Method for isolating spermatogonial stem cells from frozen testicular tissue

Abstract

This invention provides a method for isolating spermatogonial stem cells from frozen testicular tissue, belonging to the field of cell biology. The isolation method provided by this invention includes the following steps: thawing frozen testicular tissue; digesting the thawed tissue with collagenase IV and DNase I until the seminiferous tubules are exposed and their walls become rough; washing with DPBS buffer, centrifuging, and culturing until cells migrate; collecting the migrated cells and purifying them for spermatogonial stem cell enrichment. The method described in this invention can maximally preserve the internal environment of spermatogonial stem cells, maintain cell viability, and reduce cell damage, and can be used for isolating spermatogonial stem cells from frozen testicular tissue.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to a method for isolating spermatogonial stem cells from frozen testicular tissue. Background Technology

[0002] Spermatogenesis comprises three stages: spermatogonial mitosis, spermatocyte meiosis, and spermatogenesis. The mitotic process involves undifferentiated spermatogonia (monotype A) s ), paired type (A) pr ) and chain-like (A) al Differentiated spermatogonia (A1, A2, A3, A4, In, and B types) are found in the spermatogonial cell pool. Undifferentiated spermatogonia contain spermatogonial stem cells that balance the self-renewal of the stem cell pool with the production of continuous spermatogenesis. Spermatogonial stem cells play a crucial role in maintaining spermatogenesis. As the only adult stem cell type that provides genetic information for the next generation, spermatogonial stem cells have broad application prospects in animal genetics, breeding, and reproduction. In 1994, Brinster used microinjection technology to transplant donor-derived mouse spermatogonial stem cells into the testes of recipient mice pre-treated with busulfan. The spermatogonial stem cells colonized in the seminiferous tubules and restarted spermatogenesis to produce offspring. This experiment pioneered the technology of allogeneic spermatogonial stem cell transplantation. Through gene transfection and homologous transplantation of spermatogonial stem cells, not only can the superior traits of excellent breeding animals be inherited, but commercially valuable transgenic animals can also be produced, accelerating the breeding process.

[0003] However, spermatogonial stem cells constitute a very small percentage of the testes, only 0.2%–0.3% in bovine spermatogonial stem cells. Therefore, it is necessary to enrich and purify isolated spermatogonial stem cells to meet the requirements for culture and transplantation. Spermatogonial stem cell isolation mainly employs a two-step enzymatic digestion method, and the main methods for enrichment and purification include immunomagnetic bead sorting, flow cytometry sorting, differential adhesion, and density gradient centrifugation. Compared with other enrichment methods, differential adhesion yielded the best results for spermatogonial stem cell enrichment, but its specificity is low. Immunomagnetic beads and flow cytometry sorting can enrich spermatogonial stem cells to high purity. For example, Li et al. used flow cytometry sorting or immunomagnetic bead separation to obtain Thy1... +Spermatogonial stem cell populations are available, but their availability is limited by cell surface-specific marker genes. Density gradient centrifugation is widely used for the enrichment and purification of spermatogonial stem cells in animals such as humans, monkeys, sheep, pigs, and cattle. In the enrichment and purification of bovine spermatogonial stem cells, Izadyar et al. used the Percoll gradient to purify a testicular cell suspension containing 25.5% bovine type A spermatogonia to 51%; Oatley et al. used the Percoll gradient plus differential adhesion method to separate highly enriched bovine spermatogonial stem cell fractions. However, trypsin in the two-step enzymatic digestion method causes significant cell damage and can impair cell viability. Especially when the tissue to be separated is frozen tissue, frozen tissue is more fragile than fresh tissue, making it difficult to effectively isolate highly viable spermatogonial stem cells. Therefore, developing a method for isolating spermatogonial stem cells that can reduce cell damage during the separation process and is suitable for frozen tissue is crucial research in this field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for isolating spermatogonial stem cells from frozen testicular tissue, which can maximize the preservation of the internal environment of spermatogonial stem cells, maintain cell activity, and reduce cell damage.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for isolating spermatogonial stem cells from frozen testicular tissue, comprising the following steps:

[0007] Frozen testicular tissue was revived, and after revival, collagenase IV and DNase I were added for digestion until the seminiferous tubules were exposed and the tubule walls became rough. The tissue was then washed with DPBS buffer, centrifuged, and cultured until the cells migrated. The migrated cells were collected and purified as spermatogonial stem cells.

