METHODS FOR ISOLATING DIFFERENT TYPES OF SINGLE CELLS FROM OVARY

Abstract

The invention provides a method for separating a mammal early follicle to obtain a single oocyte and a single granulocyte thereof. The method is capable of separating a mammal early follicle to obtain an active single oocyte and a corresponding granulocyte thereof. The invention further provides a kit for obtaining a single oocyte and a single granulocyte thereof from a mammal early follicle.

Claims

1. A method of obtaining a single oocyte of an early follicle of a mammal and single granulosa cell thereof, comprising the steps of: (1) blunt separating an ovarian tissue of the mammal; (2) digesting the mammalian ovarian tissue fragments in the first digestive solution at about 37 C. for about 20-60 minutes, wherein said first digestive solution contains collagenase I, collagenase II, collagenase IV or a mixture thereof; (3) passing the digestive solution mixture obtained in the step (2) through a first pore size cell strainer having a pore diameter of about 40 to 100 m and then passing the filtrate through a second pore size cell strainer having a pore size of about 8-12 m; (4) rinsing the precipitate in the second pore cell strainer with a culture solution, and then resuspending the precipitate with a culture solution; (5) aspirating a single early follicle in the resuspended solution; (6) after washing the obtained single early follicle, digesting it in the second digestive solution for about 3-10 minutes, the second digestive solution contains trypsin or Accutase; (7) transferring the digestive solution mixture obtained in step (6) to a culture solution, then separating and obtaining a single oocyte and single granulosa cells thereof.

2. The method of claim 1, wherein it further comprises the following step: (8) using the single oocyte and/or single granulosa cells thereof for single cell assay.

3. The method of claim 1, wherein the early follicle is a primordial follicle or a primary follicle.

4. The method of claim 1, wherein said first digestive solution comprises a mixture of Liberase and collagenase IV.

5. The method of claim 1, wherein said first digestive solution further comprises a neutral protease and/or a metalloprotease.

6. The method of claim 1, wherein said second digestive solution comprises Accutase.

7. The method of claim 1, wherein in step (6), the second digestion is carried out for about 3-5 minutes.

8. The method of claim 1, wherein said second pore size cell strainer is a Transwell.

9. The method of claim 1, wherein the mammal is a small and medium size mammal.

10. A kit for using in a method of obtaining a single oocyte of an early follicle of a mammal and single granulosa cell thereof as defined in claim 1, which comprising a first digestive solution and a second digestive solution, wherein said first digestive solution contains collagenase I, collagenase II, collagenase IV or a mixture thereof, and said second digestive solution contains trypsin or Accutase.

11. The kit of claim 10, wherein the early follicle is a primordial follicle or a primary follicle.

12. The kit of claim 10, wherein said first digestive solution comprises a mixture of Liberase and collagenase IV.

13. The kit of claim 12, wherein said first digestive solution further comprises a neutral protease and/or a metalloprotease.

14. The kit of claim 10, wherein said second digestive solution comprises Accutase.

15. The kit of claim 10, wherein the mammal is a small and medium size mammal.

16. The method of claim 4, wherein said first digestive solution comprises a mixture of Liberase and collagenase IV, wherein the ratio of Liberase to collagenase IV in said first digestive solution is about 1:4.

17. The method of claim 5, wherein said first digestive solution further comprises DNase.

18. The method of claim 9, wherein the mammal is a human being, a monkey, a caprine, a porcine, a bovine, a mouse, a rat, a guinea pig, a rabbit, a cat or a dog.

19. The kit of claim 12, wherein said first digestive solution comprises a mixture of Liberase and collagenase IV, wherein the ratio of Liberase to collagenase IV in said first digestive solution is about 1:4.

20. The kit of claim 13, wherein said first digestive solution further comprises DNase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows separated mice early follicles and their corresponding oocytes and granulosa cells observed under microscope. FIG. 1A shows separated early follicles. FIG. 1B shows separated oocytes and granulosa cells.

[0030] FIG. 2 shows oocytes observed under microscope. FIG. 2A shows single oocytes from mouse primordial follicles based on the method in Example 2. FIG. 2B shows single oocytes from mouse primary follicles based on the method in Example 2.

[0031] FIG. 3 shows the real-time PCR amplification curve and dissolution curve of the Gapdh gene of a single early oocyte. A: Amplification curve of the single oocyte, B: Dissociation curve of the single oocyte.

