and remodeling activity.

[0048] 2. Adherent culture: This refers to a primary cell culture method, specifically the tissue block adhesion method. The specific procedure involves: evenly spreading chopped tissue blocks at the bottom of a culture dish, briefly inverting the dish to allow the tissue blocks to physically adhere to the culture surface, then adding complete culture medium and inverting the dish for culture. Cells then migrate and proliferate from the edges of the adhered tissue blocks.

[0049] 3. Complete culture medium: refers to a culture medium suitable for the growth of Emei bearded toad cells. In this application, it specifically refers to a culture medium in which commercially available DMEM / F12 basal medium is diluted with sterile water to a final concentration of 55% to 65% (v / v), and 10%-15% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin dual antibiotic solution are added to the diluted medium to adjust its osmotic pressure to approximately 155-190 mOsm / kgm / kg. Furthermore, in the embodiments of this application, the working concentration of penicillin in the complete culture medium is 100 U / ml, and the working concentration of streptomycin is 0.1 mg / ml.

[0050] 4. Cell conditioned medium: refers to the cell-free supernatant collected from the ovarian epithelial cell culture of *Toadoptera exigua* in the logarithmic growth phase with a confluence of more than 70%. After centrifugation and sterilization by filtration, this supernatant contains active substances such as cell-secreted growth factors, cytokines, and extracellular matrix proteins.

[0051] 5. Consolidation: This refers to the percentage of the area occupied by adherent cells to the total usable area at the bottom of the culture dish when observed under a microscope.

[0052] 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.

[0053] 7. Karyotype formula 2n=26(11M+2SM): This indicates that the cell line contains 26 chromosomes (diploid). According to Levan's (1964) centromere classification criteria, "M" represents mesocentric chromosomes, and "SM" represents submesocentric chromosomes. This formula indicates that it contains 11 pairs of mesocentric chromosomes and 2 pairs of submesocentric chromosomes.

[0054] 8. 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.

[0055] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0056] The main materials / reagents used in the following examples are shown in Table 1 below.

[0057] Table 1. Information on the main materials and reagents used in the examples.

[0058]

[0059] Example 1: Primary culture and establishment of ovarian epithelial cell lines from Emei bearded toad

[0060] This embodiment details the entire process of the Emei bearded toad ovarian epithelial cell line, from donor selection to successful primary culture.

[0061] 1. Experimental Methods

[0062] (1) Donor selection

[0063] Using amphibian larvae as subjects, a preliminary experiment was conducted to investigate the cell migration rate of ovarian tissue at different developmental stages. The results are shown in Table 2 below.

[0064] Table 2. Record of relative migration of primary cells

[0065]

[0066] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative amount of migration; "++" indicates cell confluence exceeds 30%, "+++" indicates cell confluence exceeds 50%, and "++++" indicates cell confluence exceeds 70%.

[0067] As shown in Table 2 above, the ovarian tissue cells of individuals in the metamorphosis stage (stages 42-45) exhibited a cell confluence exceeding 70% (meeting the passage standard) by day 9 of culture, demonstrating the most vigorous proliferation. Therefore, Emei bearded toad larvae at this stage were ultimately selected as cell donors.

[0068] (2) Ovarian tissue acquisition and pretreatment

[0069] ① Disinfection of body surface: Select well-developed, healthy, and disease-free Emei bearded toad larvae in the metamorphosis stage (stages 42-45). Disinfect the whole body with 0.01%-0.05% (mass fraction) potassium permanganate solution or 75% (volume fraction) alcohol for 1-2 minutes. Then, transfer them to 60% PBS solution (60% PBS + 30% sterile water + 10% penicillin-streptomycin-amphoteric B) for thorough soaking and washing. Repeat 3-4 times before dissection.

[0070] ② Obtain complete ovarian tissue through aseptic dissection. Since the ovarian tissue is located near the intestines, care must be taken to avoid intestinal rupture and the release of excrement during the procedure. If this occurs, immediately replace the sterilized instruments or discard the individual.