[0008] Preferably, the thawing method includes: placing the cryovial in a water bath at 36–38°C for thawing until it is completely thawed.

[0009] Preferably, the frozen testicular tissue includes frozen bovine testicular tissue.

[0010] Preferably, the testicular tissue is digested by sequentially adding equal volumes of DPBS buffer, collagenase IV solution, and DNase I solution.

[0011] Preferably, the concentration of the collagenase IV solution is 0.5–2 mg / mL, and the concentration of the DNAase I solution is 0.5–2 mg / mL.

[0012] Preferably, the digestion conditions are: water bath incubation at 36–38°C.

[0013] Preferably, the culture conditions are: 36-38℃, cultured in a 5% CO2 incubator, and the culture medium is DFS medium.

[0014] Preferably, the method for enriching and purifying spermatogonial stem cells is as follows: Percoll gradient centrifugation is used to purify and obtain spermatogonial stem cells.

[0015] Preferably, 40% Percoll solution, 20% Percoll solution and effluxed cell suspension are added sequentially to a centrifuge tube. After centrifugation, the intermediate phase between the 40% Percoll solution and the 20% Percoll solution is taken to obtain spermatogonial stem cells.

[0016] Preferably, the volume ratio of the 40% Percoll solution, the 20% Percoll solution, and the emigrating cell suspension is 1:1:2.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention maintains the stability of the internal environment of spermatogonial stem cells through short-term culture of seminiferous tubules, while allowing cells to migrate naturally. This reduces the damage to cells caused by trypsin in the two-step enzymatic digestion method, maintains cell viability, and is more suitable for the isolation of spermatogonial stem cells from frozen testicular tissue. It not only solves the problem of long-term preservation of tissues used for cell isolation, but also provides important support for the preservation of genetic information and individual restoration and renewal of rare and endangered species.

[0019] The experimental method described in this invention is simple, convenient, easy to operate, and conducive to promotion and implementation. Attached Figure Description

[0020] Figure 1 These are seminiferous tubules exposed after enzyme digestion.

[0021] Figure 2 This facilitates the migration and adhesion of seminiferous tubule cells.

[0022] Figure 3 The spermatogonial stem cells are growing in clusters, with black arrows indicating the spermatogonial stem cells;

[0023] Figure 4 The results are for the identification of spermatogonial stem cells by fluorescent staining. Detailed Implementation

[0024] This invention provides a method for isolating spermatogonial stem cells from frozen testicular tissue, comprising the following steps:

[0025] Frozen testicular tissue was revived, and after revival, collagenase IV and DNase I were added for digestion until the seminiferous tubules were exposed and the tubule walls became rough. The tissue was then washed with DPBS buffer, centrifuged, and cultured until the cells migrated. The migrated cells were collected and purified as spermatogonial stem cells.

[0026] The frozen testicular tissue described in this invention is preferably frozen bovine testicular tissue.

[0027] The frozen testicular tissue described in this invention needs to be thawed before being used for spermatogonial stem cell separation. The thawation method can be as follows: the cryopreservation tube is removed from liquid nitrogen and immediately placed in a water bath at 36-38°C for thaw. After complete thawing, subsequent cell separation experiments are performed. A more preferred water bath temperature is 37°C.

[0028] This invention first separates the seminiferous tubules from the revived testicular tissue. Preferably, the thawed testicular tissue is washed with DPBS buffer before separation. As an optional implementation, the thawed testicular tissue is placed in a centrifuge tube, DPBS buffer containing 10% penicillin and streptomycin solution (P / S) is added, and the mixture is incubated on ice for 4–6 minutes. The supernatant is then removed, and this process is repeated 2–3 times.

