Name: two methods for establishing primary human endometrial stromal cells from hysterectomy specimens
Uploaded: 2014-05-23T14:00:00.000+00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

We thank the collaborative efforts of Dr. Thao Dang and members of her lab for use of their imaging and microscope equipment. We also thank the Biorepository and Tissue Research Facility (BTRF) core, Jeff Harper, and the residents at the University of Virginia for providing us with uterine tissue. We thank Karol Szlachta for the help with Schematic Overview.

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
<ol>
	<li>Obtain government and institution-based ethical guidelines and approval documentation before beginning.</li>
	<li>Conduct all steps in sterile conditions.</li>
	<li>Preserve patient-derived tissue in media (RPMI or DMEM/High Glucose) in a 50 ml tube at 4 &#176;C if the sample cannot be processed in culture immediately. Samples can be stored in this state for a maximum of 24 hr.</li>
	<li>Wash tissue sample three times with 1X sterile phosphate buffered saline (PBS) and discard the solution between washes.</li>
</ol>
2. Preparation of Primary Cell Lines using the Scraping Method
<ol>
	<li>Add 4-10 ml of growth media (RPMI or DMEM/High Glucose supplemented with 10% Fetal Bovine Serum and 1% Penicillin-streptomycin) to tissue and add Fungizone (0.25 &#181;g/ml final concentration) for 30 min.</li>
	<li>Discard the growth media.</li>
	<li>Wash tissue twice with 1X PBS.</li>
	<li>Place the tissue on a 6 cm cell culture plate to distinguish between myometrium and endometrium layers (refer to Figures 2A-2C for further description). Separate the endometrium.</li>
	<li>Using a scalpel or razor blade, transect the tissue into small pieces while scratching onto the 6 cm cell culture dish. These scratches facilitate the attachment of the emerging primary endometrial cells. Compared to the trypsin method, more tissue is needed (see Figure 2D for an approximation).</li>
	<li>Gently, add 2 ml of growth media to scratched dish (tissue fragments will be visible and ideally immobilized from the scratching motion).</li>
	<li>Transfer the plate(s) to a designated cell culture incubator (37 &#176;C and 5% CO2). Designate a place for primary cell lines, away from other cell culture dishes. Primary cultures are more sensitive to infection and contamination.</li>
	<li>Examine the cells under a light microscope daily. Small populations of cells should emerge by day two or three from around the sliced tissues (see Figure 3B).</li>
	<li>To maintain cultures, wash gently with 1X PBS and add fresh growth media (2ml) every three days. When proliferation rate increases, additional growth media (3-4 ml) can be added to the 6 cm culture plate(s). 
	Note: During washing and media changes, pieces of tissue will likely be aspirated. This will not affect growth of adherent cells. Pieces of tissue should be aspirated by the subsequent step (2.10) as new colonies are unlikely to emerge after one week.</li>
	<li>When the 6 cm plate is approximately 75 - 80% confluent (see Figure 3B), passage using 0.05% trypsin. Primary cells cannot be passaged indefinitely - freeze down one plate of cells as soon as two or three plates are maintained. If more passages are required, follow an immortalization protocol (reviewed in Ref 3).</li>
</ol>
3. Preparation of Primary Cell Lines using the Trypsin Method
<ol>
	<li>Place a small piece of endometrial tissue (see Figure 2D for an approximation) in 2 ml of 0.25% trypsin supplemented with Kanamycin (0.03 mg/ml final concentration).</li>
	<li>Incubate tissue at 37 &#176;C on rotating platform for 30 min.</li>
