CN114181891A

CN114181891A - Efficient culture method of mouse testicular organoid

Info

Publication number: CN114181891A
Application number: CN202111469549.1A
Authority: CN
Inventors: 李向东; 王盛鸿
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-03-15
Anticipated expiration: 2041-12-03
Also published as: CN114181891B

Abstract

The invention relates to a high-efficiency culture method of mouse testicular organoids, belonging to the field of bioengineering. The method comprises the following steps: (1) a mould for preparing organoid culture is designed by itself, and on the basis of the mould, a gel mould for organoid culture is prepared. (2) Separating primary testis cells, and adding key factors for inducing differentiation step by step, (3) adding the separated testis single cell suspension to the gel mold obtained in the step (1) for in vitro induction culture, so as to obtain testis organoids which have considerable quantity, testis lumen structure and can start meiosis. The application provides an excellent model for researching the interaction of each cell group of the testis and spermatogenesis in vitro, and can provide a reference method for culturing human testis organoids.

Description

Efficient culture method of mouse testicular organoid

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for efficiently culturing mouse testicular organoids with meiosis in vitro.

Background

Global statistics indicate that approximately 8-15% of couples worldwide are faced with infertility problems, more than half of which are caused by male factors. In some populations, the rate of infertility is as high as 30%. In China, the disease of infertility is gradually aggravated due to social problems such as "late marriage and late rearing". Non-obstructive infertility, which is the most serious type of male infertility, is caused by abnormalities in spermatogenesis in the testes. The study of spermatogenesis in humans has been very limited due to legal and ethical constraints. The establishment of the human testicular organoid culture method provides a powerful tool for solving the problem. By utilizing testis organoid, the research on spermatogenesis mechanism and interaction of each cell group of testis can be carried out in vitro, and the generation of various infertility can be simulated, thereby providing reference for formulating personalized treatment scheme. At present, a plurality of methods for culturing testicular cells in vitro have been established, however, a method for culturing testicular organoids which is efficient and can initiate meiosis is still lacking.

Takehiko Ogawa et al establishes a testis tissue block in-vitro culture method based on an air-liquid culture interface in 2011, in their system, an in-vitro testis tissue block can continue to develop and survive, further spermatogenesis is induced, sperm with fertilization capability is generated, and further, by adding a microfluidic device, the tissue block can survive for a longer period of time (Sato et al, 2011). Their approach, while achieving induction of complete spermatogenesis in vitro, starts with immature testicular tissue mass rather than individual testicular cells, which limits the utility of this approach. In addition, the cultured testicular tissue mass cannot be further proliferated, and the number of testicular tissue masses that can be simultaneously cultured is limited. In another study in 2013, the main structure of testis was reconstructed in vitro using testis single cells and the occurrence of haploid sperm cells was observed (Yokonishi et al, 2013), but here the obtained haploid cells were not evaluated for fertilization potential and the presence of spermatogonial stem cells in the reconstructed testis tissue was not examined.

In 2016, Hanchun et al, meiosis of spermatogonial stem cells was induced in vitro by co-culturing spermatogonial stem cells and supporting cells and addition of retinoic acid, with an induction efficiency of 28% (Wang et al, 2016), but only up to the spermatocyte stage, but no haploid cells were produced, and in this system, co-culturing of two types of cells lacked the three-dimensional structure of testis.

Jan-Bernd Stukenborg et al constructed an in vitro culture system of testis organoids in 2017 in rats by Matrigel, successfully obtained testis organoids with blood barrier structure and spermatogenic cell survival (Alves-Lopes et al, 2017). Although survival of the major testicular cell population has been observed in their system, no meiosis was observed.

Geert Hamer et al also induced a co-culture system of spermatogonial stem cells and supporting cells in 2020 by addition of a series of induction factors, which in addition to obtaining cells in the metaphase of meiosis, also obtained sperm-like cells (Lei et al, 2020), but they did not evaluate this sperm-like cell functionally.

Terea K.Woodrff et al realized that multiple testicular organoids could be cultured simultaneously in 2020 by microwell culture, that they obtained a very good testicular luminal structure and secreted testosterone and inhibin B in response to gonadotropins (Edmonds and Woodrff, 2020), but too few spermatogenic cells remained in their testicular organoids and the onset of meiosis was difficult to observe.

In the research of testis in vitro reconstruction at present, partial research can realize the induction of in vitro spermatogenesis, but the induction efficiency is generally low, and the tissue structure of in vivo testis is not available; some studies can form the physiological structure of the testis, but lack the presence of spermatogenesis; at present, no research is available for efficiently realizing both testicular tissue structure and spermatogenesis in vitro. There is a need to improve existing methods and to propose new culture strategies.

