Isolation, Expansion and Characterization of Mesenchymal Stromal Cells from Fresh and Cryopreserved Umbilical Cord Tissues in Horse and Dog.

Research Overview

• This study investigated whether mesenchymal stromal cells (MSCs) can be successfully isolated from fresh and cryopreserved umbilical cord tissues in horses and dogs.
• It compared the biological properties of MSCs derived from fresh versus frozen (cryopreserved) umbilical cord samples to assess the impact of freezing on their quality and functionality.

Background

• Mesenchymal stromal cells (MSCs) have valuable regenerative properties including the ability to proliferate, modulate immune responses, and differentiate into various cell types.
• Perinatal tissues, such as umbilical cord, are rich sources of MSCs that are easily accessible and ethically acceptable for collection.
• Using umbilical cord tissues for MSC extraction offers a non-invasive source for regenerative therapies in veterinary medicine.
• Cryopreservation (freezing) of such tissues would facilitate biobanking and enable future use without compromising cell quality, but its effects needed evaluation in canine and equine species.

Objectives

• To assess if cryopreserved whole umbilical cord tissues from dogs and horses yield MSCs with similar properties to those obtained from fresh tissues.
• To compare two different freezing media for tissue cryopreservation and their effects on MSC isolation and function:
• 10% dimethyl sulfoxide (DMSO) + 10% fetal bovine serum (FBS) (DF)
• 0.05 M glucose + 0.05 M sucrose + 1.5 M ethylene glycol (GSE)

Methodology

• Umbilical cord tissues were collected from dogs (canine) and horses (equine), specifically from Wharton’s jelly in horses.
• Tissues were either processed immediately (fresh) or after cryopreservation using the two freezing media detailed above.
• After thawing, MSCs were isolated and cultured from all groups.
• Isolated MSCs underwent multiple assessments to characterize their biological properties:
• Proliferation assays to measure growth capacity.
• Clonogenicity assays to test colony-forming ability.
• Adhesion and migration assays to evaluate cell behavior and mobility.
• Trilineage differentiation to test their ability to differentiate into bone, cartilage, and fat cells.
• Molecular marker analysis to confirm expression of typical mesenchymal markers.

Key Findings

• MSCs were successfully isolated from both fresh and cryopreserved umbilical cord tissues in dogs and horses.
• The cells maintained typical spindle-shaped morphology and adhered to plastic surfaces similarly to standard MSC populations.
• Proliferation rates and cloning efficiencies showed no significant differences between fresh and cryopreserved groups.
• MSC migration and adhesion properties remained intact after cryopreservation.
• Trilineage differentiation potential was preserved, confirming multipotency of MSCs from frozen tissues.
• Molecular analysis revealed stable expression of mesenchymal surface markers, indicating maintenance of characteristic MSC identity post-freezing.
• No substantial differences were observed between the two freezing media (DF versus GSE) in terms of cell quality outcomes.

Implications and Conclusions

• The study demonstrates the feasibility of using cryopreserved whole umbilical cord tissues from horses and dogs as reliable sources for MSC isolation.
• Cryopreservation did not significantly impair the biological or functional properties of MSCs, supporting their use in future regenerative medicine applications.
• This supports the development of veterinary biobanks storing cryopreserved perinatal tissues for later use in cell therapies.
• The ability to use different freezing media effectively increases flexibility for tissue preservation protocols.
• Overall, the findings promote the ethical and practical harvesting of MSCs from umbilical cord tissues to support treatments for various veterinary conditions.