Spermatogonial stem cell transplantation recipient cell as well as preparation method and application thereof
By using NTSG-PEG-CaCl2 reagent to remove spermatogenetic cells and retain support cells and trichosperm structures, the problems of high toxicity and unstable effects in the prior art are solved, and the success rate and safety of spermatogenetic stem cell transplantation are improved.
Patent Information
- Application Number
- CN202411633276.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-03-11
AI Technical Summary
The existing spermatogenic stem cell transplant recipient preparation methods have problems such as high toxicity, unstable effect and difficulty in controlling, which affects the success rate and safety of transplantation.
The NTSG-PEG-CaCl2 reagent was used as the preparation method for the receptor cells, and the spermatogenesis cells were removed and the support cells and trispersus structures were retained by injecting them into testicular tissue or as a component of the culture medium.
It achieves low toxicity and efficient removal of spermatogenetic cells, retains the integrity of support cells and trichospermia structure, and improves the success rate and safety of spermatogenetic stem cell transplantation.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a spermatogonial stem cell transplantation recipient cell and a preparation method and application thereof. Background Art
[0002] The production of sperm in males is regulated by the supporting cells of the testis and other testicular somatic cells. SSC (spermatogonial stem cell) transplantation involves injecting donor SSC into the recipient rete testis, allowing them to re-migrate and colonize in the spermatogonial stem cell "niche", thereby establishing a stable sperm production process derived from the donor. Spermatogonial stem cells are a group of diploid cells located near the basement membrane of the seminiferous epithelium. They have a high degree of self-renewal and multidirectional differentiation potential and are the only adult stem cells in adult animals that can pass genetic information to the next generation. These stem cells can not only continuously self-renew to maintain a stable population, but also differentiate into spermatogonia of various levels and eventually form sperm. Spermatogonial stem cell transplantation is the initial step in stem cell breeding, and its main challenge is the lack of mature and efficient recipient preparation methods. In order to ensure that donor spermatogonial stem cells will not be ablated by endogenous germline cells after transplantation in the recipient rete testis, it is necessary to ensure that the recipient model has normal somatic cell function and seminiferous tubule structure.
[0003] Since 1994, the Br inster team transplanted SSC cell suspension into the seminiferous tubules of fertility-deficient mice for the first time and observed the complete sperm production process. These mice successfully produced normal offspring through natural mating, opening up new possibilities for male reproductive medicine and theoretical research on human spermatogenesis.
[0004] Recipient preparation is a key part of spermatogonial stem cell transplantation technology and a necessary condition and basis for successful transplantation. The efficiency of receptor preparation directly affects the transplantation effect. At present, receptor preparation techniques such as intraperitoneal injection of busulfan and testicular injection of busulfan have been developed. These techniques induce apoptosis of spermatogenic cells at all levels of the recipient testis, forming cavities in the abluminal and basal chambers surrounded by the supporting cells of the seminiferous tubules, which facilitates the migration and implantation of donor spermatogonial stem cells after transplantation, thereby completing regeneration, differentiation and spermatogenesis. Although relatively mature receptor preparation methods have been established, the toxicity of busulfan can cause serious damage to the body of experimental animals. With the renewal of blood circulation, busulfan is gradually metabolized, and its effect will also weaken. In addition, due to the influence of individual differences, injection techniques and locations, it is difficult to accurately judge the actual concentration reaching the spermatogonial stem cell niche. Excessive concentration may damage the body or even cause animal death.
[0005] At present, the preparation method of spermatogonial stem cell transplantation recipients is being continuously improved. Although the busulfan injection method is simple and easy to perform and is currently the most commonly used method for preparing recipients, how to reduce the impact of blood renewal on busulfan metabolism and reduce its toxic effects on the body is still an urgent problem to be solved. Therefore, it is particularly important to establish a simple, efficient and basically side-effect-free spermatogonial stem cell transplantation recipient method.