	<li>Briefly vortex the tissue.</li>
	<li>Centrifuge the sample at 200-400 x g for 2 min&#160;and discard the supernatant.</li>
	<li>Add fresh trypsin and Kanamycin.</li>
	<li>Incubate the tissue at 37 &#176;C while rotating for an hr.</li>
	<li>Vortex the tissue for 5-10 sec.</li>
	<li>Add 2ml of growth media (supplemented with 10% Fetal Bovine Serum and 1% Penicillin-streptomycin) to deactivate the trypsin.</li>
	<li>Centrifuge cells at 200-400 x g for 2-3 min.</li>
	<li>Discard the supernatant and add 2 ml of growth media to the pelleted cells.</li>
	<li>Plate onto a 6 cm cell culture dish. Scraping the plate as in Step 2.5 of Preparing Primary Cell Lines using the Scraping Method is not necessary, but enhances the attachment and outgrowth of primary cultures.</li>
	<li>Monitor cells under a light microscope daily. Cells are usually visible under a light microscope after 24-48 hr&#160;(see Figure 3C).</li>
	<li>To maintain the cultures, monitor the cells and change media every 2-3 days as in Step 2.9.</li>
	<li>To passage the cells, use 0.05% or 0.25% Trypsin when cells reach 75-80% confluency (see Figure 3C).</li>
</ol>
4. Saving Extra Tissue for Analysis (Snap Freezing and Formalin Fixation)
<ol>
	<li>Wash sample twice with 1X PBS.</li>
	<li>Aspirate as much liquid as possible.</li>
	<li>For Snap Freezing, place endometrial tissue sample in a 1.7 centrifuge tube (see Figures 2F and 2G). Place the 1.7 centrifuge tube in liquid nitrogen for 10 sec&#160;or until it can be seen that the tissue has frozen down. 
	Note: Take care not to directly touch liquid nitrogen when preparing samples. Samples can be stored at -80 &#176;C until further processing or analysis.</li>
	<li>For formalin fixation, cut a small piece of endometrial tissue (see Figure 2H), and submerge in 10% buffered zinc formalin. After 24 hr, discard formalin and add 70% ethanol to tissue samples until further processing.</li>
</ol>
5. Immunofluorescence (IF)
<ol>
	<li>Cell fixation
	<ol>
		<li>Prepare cells on a culture slide or plate.</li>
		<li>Gently, wash cells with 1X PBS.</li>
		<li>Add pre-chilled (4 &#176;C) Methanol and Acetone (mixed at a 1:1 ratio) for 5 min.</li>
		<li>Air dry the slide before proceeding. Slide can be stored at 4 &#176;C for 1-2 weeks if needed.</li>
	</ol>
	</li>
	<li>Processing IF
	<ol>
		<li>Rehydrate cells with 1X PBS for 10 min. For the following Steps 1-6, conduct all washes and incubations on a rotating platform.</li>
		<li>Block using 1X PBS supplemented with 1% Bovine Serum Albumin (BSA) and 1% species specific serum (source of 2nd antibody) for 30 min&#160;at room temperature (RT).</li>
		<li>Incubate in primary antibody (see manufacturers recommendations for antibody dilutions). Refer to Materials for specific antibodies. Dilute the primary antibody in fresh blocking buffer as in Step 5.2.2. This incubation can be conducted for at least 2 hr&#160;at RT or overnight at 4 &#176;C.</li>
		<li>Wash 3 times with 1X PBS for 5 min&#160;each.</li>
		<li>Incubate in secondary antibody (see manufacturers recommendations for antibody dilutions). Dilute the secondary antibody in fresh blocking buffer as in Step 5.2.2. To prevent photo-bleaching, it is important to conduct this and subsequent steps in the absence of light.</li>
		<li>Wash 3 times with 1X PBS for 5 min&#160;each.</li>
		<li>Thoroughly dry the slides.</li>
		<li>Add mounting media and DAPI counterstaining solution.</li>
		<li>Apply coverslip and seal the edges using sealant(s). Slides can be stored at 4 &#176;C in the dark for up to 2 weeks.</li>
	</ol>
	</li>
</ol>
6. RNA Extraction
<ol>
