Isolation, culture and, characterization of endometrial mesenchymal stromal cells (endMSCs)

doi:10.21203/rs.3.pex-1457/v1

Method Article

Isolation, culture and, characterization of endometrial mesenchymal stromal cells (endMSCs)

https://doi.org/10.21203/rs.3.pex-1457/v1

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Endometrial Mesenchymal Stromal Cells (endMSCs) can be isolated from menstrual blood of healthy women and expanded under standard culture conditions. Once the cell culture is established, the multipotency of endMSCs can be in vitro evaluated using specific differentiation media for 21 days. Cell differentiation state can be observed by optical microscopy. On the other hand, the phenotypical characterization can be performed by flow cytometry targeting a combination of surface markers.

This protocol is suitable for establishing an endMSCs culture for multiple purposes: in vitro assays, preclinical studies, “omic” analyses, etc.

Cell Biology

Stem cells

endometrial

mesenchymal stromal cell

isolation

culture

characterization.

The isolation of MSCs from the endometrial decidual tissue in menstrual blood is a non-invasive and reproducible method. This methodology does not require any painful procedure for donors. In terms of laboratory processing, these cells can be selected by plastic adherence and in vitro expanded under standard culture conditions. The menstrual blood-derived stromal cells display multipotent capabilities and immunomodulatory effects¹.

The present isolation protocol of endMSCs is based on previous publications^2,3. Slight modifications have been performed by our group^4,5. Reagents, equipment, and methodology are described in the cited publications.

Reagents:

- Phosphate buffer saline (PBS).

- Trypsin

- Fetal bovine serum (FBS)

- Penicillin-streptomycin

- DMEM

- L-glutamine

- Monoclonal antibodies against CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD56, CD73, CD90, CD105, and HLA-DR

- Stem Pro Adipogenesis, Chondrogenesis and Osteogenesis Differentiation Kits (Gibco, Thermo Fisher Scientific, USA)

- Oil Red O Staining

- Alcian Blue 8GX staining

- Alizarin Red S staining

- CO₂ incubator

- Laminar flow chamber

- FACScalibur cytometer (BD Biosciences)

- Optical microscope

- Spectrophotometer

Isolation and culture of endMSCs:

1- Healthy pre-menopausal women, without infections or immune disorders and who are not undergoing hormonal therapy, can be eligible as potential menstrual fluid donors. Menstrual fluid can be collected by the use of a menstrual cup or in a sterile urine container on day 2 or 3 of the menstrual cycle.

2- Dilute blood samples in PBS. If endometrial tissue fragments are retrieved, mechanical disaggregation is required. Transfer the recovered tissue to a sterile Petri dish and use a scalpel blade to obtain smaller pieces. Add the disaggregated tissue to the diluted blood. Centrifuge at 450 × g for 10 minutes.

3- Discard the supernatants to remove the residues of cervical mucus and resuspend the cells in DMEM containing 10 % fetal bovine serum (FBS), 1% penicillin/streptomycin and 1 % glutamine. Seed the resuspended cells onto tissue culture flasks and incubate at 37 ˚C in 5 % CO₂ atmosphere.

4- After 24 hours, remove non-adherent cells by washing with PBS. Once some colonies are formed, detach the adherent cells using PBS containing 0.25 % trypsin (v/v), seed them at a density of 5 000 cells/cm², and expand to 80% confluency.

5- Change culture medium every three days.

Phenotypical characterization:

1- Stain 2 × 10⁵ endMSCs with the appropriate concentrations of monoclonal antibodies against CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD56, CD73, CD90, CD105, and HLA-DR and incubate for 30 min at 4 ºC in the presence of PBS containing 2% FBS.

2- After incubation, wash the cells and resuspend in PBS.

3- Use isotype-matched antibodies as negative controls.

4- For flow cytometric analyses, acquire 10⁵ events on a FACScalibur cytometer (BD Biosciences, CA, USA).

Functional characterization:

1- Culture cells with an 80 % of confluence in a differentiation media for 21 days (Stem Pro Adipogenesis, Chondrogenesis and Osteogenesis Differentiation Kits, Gibco, Thermo Fisher Scientific, MA, USA).

2- After 21 days, fix cells with a paraformaldehyde 4 % solution for 20 minutes.