[0006] Although there are many compounds, such as busulfan, bisphenol A and omeprazole, which can be used to inhibit endogenous germ cells, there are still many deficiencies in preparing spermatogonial stem cell transplantation recipients, and it is difficult to successfully prepare ideal receptors. Therefore, inventing a new spermatogonial stem cell transplantation recipient cell and its preparation method and application are the key to solving the existing technical problems. Summary of the invention
[0007] In view of this, the present invention proposes a spermatogonial stem cell transplantation recipient cell and its preparation method and application, aiming to provide a spermatogonial stem cell transplantation recipient cell preparation method that is safer, more efficient and has less impact on the body, as well as a new recipient cell, so as to improve the success rate and safety of spermatogonial stem cell transplantation.
[0008] The present invention provides a method for preparing a NTSG-PEG-CaCl2 reagent, the preparation method comprising:
[0009] The polyethylene glycol is dissolved in a buffer solution, and detosylglycine is added. After the polyethylene glycol is completely dissolved, calcium chloride is added, and then glycerol is added to obtain the NTSG-PEG-CaCl2 reagent.
[0010] Preferably, the preparation method further comprises:
[0011] The polyethylene glycol is dissolved in a buffer solution and the concentration is adjusted to 10% (w / v), destoluenesulfonyl glycine is added at a ratio of 1% (w / v), and after complete dissolution, calcium chloride is added until the concentration reaches 0.5 mM, and then glycerol is added to a concentration of 0.5% (v / v) to obtain the NTSG-PEG-CaCl2 reagent.
[0012] Preferably, the buffer is a phosphate buffer.
[0013] The present invention also provides a NTSG-PEG-CaCl2 reagent, and the NTSG-PEG-CaCl2 reagent is prepared by the above-mentioned preparation method.
[0014] The present invention also proposes an application of the NTSG-PEG-CaCl2 reagent in preparing spermatogonial stem cell transplantation receptors.
[0015] The present invention also provides a method for preparing spermatogonial stem cell transplant recipient cells, and the spermatogonial stem cell transplant recipient cells are obtained by injecting the above-mentioned NTSG-PEG-CaCl2 reagent into the testicular tissue of a ram.
[0016] Preferably, the concentration of the NTSG-PEG-CaCl2 reagent is 0.02-0.03%.
[0017] The present invention also provides a method for preparing spermatogonial stem cell transplant recipient cells, using the above-mentioned NTSG-PEG-CaCl2 reagent as a culture medium component to culture cells to obtain the spermatogonial stem cell transplant recipient cells.
[0018] Preferably, the cells are spermatogenic cells.
[0019] Preferably, the concentration of the NTSG-PEG-CaCl2 reagent is 0.02-0.03%.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1. Low toxicity and side effects
[0022] NTSG-PEG-CaCl2 solution is less toxic than busulfan and will not cause serious damage to the body. Busulfan is often accompanied by toxic side effects when used, such as damage to the blood system and other organs, and may cause death of experimental animals. The low toxicity of NTSG-PEG-CaCl2 makes receptor preparation safer, reduces harm to experimental animals, and improves the controllability of receptor preparation.
[0023] 2. Higher selectivity
[0024] NTSG-PEG-CaCl2 has a good selective removal effect on spermatogenic cells and supporting cells. It can efficiently remove spermatogenic cells while protecting the survival and function of supporting cells to a large extent. This selectivity avoids the destruction of supporting cells and ensures the integrity of the spermatogenic environment after donor spermatogonial stem cell transplantation, while traditional reagents often lack this cell selectivity.
[0025] 3. The integrity of the seminiferous tubule structure is preserved
[0026] This reagent effectively removes spermatogenic cells while basically maintaining the integrity of the seminiferous tubule structure. In contrast, agents such as busulfan are prone to damage or deformation of the seminiferous tubules, affecting the attachment and functional recovery of subsequent transplanted cells. The integrity of the seminiferous tubules is an important prerequisite for the successful reconstruction of spermatogenic function after transplantation.
[0027] 4. No rapid metabolism problem in the body
[0028] The effect of busulfan is easily affected by the blood circulation metabolism in the body, which makes its effect short-term and the concentration difficult to control. In actual application, it is often difficult to achieve the ideal concentration due to individual differences. However, NTSG-PEG-CaCl2 is metabolized more slowly in the body and has a longer-lasting effect, which reduces the need for frequent intervention of experimental animals and facilitates more precise control of the action time and effect of the reagent.