	<li>Pellet 1 x 107 cells by centrifugation. All materials and reagents in this protocol should be RNAse free.</li>
	<li>Lyse cells in 1 ml TRIZOL reagent, and incubate the homogenized samples for 10 min&#160;at room temperature to complete dissociation of nucleoprotein complexes.</li>
	<li>Add 200 &#181;l of chloroform per 1 ml of TRIZOL. Shake vigorously by hand for 15 sec&#160;and incubate for 2-3 min&#160;at RT.</li>
	<li>Centrifuge at maximum speed (15,000 x g) for 15 min&#160;at 4 &#176;C. Three layers will result.</li>
	<li>Transfer the top aqueous phase into a fresh microcentrifuge tube. Add 1 &#181;l glycogen to increase the RNA yield; however, this step is only necessary when a small amount of RNA is anticipated.</li>
	<li>Add 0.5 ml of isopropyl alcohol to the aqueous layer, and invert the samples 3-5 times. Incubate samples at RT for 10 min. To increase yield, samples can be placed in -20 &#176;C or -80 &#176;C for 30 min.</li>
	<li>Centrifuge at maximum speed for 10 min&#160;at 4 &#176;C.</li>
	<li>At this point, a small pellet of RNA should be visible. Discard the supernatant.</li>
	<li>Wash the RNA with 1 ml of 75% ethanol.</li>
	<li>Centrifuge at maximum speed for 5 min&#160;and discard the supernatant. Samples can be washed once more to rid inorganic contaminants, but this step is not necessary.</li>
	<li>Briefly, dry the RNA pellet until RNA pellet is dry (usually takes 7-10 min). 
	Note: One additional step can be used to decrease inorganic matter and decrease drying time. After discarding the supernatant from the ethanol wash, spin samples at maximum speed for an additional minute and aspirate residual liquid. Take care not to aspirate pellet.</li>
	<li>Dissolve RNA in RNase-free water, depending on the size of the RNA pellet. Volumes typically range from 10-50 &#181;l.</li>
	<li>Incubate samples at 55 &#176;C for 10 min, and measure the concentration of RNA.</li>
	<li>Store samples at -20 &#176;C until further processing.</li>
</ol>
7. Reverse Transcription
<ol>
	<li>Bring the sample RNA (1-3 &#181;g) to a volume of 9 &#181;l with H2O.</li>
	<li>Heat RNA to 70 &#176;C for 10 min.</li>
	<li>To generate an AMV master mix, combine the following reagents: 5 &#181;l of dNTP (working concentration of 50 &#181;M), 5 &#181;l of N6 DNA oligos (working concentration of 80 &#181;M), 5 &#181;l of 5X AMV buffer, and 1 &#181;l of AMV. This master mix solution is designed per one sample.</li>
	<li>Add 16 &#181;l of the master mix to the heated RNA. The final reaction volume will be 25 &#181;l reaction.</li>
	<li>Use the following PCR conditions for reverse transcription: (Stage 1) 42 &#176;C for 90 min, (Stage 2) 95 &#176;C for 5 min, and (Stage 3) 4 &#176;C until further processing.</li>
</ol>
8. Real Time PCR
<ol>
	<li>Conduct PCR reactions using the primers, annealing temperatures, cycle numbers, and reagents found in Table 1. 
	Note: It is ideal to follow manufacturer instructions when using PCR reagents (refer to company and catalog numbers in Materials).</li>
</ol>
9. In vitro Decidualization Protocol (Derived from Ref 4 and 5)
<ol>
	<li>Plate endometrial cells.</li>
	<li>Grow to a confluency of 75-85%.</li>
	<li>After cells become confluent, wash once with 1X PBS.</li>
	<li>Quickly, add hormone-free media (Phenol free RPMI supplemented with 5% charcoal strip FBS and 1% Penicillin-streptomycin).</li>
	<li>After 24 hr, add hormone free media supplemented with medroxyprogesterone acetate (MPA) at a final concentration of 1 &#181;M and 8-bromoadenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP) at a final concentration of 0.5 mM.</li>
	<li>After at least 48 hr, stop the reaction and save the plate(s) for further processing.</li>
</ol>