[0029] In this invention, DPBS buffer, collagenase IV solution, and DNase I solution are sequentially added to testicular tissue for digestion. The preferred volume ratio of the DPBS buffer, collagenase IV solution, and DNase I solution is 1:1:1. The concentration of the collagenase IV solution is 0.5–2 mg / mL, preferably 1 mg / mL; the concentration of the DNase I solution is 0.5–2 mg / mL, preferably 1 mg / mL. The digestion conditions are: incubation in a water bath at 36–38°C. As an optional embodiment, the digestion system is incubated in a water bath at 37°C, with repeated shaking and observation under a microscope until the seminiferous tubules are exposed and their walls become rough, at which point digestion is terminated.

[0030] In this invention, DPBS buffer is added to the separated seminiferous tubules to terminate digestion, and the mixture is incubated on ice for 8–15 min. The supernatant is discarded, and this process is repeated 2–3 times to remove mesenchymal cells. The precipitate is then resuspended in DPBS buffer, centrifuged, and resuspended in DFS medium for cell culture until cell migration occurs. The culture conditions described in this invention are 36–38°C, preferably 37°C, and the culture time is preferably 22–24 h. As an optional embodiment, the cells are resuspended in DFS medium and seeded into 6-well plates, then cultured in a 5% CO2 incubator.

[0031] The preferred formulation of the DFS culture medium of the present invention is: 88.5%–90% DMEM culture medium + 8.5%–10.5% FBS + 1%–1.5% penicillin and streptomycin solution (P / S).

[0032] In this invention, a culture plate inoculated with seminiferous tubules is removed, and cell migration is observed under a microscope. After cell migration is observed, the cells are pipetted and the cell suspension is collected. The suspension is allowed to stand for 8–12 minutes, and the supernatant is passed through a 40 μm cell sieve. The cells after sieving are collected.

[0033] In this invention, the collected cells are washed to prepare a DPBS cell suspension. As an optional embodiment, the sieved cells are centrifuged, the supernatant is removed, and the cells are washed again with DPBS buffer, centrifuged, the supernatant is removed, and the cells are resuspended in DPBS buffer to obtain a DPBS cell suspension; the preferred centrifugation conditions are 600g for 5 minutes.

[0034] The method for enriching and purifying spermatogonial stem cells described in this invention is as follows: Percoll gradient centrifugation is used to purify and obtain spermatogonial stem cells.

[0035] This invention employs Percoll gradient centrifugation on DPBS cell suspensions. The Percoll solution comprises 20% Percoll and 40% Percoll. In this invention, 40% Percoll, 20% Percoll, and the effluxed cell suspension are added sequentially to a centrifuge tube, with a volume ratio of 1:1:2. After centrifugation, the intermediate phase between the 40% and 20% Percoll solutions is collected (due to density differences after centrifugation, stratification occurs, with spermatogonial stem cells located in the intermediate phase), yielding spermatogonial stem cells. The centrifugation conditions can be selected as 600g for 7 minutes.

[0036] In this invention, the preparation methods for 40% Percoll and 20% Percoll are as follows:

[0037] 40% Percoll: 10% 10×PBS buffer, 50% ultrapure water, 40% Percoll solution, add 20 μL phenol red solution, and adjust the pH to 7.0 with concentrated hydrochloric acid.

[0038] 20% Percoll: 10% 10×PBS buffer, 70% ultrapure water, 20% Percoll solution, add 20 μL phenol red solution, and adjust the pH to 7.0 with concentrated hydrochloric acid.

[0039] Preferably, the present invention involves washing the intermediate phases of 40% Percoll solution and 20% Percoll solution 2-3 times with DPBS buffer, and then resuspending the cells in spermatogonial stem cell culture medium to obtain a cell suspension. Alternatively, after adding DPBS buffer, the cells are thoroughly mixed by pipetting, centrifuged, the supernatant is discarded, and the washing is repeated; the centrifugation conditions can be selected as 600g for 5min.

[0040] In this invention, the obtained spermatogonial stem cell culture medium resuspension is seeded into a culture plate coated with a feeder layer of cells for culture. Preferably, the cells are cultured in an incubator, with the culture medium changed every 2-3 days, and passaged every 5-7 days.