[0071] ③ Tissue Cleaning. After cutting the tissue, it is placed in a tissue cleaning solution (60% L-15 + 30% sterile water + 10% penicillin-streptomycin-amphoteric B) for rapid cleaning and disinfection, repeated 3-4 times until the solution is clear, to remove blood cells and residual body cavity fluid. Subsequently, the tissue is trimmed to approximately 1 mm using sterile ophthalmic scissors. 3 The tissue blocks are of a specific size to facilitate subsequent digestion. The tissue washing solution is diluted with sterile water to maintain the same osmotic pressure as the culture medium.

[0072] (3) Tissue enzyme digestion

[0073] ① Preparation of digestive enzyme solution: Weigh collagenase type I and dissolve it in 60% DMEM / F12 solution preheated to 26 °C.

[0074] The culture medium was prepared into a digestion solution with a concentration of 1.0 mg / mL, and then filtered through a 0.22 μm filter for sterilization before use. The 60% DMEM / F12 basal medium consisted of 60% (v / v) DMEM / F12 + 40% (v / v) sterile water.

[0075] ② Tissue block digestion: Transfer tissue fragments to centrifuge tubes containing digestive enzymes and place them in an incubator at 26 °C for digestion. First, explore the enzyme digestion time through preliminary experiments, as shown in Table 3 below.

[0076] Table 3. Record of relative migration amount and migration rate of primary cells under different enzyme digestion times.

[0077]

[0078] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative amount of migration; "++" indicates cell confluence exceeds 30%, "+++" indicates cell confluence exceeds 50%, and "++++" indicates cell confluence exceeds 70%.

[0079] Based on the results of the above preliminary experiments, a digestion time of 1 hour was selected as the optimal time. At this time, the cell migration amount was the largest (++++) and the migration speed was the fastest.

[0080] (4) Tissue block adhesion and initial culture

[0081] Pre-wet the bottom of the culture bottle with a small amount of complete culture medium, evenly spread the digested tissue block on the bottom of the bottle, invert the culture bottle, and place it in an incubator set to 26 °C for 24 hours to allow the tissue block to fully adhere. After 24 hours, add sufficient complete culture medium to the culture bottle, invert the culture bottle, and continue to incubate at 26 °C. Since the osmotic pressure differs between amphibians and mammals, and the osmotic pressure of currently used DMEM / F12 basal medium is mainly designed for mammals, the complete culture medium used in this application embodiment was obtained by diluting DMEM / F12 basal medium with sterile water, and then adding 10%-15% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution (100X). Specific dilution ratios are shown in Table 4 below.

[0082] Table 4. Suitable osmotic pressure range of ovarian tissue under different culture conditions.

[0083]

[0084] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; "++" indicates cell confluence exceeds 30%, "+++" indicates cell confluence exceeds 50%, and "++++" indicates cell confluence exceeds 70%.

[0085] As shown in Table 4, the best complete medium was prepared by diluting DMEM / F12 basal medium with sterile water at a 60% dilution ratio, followed by adding 0-15% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution (100X). This resulted in the highest cell migration (++++), with an osmotic pressure of 155-190 mOsm / kg. Therefore, the complete medium prepared under these conditions was selected.

[0086] (5) Primary culture and maintenance

[0087] After the tissue blocks have adhered to the culture medium and been cultured for 48-72 hours, perform the first half-volume medium replacement to remove unadhered dead cells and debris. Thereafter, depending on the color change of the medium (e.g., turning yellow), replace 1 / 2 or 2 / 3 of the volume with fresh complete culture medium every 3-5 days. Using a partial medium replacement method preserves the growth factors secreted by the cells themselves, which is beneficial for cell adaptation and growth.

