Isolation and In Vitro Decidualization of Mouse Primary Endometrial Stromal Cells

Summary

We aim to provide a method for the isolation and culturing of mouse endometrial stromal cells by enzymatic digestion. The isolated stromal cells are of high purity and can be decidualized by hormones in vitro. This protocol represents a feasible and stable method for stromal cell culture and for the further study of decidualization.

Abstract

A successful pregnancy relies on the establishment of decidualization, which involves the morphological and functional reprogramming of the progesterone-primed endometrial stromal cells under the influence of estrogen. In this protocol, we present a method for acquiring highly purified stromal cell isolated from the mouse uterus on day 4 of early pregnancy. Cultured primary stromal cells are then subject to further applications, such as RNA interference, overexpression, pharmaceutical treatment, immunoprecipitation, chromatic immunoprecipitation, and so on. Additionally, we provide a technique for the in vitro decidualization of cultured stromal cells using estrogen and progesterone. The in vitro decidualization method allows for the physically significant study of decidualization-related molecules. Altogether, this protocol provides a reliable and efficient method to facilitate further studies to define the molecular mechanism of decidualization.

Introduction

Decidualization is a prerequisite for the establishment of pregnancy. In mice, decidualization occurs after the attachment of the embryo to the uterine luminal epithelium during the receptivity stage¹. However, the mechanism underlying decidualization is still not completely understood². Considering the complex compositions of many cell types and the cell-specific expression of different genes in the uterus, there is a high demand for the purification of different cell types to investigate the potential function of each gene in different compartments.

The wall of the uterus consists of three layers: the outer perimetrium; the intermediate myometrium; and, most importantly, the innermost endometrium. The endometrium is a specialized mucosa consisting of simple columnar luminal and glandular epithelia and stroma. Maghsoudlou et al. compared three different methods to separate the esophageal epithelial cells and found that intact mucosal sheets yield a single-cell suspension under the influence of a digestive enzyme. The isolated epithelial cells are of high viability and have minimal contamination with other types of cells³. Kalabis et al. and Saxena et al. also employed trypsin and dispase to collect cells from the rodent esophagus^4,5. Accordingly, we use trypsin and dispase as the digestive enzymes to dislodge the epithelial sheets from the integrated uterus. The layer of stroma contains various kinds of matrix metalloproteinases that play a crucial role in the remodeling of connective tissue. Collagenase is widely used in isolating stromal cells from the human endometrium^6,7. Taken together, a modified method of isolating mouse stromal cells has come into being.

The mouse appears to be the most commonly used and powerful animal model for scientific research. Estrogen and progesterone are the two main steroid hormones during pregnancy, and they are vital for implantation and decidualization Therefore, they are employed to mimic the in vivo hormone environment in order to induce the cultured stromal cells to differentiate into decidual cells in vitro, according to Li Q's method⁸.

Protocol

All animal procedures were approved by the Animal Care and Use Committee of South China Agricultural University.

1. Preparation

1. Animals and treatments
   1. House adult mice (CD-1 strain) in a specific, pathogen-free room with a temperature- and light-controlled environment (12 h light/12 h dark).
   2. Mate mature female mice with fertile males at 16:00 h.
      NOTE: The day the vaginal plug is formed is considered day 1 of pregnancy.
   3. Sacrifice the mice by cervical dislocation at 10:00 h on day 4 to collect the uterus for the isolation of mouse stromal cells.
2. Laboratory supplies
   1. Soak all glass bottles, pipettes, and 15- and 50-mL centrifuge tubes in chromic acid cleaning solution (1,000 g of potassium dichromate, 1,000 mL of concentrated sulfuric acid, and 9,000 mL of H₂O) overnight and then rinse them with distilled water. Bake all of the glassware at 180 °C for 6 h and sterilize all plastic-ware in an autoclave for 30 min.
   2. Prepare the basal culture medium DMEM/F12 and HBSS according to the manufactures' instructions. Filter them using a 0.22-µm strainer. Store at 4 °C.
   3. Prepare 250 mL of the medium for stromal cell culture: DMEM/F12 supplemented with 10% charcoal-stripped fetal bovine serum (cFBS) and 1% penicillin-streptomycin.
   4. Prepare the digestive enzymes. Dissolve 2 g of trypsin in 40 mL of HBSS for a 5X storage concentration and aliquot 1 mL/tube. Dissolve 1 g of dispase in 16.7 mL of HBSS at the concentration of 60 mg/mL and aliquot 0.5 mL/tube. Dissolve 35 mg of collagenase in 7 mL of HBSS at a concentration of 0.5% and aliquot 180 µL/tube. Store all of the digestive enzymes at 20 °C.
   5. Dissolve estradiol-17β (10 µM) and progesterone (1 mM) in alcohol and store at -20 °C.