[0029] 5. Easy to operate
[0030] The preparation and use of NTSG-PEG-CaCl2 solution is relatively simple, and its operation is convenient and suitable for laboratory environment, which reduces the difficulty of operation. Compared with other more complicated receptor preparation methods, it has short operation time and low difficulty, which helps to improve the success rate of experiments, especially for large-scale or repetitive experiments.
[0031] In summary, NTSG-PEG-CaCl2 solution has the advantages of high efficiency, safety, strong selectivity, and good protection of the seminiferous tubule structure in the preparation of spermatogonial stem cell transplantation receptors, providing a more ideal receptor preparation solution for spermatogonial stem cell transplantation technology. DETAILED DESCRIPTION
[0032] The exemplary embodiments of the present disclosure will be described in more detail below. Although the exemplary embodiments of the present disclosure are shown below, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.
[0033] Example 1 Preparation of NTSG-PEG-CaCl2 Reagent
[0034] 1. Reagent composition
[0035] 1. Polyethylene glycol (PEG): This ingredient is known for its excellent biocompatibility and can effectively regulate the permeability of the solvent, thereby promoting deeper penetration of the reagent into the tissue.
[0036] 2. Tosylglycine (NTSG): As a non-cytotoxic detergent, NTSG can work synergistically with PEG to destroy the spermatogenic cell structure in the testis while minimizing damage to the supporting cells and matrix of the seminiferous tubules.
[0037] 3. Low-dose calcium chloride (CaCl2): This ingredient helps to improve the reagent's efficiency in removing spermatogenic cells and reduce intercellular adhesion.
[0038] 4. A small amount of glycerol: The addition of glycerol can increase the permeability of cell membranes and enhance the solubility of drugs, ensuring that they act evenly on tissues.
[0039] 2. Preparation steps of reagents
[0040] 1. Solution preparation: Dissolve PEG in sterile phosphate buffered saline (PBS) and adjust the concentration to 10% (w / v).
[0041] 2. Add NTSG: Add NTSG at a ratio of 1% (w / v) and ensure that the solution is completely dissolved.
[0042] 3. Add CaCl2: Slowly add CaCl2 powder until the concentration reaches 0.5mM.
[0043] 4. Add glycerol: Add glycerol to a concentration of approximately 0.5% (v / v).
[0044] 5. Filtration sterilization: Filter the mixed reagents through a 0.22 μm filter membrane to ensure its sterility.
[0045] Example 2 Effect of different concentrations of NTSG-PEG-CaCl2 reagent testicular injection on the preparation of ram receptors
[0046] 1. Experimental Animals and Grouping
[0047] Ten 4-month-old rams were selected and randomly divided into three groups:
[0048] Experimental group 1: 4 rams were injected with 0.02% concentration of NTSG-PEG-CaCl2 complex solution (NTSG-PEG-CaCl2 reagent for short).
[0049] Experimental Group 2: 4 rams received injection of NTSG-PEG-CaCl2 reagent at a concentration of 0.03%.
[0050] Blank control group: 2 rams were injected with an equal volume of 50% dimethyl sulfoxide (DMSO) solution.
[0051] 2. Injection Method
[0052] 1. Anesthesia: The rams were generally anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution at a dose of 60 mg / kg body weight. After the animals were fully anesthetized, they were kept in a supine position.
[0053] 2. Testicular exposure: While the animal is fixed, gently push the ram's testicles from the abdominal cavity into the scrotum to expose the injection site.
[0054] 3. Disinfection: Use 75% alcohol to wipe and disinfect the surface of the scrotum, and use the index finger and thumb of the left hand to gently fix the unilateral testicle to ensure stability during the injection process.
[0055] 4. Reagent injection: Use a 1.5mL sterile syringe to draw the NTSG-PEG-CaCl2 solution of the corresponding concentration, avoid the blood vessels on the testicular tunica albuginea, directly penetrate the scrotal skin, and slowly inject into the testicular tissue. The injection dose is determined by converting the adult dosage formula to ensure the appropriate dosage. After the injection is completed, the same concentration of reagent is injected into the other testicle using the same method.
[0056] 5. Vital sign monitoring: After the injection, closely observe the vital signs of the test ram, including respiration, heart rate and body temperature, to ensure the safety of the injection process and anesthesia effect.
