Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Summary

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients. 

Abstract

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as “the scraping method” and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed “the trypsin method.” This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Introduction

The human uterus corpus is comprised of three layers, the perimetrium (or serosa), the myometrium, and the endometrium. Distinguishing each of these layers is an important step to establish endometrial cell lines. The perimetrium is the outer most layer of the uterus and composed of thin, serous cells. The myometrium is the thick, middle layer of the uterus and comprised of smooth muscle cells. The endometrium is identified as the inner layer of the uterus and includes epithelial and stromal cell populations.

The endometrium is further subdivided into the basalis layer whose stem cell population is hypothesized to repopulate the functionali....

Protocol

Hysterectomy specimens used in this manuscript were collected in concordance with a University IRB-approved ethics protocol numbered IRB-HSR #14424.

1. Sample Acquisition from Clinical Source

1. Obtain government and institution-based ethical guidelines and approval documentation before beginning.
2. Conduct all steps in sterile conditions.
3. Preserve patient-derived tissue in media (RPMI or DMEM/High Glucose) in a 50 ml tube at 4 °C if the sample cannot be processed in culture immediately. Samples can be stored in this state for a maximum of 24 hr.
4. Wash tissue sample three times with 1X sterile phosph....

Results

As emphasized in the Protocol section, be sure to conduct all methods under government, institutional, and ethical guidelines when handling and preparing human tissue.

Included in this manuscript is an illustration of the general workflow of "the scraping method" (Figure 1A) and "the trypsin method" (Figure 1B) used to establish primary endometrial cultures. These methods are described in detail in the Protocol section (see pa.......

Discussion

Other groups have described and adapted methodology for the preparation of endometrial stromal cultures, most of which utilize collagenase ^{4,12,13,15-18}. In this manuscript, we have provided methodology and evidence for two simplified primary endometrial stromal culture methods, both of which are utilized by our lab for economical reasons and the convenient availability of trypsin and/or a razor blade.

When comparing our two methods, both successfully generate viable primary cu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25 Trypsin or 0.05% Trypsin	Hyclone	SH3023602 or SH30004202
1.7 micro Centrifuge Tube	Genesee Scientific	22-272A
1 µl, 20 µl, 200 ml and 1,000 µl Pipette	Genesee Scientific	24-401,24-402, 24-412, 24-430
15 ml Conical Tube	Hyclone	339650
50 ml Conical Tube	Hyclone	339652
6 cm Cell Culture Dish	Thermo scientific	12-556-002
8 well Chambers	Thermo Scientific	AB-4162
Acetate	Fisher scientific	C4-100
AMV RT Enzyme/Buffer	Bio Labs	M077L
Bovine Serum Albumin (BSA)	Fisher Scientific	BP-1605-100
Buffered Zinc Formalin	Thermo	59201ZF
Charcoal strip FBS	Fisher	NC9019735
Chloroform	Fisher Scientific	BP1145-1
Cover slip	Fisher Brand	12-544D
Cyclic AMP (cAMP)	Sigma	B7880
DMEM/High Glucose	Hyclone	SH30243FS
dNTP	Bioline	BIO-39025
Donkey Anti Goat -TRITC	Santa Cruz	SC-3855
Donkey Serum	Jackson’s lab	017-000-002
E Cadherin Antibody	Epitomics	1702-1
Ethanol	Fisher Scientific	BP2818-1
Fetal Bovine Serum (FBS)	Fisher Scientific	03-600-511
Fungizone Amphotericin B	Gibco	15290-018
GAPDH Probe	Life Technologies	HS99999905
Glycogen	5Prime	2301440
Goat Anti Mouse - FITC	Jackson’s Lab	115-096-003
Isopropanol	Fisher Scientific	BP2618-1
Kanamycin	Fisher Scientific	BP906-5
Medroxyprogesterone acetate (MPA)	Sigma	M1629
MeOH (Methanol)	Fisher Scientific	A4-08-1
Mounting Media (w/DAPI)	Vector Labratories	H-1500
N6 DNA Oligos	Invitrogen
Number 15 Scraper	BD	371615
Pan Cytokeratin  Mouse mAB	Cell Signaling	4545
PBS (phosphate buffered saline)	Fisher Scientific	BP-399-4
Penicillin-Streptomycin Glutamine Solution 100X	Hyclone	SV30082.01
PML Anti Goat Anti body	Santa Cruz	SC-9862
Primer(s)	Eurofins
RPMI	Hyclone	SH30027FS
RPMI (Phenol free)	Gibco	11835
Sybr Green	Thermo Scientific	AB-4162
Taqman	Thermo	AB-4138
Trizol	Life Technologies	15596018
Vimentin Antibody	Epitomics	4211-1