Disclosure of Invention

By improving the micropore culture method and optimizing the components of the culture medium, dozens or hundreds of testicular organoids can be simultaneously cultured in one culture process, and besides the testicular tubular cavity structure, the testicular tissue can start meiosis and has the endocrine function.

The invention adopts the following technical scheme:

the invention provides a method for efficiently culturing mouse testicular organoid with meiosis in vitro, which comprises the following steps:

(1) designing parameters of a culture mold, and manufacturing the culture mold by using a 3D printing technology;

(2) under the premise of culturing by using a mould, a step-by-step culture method is adopted: separating to obtain testis single cell suspension, inoculating into culture mold, culturing in 34 deg.C incubator, and using culture medium I from 1 st to 3 rd days;

(3) changing to culture medium II on the 4 th to 9 th days of culture;

(4) on days 10-16 of culture, medium III was replaced.

Preferably, the mold in step (1) comprises three parts, namely a bottom part, a circular groove and bases fully distributed with bulges, wherein the cylinder at the bottom part is 15-30mm and 3-52mm in height, the diameter of the outer edge of the groove is 10-3020mm, the diameter of the inner edge of the groove is 10-3015mm and 5-105mm in height, the bases of the bulges are cuboids with the length of 5-107mm, the width of 5-105mm and the height of 1-52.5mm, the lower half part of each bulge is a cylinder with the diameter of 0.5-1.00.8mm and the height of 0.1-0.50.4mm, the radius of the upper half part of the hemisphere is 0.1-0.50.4mm, and the number of bulges on each base is 25-80 and the bulges are arranged in a rectangular manner.

Preferably, the mold of step (1) has a biocompatible material as a printing material.

Preferably, the material of the mould in the step (1) is a mixed solution of 1-3% agarose solution and 1-5% F127 polylactic acid nano particles.

Preferably, the mouse used for isolating testicular single cells in step (2) is aged from 1 to 7 days after birth.

Preferably, the testicular single cells are seeded in the culture molds in the number of 10 to 100 ten thousand cells per gel mold in the step (2).

Preferably, the culture media I, II and III in the steps (2), (3) and (4) are all added with corresponding components on a basic culture medium, and the components of the basic culture medium consist of alpha-MEM, NaHCO3, KSR, penicillin and streptomycin.

Preferably, the medium I in step (2) is added to a basic medium to obtain transferrin, bovine serum albumin, insulin, L-glutamine, vitamin C, 2-mercaptoethanol, a fatty acid mixture, human GDNF, human bFGF, N2 supplement, mouse EGF and human platelet lysate. Further, the transferrin concentration is between 1-100 mug/mL, the bovine serum albumin concentration is between 1-10mg/mL, the insulin concentration is between 1-100 mug/mL, and the vitamin C concentration is 10^-3-10^-5Between mol/L, human GDNF concentration is between 1-100ng/mL, human bFGF concentration is between 1-10ng/mL, human platelet lysate is between 1-5%, and mouse EGF concentration is 1-10 ng/mL.

Preferably, the medium II in step (3) is a basic medium supplemented with retinoic acid, BMP4 and activin A, testis extract. Further, the concentration of retinoic acid is 10^ one-⁶-10^-⁷The concentration of mouse BMP4 is between 1 and 100ng/mL, the concentration of mouse activin A is between 1 and 100ng/mL, and the concentration of testis extract is 500 mug/mL.

Preferably, the medium III in step (4) is supplemented with testosterone, FSH, hCG, BPE and mouse testis extract on a basal medium. Further, testosterone concentration is between 1-10 μmol/L, mouse FSH concentration is between 1-200ng/mL, hCG concentration is between 1-5IU/mL, mouse BPE concentration is between 1-100 μ g/mL, and mouse testis extract concentration is 500 μ g/mL.