[0057] 6. Recovery and feeding: After the test animals are fully awake, they are transferred to the feeding house and raised in a single pen, and their health status is monitored during the period. The test sheep are raised in a single pen for 18-60 days, so that the effects of different concentrations of NTSG-PEG-CaCl2 reagent on receptor preparation can be tested later.
[0058] 3. Treatment of blank control group
[0059] For the blank control group, an equal volume of 50% DMSO solution was injected into both testicles according to the above method to ensure the consistency of the control conditions.
[0060] 4. Subsequent testing
[0061] Effect of recipient preparation: After 18-60 days of single-pen rearing, samples were taken to detect histological changes in the recipient testes to evaluate the removal effect of endogenous spermatogenic cells and the retention of supporting cells and stromal cells.
[0062] Effect analysis: Analyze the decellularization effect of different concentrations of NTSG-PEG-CaCl2 reagent in the testis and its influence on the structure of seminiferous tubules, so as to provide experimental basis for optimizing the preparation method of spermatogonial stem cell transplantation recipients.
[0063] V. Results Analysis
[0064] The results are shown in Table 1.
[0065] Table 1
[0066]
[0067] Notes:
[0068] Spermatogenic cell apoptosis rate: the percentage of apoptotic spermatogenic cells in the total number of spermatogenic cells.
[0069] Supporting cell survival rate: the percentage of surviving supporting cells to the total supporting cells.
[0070] The structural integrity of the seminiferous tubules was scored: 1 indicated severe damage and 5 indicated structural integrity.
[0071] As shown in Table 1, different concentrations of NTSG-PEG-CaCl2 reagent had different effects on ram testicular tissue. 18 days after injection, the average apoptosis rate of spermatogenic cells in test group 1 was 67.25%, while the average apoptosis rate of spermatogenic cells in test group 2 was 80.75%, both higher than the apoptosis rate of spermatogenic cells in the blank control group (11%). This indicates that NTSG-PEG-CaCl2 reagent can effectively induce apoptosis of spermatogenic cells, and the higher the concentration, the higher the apoptosis rate.
[0072] 60 days after injection, the average survival rate of supporting cells in test group 1 was 82.5%, and the average survival rate of supporting cells in test group 2 was 77.25%, both lower than the survival rate of supporting cells in the blank control group (94%). This shows that the NTSG-PEG-CaCl2 reagent has a certain effect on the survival of supporting cells, but most supporting cells can still survive.
[0073] The scores of the seminiferous tubules structural integrity showed that the scores of the seminiferous tubules structural integrity of experimental groups 1 and 2 remained at 3-4 points at 18 days and 60 days after injection, while that of the blank control group was 5 points, indicating that the structural integrity of the testicular seminiferous tubules decreased after treatment with NTSG-PEG-CaCl2 reagent, but still maintained a certain structural integrity.
[0074] Comprehensive analysis showed that NTSG-PEG-CaCl2 reagent had a better removal effect on spermatogenic cells at a higher concentration (0.03%), but it also had a greater impact on the survival of supporting cells and the integrity of the seminiferous tubule structure.
[0075] Example 2
[0076] Effects of different concentrations of NTSG-PEG-CaCl 2 reagent on spermatogenic cells and supporting cells in vitro 1. Experimental materials
[0077] Test reagents: NTSG-PEG-CaCl2 composite solution was used, and the concentrations were prepared into 0.02% and 0.03% respectively.
[0078] Cell source: Spermatogenic cells and supporting cells (Serto li cells) isolated from the testicles of healthy rams at 4 months of age.
[0079] Culture medium: DMEM / F12 culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used.
[0080] 2. Cell Culture
[0081] 1. Cell separation: Spermatogenic cells and supporting cells are extracted from testicular tissue by enzymatic digestion.
[0082] 2. Cell culture: The isolated cells were suspended in DMEM / F12 culture medium and cultured at 37°C and 5% CO2 until the cells were stable.
[0083] 3. Reagent Handling
[0084] 1. Grouping treatment: The cultured spermatogenic cells and supporting cells are divided into three groups:
[0085] Experimental group 1: treated with 0.02% NTSG-PEG-CaCl2 solution.
[0086] Experimental group 2: treated with 0.03% NTSG-PEG-CaCl2 solution.