[0088] (6) Purification of primary cells

[0089] Cells obtained through tissue block adherent culture include tissue-derived fibroblasts and epithelial cells. Epithelial cells have different adhesion abilities than non-epithelial cells (such as fibroblasts and blood cells), and cell purification can be achieved by controlling the trypsin digestion time. Epithelial cells have a stronger adhesion ability than other cells. By culturing cells on the surface of the culture dish for a period of time, cells attached to the culture medium surface can be digested and separated. Non-epithelial cells will detach in a short time, while epithelial cells can attach and proliferate. Other non-adherent cells are removed when the culture medium (complete medium) is changed.

[0090] 2. Results

[0091] Observe and record daily under a microscope. For example... Figure 2 As shown, during 2-4 days of culture, cells began to migrate from the edges of the tissue block, with cell morphologies including irregular polygonal (epithelial-like cells) and spindle-shaped (fibroblast-like cells). During 8-10 days of culture, the cell confluence in the ovarian tissue culture flask reached 80%, with epithelial-like cells exhibiting dominant growth. In a typical cell line establishment experiment, four culture flasks were used for primary ovarian tissue culture. One flask failed due to contamination, while the other three successfully yielded epithelial and fibroblast cells. A total of six tubes of primary cells were ultimately cryopreserved.

[0092] Example 2: Passage culture, purification, and application of conditioned medium for ovarian epithelial cells

[0093] This example illustrates how to passage and purify primary cells to obtain a more homogeneous epithelial cell population and demonstrates the promoting effect of conditioned medium.

[0094] 1. Optimization of cell passage and digestion time

[0095] (1) Subculture timing: Subculture should be performed when the confluence of primary cultured cells reaches 70% or more.

[0096] (2) Pretreatment: Remove the cell culture flask and discard the old culture medium. Add phosphate-buffered saline (PBS) to the flask, gently shake to wash the cell surface and then discard the flask to remove residual serum.

[0097] (3) Key digestion steps: Add 0.25% (w / v) trypsin-EDTA digestion solution to the flask and gently shake to ensure the digestion solution fully covers the cell layer. Immediately place the culture flask in an incubator at 25-27 °C for digestion.

[0098] (4) Optimization and determination of digestion time: To determine the optimal digestion time, six time gradients (0.5, 1.0, 1.5, 2.0, 3.0, 5.0 min) were set for the experiment. The results are shown in Table 5 below.

[0099] Table 5 Cell states at different trypsin digestion times

[0100]

[0101] Note: "-" indicates no cell digestion and shedding; "+" indicates cell digestion and shedding; "++" indicates a cell digestion and shedding rate exceeding 30%; "+++" indicates a cell digestion and shedding rate exceeding 50%; "++++" indicates a cell digestion and shedding rate exceeding 70%.

[0102] The results in Table 5 show that when digestion takes 1-2 minutes, most cells shrink, become round, and detach, resulting in the largest number of digested cells (++++). Furthermore, when the operation is performed within this time window, cell damage is minimal, subsequent adhesion and survival rates are high, and the cells are in good condition. Therefore, the digestion time for passage culture should be selected at 1-2 minutes.

[0103] (5) Termination of digestion and cell collection: After digestion for 1-2 minutes, observe under a microscope. When most cells become round and detach, immediately add an equal volume of complete culture medium (composition as in Example 1) to terminate digestion. Gently pipette the bottom of the flask to transfer the cell suspension to a centrifuge tube. Centrifuge at 800 rpm for 8 minutes and discard the supernatant.

[0104] 2. Optimization and Determination of the Subculture System

[0105] (1) Preparation of cell conditioned medium (CCM):

[0106] In a laminar flow hood, cell-free supernatant was collected from ovarian epithelial cell culture flasks of *Toadoptera exigua* in a confluent state (confluence >70%). The collected supernatant (i.e., crude CCM) was transferred to centrifuge tubes and centrifuged at 1000×g for 10 min to precipitate large particles such as cell debris. Inside the laminar flow hood, the centrifuged supernatant was filtered through a 0.22 μm pore size filter membrane (a bottle-top filter or syringe filter can be used) to obtain sterile CCM. The prepared sterile CCM can be stored at 4 °C.