[0041] As an optional implementation, the method for preparing trophoblast cells according to the present invention is as follows: Mouse embryonic fibroblast (MEF) cells are cultured in DFS medium. When the cell confluence reaches 70%–80%, MEF cells are treated with mitomycin C (10 ng / mL) for 2 hours. After digestion with 0.25% trypsin for 3 minutes, DFS medium is added to terminate the digestion. The cells are collected by centrifugation at 600g for 5 minutes. The MEF cells are resuspended in DFS medium and cell counted at 5 × 10⁻⁶ cells / mL. 5 Cells were seeded into 6-well plates at a concentration of 1 / mL.

[0042] The spermatogonial stem cells isolated by this invention can be passaged. The passaged culture method can be as follows: when the cell confluence in the culture plate reaches 70% to 80%, the culture medium is discarded, 0.25% trypsin is added for digestion, and after about 3 minutes, DFS culture medium is added to stop the digestion. The cells are pipetted into a centrifuge tube, centrifuged at 600g for 5 minutes, the supernatant is discarded, spermatogonial stem cell culture medium is added for resuspension, and the cells are seeded into a culture plate pre-coated with feeder cells for culture.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] In a specific embodiment of the present invention,

[0045] DFS medium: 89% DMEM medium + 10% FBS + 1% penicillin and streptomycin solution (P / S);

[0046] 40% Percoll: 10% 10×PBS buffer, 50% ultrapure water, 40% Percoll solution, add 20 μL phenol red solution, and adjust the pH to 7.0 with concentrated hydrochloric acid;

[0047] 20% Percoll: 10% 10×PBS buffer, 70% ultrapure water, 20% Percoll solution, add 20 μL phenol red solution, and adjust the pH to 7.0 with concentrated hydrochloric acid;

[0048] Feeder cell preparation: Mouse embryonic fibroblasts (MEF) cells were cultured in DFS medium. When the cell confluence reached 70%–80%, MEF cells were treated with mitomycin C (10 ng / mL) for 2 h. After digestion with 2 mL of 0.25% trypsin for 3 min, digestion was terminated with 2 mL of DFS medium. Cells were collected by centrifugation at 600 g for 5 min, resuspended in 1 mL of DFS medium, and cell counts were performed at 5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of [cells / mL] into 6-well plates;

[0049] Spermatogonial stem cell culture medium: MEM-Alpha basal medium (Gibco, 12561-049), bovine serum albumin (Sigma, A6003) 5 mg / mL, total transferrin (Sigma, T0665) 100 μg / mL, insulin (Sigma, 12643) 25 μg / mL, L-glutamine solution 100× (Pronosine, PB180419) 1×, putrescine (Sigma, P5780) 20 μg / mL, sodium selenite (Sigma, 214485) 0. 3 nM, 2-thioethanol (Gibco, 21985) 55 μM, MEM non-essential amino acid 100× solution (Gibco, 11140) 1×, MEM vitamin 100× solution (Gibco, 11120052) 1×, StemPro™ additive 50× solution (Gibco, A1050801) 1×, GDNF (Peprotech, AF-450-10) 20 ng / mL, FGF (Peprotech, AF-100-18B) 5 ng / mL.

[0050] Unless otherwise specified, the following embodiments are all conventional methods.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] Example 1

[0053] This embodiment provides a method for isolating and culturing spermatogonial stem cells from cryopreserved bovine testicular tissue:

[0054] (1) Cryopreservation of testicular tissue

[0055] Preparation of cryopreservation solution:

[0056] DMEM, FBS and DMSO were mixed in a ratio of 7:2:1 to prepare a cryopreservation solution, which was then filtered through a 0.22 μm filter for later use.

[0057] Testicular tissue collection and cryopreservation:

[0058] Remove the tunica albuginea from the collected 3-5 month old testicular tissue, make a transverse cut in the middle of the testis, and slowly scrape thin slices (the size of a soybean) of testicular tissue along the transverse cut with a blade. Place the slices into cryovials, add about 1.5 mL of cryopreservation solution and mix well. Then, use a gradient cooling method to place the cryovials horizontally at 4℃ for 1-2 hours, -20℃ for 1-2 hours, and -80℃ for 12-16 hours. Finally, preserve them in liquid nitrogen for long-term storage.