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Other groups have described and adapted methodology for the preparation of endometrial stromal cultures, most of which utilize collagenase&#160;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...

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 functionalis layer approximately every 28 days&#160;1. The functionalis layer of the human endometrium undergoes significant biochemical and morphological changes in response to circulating hormones. These hormones are derived from the pituitary gland and the ovaries.
The coordinated production and release of hormones results in a reproductive cycle. The reproductive cycle is designed to prepare the endometrium for potential embryo implantation events. In humans, the reproductive cycle is known as &#8220;the menstrual cycle&#8221; and divided into three phases &#8211; proliferative, secretory, and menstrual. The proliferative phase involves the proliferation of the functionalis endometrial layer whereas the secretory phase is marked by functionalis maturation. Specifically, extracellular alterations, secretions, and cellular differentiation signal a potential implantation. If implantation does not occur before the end of the secretory phase, the functionalis endometrial layer is shed during the menstrual phase. The importance of menstruation and the events that trigger the shedding of the functionalis layer are still being debated. In humans, it has been posed that menstruation is the result of a specific mid-secretory phase differentiation event known as &#8220;spontaneous decidualization&#8221;&#160;2. In this manuscript, we provide detailed methodology for both endometrial stromal cell isolation methods, and use a combination of immunofluorescence and digital images to demonstrate efficacy of these approaches. In addition, we apply a commonly used in vitro model of spontaneous decidualization to confirm endometrial stromal cell isolation.

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

two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

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 &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; 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).

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...

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

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.&#160;

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 ...

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Research

JoVE Journal

Medicine

22.7K Views.  University of Virginia.  Oprichting primaire endometrium stromale celkweek systemen van hysterectomie exemplaren is een waardevolle biologische techniek en een cruciale stap voorafgaand aan het nastreven van een breed scala van onderzoek wil. We beschrijven hier twee methoden om stroma culturen chirurgisch weggesneden endometriale weefsel van menselijke patiënten vastgesteld. 

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Developmental Biology

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Cancer Research

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Biology

Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.

Here, we present a protocol to isolate and culture rat endometrial epithelial stem cells (reESCs), generating rat endometrial organoids. This method facilitates in vitro studies of endometrial diseases, enabling gene editing and other cellular manipulations.

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. ...

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

Pelvic organ prolapse is a disorder that seriously impacts the quality of life of women. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina.&#160;Surgery to correct pelvic organ prolapse has been a mainstay treatment. Recently, there has been growing interest in studying the tissue composition of patients with prolapse at the cellular level.
There is currently little consensus regarding the effect of donor or patient age on cell-based therapies. Current published protocols for vaginal fibroblast isolation either concentrate on premenopausal tissue or neglect to comment on the age of donor tissue. Most existing protocols use animal models. The consistency of human vaginal tissue is denser than the tissues used in most protocols. In this study, human vaginal tissue was obtained primarily from older donors, which likely contributed to the failure of existing protocols.
The aim of this study is to describe a standard protocol for reliably acquiring human vaginal fibroblasts, regardless of donor age and menopausal status. Results were reproduced using tissue from nine separate donors who underwent pelvic organ prolapse surgery. Six patients were postmenopausal, with the oldest donor being 78 years old. The median age of the tissue donors was 59.
Here, we describe a reliable method for generating a fibroblast-enriched single-cell suspension using a combination of enzymatic and mechanical dissociation and cell suspension pooling of multiple vaginal biopsies from a single donor. Reliable isolation of human vaginal primary fibroblasts may be useful in the study of pelvic organ prolapse as well as microbiome-host interactions.

This protocol demonstrates a reliable and effective technique to isolate primary fibroblasts from either premenopausal or postmenopausal human vaginal tissue. Existing protocols for vaginal fibroblast isolation do not consider the challenges of cell isolation from senescent tissue. Vaginal tissue was obtained from women after pelvic organ prolapse surgery.

Pelvic organ prolapse is a disorder that seriously impacts the quality of life of women. It occurs when muscles and ligaments weaken and cause pelvic ...

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

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