[0107] (2) Determination of optimized subculture medium formulation:

[0108] Cell culture medium supplemented with CCM was selected to verify its growth-promoting effect. A comparative experiment was established: the experimental group used a mixture of 70% (v / v) complete culture medium and 30% (v / v) CCM; the control group used only 100% complete culture medium. Both groups were seeded with cells at an initial confluence of 30%.

[0109] The results of the efficacy verification are shown in Table 6. By day 6 of culture, the cell confluence in the experimental group exceeded 70% (++++), meeting the standard for further passage; while the cell confluence in the control group was only about 50% (+++). This result clearly demonstrates that adding 30% CCM can significantly promote the proliferation rate of ovarian epithelial cells of *Toadoptera exigua*. Based on the above experimental results, the optimized culture medium formula was determined to be: 70% (v / v) complete culture medium + 30% (v / v) CCM.

[0110] Table 6. Experiments investigating how conditioned medium (CCM) promotes cell growth.

[0111]

[0112] Note: "-" indicates no cell migration or traces; "+" indicates cell migration and the number represents the relative migration amount; ++ indicates cell confluence exceeds 30%, +++ indicates cell confluence exceeds 50%, and ++++ indicates cell confluence exceeds 70%.

[0113] 3. Cell seeding and passage markers

[0114] (1) Cell resuspension: Add passage culture medium to the cell pellet after centrifugation and gently pipette to resuspend the cells.

[0115] (2) Cell seeding: Prepare a new culture flask, add an appropriate amount of passage medium, and then add the cell suspension in a 1:1 ratio (i.e., the cell suspension and the pre-stored medium in the flask are of equal volume). Gently shake or draw a cross to distribute the cells evenly.

[0116] (3) Marking and culturing: Mark the passage number on the bottle (e.g., P1, P2...) and place it in an incubator at 25-27 °C for further culturing.

[0117] 4. Results

[0118] Ovarian tissue cells from *Toadoptera exigua* were passaged and cultured. Cell growth was recorded by photographing each passage. Currently, the cells have reached P5. Figure 3 As shown in the figure, after cell passage, most ovarian tissue cells exhibit a strong three-dimensional morphology, displaying a peak-and-valley pattern. A small portion of the cells show a flattened, irregular polygonal shape. With increasing cell culture passages, the time required for cell confluence to reach over 70% increases, and the cell morphology gradually changes from three-dimensional triangular to flattened, irregular polygons. Cell debris in the culture medium also increases with each cell passage.

[0119] Example 3: Cryopreservation, thawing, and identification of biological characteristics of cell lines

[0120] This embodiment demonstrates that the Emei bearded toad ovarian epithelial cell line constructed in this application can be preserved for a long time and has stable biological characteristics.

[0121] 1. Cell cryopreservation

[0122] (1) Collect cells in good growth condition with confluence exceeding 70% (P3 generation) for cryopreservation. Discard the old culture medium and wash once with PBS to remove residual serum. Add 1 mL of 0.25% (w / v) trypsin-EDTA digestion solution, gently shake to cover the cell layer, and incubate in an incubator for 1-2 min. Add complete culture medium to stop digestion, gently pipette the bottom of the flask to detach the cells, and transfer the cell suspension to a centrifuge tube. Centrifuge at 800 rpm for 8 min and discard the supernatant.

[0123] (2) Resuspend the cells in an appropriate amount of complete culture medium and adjust the cell density. Mix the cell suspension with the cryopreservation solution at a volume ratio of 1:1, gently invert and mix 5-6 times, and dispense into cryovials. The cryopreservation solution is prepared as follows: DMEM / F12 and sterile water are mixed at a volume ratio of 6:4 to prepare 60% DMEM / F12 basal culture medium; 60% DMEM / F12 basal culture medium and serum are mixed at a volume ratio of 10:3 to prepare culture medium; then the culture medium and DMSO are mixed at a volume ratio of 8:2 to prepare the cryopreservation solution.