[0059] (2) Frozen tissue revival

[0060] Remove the cryopreservation tube of testicular tissue stored in liquid nitrogen and immediately place it in a 37°C water bath to thaw. After it has completely thawed, transfer the tissue from the cryopreservation tube to a 50mL centrifuge tube.

[0061] (3) Separation and culture of spermatogonial stem cells

[0062] Semen fraction isolation and culture:

[0063] Wash with DPBS buffer, adding 10 mL of DPBS buffer (containing 10% P / S) each time, washing 3 times, and repeatedly pipetting during the process to disperse the tissue as much as possible. Then add 5 mL of DPBS buffer, 5 mL of collagenase IV solution (1 mg / mL), and 5 mL of DNase I solution (1 mg / mL), and incubate at 37°C in a water bath for 15 minutes, mixing repeatedly during the process to completely disperse the tissue, until the seminiferous tubules are exposed and the tubule walls are roughened (see...). Figure 1 Then, add 30 mL of DPBS buffer to stop the digestion. Place the centrifuge tube on ice and allow it to settle by gravity for 10 min. Discard the supernatant, add 10 mL of DPBS buffer to wash, and repeat this process 3 times to remove as many stromal cells as possible, retaining only the seminiferous tubules. Centrifuge at 400 g for 5 min and discard the supernatant. Resuspend the seminiferous tubules in 3 mL of DFS medium, seed them into 6-well plates, and incubate at 37°C in a 5% CO2 cell culture incubator for 24 h.

[0064] Enrichment and purification of spermatogonial stem cells:

[0065] After 24 hours, the culture plate was removed. At this time, the seminiferous tubules adhered to the wall, and a large number of cells migrated out of the tubules (see...). Figure 2Collect the cell suspension by pipetting, let it stand for 10 min, and then pass the supernatant through a 40 μm cell sieve. Collect the sieved cells together, centrifuge at 600 g for 5 min, discard the supernatant, wash with DPBS buffer, centrifuge at 600 g for 5 min, and then resuspend in 2 mL of DPBS buffer. Take a 15 mL centrifuge tube and add 1 mL of 40% Percoll solution, 1 mL of 20% Percoll solution, and 2 mL of cell suspension in sequence. Centrifuge at 600 g for 7 min, take the intermediate phase of 40% Percoll solution and 20% Percoll solution, add 8 mL of DPBS buffer, mix well by pipetting, and centrifuge at 600 g for 5 min. Discard the supernatant, repeat twice, resuspend in 3 mL of spermatogonial stem cell culture medium, and then seed into a culture plate with feeder cells. Incubate in an incubator, change the culture medium every 2-3 days, and passage after 5-7 days.

[0066] (4) Passaging of spermatogonial stem cells

[0067] When cell confluence reaches 70%–80%, discard the culture medium, add 2 mL of 0.25% trypsin for digestion, and when the cells become rounded after digestion, add 2 mL of LDF medium to stop digestion. Transfer the cells to a 15 mL centrifuge tube, centrifuge at 600 g for 5 min, discard the supernatant, resuspend in spermatogonial stem cell culture medium, and seed into culture plates pre-coated with feeder cells. At passage P1, spermatogonial stem cells begin to grow in clusters (see...). Figure 3 ).

