Stem Cell Isolation

Overview

Menstrual blood represents a unique, non-invasive source of mesenchymal stem cells (MSCs) that can be obtained without surgical intervention. Our research focuses on developing and optimizing isolation protocols that maximize cell yield while maintaining viability and multipotency.

                Key Advantages of MenSCs
                Non-invasive collection method
High proliferation rate compared to bone marrow MSCs
Minimal ethical concerns
No risk to donor
Can be collected monthly

            

Isolation Methods

1. Density Gradient Centrifugation (Recommended)

This method uses Ficoll-Paque density gradient media to separate mononuclear cells from red blood cells and other components. The buffy coat layer, containing nucleated cells, is collected and cultured.

Protocol Highlights

Dilute sample 1:1 with PBS
Layer over Ficoll-Paque (density 1.077 g/mL)
Centrifuge at 400 x g for 30 minutes (no brake)
Collect buffy coat interface
Wash and plate at 1-2 × 10⁵ cells/cm²

View Full Protocol →

2. Red Blood Cell Lysis

An alternative method using ammonium chloride-based lysis buffer to remove RBCs, followed by washing and plating of the remaining cell population.

Method	Yield	Purity	Time	Best For
Density Gradient	1-5 × 10⁶ cells/10mL	High (>90%)	2 hours	Research, clinical
RBC Lysis	2-8 × 10⁶ cells/10mL	Moderate (70-85%)	30 minutes	Rapid screening

Characterization

ISCT Standard Markers

MenSCs must meet International Society for Cell & Gene Therapy (ISCT) criteria for MSC identification:

Positive Markers (≥95%)

CD73, CD90, CD105, CD29, CD44 — cell adhesion and signaling molecules characteristic of MSCs

Negative Markers (≤2%)

CD34, CD45, CD14, CD19, HLA-DR — hematopoietic and immune cell markers

Tri-lineage Differentiation

Osteogenic, adipogenic, and chondrogenic differentiation capability confirmed

Plastic Adherence

Fibroblast-like morphology and adherence to tissue culture plastic

Growth Characteristics

Parameter	Typical Value	Range
Population Doubling Time	24-48 hours	18-72 hours
Time to Confluence (P0)	7-14 days	5-21 days
Maximum Passages	10-12 passages	8-15 passages
CFU-F Frequency	1 in 100-500 cells	Donor dependent

Current Research Projects

Project: Optimization of Collection Timing

Investigating the relationship between menstrual cycle day and cell yield/viability. Preliminary data suggests Days 1-3 provide optimal cell recovery.

Status: Data collection ongoing | Expected completion: Q2 2026

Project: Comparative Analysis of Isolation Methods
Head-to-head comparison of density gradient, RBC lysis, and novel microfluidic approaches for MenSC isolation.

Status: Protocol development | Expected completion: Q3 2026

Project: Donor Variability Study

Characterizing inter-donor variability in MenSC yield, proliferation, and differentiation potential across diverse populations.

Status: IRB approval pending | Expected start: Q2 2026

Publications & Resources

Key Literature

Meng X, et al. (2007) "Endometrial regenerative cells: A novel stem cell population." Journal of Translational Medicine. 5:57.
Patel AN, et al. (2008) "Multipotent menstrual blood stromal stem cells: Isolation, characterization, and differentiation." Cell Transplantation. 17(3):303-311.
Hida N, et al. (2008) "Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells." Stem Cells. 26(6):1695-1704.
Dominici M, et al. (2006) "Minimal criteria for defining multipotent mesenchymal stromal cells." Cytotherapy. 8(4):315-317.