[0124] (3) Place the cryovials in a programmed cryopreservation box and place them in a -80 °C freezer overnight to achieve gradient cooling. The next day, transfer the cryovials to liquid nitrogen for long-term storage.

[0125] 2. Cell resuscitation and survival rate determination

[0126] (1) Remove the cryovial from the liquid nitrogen and quickly transfer it to a water bath preheated to 30 °C. Shake it rapidly to thaw it completely within 1-2 minutes.

[0127] (2) Add an appropriate amount of cooled (4 °C) complete culture medium to a centrifuge tube beforehand, transfer all the thawed cell suspension to the centrifuge tube, and mix well. Centrifuge at 800 rpm for 8 min, and carefully discard the supernatant containing the cryopreservation solution.

[0128] (3) Resuspend the cell pellet in fresh complete culture medium, inoculate it into the labeled culture flask, gently shake to distribute the cells evenly, and incubate in a 25-27 °C incubator.

[0129] (4) Resuscitation survival rate determination: The cells were stained with trypan blue staining and the number of live and dead cells was counted under a microscope using a hemocytometer to calculate the cell resuscitation survival rate.

[0130] Results: Table 7 shows the cell viability results. The resuscitation survival rates of the three independent parallel experiments were 72.73%, 78.95%, and 80.77%, respectively, with a mean resuscitation survival rate of 77.48% ± 4.22%.

[0131] Table 7 Cell survival rate after resuscitation

[0132]

[0133] Figure 4 The image shows P3 cells from the ovary of a Toad leucantha 24 hours after resuscitation. As can be seen from the image, the cells adhere well to the wall and have normal morphology.

[0134] 3. Determination of cell growth curves in vitro

[0135] To characterize the cell's proliferative capacity, growth curves were plotted for cells at different passages. The specific steps are as follows:

[0136] P3, P4, and P5 generation cells in good growth condition were collected, digested with trypsin, and prepared into single-cell suspensions for counting using a hemocytometer. 3.6 × 10⁶ cells were collected. 4 Cells were prepared by mixing with complete culture medium to form a cell suspension. 1.5 × 10⁶ cells were seeded per well in a 24-well cell culture plate. 4 Divide the cells into 8 groups of 3 cells each, and place the 24-well cell culture plate in a CO2 incubator for culture. After the culture begins, digest the cells in the 3-wells at regular intervals and change the medium in the remaining wells.

[0137] After 24 hours of cell culture, cell counts were performed using a hemocytometer, and the average value was taken. The number of cells in one group was counted daily at regular intervals, and the total number of cells corresponding to the growth days was calculated. This method was repeated for 7 consecutive days, and the data was statistically analyzed. The cell count (×10⁻⁶) was plotted on the x-axis as culture time. 4 Using the number of cells as the ordinate, plot the cell growth curve.

[0138] Results and Analysis: Growth curves of ovarian cells from *Toadus emeiensis* at different passages were statistically analyzed and plotted. Figure 5 As shown in the figure. The cell counts of ovarian cells at passages P3, P4, and P5 were counted at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, respectively, and the changes in the density of viable cells in the environment were observed within an 8-day period.