[0032] FIG. 4 shows gene expression pattern of single oocytes derived from mouse primordial follicles and primary follicles. A: Hierarchical cluster analysis, B: Principal component analysis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The nature and benefits of the invention are further described in the following examples, which are intended to illustrate the invention and not to limit the invention.

Example 1 Preparing Experimental Materials and Animals

[0034] Reagent Preparation:

[0035] Dissection medium: Leibowitz L15 medium supplemented with 10% fetal bovine serum (FBS) and 100 ug/ml Penicillin-Streptomycin;

[0036] Digestion medium 1:

[0037] MEM medium supplemented with 0.05 mg/ml Liberase TL Research Grade (Sigma, catalog number: 05401020001, product information: http://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=zh&region=CN), 0.2 mg/ml collagenase type IV (ThermoFisher, 17104-019) and 5% DNase I Solution (STEMCELL Technologies, 07900).

[0038] Digestion medium 2: StemPro Accutase Cell Dissociation Reagent (ThermoFisher, catalog number: A1110501, product information: https://www.thermofisher.com/order/catalog/product/A1110501). Accutase is a commonly used digestive enzyme reagent in recent years. It has proteolytic and collagenolytic enzymes activities and does not contain mammalian or bacterial derived components. Accutase is a modest digestive reagent and has low damage to cells. Accutase is often used as a replacement for trypsin.

[0039] Culture Medium:

[0040] MEM medium: comprises 10 mIU/mL follicle stimulating hormone (FSH), 3 mg/mL bull serum albumin (BSA), 1 mg/mL Fetuin (Sigma, F3385), 5 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/ml selenium (ITS, Sigma, I3146).

[0041] Animals:

[0042] One- or two-week female C57 mice

Example 2 Isolation of Single Oocytes and their Corresponding Granulosa Cells from Mouse Primordial and Primary Follicles

[0043] Procedures:

[0044] (1) Remove the ovary from mice and suspend in dissection medium and wash 3 times.

[0045] Use sterilized needles to mechanically dissociate the ovarian tissue into fragments of approximately 110.sup.6-410.sup.6 m.sup.3 under a dissecting microscope. Transfer to a culture dish containing 2 ml digestion medium 1.

[0046] (2) Place the culture dish into a sterile cell incubator and digest at 37 C. for 30 minutes. Mix the digestion medium by pipetting up and down every 10 minutes during digestion. Observing follicle separation by a microscope, when most follicles are in a free state and no large tissue fragments are observed, the next step could be performed.

[0047] (3) Filter the medium twice with a 70 m cell strainer (Falcon 70 m Cell Strainer, Corning, 352350). Transfer the filtrate to an 8-m-pore-size culture insert Transwell (SPLInsert Hanging, SPL Life Sciences, 35206) for a further filtration. Discard the filtrate.

[0048] (4) Wash the precipitate inside the Transwell 3 times with the culture medium. Then resuspend the precipitate above the membrane with 1 ml of culture medium (Note: during resuspending, the bottom of Transwell needs to be suspended above the liquid level of the culture dish.).

[0049] The resuspended culture medium contains follicles and cells of 8 m and 70 m in diameter.

[0050] The early follicle morphology is as follows:

[0051] Primordial follicles (20-40 m): follicles with an oocyte of about 20 m in diameter surrounded by a single layer of flattened granulosa cells;

[0052] Primary follicles (50-70 m): follicles with an oocyte of about 40 m in diameter surrounded by one layer of cubical granulosa cells.

[0053] The method of the present invention uses a culture insert with a pore size of 8 mTranswell. When the culture insert is placed in a Petri dish, the liquid can easily pass through the membrane, however when the culture insert is suspended right above the medium below, the liquid can stay on the membrane of the culture insert due to surface tension. Using this feature, the method of the present invention is able to re-filter the mixture after the first digestion and first filtration by passing the Transwell and recover the cells retain in the Transwell. The advantages of the method of the present invention include: a. the medium containing digestive enzyme is filtered, digestion can be stopped directly without a step of centrifugation; b. by using a culture insert with a pore size of 8 m, cells of smaller size such as membrane cells pass through the filter and are discarded; only the early follicles in need are retained. The early follicles can be then resuspended and obtained by aspiration easily without damaging the cells because the liquid staying on the membrane due to surface tension.

[0054] (5) Use PicoPipet Micro Pick and Place systems (NEPA GENE) to aspirate the single follicles.