2. Isolation and culture of mouse endometrium stromal cells

1. Preheat the HBSS at 37 °C in a water bath. Prepare the workbench with ultraviolet light for at least 15 min. Preheat HBSS containing 1% penicillin-streptomycin in 6-cm petri dishes.
2. Split the uteri longitudinally to expose the uterine lumen. Wash them 3 times in the prepared HBSS in 6-cm culture dishes to clean out blood, fat, and small pieces of tissue.
3. Prepare the epithelium digestive enzyme solution. Mix 1 mL of trypsin, 0.5 mL of dispase, and 3.5 mL of HBSS in a 15-mL centrifuge tube.
4. Digest the uteri in the enzyme solution at 4 °C for 1 h, at room temperature for 1 h, and at 37 °C for 10 min.
5. Pour out the digested uteri into a new dish and wipe off the sheets of luminal epithelial cells in the HBSS by swinging each uterine horn about 10 times.
6. Prepare the stroma digestive enzyme. Mix 150 µL of collagenase with 5 mL of HBSS.
7. Incubate the remaining uterine tissue at 37 °C for 30 min. Vigorously shake the digested tissue 40 times to disperse the stromal cells.Filter the digested tissue through a 70-µm filter to eliminate the residual tissue.
8. Collect the filterable cell dissociation solution in a new centrifuge tube and centrifuge at 1,500 x g for 5 min. Discard the supernatant, resuspend the cell sediment with HBSS, and centrifuge again.
9. Discard the HBSS and resuspend the cells with 4 mL of DMEM/F12 medium containing 2% cFBS. Mix 100 µL of the resuspended medium with 100 µL 0.4% trypan blue and then count the live cells under the microscope.
10. Dilute the cells with the culture medium to 2x10⁵ cells/mL and seed the cells into different culture plates as required. Incubate the cells at 37 °C for 30 min and then change to culture medium with 10% cFBS to eliminate the unattached epithelial cells. Culture the stromal cells overnight for further study.

3. Induction of decidualization in vitro

1. Preheat the HBSS and DMEM/F12 medium containing 2% cFBS. Wash the cultured stromal cells with HBSS.
2. Prepare the decidualization culture medium using DMEM/F12 with 2% cFBS and dilute the stored estradiol-17β and progesterone to final concentrations of 10 nM and 1 µM, respectively. Supplement the control group with an appropriate volume of alcohol.
3. Add the decidualization culture medium to the culture plates in triplicate and culture the cells for 24 h, 48 h, and 72 h. Replace the working solution every 2 days.
4. Confirm the decidualization by observing cell morphology changes and the expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable decidualization marker.

Results

The workflow of procedures are shown in Figure 1.

The purity of isolated mouse endometrium stromal cells

The main morphological feature of the isolated stromal cells was a spindle-like appearance (Figure 2A). To confirm the purity of the isolated stromal cells, immunofluorescence was performed to detect the expression of the stromal cell marker Vimenti...

Discussion

The uterus, the residence of the conceptus, is of great importance for a successful pregnancy. A tight interaction between the conceptus and the maternal uterine endometrium initiates implantation and then the proliferation and differentiation of the stromal cells around the implantation sites, which will turn into specialized decidual cells under the influence of ovarian hormones. As a result of differentiation, decidual cells acquire various new functions during this transformation, such as providing a nutritional supp...

Materials

Name	Company	Catalog Number	Comments
Hanks’ Balanced Salts	Sigma	H4891	For washing the tissues or cells
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham	Sigma	D2906	For mESC culture
Trypsin	Amresco	0 458	For epithelial sheets dissociation
Dispase	Roche Applied Science	0 4942078001	For epithelial sheets dissociation
Collagenase I	Invitrogen	17100-017	For stromal cells dissociation
Charcoal Stripped  Foetal Bovine Serum	Biological Industries	04-201-1A	For the culture of mESC
Progesterone	Sigma	850454	For in vitro decidualization
Estradiol-17	Sigma	E1024	For in vitro decidualization
Vimentin	Cell Signaling Technology	3932s	Marker of stromal cells
0.22 μm Sterile Filter	Millipore	SLGP033RB	For filtration medium
CO2 Incubator	Thermo	3111	For the culture of mESC