[0068] (5) Spermatogonial stem cell identification

[0069] Remove the culture plate and carefully and slowly pipette to detach some cells. Aspirate the culture medium and transfer it to a 15 mL centrifuge tube. Add fresh spermatogonial stem cell culture medium to the culture plate. Centrifuge the detached cells at 600 g for 5 min, discard the supernatant, resuspend in DPBS buffer, centrifuge again at 600 g for 5 min, discard the supernatant, resuspend the pellet in 4% paraformaldehyde fixative, transfer to an adhesive slide, and incubate on ice for 20 min. When the cells settle to the surface of the slide, discard the fixative, add DPBS buffer, and wash gently on a shaker for 5 min each time. Add 0.1% Triton X-100 solution, permeate for 20 min, discard, and wash three times with DPBS buffer for 5 min each time. Discard the DPBS buffer, add 10% donkey serum for blocking for 1 hour, then add diluted primary antibodies (mouse-derived UCHL1 and rabbit-derived VASA), incubate at 4°C for 16 hours, wash three times with DPBS buffer for 10 minutes each time, add secondary antibodies (donkey anti-rabbit 488 and donkey anti-mouse 594), incubate at room temperature for 2 hours, then wash three times with DPBS buffer for 10 minutes each time, add DAPI staining solution, stain for 1 minute, then aspirate, mount with 50% glycerol, and observe under a fluorescence microscope (see...). Figure 4 ).

[0070] Depend on Figure 4 It can be seen that, through immunofluorescence staining, the germ cell marker gene VASA (green) and the spermatogonial stem cell marker gene UCHL1 (red) can both clearly stain the isolated and cultured cells, therefore the isolated and cultured cells are spermatogonial stem cells.

[0071] Currently, fresh testicular tissue is used for the isolation and culture of bovine testicular tissue, and no method has been found for the isolation and culture of spermatogonial stem cells using frozen bovine testicular tissue. This invention innovatively proposes a method applicable to cryopreserved bovine testicular tissue, followed by thawing and culture of spermatogonial stem cells. Furthermore, this invention also innovatively proposes a bovine seminiferous tubule fragment culture method, allowing cells to naturally migrate and enrich and purify spermatogonial stem cells. Bovine spermatogonial stem cells isolated and cultured using the method described in this invention showed clustered proliferation of spermatogonial stem cells in the P1 generation.

[0072] Example 2

[0073] The difference between this embodiment and embodiment 1 is that in step (3), the concentration of collagenase IV solution is 0.5 mg / mL and the concentration of DNAase I solution is 0.5 mg / mL.

[0074] Example 3

[0075] The difference between this embodiment and embodiment 1 is that in step (3), the concentration of collagenase IV solution is 2 mg / mL and the concentration of DNAase I solution is 2 mg / mL.

[0076] Example 4

[0077] The difference between this embodiment and Embodiment 1 is that the food is digested in a 38°C water bath for 13 minutes.

[0078] Example 5

[0079] The difference between this embodiment and Embodiment 1 is that the food is digested in a 36°C water bath for 16 minutes.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for isolating spermatogonial stem cells from frozen testicular tissue, characterized in that, Includes the following steps: Frozen testicular tissue was revived, and after revival, collagenase IV and DNase I were added for digestion until the seminiferous tubules were exposed and the tubule walls became rough. Wash with DPBS buffer, centrifuge, and culture until cells migrate. Collect emigrating cells and enrich and purify spermatogonial stem cells; The thawing method includes: placing the cryovials in a water bath at 36–38°C for thawing until they are completely thawed; The frozen testicular tissue includes frozen bovine testicular tissue; The testicular tissue was digested sequentially with equal volumes of DPBS buffer, collagenase IV solution, and DNase I solution; the concentration of collagenase IV solution was 0.5–2 mg / mL, and the concentration of DNase I solution was 0.5–2 mg / mL; the digestion conditions were: incubation in a water bath at 36–38°C. The culture conditions are as follows: cultured at 36-38℃ in a 5% CO2 incubator, using DFS medium; culture time is 22-24 hours; the DFS medium is prepared as follows: 88.5%-90% DMEM medium + 8.5%-10.5% FBS + 1%-1.5% penicillin and streptomycin solution; The method for enriching and purifying spermatogonial stem cells is as follows: Percoll gradient centrifugation is used to purify and obtain spermatogonial stem cells; 40% Percoll solution, 20% Percoll solution and efflux cell suspension are added sequentially to a centrifuge tube, and after centrifugation, the intermediate phase of 40% Percoll solution and 20% Percoll solution is taken to obtain spermatogonial stem cells; the volume ratio of 40% Percoll solution, 20% Percoll solution and efflux cell suspension is 1:1:2.

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