[0087] Blank control group: add an equal volume of 50% dimethyl sulfoxide (DMSO) solution.
[0088] 2. Add reagents: According to the experimental grouping, add the corresponding concentration of reagents to the culture medium, dilute to the appropriate concentration at a ratio of 1:10, and mix gently.
[0089] 3. Action time: Allow the reagent to act for 1 hour, 2 hours, 4 hours and 6 hours respectively, and observe the effects of different action times on cells.
[0090] 4. Experimental testing
[0091] 1. Cell viability detection: After each action time point, take the cells in the culture plate and use CCK-8 or MTT reagent to detect cell viability to evaluate the toxic effect of the reagent on the cells.
[0092] 2. Cell apoptosis and necrosis detection: Annexin V / PI staining method was used to detect the ratio of cell apoptosis and necrosis, so as to distinguish the survival status of each cell after reagent treatment.
[0093] 3. Microscope observation: Observe the changes in cell morphology under an inverted microscope, record the status of spermatogenic cells and supporting cells, and evaluate their morphological changes after being affected by the reagents.
[0094] 4. Data analysis: Record the cell survival rate, apoptosis rate and necrosis rate at each time point and reagent concentration, and perform statistical analysis to evaluate the selective effect of different concentrations of NTSG-PEG-CaCl2 on cells.
[0095] 5. Results Analysis
[0096] By comparing the cell viability and apoptosis rate at different concentrations and different action times, the removal effect of NTSG-PEG-CaCl2 reagent on spermatogenic cells and its selective effect on supporting cells were determined.
[0097] The results are shown in Table 2.
[0098] Table 2
[0099]
[0100]
[0101] Notes:
[0102] Cell viability: the proportion of cells that did not undergo apoptosis or necrosis in the experimental group.
[0103] Cell apoptosis rate: the proportion of apoptotic cells detected after reagent treatment.
[0104] As shown in Table 2, with the increase of NTSG-PEG-CaCl2 reagent concentration and the extension of action time, the activity of spermatogenic cells gradually decreased, while the activity of supporting cells decreased to a smaller extent. Specifically, in the 0.02% NTSG-PEG-CaCl2 treatment group, the activity of spermatogenic cells and supporting cells remained at a high level at 1 hour and 2 hours of action time, but as the action time was extended to 4 hours and 6 hours, the activity of spermatogenic cells decreased significantly and the apoptosis rate increased significantly. In the 0.03% concentration treatment group, the activity of spermatogenic cells decreased more significantly and the apoptosis rate was also higher, while although the activity of supporting cells decreased, the increase in apoptosis rate was relatively small.
[0105] This indicates that the NTSG-PEG-CaCl2 reagent has a certain toxic effect on spermatogenic cells and can induce spermatogenic cell apoptosis, and this effect is concentration- and time-dependent. At the same time, the tolerance of supporting cells to NTSG-PEG-CaCl2 is relatively good, and the increase in apoptosis rate is lower than that of spermatogenic cells, indicating that NTSG-PEG-CaCl2 has a certain selective effect on spermatogenic cells and supporting cells.
[0106] In summary, NTSG-PEG-CaCl2 reagent can effectively reduce the viability of spermatogenic cells and induce their apoptosis in vitro, while the effect on supporting cells is relatively small.
[0107] In summary, based on in vitro and in vivo test data, NTSG-PEG-CaCl2 solution has the following characteristics and effects in the preparation of spermatogonial stem cell transplantation recipients:
[0108] 1. Significant effect of spermatogenic cell removal
[0109] In the in vivo test, NTSG-PEG-CaCl2 solutions at concentrations of 0.02% and 0.03% can effectively promote the apoptosis of spermatogenic cells. As the concentration increases, the apoptosis rate of spermatogenic cells increases significantly, indicating that NTSG-PEG-CaCl2 has a concentration-dependent removal effect on spermatogenic cells.
[0110] In the in vitro experiment, as the action time prolonged, the activity of spermatogenic cells decreased significantly and the apoptosis rate increased accordingly, which further confirmed the continuous removal effect of NTSG-PEG-CaCl2 on spermatogenic cells.