[0055] Different diameter S-shaped glass capillaries are selected to pick up early follicles and cells of different sizes: primordial follicles and their corresponding oocytes and granulosa cells need to be picked using a 30 m S-shaped glass capillary, while primary follicles and their oocytes need to use 50 m glass capillary. The suction is generally controlled between 0.5 and 1.5V when sucking early follicles and the fine-tuning amplitude should be set to 0.05V. The suction power is increased by 0.5V for cell transferring after the cells reach equilibrium.

[0056] (6) After washing the aspirated follicles in the clean medium, transfer them to a container containing digestion medium 2, and then digest them in a cell culture incubator at 37 C. for 5-10 min.

[0057] The digestive medium 2 in the method of the present embodiment uses StemPro Accutase Cell Dissociation Reagent. Accutase digestive enzyme formulation has a modest and effective digestive enzyme activity that minimizes damage to cells while efficiently digesting and isolating oocytes and granulosa cells in follicles. At the same time, the inventors have found that subjecting the follicles to digestion for a short period of time, for example, within 3 to 5 minutes, is able to minimize damage to the cells, thereby facilitates the direct use of single oocytes and their corresponding granulosa cells after they are separated from follicles in the subsequent experiments on single cells, such as cell culture, nucleic acid extraction and amplification. Longer digestion can easily reduce the activity of oocytes, or even lead to cell degradation.

[0058] It was found in the experiments that using trypsin, such as trypsin-EDTA solution (0.25%: 0.02%), can digest follicles and isolate oocytes and granulosa cells. However, after the oocyte and its corresponding granulosa cells are separated into single cells, the step of stopping digestion is required for subsequent single cell experiments, such as termination of trypsin digestion by addition of culture medium for dilution or trypsin inhibitor. Additional steps include washing the cells, or filtering again through a suitable cell strainer.

[0059] It has been found that when other digestive enzymes, such as collagenase, are used in the digestive medium 2, it is not possible to effectively digest and separate follicles to oocytes and granulosa cells within a short period of time (for example, within half an hour, especially within 3-10 minutes) required to maintain cell viability.

[0060] (7) After the digestion, the mixture in the container holding the digestive medium 2 is pipetted up and down under a microscope (for example, using a capillary tube or a pipette, etc.) until the oocyte and its corresponding granulosa cells are separated into free and single cells.

[0061] (8) Single oocytes and/or the granulosa cells thereof are directly transferred to the culture medium for subsequent single cell experiments.

Example 3 Characterization of Single Oocytes and their Corresponding Granulosa Cells from Mouse Primordial and Primary Follicles

[0062] Based on the cell morphology and size of the cells obtained in Example 2, it is confirmed that single oocytes and their corresponding granulosa cells from mouse primordial and primary follicles are obtained.

[0063] FIG. 1 shows separated mice early follicles and their corresponding oocytes and granulosa cells observed under microscope. FIG. 1A shows separated early follicles. FIG. 1B shows separated oocytes and granulosa cells.

[0064] As FIG. 2 shows, single oocytes from mouse primordial and primary follicles are obtained. FIG. 2A shows single oocytes from mouse primordial follicles based on the method in Example 2. FIG. 2B shows single oocytes from mouse primary follicles based on the method in Example 2.

Example 4 Gene Expression and Hierarchical Cluster Analysis and Principal Component Analysis of Mouse Single Oocytes from Primordial Follicles and Primary Follicles

[0065] 1. gene expression analysis of mouse single oocytes from primordial follicles and primary follicles obtained in embodiment 2.

[0066] Experimental materials: Single Cell Lysis Kit (ThermoFisher, 4458235); SuperScript VILO cDNA Synthesis Kit (ThermoFisher, 1754-050); Platinum Taq DNA Polymerase (ThermoFisher, 10966-034); PowerUp SYBR Green Master Mix (ThermoFisher, A25777).

[0067] Experimental Steps:

[0068] (1) Transfer the 7 primordial follicle oocytes and 7 primary follicle oocytes isolated in Example 2, respectively, to a PCR tube containing 10 l Cell Lysis Solution (Single Cell Lysis Kit). Lysis for 5 minutes at room temperature according to the instructions of the kit's manual. The lysate can be stored at 20 C. after lysis.

[0069] (2) Add 1 l of Stop Solution to each PCR tube and stand for 2 minutes at room temperature.