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

in previous article reports, the testis organoid spermatogenic cells obtained by the micropore culture method have very low content and do not start meiosis (Edmonds and Woodruff, 2020; Sakib et al, 2019), on the basis, the proportion of spermatogenic cells is improved by optimizing and improving the component proportion of the culture medium, and 80% of spermatogenic cells can be observed to carry out meiosis, and the organs have endocrine function. By the method, the mouse testicular organoid which is efficiently cultured and has a testicular tissue structure is established, so that an in-vitro research method can be provided for basic research of mouse male reproduction, a referential experimental scheme is provided for establishing the human testicular organoid in the future, and the method has important application values for understanding spermatogenesis and researching the pathogenesis of male infertility.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a flow chart of the culture;

FIG. 2 is a 3D schematic diagram and three-dimensional view of a model for designing culture;

FIG. 3 shows that 35 testicular organoids can be cultured simultaneously in a organoid culture gel culture mold;

FIG. 4 is a plot of testicular organoid morphology in micropores on different days;

FIG. 5 is a 16-day cultured testicular organoids obtained by hematoxylin and eosin staining;

FIG. 6 is the localization of perivascular myoid cell marker α -SMA as shown by immunofluorescence staining (red fluorescence), with the right panel being the combined colocalization map DAPI stained nuclei, blue;

FIG. 7 is a plot of the co-localization of SOX9(Sertoli cell biomarker, red fluorescence) with DDX4 (spermatogenic cell biomarker, green fluorescence) (as a composite plot, yellow fluorescence) showing the distribution of Sertoli cells and spermatogenic cells in organoids and DAPI showing the nucleus;

FIG. 8 is a co-localization of SYCP3 (meiotic biomarker, red fluorescence) with DDX4 (spermatogenic cell biomarker, green fluorescence) (as a yellow fluorescence plot) showing primary spermatocytes entering meiosis;

FIG. 9 shows that the organ has endocrine function, and can produce Testosterone (Testosterone) and Inhibin (Inhibin B).

Detailed Description

The invention discloses a method for efficiently culturing mouse testicular organoid with meiosis in vitro, which can be realized by a person skilled in the art by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the whole process of in vitro induction culture of testicular organoids, ICR or C57 male mice within one week are used, testicular tissues are digested into single cell suspensions by a two-step enzymatic digestion method, cells are transferred into micropores poured by gel at a certain concentration, and stem cell maintenance factors and meiosis inducing factors are added step by step for culture, wherein the outline of the steps is shown in figure 1. In addition, a high proportion of spermatogenic cells are left in vitro, and the meiosis process is started. When the testis organoids are cultured for a suitable number of days, the tissue structure is observed by HE staining, and the presence and location of the corresponding cells are analyzed by immunofluorescence.

EXAMPLE 1 preparation of culture molds

(1) Designing the parameters of the die: in reference to the pore size of the micropore culture system in the literature, three different sizes of pore sizes were designed for testing, and parameters having the best effect of single cell agglomeration were selected, wherein the diameter of the micropore is 0.5-0.8mm, the height is 0.5-0.8mm, the lower half part is a hemisphere, and the upper half part is a cylinder, and a 3D diagram and a three-view diagram thereof are shown in FIG. 1.

(2) Pouring of the mold: soaking the mold in 75% alcohol and ultraviolet irradiating for 30min, preparing 3% agarose with ultrapure water, sterilizing with high pressure steam, sucking 600 μ L of the melted mixed solution into the mold groove, rapidly transferring into a-20 deg.C refrigerator, and standing for 5 min;

(3) inserting the tip of a tweezers into the edge of the gel block, carefully taking out the gel block, placing the gel block in a new culture dish, pouring PBS (containing 1% penicillin and streptomycin mixed solution) to cover the gel block, clamping the gel block in a 24-hole cell culture plate by the tweezers, adding 1mL of a basal medium, and placing the cell culture plate in an incubator at 37 ℃ for balancing for 30 min; is used for cell inoculation. As shown in fig. 2, 35 testicular organoids can be cultured simultaneously in one gel well of one 24-well plate.

EXAMPLE 2 culture of testis organoids

A simple culture circuit is shown in FIG. 3. The method comprises the following specific steps:

(1) killing newborn mice (no more than one week old) by removing neck, soaking in 75% alcohol for sterilization for 1min, cutting open abdominal cavity of mice, and cleaning testis in PBS for at least 3 times;

(2) peeling the adhered epididymis tissue with fine forceps, tearing off the tunica albuginea, extruding out the seminal tubules, and cutting the testis tissue to pieces smaller than 2mm 3;

(3) transferring the testis tissues in the step (2) into a digestive juice I (consisting of 1mg/mL collagenase IV and 0.5mg/mL DNase I), carrying out water bath at 37 ℃ for 10min, and digesting 10 testis tissues by 1mL digestive juice at most;

