阅读说明：本技术 一种大鼠原代生精细胞体外分离的方法 (Method for in-vitro separation of primary rat sperm cells ) 是由 周党侠 葛攀 张健 杨妍琪 吕茉琦 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种大鼠原代生精细胞体外分离的方法,采用的方案包括：1)取新鲜离体大鼠双侧睾丸,剥除被膜及血管,获得睾丸组织；2)将睾丸组织消化处理制备单细胞悬液；3)将单细胞悬液培养直至非生精细胞贴壁后,吸出未贴壁的悬浮细胞,对悬浮细胞采用Percoll法分离获得生精细胞。本发明为体外研究大鼠睾丸生殖功能奠定基础,该方法实现了体外分离和培养大鼠睾丸生精细胞,并证实这种方法分离和培养的生精细胞体外存活率高,且存活时间较长。利用该方法能获得正常的大鼠睾丸生精细胞,花费少,操作简单,可重复度高,能满足多种实验需求,值得大范围推广。(The invention discloses a method for separating primary rat sperm cells in vitro, which adopts the following scheme: 1) taking fresh in-vitro rat bilateral testicles, and stripping tunica capsularis and blood vessels to obtain testicular tissues; 2) digesting testis tissue to prepare single cell suspension; 3) culturing the single cell suspension until the non-spermatogenic cells are attached to the wall, sucking the non-attached suspension cells out, and separating the suspension cells by adopting a Percoll method to obtain the spermatogenic cells. The invention lays a foundation for in vitro research of the reproductive function of the testis of the rat, realizes the in vitro separation and culture of the spermatogenic cells of the testis of the rat, and proves that the spermatogenic cells separated and cultured by the method have high in vitro survival rate and longer survival time. The method can be used for obtaining normal rat testicular spermatogenic cells, is low in cost, simple to operate, high in repeatability, capable of meeting various experimental requirements and worthy of being popularized in a large range.)

1. A method for in vitro isolation of primary rat sperm producing cells comprising the steps of:

1) taking fresh in-vitro rat bilateral testicles, and stripping tunica capsularis and blood vessels to obtain testicular tissues;

2) digesting testis tissue to prepare single cell suspension;

3) culturing the single cell suspension until the non-spermatogenic cells are attached to the wall, sucking the non-attached suspension cells out, and separating the suspension cells by adopting a Percoll method to obtain the spermatogenic cells.

2. The method for separating rat primary spermatogenic cells in vitro according to claim 1, wherein in step 3), spermatogenic cells are separated by Percoll method, which comprises the following specific operations:

respectively diluting the Percoll cell separation solution into Percoll gradient solutions with volume fractions of 10%, 25%, 35% and 50% by using NaCl with the mass concentration of 8.5%;

sequentially adding the same volume of Percoll gradient liquid into a centrifuge tube from high concentration to low concentration, slowly adding the cultured cell suspension into the Percoll gradient liquid at the uppermost layer, centrifuging, and collecting cells in layers.

3. The method for in vitro separation of rat primary spermatozoa according to claim 2, wherein the centrifugation is carried out at 1200-1600r/min for 15-30 min.

4. The method for in vitro isolation of rat primary spermatogenic cells according to claim 2, wherein spermatogenic cells are obtained by collecting cells between the interfaces of a 25% volume fraction Percoll gradient and a 35% volume fraction Percoll gradient when collecting cells in layers.

5. The method for separating primary spermatozoa of rats according to any one of claims 1 to 4, wherein in the step 2), the digestion treatment is to shear the testicular tissue into a minced shape, add a collagenase IV solution with a concentration of 1mg/ml which is 10 times the volume of the minced testicular tissue, shake the minced testicular tissue in a constant temperature water bath box at 37 ℃ for 20min, then centrifuge the minced testicular tissue at 900r/min for 8min, remove the supernatant, precipitate, add a digestion solution of complex enzyme, shake the digestion solution in a water bath at 37 ℃ for 30min, and finally add 10% FBS to terminate the digestion.

6. The method of claim 5, wherein the rat primary spermatozoa cells are separated in vitro by passing through a 400 mesh screen after digestion, collecting the filtrate, centrifuging at 900 rpm for 8min, discarding the supernatant, and adding high-sugar DMEM complete medium for resuspension to obtain a single cell suspension.

7. The method for the in vitro isolation of rat primary spermatozoa according to claim 6, characterized in that the high-sugar DMEM complete medium is a 10% FBS-containing high-sugar DMEM medium.

8. Rat primary fineness generator according to claim 1The method for in vitro separation of cells is characterized in that in the step 3), the single cell suspension culture is processed at 37 ℃ and 5% CO₂The culture box is used for culturing for 2 hours.

Technical Field

The invention belongs to the technical field of cell culture separation, and relates to a method for separating primary rat sperm cells in vitro.