[0111] 2. Protective effect on supporting cells
[0112] In the in vivo test, the survival rate of supporting cells at different concentrations remained at a high level, especially at a concentration of 0.02%, the survival rate of supporting cells exceeded 80%, and the structure of the seminiferous tubules remained well. This shows that NTSG-PEG-CaCl2 minimized the damage to supporting cells while removing spermatogenic cells.
[0113] In in vitro experiments, supporting cells showed higher tolerance than spermatogenic cells. Even when the exposure time was prolonged, the apoptosis rate of supporting cells was significantly lower than that of spermatogenic cells, indicating that NTSG-PEG-CaCl2 has a certain selective protective effect on supporting cells.
[0114] 3.Maintenance of the structure of seminiferous tubules
[0115] After the NTSG-PEG-CaCl2 solution was applied in vivo, the structure of the seminiferous tubules remained good, especially at a lower concentration (0.02%), with a high survival rate of supporting cells and sufficient apoptosis of spermatogenic cells, creating an ideal "cavity" environment for the transplantation and colonization of donor spermatogonial stem cells. This is crucial to ensure that donor cells can reconstruct spermatogenic function after transplantation.
[0116] 4. Analysis of the advantages and disadvantages of in vivo and in vitro experiments
[0117] Advantages of in vivo experiments: The injection operation of NTSG-PEG-CaCl2 solution is relatively simple, and it can effectively remove spermatogenic cells while maintaining the structure of the seminiferous tubules, providing an ideal colonization environment for donor spermatogonial stem cells.
[0118] Advantages of in vitro experiments: In vitro experiments facilitate direct observation of cell responses to reagents and can precisely control time and dosage, further confirming the selective removal effect of the reagent and providing a reference for dosage and duration for in vivo applications.
[0119] 5. Overall evaluation
[0120] As a novel reagent, NTSG-PEG-CaCl2 solution provides an efficient and low-side-effect alternative method for the preparation of spermatogonial stem cell transplantation recipients by selectively removing endogenous spermatogenic cells while basically retaining the function of supporting cells and the structure of seminiferous tubules.
[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing a NTSG-PEG-CaCl2 reagent, characterized in that: The preparation method comprises: The polyethylene glycol is dissolved in a buffer solution, and detosylglycine is added. After the polyethylene glycol is completely dissolved, calcium chloride is added, and then glycerol is added to obtain the NTSG-PEG-CaCl2 reagent.
2. The preparation method according to claim 1, characterized in that: The preparation method further comprises: The polyethylene glycol is dissolved in a buffer solution and the concentration is adjusted to 10% (w / v), destoluenesulfonyl glycine is added at a ratio of 1% (w / v), and after complete dissolution, calcium chloride is added until the concentration reaches 0.5 mM, and then glycerol is added to a concentration of 0.5% (v / v) to obtain the NTSG-PEG-CaCl2 reagent.
3. The preparation method according to claim 1, characterized in that: The buffer is phosphate buffer.
4. A NTSG-PEG-CaCl2 reagent, characterized in that The NTSG-PEG-CaCl2 reagent is prepared by the preparation method described in any one of claims 1-3.
5. Use of the NTSG-PEG-CaCl2 reagent according to claim 4 in preparing a spermatogonial stem cell transplantation recipient.
6. A method for preparing spermatogonial stem cell transplantation recipient cells, characterized in that: The spermatogonial stem cell transplantation recipient cells are obtained by injecting the NTSG-PEG-CaCl2 reagent described in claim 4 into the testicular tissue of a ram.
7. The method for preparing spermatogonial stem cell transplantation recipient cells according to claim 6, characterized in that: The concentration of the NTSG-PEG-CaCl2 reagent is 0.02-0.03%.
8. A method for preparing spermatogonial stem cell transplantation recipient cells, characterized in that: The spermatogonial stem cell transplantation recipient cells are obtained by culturing cells using the NTSG-PEG-CaCl2 reagent described in claim 4 as a culture medium component.
9. The method for preparing spermatogonial stem cell transplantation recipient cells according to claim 8, characterized in that: The cells are spermatogenic cells.
10. The method for preparing spermatogonial stem cell transplantation recipient cells according to claim 8, characterized in that: The concentration of the NTSG-PEG-CaCl2 reagent is 0.02-0.03%.