(4) blowing and beating the digested testis tissue in the step (3) for 50 times by using a 1000-microliter pipette to ensure that no obvious tissue block is formed, if the tissue block is remained, continuing digestion in a water bath at 37 ℃ for 5min, blowing and beating for 50 times by using a 1000-microliter pipette, adding a digestive juice II (consisting of 1mg/mL collagenase IV, 0.5mg/mL DNase I, 1mg/mL hyaluronidase and 0.25% trypsin-EDTA), and performing water bath at 37 ℃ for 5 min;

(5) blowing and beating the testis tissue digested in the step (4) for 50 times by using a 200 mu L pipette to ensure that no obvious tubular tissue is visible, if the obvious tubular tissue is still visible, continuing digesting for 5min in water bath at 37 ℃, blowing and beating for 50 times by using a 200 mu L pipette, and adding 10% FBS (fetal bovine serum) to stop digesting;

(6) filtering the cell suspension obtained in the step (5) by using a 400-mesh cell screen, collecting filtrate, and centrifuging at 800rpm for 5 min;

(7) after centrifugation, discarding the supernatant, resuspending the supernatant with a fresh culture medium, counting cells, determining the survival rate of the cells by a trypan blue staining method, wherein the survival rate of the cells is at least 80% and can be used for subsequent experiments, and centrifuging the cell suspension at 800rpm for 5 min;

(8) centrifuging, removing supernatant, and resuspending with appropriate amount of culture medium I to make its cell concentration be 5 KHz 10⁶Per mL;

TABLE 1 Medium I composition and concentration

Composition (I)	Final concentration
		Basic culture medium	1Х
Transferrin	100μg/mL
		BSA	2mg/mL
Insulin	20μg/mL
		L-Glutamine	2mM
Vitamin C	10^-4M
		2-mercaptoethanol	55μM
Fatty acid mixed solution	100Х
		Human GDNF	20ng/mL
Human bFGF	5ng/mL
		Mouse EGF	10ng/mL
Human platelet lysate	3％
		N2 supplement	100Х

(9) Absorbing and abandoning the basic culture medium in the gel culture hole and the gel block, absorbing 60 mu L of the cell suspension in the step (8) into the groove of the gel block, blowing and uniformly mixing, and adding 200 mu L of the culture medium I into the gap between the gel block and the culture hole;

(10) culturing the cell suspension in the step (9) in an incubator at 34 ℃ overnight, adding 800 μ L of fresh medium I into each culture well, continuing culturing, changing the culture solution at the 2 nd day of culture, and changing half of the volume, wherein the microsphere is formed after overnight by the single cell suspension, and becomes denser with the addition of days as shown in FIG. 4.

(11) When the culture is carried out till the 4 th day, the culture medium I is sucked and removed, the culture medium I is replaced by the culture medium II, and half of the culture medium is replaced every two days;

TABLE 2 Medium II composition and concentration

Composition (I)	Final concentration
		Basic culture medium	1Х
Retinoic acid	10^-6M
		Mouse BMP4	20ng/mL
Mouse activin A	100ng/mL
		Testis extract	300μg/mL

(12) When the culture is carried out till the 10 th day, the culture medium II is sucked and removed, the culture medium II is replaced by the culture medium III, and half of the culture medium is replaced every two days;

TABLE 3 Medium 3 composition and concentration

Composition (I)	Final concentration
		Basic culture medium	1Х
Testosterone	10μM
		FSH	200ng/mL
hCG	4.5IU/mL
		BPE	50μg/mL
Testis extract	300μg/mL

(13) When the culture is carried out for 16 days, the gel block is placed in a culture dish containing PBS by tweezers, the gel block is slightly shaken to lead the organoid to fall into the culture dish, the organoid is collected in a centrifuge tube, and the organoid is kept stand to naturally settle;

example 3 testicular organoid production and HE staining

The testis organoids fixed by paraformaldehyde are dehydrated by 30% sucrose, are embedded into frozen sections by OCT, are sliced into 5 μm, and are observed under a light microscope after being stained by conventional HE, as shown in figure 5, the cavity structure of the testis organoids can be observed, and the testis organoids have seminal tubules, peritubular muscle-like cells and interstitial cells.

Example 4 testing physiological function of testis organoids

The testes have two of the most important physiological functions: spermatogenesis and hormone production. To identify whether cultured organoids have the above two functions, we performed the following experiments:

(1) immunofluorescence biomarker for detecting various cells in testis organoid

Frozen sections were blocked with 10% goat serum, incubated with α -SMA (peritubular myoid cells), SOX9(Sertoli cells), DDX4 (spermatogonium), SYCP3 (meiosis) antibody, incubated with the corresponding fluorescent secondary antibody, and observed using a fluorescent microscope. As shown in fig. 6, which shows the annular distribution of perimyoid cells, fig. 7 shows the distribution of Sertoli cells and spermatogenic cells, and fig. 8 shows the presence of spermatocytes entering the meiotic first mitotic prophase.