Background

Nearly 10% -15% of fertile couples worldwide are afflicted by infertility, with about 50% of infertility caused by male factors. Several studies have shown that despite regional and ethnic differences, human sperm concentration has decreased dramatically and sperm quality and fertility have decreased significantly over the past few decades. Several studies have shown that disorders of the associated genetic material may be the major cause of dysspermia. Therefore, the research on the related genetic molecular mechanism of the spermatogenesis abnormality lays a foundation for the elucidation of the causes of idiopathic male infertility and the promotion of the development of related diagnosis and treatment technologies.

Spermatogenesis is a complex and unique cell differentiation process, which can be roughly divided into three stages: first, spermatogenic cells enter the mitotic phase, where spermatogonial cells differentiate into primary spermatocytes; then entering a meiosis stage, wherein the primary spermatocyte carries out chromosome replication, then homologous chromosomes are united and separated, two secondary spermatocytes are formed after the first meiosis, and then the secondary spermatocyte enters the second meiosis and sister chromosome separation to form a haploid round spermatocyte; finally, in the sperm formation stage, the round sperm cells form mature sperms after a series of complex processes such as complex morphological changes, nucleoprotein transformation and modification and the like. In the process of spermatogenesis, interference and influence on any link can possibly cause spermatogenesis failure, so that the research on the possible molecular mechanism of spermatogenesis is also a research hotspot in the current reproductive medicine field.

Because the mature animal spermatogenic cell lines in the current market are single in type and difficult to meet all experimental requirements, the current related research on spermatogenesis mostly focuses on the in vivo level of animals, and is difficult to extend to the in vitro cell level to carry out related experimental research. Although there have been in vitro culture methods for rat spermatogenic cells, most methods for extracting rat spermatogenic cells have been limited to the use of single enzyme digestion. The extraction method has the advantages of small number of spermatogenic cells obtained by a single experiment, difficulty in meeting experiment requirements, time consumption and labor consumption.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a method for separating rat primary spermatogenic cells in vitro, which has the advantages of high number of separated spermatogenic cells, high in vitro survival rate, longer survival time and capability of meeting experimental requirements.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a method for separating primary rat sperm cells in vitro, which comprises the following steps:

1) taking fresh in-vitro rat bilateral testicles, and stripping tunica capsularis and blood vessels to obtain testicular tissues;

2) digesting testis tissue to prepare single cell suspension;

3) culturing the single cell suspension until the non-spermatogenic cells are attached to the wall, sucking the non-attached suspension cells out, and separating the suspension cells by adopting a Percoll method to obtain the spermatogenic cells.

Preferably, in the step 3), the Percoll method is adopted to separate and obtain spermatogenic cells, and the specific operations are as follows:

respectively diluting the Percoll cell separation solution into Percoll gradient solutions with volume fractions of 10%, 25%, 35% and 50% by using NaCl with the mass concentration of 8.5%;

sequentially adding the same volume of Percoll gradient liquid into a centrifuge tube from high concentration to low concentration, slowly adding the cultured cell suspension into the Percoll gradient liquid at the uppermost layer, centrifuging, and collecting cells in layers.

Further preferably, the centrifugation treatment is carried out for 15-30 min under the condition of 1200-1600 r/min.

Preferably, when the cells are collected in layers, the cells between the interfaces of the Percoll gradient liquid with the volume fraction of 25% and the Percoll gradient liquid with the volume fraction of 35% are collected to obtain spermatogenic cells.

Preferably, in the step 2), the digestion treatment is to cut testis tissues into paste, add a 10-fold volume of type IV collagenase solution with the concentration of 1mg/ml, shake and digest the testis tissues in a 37 ℃ constant temperature water bath box for 20min, then centrifuge the testis tissues for 8min at 900r/min, remove supernatant, precipitate the testis tissues, add complex enzyme digestion solution, shake and digest the testis tissues in a 37 ℃ water bath for 30min, and finally add 10% fetal bovine serum FBS to stop digestion.

Further preferably, after the digestion treatment, the mixture is screened by a 400-mesh screen, the filtrate is collected, the mixture is centrifuged at 900r/min for 8min, the supernatant is discarded, and then high-glucose DMEM is added to complete the culture medium for resuspension to obtain a single-cell suspension.

Preferably, the high-glucose DMEM complete medium is 10% FBS-containing high-glucose DMEM medium.

Preferably, in step 3), the single cell suspension culture is treated at 37 ℃ with 5% CO₂The culture box is used for culturing for 2 hours.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for separating primary spermatogenic cells in vitro by using a rat model, lays a foundation for in vitro research of reproductive function of a rat testis, realizes in vitro separation of spermatogenic cells of the rat testis, and proves that the number of spermatogenic cells separated and cultured in one time by the method can exceed 1 multiplied by 10⁸And the cell morphology and the trypan blue counting cell survival rate are observed through Giemsa staining, the spermatogenic cells can survive for at least 5 days, the cell survival rate can reach 75% on the fifth day, and the cell morphology is circular and the edge halo is obvious. The method can be used for obtaining normal rat testicular spermatogenic cells, is low in cost, simple to operate, high in repeatability, capable of meeting various experimental requirements and worthy of being popularized in a large range.