[0139] from Figure 5 It can be seen that the cell growth curves of the ovarian epithelial cells of *Bufo emblica* in P3 and P4 generally conform to an "S" shape, with cells experiencing three phases during growth: a latent phase, a logarithmic growth phase, and a plateau phase. The number of cells increases significantly in the initial stage after inoculation, exhibiting logarithmic growth. The cell number enters the plateau phase around 72 hours, reaching its maximum around 96-120 hours. Afterward, cell shedding due to contact inhibition and other factors leads to a slight decrease in cell number. Compared to the growth curve in P3, the growth curve in P4 is flatter in the logarithmic growth phase, with a similar time to the plateau phase, but the time to reach the maximum cell number is earlier, and the maximum cell number is lower. In P5, the cell proliferation and doubling capacity are lower than in previous generations, and cell viability decreases significantly. That is, as the cell culture passages increase, the growth curve tends to flatten, and the maximum proliferation number also decreases significantly. This may indicate that as the culture passages increase, the cells gradually age, resulting in decreased cell viability and slower proliferation.

[0140] 4. Chromosome karyotype analysis

[0141] To determine the genetic stability and species characteristics of the cell line established in this application, chromosome karyotype analysis was performed. The specific steps are as follows:

[0142] (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 to 4 hours.

[0143] (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.

[0144] (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, so that the fixative solution fills the bottom of the culture dish, but the liquid surface does not touch the glass slide. Cover the culture dish and perform steam fixation at 24~28 °C for 120 min.

[0145] 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.

[0146] 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.

[0147] (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.

[0148] (5) Karyotype analysis: Observe and photograph clear metaphase chromosome divisions under an oil immersion microscope. Use image analysis software (such as ImageJ) to measure the total length, long arm length, and short arm length of each chromosome. Calculation formula:

[0149] Relative chromosome length = length of a single chromosome / (length of haploid chromosome + length of sex chromosome) × 100%;

[0150] Chromosome arm index = Long arm length / Short arm length;

[0151] The centromere index of chromosomes = (short arm length / total chromosome length) × 100%; according to Levan (1964) criteria (Table 8), the centromere type is determined based on the arm ratio.

[0152] Table 8 Chromosome arm ratio and centromere position parameters

[0153]

[0154] Results: Using the above method, clear images of male and female chromosome division were successfully obtained, such as... Figure 6 As shown in Table 9, chromosomes of the Emei Bearded Toad were measured using a microscope and photographed. Based on the measurement data from 11 female mitotic phases and 13 male mitotic phases, the mean and standard deviation of the relative length (RL), arm ratio (AR), and centromere index (CI) of 13 pairs of chromosomes in both female and male Emei Bearded Toads were obtained.

[0155] Table 9. Karyotype results of male and female Emei bearded toads.

[0156]

[0157] According to the results in Table 9, the number of chromosomes in the established cell line is 2n=26. Among them, chromosomes 1 to 5 are large chromosomes (RL>9%), and chromosomes 6 to 13 are small chromosomes (RL<7%).

[0158] Following Levan's (1964) criteria, chromosome pairs were classified according to arm ratio (AR). The results showed that the karyotype formula of this cell line was 2n=26(11M+2SM), containing 11 pairs of mesocentric chromosomes (M) and 2 pairs of submesocentric chromosomes (SM). Chromosomes 3 and 13 were submesocentric chromosomes (SM).

[0159] Karyotype analysis confirmed that the chromosome number and morphological classification (karyotype formula) of the Emei bearded toad ovarian epithelial cell line constructed according to the method of this application are consistent with the cytogenetic characteristics of the Emei bearded toad species reported in Li Shushen. Cytologica Sinica [M]. Science Press, 2007., which proves the accuracy of the genetic background of the cell line and the stability of the karyotype during the culture process.

[0160] 5. HE staining

[0161] To morphologically identify the cell types of the constructed Emei bearded toad ovarian cell line, hematoxylin-eosin (HE) staining was used for observation. The specific steps are as follows:

[0162] (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.

[0163] (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%.

[0164] (3) In a 24-well plate, the cell smears were rinsed three times with PBS solution and excess solution was removed.

[0165] (4) Fixation: Fix with 4% paraformaldehyde for 10 min, then remove excess solution.

[0166] (5) Hematoxylin staining: Add hematoxylin to the slide, stain for 15 min, and wash away the excess stain with distilled water.

