Serum-free medium modulates the immunomodulatory and anabolic function of equine bone marrow-derived mesenchymal stromal cells.

Overview

• This study investigates how using a chemically defined serum-free medium (SFM) versus traditional serum-containing medium (SCM) affects the growth, characteristics, and function of equine bone marrow-derived mesenchymal stromal cells (eqBM-MSCs).
• It finds that SFM influences cellular phenotype and protein expression related to immune and repair functions but may reduce expansion potential compared to SCM.

Background and Purpose

• Adult bone marrow-derived mesenchymal stromal cells (BM-MSCs) are important for therapies in humans and animals due to their regenerative and immunomodulatory properties.
• Typically, culture media for BM-MSCs contain fetal bovine serum (FBS), which helps cell growth but presents risks such as batch variability and contamination from non-species sources (xenogeneic contamination).
• Serum-free media (SFM) offers a defined and potentially safer alternative, already optimized for human MSCs, but less is known about their effects on veterinary species like horses.
• The research aims to compare eqBM-MSC isolation and growth in SFM versus traditional serum-containing medium (SCM), focusing on cellular behavior and functional protein expression.

Methodology

• A chemically defined, customizable serum-free medium was tested alongside conventional serum-containing medium for the culture of equine BM-MSCs.
• Both media were assessed for their ability to support:
  • Cell adherence to culture plates
  • Trilineage differentiation potential (ability to become bone, cartilage, and fat cells)
  • Colony-forming unit (CFU) output as a growth measure
  • Surface marker expression specific to MSCs (e.g., CD29)
  • Cell proliferation rates
• Quantitative proteomic analysis was conducted to identify differences in protein abundance and pathways enriched in cells grown in each medium.
• Cytokine profiling measured levels of key secreted molecules linked to immune function and tissue repair.

Key Findings

• Growth and morphology:
  • Both SFM and SCM allowed cells to adhere and differentiate into multiple lineages.
  • SFM cultures displayed colony-based growth and produced up to three times more colonies than SCM.
  • Cells grown in SFM showed slower proliferation with roughly double the population doubling time compared to SCM.
  • This slower growth led to reduced total expansion across passages in SFM.
  • SFM cells were enriched for the CD29 surface marker, a characteristic MSC marker, suggesting possible phenotypic differences.
• Proteomic and pathway differences:
  • SFM-grown cells showed increased abundance of proteins involved in immune signaling and tissue repair mechanisms such as:
    • Neutrophil degranulation
    • Interferon-associated signaling
    • Hemostasis and angiogenesis pathways
  • SCM-grown cells had higher levels of proteins linked to cell proliferation and metabolic processes.
• Cytokine secretion:
  • SFM cultures secreted higher levels of inflammatory and growth-related cytokines, including KC-GRO, IL-8, and fibroblast growth factor (FGF).

Implications and Conclusions

• The serum-free medium can significantly modulate the phenotype and secretory profile of equine BM-MSCs, potentially enhancing their immunomodulatory and repair-related functions.
• There is a trade-off with SFM between maintaining a desirable MSC phenotype and the overall cell expansion yield due to slower proliferation.
• These findings highlight the importance of further optimizing serum-free culture conditions to increase scalability while preserving beneficial cell functions, which is crucial for veterinary therapeutic applications.
• Future work is needed to confirm how these in vitro differences affect MSC function in vivo and therapeutic efficacy.