Drawings

FIG. 1 is a primary rat spermatogenic cell not stimulated with formaldehyde; wherein A is primary rat spermatogenic living cells of all levels cultured in vitro for 24 hours under a microscope; b, performing in-vitro culture on the primary rat spermatogenic cell Giemsa stain for 24 hours; c, performing in-vitro culture for 72 hours, and staining primary rat spermatogenic cells Giemsa; d is the Giemsa staining of the primary rat spermatogenic cells cultured in vitro for 96 hours. (scale bar 50 μm)

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

1. laboratory animal

One male sex rat of sexual maturity (6-10 weeks old) from the experimental animal center of the university of Xian traffic.

2. Isolation of testis tissue

After the SD rat is sacrificed, soaking the SD rat in 75% alcohol for 10 minutes; bilateral testes were separated in a clean bench, carefully stripped of testicular capsule and blood vessels on tissues, and washed 3 times with sterile HBSS solution.

3. Digesting tissue mass

Cutting testis tissue into paste, adding 10 times volume of 1mg/ml collagenase IV (DIYIBIO, batch No. 10312) solution (Hank's balanced salt solution (prepared from Shanghai cultured organism, batch No. F40508)), and digesting at 37 deg.C in a constant temperature water bath for 20 min; centrifuging at 900r/min for 8min, and discarding the supernatant. Adding 5ml of compound protease digestive juice (manufacturer: Solarbio, lot: 606F031), digesting with shaking in water bath at 37 deg.C for 30min, and adding 5ml of 10% Fetal Bovine Serum (FBS) (manufacturer: fructus Ilicis Purpureae, lot: 20050503) to stop digestion.

4. Preparation of Single cell suspensions

Filtering with a 400-mesh screen, and collecting filtrate; centrifuging at 900r/min for 8min, and discarding the supernatant. The suspension was resuspended in high-glucose DMEM medium (product: Basalmedia, batch No. J210803) containing 10% FBS to obtain a single-cell suspension.

5. Removal of non-sperm cells

The cell suspension was transferred to a 10cm petri dish at 37 ℃ with 5% CO₂The culture box is used for culturing for 2 hours, and after other non-spermatogenic cells are attached to the wall, non-attached suspension cells are carefully sucked out.

6. Isolation of spermatogenic cells

Percoll cell separating medium (manufacturer: Gentihold, batch number: P1068-100ml) is respectively diluted into 10%, 25%, 35% and 50% gradient solutions by 8.5% NaCl, 2ml of each gradient solution is slowly added from high to low concentration from the bottom of a centrifuge tube, newly collected cell suspension is slowly added to the uppermost layer of the Percoll cell separating medium, 400g of the Percoll cell separating medium is centrifuged for 30min, and then cells are collected in layers. According to previous experimental results, cells on the 25% -35% Percoll interface are mainly spermatogenic; the collected cells were resuspended in 10% FBS-containing high-glucose DMEM medium and then incubated at 37 ℃ in 5% CO₂For subsequent experiments.

And (3) detection results:

referring to fig. 1, a in fig. 1: primary rat spermatogenic living cells at all levels under a mirror; b in FIG. 1: culturing in vitro primary rat spermatogenic cells for 24 hours, performing Giemsa staining after cell smear, and observing spermatogenic cells and cell aggregation at different levels under a light microscope; c in FIG. 1: carrying out in vitro culture on primary rat spermatogenic cells for 72 hours, and carrying out Giemsa staining; d in FIG. 1: primary rat spermatogenic cells were stained with Giemsa in vitro for 96 hours. It is clear from Giemsa staining that in the spermatogenic cells of primary rats cultured in vitro at different times, the nuclear structure of most cells is intact, and the cell morphology is normal, which indicates that most cells are not obviously damaged or dead. (scale bar 50 μm)

Meanwhile, the number of spermatogenic cells separated and cultured by the method of the invention in one time can exceed 1 × 10⁸Each cell per ml, and cell morphology observation and trypan blue counting cell viability rate through Giemsa staining find that spermatogenic cells can survive for at least 5 days, and the cell viability rate can reach 75% on the fifth day, and meanwhile, the cell morphology is circular, and the edge halo is obvious。

Compared with the prior art, the literature reports of Shandong university journal (medical edition) 2014.52(z1):8 and 23 show that the total number of spermatogenic cells obtained by adopting a single enzyme digestion method is only 1 multiplied by 10⁶One cell per ml, and the number of dead cells can reach 1 × 10⁵One per ml.

Therefore, it can be shown that the invention successfully constructs a rat primary spermatogenic cell model. Rat primary spermatogenic cells cultured according to the method of the invention survived for at least 96 hours without any intervention. As fully demonstrated above, the method for separating and culturing primary rat spermatogenic cells provided by the invention is feasible and has great significance for studying the reproductive function of rat testis in vitro.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.