[0167] (6) Differentiation: Differentiate with differentiation solution for 20 seconds, then add tap water or rinse twice, each time for 3-5 minutes.

[0168] (7) Eosin staining: stain with eosin for 5 min, pour off the excess staining solution and dehydrate quickly.

[0169] (8) Dehydration: Wash with gradient ethanol (75%, 85%, 95%, 100%) for 2-3 seconds each, and finally wash with 100% ethanol for 1 minute.

[0170] (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.

[0171] (10) Mounting: Add a drop of neutral resin to the slide and seal it. After the resin has solidified, it can be observed under a microscope.

[0172] result: Figure 7 This image shows the HE staining results of the ovarian cell line of *Bubble Tetracentron sinense*. As can be seen from the image, after HE staining, the cell nuclei appear blue-purple, and the cytoplasm appears pale red. The cells are irregularly polygonal, with round nuclei and tight intercellular connections, typical of epithelial cell morphology. At this stage, the cells are in good condition; the eosin staining may be faint, but it is uniform and consistent, without vacuolation, granular degeneration, or abnormalities.

[0173] 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 method for constructing an ovarian epithelial cell line from the Emei bearded toad, characterized in that, Includes the following steps: S1. Donor processing: Take the larvae of Emei Bearded Toad in the metamorphosis stage 42-45, disinfect the body surface, and dissect and remove the ovarian tissue under aseptic conditions; S2. Tissue processing: The ovarian tissue is placed in an antibiotic-containing cleaning solution for cleaning and then cut into pieces to obtain tissue blocks; the antibiotic-containing cleaning solution is composed of L-15 culture medium and sterile water at a ratio of 60%:40% by volume, and then 10% of penicillin-streptomycin-amphoteric B antibiotic is added to the mixture. S3. Enzymatic digestion: The tissue block obtained in step S2 was placed in a collagenase I digestion solution with a concentration of 1.0 mg / mL and digested at 26 °C for 1 hour; S4. Primary culture: The enzyme-digested tissue blocks are placed in a complete culture medium and cultured at 25 °C to 27 °C. The complete culture medium is prepared by diluting DMEM / F12 basal medium to 60% with sterile water, and then adding 10% to 15% of fetal bovine serum and 1% penicillin-streptomycin by volume. The osmotic pressure of the complete culture medium is 155-190 mOsm / kg. S5. Subculture: When the confluence of primary cultured cells reaches 70% or more, digest with trypsin-EDTA digestion solution for 1 to 2 minutes, collect cells by centrifugation, resuspend in subculture medium and inoculate; wherein, the subculture medium, by volume, consists of 70% of the complete medium and 30% of the cell conditioned medium; the cell conditioned medium is obtained by collecting the supernatant of Emei bearded toad ovarian epithelial cell culture with a confluence of 70% or more, and sterilizing it by centrifugation and filtration through a 0.22 μm filter membrane; S6. Cryopreservation and thawing: The cells obtained in step S5 are mixed with the cryopreservation solution at a volume ratio of 1:1, and then cryopreserved after programmed cooling; during thawing, the cryopreservation tubes are thawed in a 30 °C water bath, centrifuged to remove the cryopreservation solution, and then resuspended and cultured in the complete culture medium.

2. A cell line of ovarian epithelial cells from the Emei bearded toad, characterized in that, The Emei bearded toad ovarian epithelial cell line was obtained by the construction method described in claim 1.

3. The Emei bearded toad ovarian epithelial cell line according to claim 2, characterized in that, The chromosome karyotype of the Emei bearded toad ovarian epithelial cell line is 2n=26, including 11 pairs of mesocentric chromosomes and 2 pairs of near-mesocentric chromosomes, of which chromosomes 3 and 13 are near-mesocentric chromosomes.

4. The use of the Emei Bearded Toad ovarian epithelial cell line as described in claim 2 or 3 in the in vitro preservation of Emei Bearded Toad germplasm resources.

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