3- Wash cells 3 times with PBS and stain differentiated cell:

- Adipogenic staining: incubate cells with isopropanol 60 % during 5 min, dry, stain with 3.5 mg/ml de Oil Red O in isopropanol 60 % for 5 min and, wash with distilled water.

- Chondrogenic staining: wash cells twice with HCl 0.1 M, stain with 10 ng/ml Alcian Blue 8GX (diluted in HCl 0.1 M) for 2 h and wash with HCl 0.1 M and distilled water.

- Osteogenic staining: stain with 20 mg/ml Alizarin Red S (pH = 4.1‐4.2) for 30 min and wash with distilled water.

4- Quantify cell differentiation by spectrophotometry determining the absorbance of the extracts at 490 nm (Oil Red O and Alizarin Red S staining) and at 600 nm (Alcian Blue 8GX).

5- Confirm the in vitro differentiations by microscopic examination at 20X magnification

In the first phases of endMSC isolation, just one centrifugation before cell pellets are seeded into culture flasks may not be enough for blood-cell removal (steps 3–4). Discard the supernatants and centrifuge again until the supernatants appear clearer. After 24 hours, multiple washings with PBS may be necessary to completely remove blood cells and observe the plastic adherent-cells under a light microscope.

Culture contamination is the most common problem in the isolation of endMSCs. For this reason, the addition of antifungal supplements, as amphotericin B (Fungizone®) and additional antibiotics, as ciprofloxacin, in the culture medium may prevent bacterial and yeast contamination, especially for the first 10 days.

Time to reach an 80 % of confluence in a culture is variable, depending on the cell division rates. Cells can be isolated 24 hours after seeding but, the expansion will depend on number of cells that is necessary for different protocols.

The phenotypic characterization can be completed in two hours, but differentiation assays and staining take 21 days.

The estimated times do not include the analysis of results.

The phenotypic analysis, demonstrated that endMSCs were negative for CD14, CD20, CD34, CD45, CD80, HLA-DR and positive for the stemness markers CD44, CD73, CD90 and CD117. Figure 1A shows the representative histograms for these markers and the numbers within the histograms represent the Mean Relative Fluorescence Intensity (MRFI) and standard deviations from different donors (n = 4). It is interesting to note that we did not observe any significant change in the phenotype of endMSCs after different passages and their phenotypes were very similar when different donors were compared.

In order to demonstrate the multipotency of isolated and in vitro expanded endMSCs, the adipogenic, chondrogenic and osteogenic differentiation potential was evaluated according to standard differentiation protocols. The endMSCs were cultured for 21 days and differentiation was evaluated with Alizarin Red, Alcian Blue and Oil Red O stainings. The stains were solubilized and the resulting absorbances were quantified by spectrophotometry (Figure 1B). As shown in the representative images, the differentiation towards adipogenic, chondrogenic and osteogenic lineages was confirmed by microscopy (Figure 1C).

1. Nikoo, S. et al. Effect of menstrual blood-derived stromal stem cells on proliferative capacity of peripheral blood mononuclear cells in allogeneic mixed lymphocyte reaction: Immunomodulatory effects of MBSCs. Journal of Obstetrics and Gynaecology Research 38, 804–809 (2012).

2. Moreno, R. et al. Human Menstrual Blood-Derived Mesenchymal Stem Cells as Potential Cell Carriers for Oncolytic Adenovirus. Stem Cells Int 2017, 3615729 (2017).

3. Zemel’ko, V. I. et al. [Neurogenic potential of human mesenchymal stem cells isolated from bone marrow, adipose tissue and endometrium: a comparative study]. Tsitologiia 55, 101–110 (2013).

4. Álvarez, V. et al. The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta. Journal of Tissue Engineering and Regenerative Medicine (2018) doi:10.1002/term.2743.

5. Blázquez, R. et al. Murine embryos exposed to human endometrial MSCs-derived extracellular vesicles exhibit higher VEGF/PDGF AA release, increased blastomere count and hatching rates. PLOS ONE 13, e0196080 (2018).

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Isolation, culture and, characterization of endometrial mesenchymal stromal cells (endMSCs)

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Isolation, culture and, characterization of endometrial mesenchymal stromal cells (endMSCs)

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Abstract

Figures

Introduction

Reagents

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Troubleshooting

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Anticipated Results

References

Associated Publications

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