(2) Radioimmunoassay measures hormone production in testicular organoids.

The testis organoid is homogenized and sent to northern biological company, which utilizes a radioimmunoassay kit to detect that the organoid can produce hormones. As shown in fig. 9, production of testosterone (Leydig cells demonstrating function in testicular organoids) and inhibin (Sertoli cells demonstrating function in testicular organoids).

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A high-efficiency culture method of mouse testicular organoids is characterized by comprising the following steps:

a) printing a culture mould with a specific size by using a 3D printing technology, b) preparing a gel mould for organoid culture on the basis of the culture mould; c) digesting mouse testis tissue into single cell suspension, re-suspending the cells with culture medium, inoculating the cells into the small hole of gel mold, and culturing in three optimized culture mediums at 34 deg.C and 5% carbon dioxide environment for 16-40 days.

2. The method according to claim 1, wherein the mold parameters are as follows:

a) the device consists of a bottom, a circular groove and a base which is fully distributed with bulges;

b) wherein the bottom cylinder is 15-30mm and the height is 3-5 mm;

c) the diameter of the outer edge of the groove is 10-30mm, the diameter of the inner edge is 10-30mm, and the height is 5-10 mm;

d) the base of the convex base is a cuboid, the length of the base is 5-10mm, the width of the base is 5-10mm, the height of the base is 1-5mm, the lower half part of the convex is a cylinder, the diameter of the convex is 0.5-1.0mm, the height of the convex is 0.1-0.5mm, and the radius of the convex is 0.1-0.5 mm;

e) the number of the bulges on each base is 25-80, and the bulges are arranged in a rectangular mode.

3. The method of claim 1, wherein the casting mold is made of a gel consisting of 1-3% agarose solution and 1-5% Pluronic F127 (polylactic acid nanoparticles).

4. The method according to claim 1, wherein the mice are aged from 1 to 7 days after birth.

5. The method of claim 1, wherein the number of cells seeded in each gel mold is between 10 and 100 ten thousand.

6. The method of claim 1, wherein three different media are used in the culturing process, and all three media are supplemented with corresponding factors on a basal medium.

7. The method of claim 1, wherein the culture is performed on days 1 to 3 using a medium I which is a composition comprising transferrin, bovine serum albumin, insulin, L-glutamine, vitamin C, 2-mercaptoethanol, a fatty acid mixture, human glial cell line-derived neurotrophic factor (human GDNF), human basic fibroblast growth factor (bFGF), N2 supplement, mouse EGF, Human Platelet Lysate (HPL) added to a basal medium.

8. The method according to claim 7, characterized in that the transferrin is added at a concentration of between 1-100 μ g/mL; the concentration of bovine serum albumin is between 1 and 10 mg/mL; the concentration of insulin is between 1 and 100 mu g/mL; vitamin C concentration is 10^-3-10^-5mol/L is between; the concentration of human GDNF is between 1 and 100 ng/mL; the concentration of human bFGF is between 1 and 10 ng/mL; HPL is between 1-5%; mouse EGF concentrations were between 1-10 ng/mL.

9. The method according to claim 1, wherein the culture is performed on days 4 to 9 using a culture medium II comprising a combination of Retinoic Acid (RA), mouse bone morphogenetic protein 4(BMP4), mouse activin A (activin A) and mouse testis extract added to a basic culture medium.

10. The method of claim 9 wherein retinoic acid is added at a concentration of between 10^ -6 to 10^ -7mol/L, mouse BMP4 at a concentration of between 1 ng/mL to 100ng/mL, mouse Activin A at a concentration of between 1 ng/mL to 100ng/mL, and mouse testicular extract at a concentration of 100 μ g/mL.

11. The method according to claim 1, wherein the culture is performed using a medium III on days 10 to 16, which is a composition comprising testosterone, mouse Follicle Stimulating Hormone (FSH), human chorionic gonadotropin (hCG) and Bovine Pituitary Extract (BPE), mouse testis extract, added to a basal medium.

12. The method according to claim 11, wherein testosterone is added at a concentration of 1-10 μmol/L, mouse FSH is at a concentration of 1-200ng/mL, hCG is at a concentration of 1-5IU/mL, mouse BPE is at a concentration of 1-100 μ g/mL, and mouse testicular extract is at a concentration of 100 μ g/mL.