Xenogen-free media provide variable equine mesenchymal stromal cell expansion after a 7-day culture period.

Overview

• This study evaluates different xenogen-free serum sources to identify which best supports the expansion and viability of equine mesenchymal stromal cells (MSCs) over a 7-day culture period while maintaining their characteristic phenotype.

Background and Objective

• Equine mesenchymal stromal cells (MSCs) are commonly cultured with serum-containing media to support cell growth and viability.
• Traditional media often contains xenogeneic serum such as fetal bovine serum, raising concerns regarding immune reactions and transmission of animal-origin pathogens.
• Investigators aim to find xenogen-free (non-animal or same-species derived) serum alternatives to minimize these risks.
• The objective was to determine which xenogen-free serum source supports optimal live MSC expansion and viability, while preserving MSC phenotype markers, after a 7-day culture.

Methods

• MSC Isolation: Bone marrow-derived MSCs were obtained from 8 horses.
• Culture Conditions: MSCs were cultured for 7 days in media supplemented with one of the following serum treatments:
  • 10% xenogeneic serum (control, e.g., bovine serum)
  • 10% commercial allogeneic equine serum (derived from other horses)
  • 20% commercial allogeneic equine serum
  • 10% autologous serum (derived from the same horse as MSCs)
  • 10% equine pooled platelet lysate (PPL)
  • A staged media reduction protocol starting with xenogeneic media
• Outcome Measures Evaluated after 7 days:
  • Number of live MSCs
  • MSC viability
  • MSC immunophenotype based on expression of surface markers (CD44, CD90, MHC I, CD45, MHC II)

Key Findings

• Live MSC Count:
  • 10% commercial allogeneic equine serum significantly decreased live MSC numbers compared to xenogeneic serum (P = 0.01), indicating poorer expansion.
  • Autologous serum, staged reduction, and PPL treatments produced similar live MSC counts to xenogeneic serum, suggesting they support comparable cell expansion.
  • Horse age (younger than 10 years vs. 10 years or older) did not significantly affect cell counts.
• MSC Viability:
  • Viability was highest in cultures grown with PPL compared to all other xenogen-free serum types, across both younger and older horses.
• Immunophenotype:
  • MSCs across all treatments consistently showed:
    • High expression of CD44, CD90, and MHC I (markers typical of MSCs)
    • Low expression of CD45 and MHC II (markers typical of hematopoietic and immune cells)
  • However, significant differences in expression levels of these markers were found when analyzed by treatment type and horse age.

Interpretation and Implications

• Using equine pooled platelet lysate (PPL) as a supplement for MSC culture media in the final 7-day expansion phase:
  • Supports adequate MSC growth comparable to xenogeneic serum.
  • Enhances MSC viability more than other xenogen-free serum sources tested.
  • Maintains key MSC immunophenotypic markers, ensuring the cells retain their expected characteristics.
• Autologous serum and staged media reduction approaches also provide comparable cell numbers, but PPL was superior in viability.
• These results support the feasibility of culturing equine MSCs in xenogen-free media without compromising cell expansion or phenotype, which is beneficial for clinical applications aiming to reduce risks associated with xenogeneic proteins.
• This approach could improve the safety and standardization of MSC therapies in veterinary medicine.

Summary

• The study reveals variable effectiveness of xenogen-free serum sources on equine MSC expansion, with PPL emerging as a promising supplement due to enhanced viability and maintained MSC characteristics.
• Culturing MSCs in nonxenogeneic media for 7 days prior to harvesting is feasible, potentially improving their safety profile for therapeutic use.