[0070] (3) Reverse transcript reaction was performed for each single cells according to the SuperScript VILO cDNA Synthesis Kit instructions. After reverse transcription, 50 l of denucleated water was added to each tube to dilute the obtained cDNA.

[0071] (4) Pre-amplification was carried out using the following specific primers, and the reaction system according to the specification of Platinum Taq DNA Polymerase is as follows:

TABLE-US-00001 10*buffer 2 l dNTP 0.4 l MgCl.sub.2 0.6 l primers mixture 2 l cDNA 1 l Taq polymerase 0.2 l H.sub.2O 13.8 l total 20 l

[0072] PCR Pre-amplification setting is as following:

TABLE-US-00002 50 C. 15 min 70 C. 2 min 95 C. 15 s {close oversize brace} 14 cycles 65 C. 4 min 4 C.

[0073] (5) the amplicon is used for single cell qPCR. The reaction mixture is as following:

TABLE-US-00003 Express SYBR GreenER qPCR SuperMix Universal 5 l primers (4 mol/l) 2 l DNA 1 l DEPC water 2 l total 10 l

[0074] qPCR is carried out according to instruction of SYBR manual.

[0075] The experimental results can be exemplified in FIG. 3. FIG. 3 shows the real-time PCR amplification curve and dissolution curve of the Gapdh gene of a single early oocyte. The experimental results show that the obtained single oocyte can generate very good qPCR results.

[0076] 2. Hierarchical cluster analysis and principal component analysis of mouse single oocytes from primordial follicles and primary follicles obtained in Example 2

[0077] To analysis the single-cell qPCR results of the oocytes from said 7 primordial follicles and 7 primary follicles, CT values obtained by qPCR require direct processing instead of normalization with the reference genes. After setting the effective threshold, deleting the false positive value and processing the missing value, then converting the CT values to the base 2 Log value. Next, import the processed data into Qlucore Omics software for analysis and mapping. The results are shown in FIG. 4. FIG. 4A is a heat map based on gene expression levels of different cells, and it can be seen that the two stages of oocytes each have the highly expressed genes. For example, ZP1 in primordial follicle-derived oocytes is highly expressed compared to primary follicle-derived oocytes. In addition, it can be observed that there is no significant difference in the expression of the housekeeping gene Gapdh in the two kinds of cells. By cluster analysis, it can be observed that the gene expression profiles of primordial follicles and primary follicles are significantly different, which can be used to distinguish between primordial follicles and primary follicles.

[0078] FIG. 4B shows the results of different sample principal component analysis. The results are similar to those of the cluster analysis. It can be seen that the principal component analysis results from the primordial follicles and the primary follicle oocytes are significantly different, so it can also be used to distinguish between primordial follicles and primary follicles.

[0079] The above results demonstrate that the method of present invention for separating different types of single cells in the ovarian tissue can correctly separate two different early oocytes, namely primordial follicles and primary follicles, and obtain active single cells. Gene expression of oocytes that have lost cell activity is significantly different from gene expression of active cells. The methods of the present invention enable researchers in the field to conduct further studies on single cells. For example, oocytes can be studied in two different developmental stages by selecting genes that are specifically expressed in a certain period of time to understand their different characteristics.

[0080] The method of the invention operates at a single follicle, separating the oocyte and corresponding granulosa cells it contains. The method of the present invention reduces the number of manipulations of cells, thereby better protecting the integrity of the cells and enabling a greater number of early follicles to be obtained from the follicles. With the development and maturity of single-cell technology, experiments at the single cell level are receiving increasing attention. Separation of oocytes and their corresponding granulosa cells from viable early follicles in ovarian tissue has been a difficult problem. The method of the present invention successfully solves this problem. It provides a platform basis for subsequent studies of cell activity, gene expression analysis, single cell sequencing and even proteomics from a single early oocyte and corresponding granule cell level.

[0081] Unless otherwise indicated, the practice of the present invention will employ common technologies of organic chemistry, polymer chemistry, biotechnology, and the like. It is apparently that in addition to the above description and examples than as specifically described, the present invention can also be achieved in other ways. Other aspects within the scope of the invention and improvement of the present invention will be apparent to the ordinary skilled in the art. According to the teachings of the present invention, many modifications and variations are possible, and therefore it is within the scope of the present invention.

[0082] Unless otherwise indicated herein, the temperature unit degrees refers to Celsius, namely C.