Influence of Follicle Number and Reproductive Cyclicity on Mesenchymal Stromal Cell Yield Isolated From Equine Ovarian Follicular Aspirates.

Overview

• This study investigates how the number of ovarian follicles and reproductive cycle status affect the ability to isolate mesenchymal stromal cells (MSCs) from follicular fluid collected via ovum pick-up (OPU) in mares.
• The research aims to identify factors that influence successful cultivation of MSCs and to establish early predictors of culture success or failure.

Background and Purpose

• Mesenchymal stromal cells (MSCs) are valuable for regenerative medicine due to their ability to differentiate and modulate immune responses.
• Equine follicular aspirates, obtained through ovum pick-up (OPU), have been proposed as an alternative source of MSCs in mares.
• However, the factors influencing successful isolation and culture of MSCs from these aspirates were not clearly understood before this study.
• The study investigates if the mare’s reproductive cyclicity (cycling versus anestrous) and the number of ovarian follicles influence the yield and success of MSC isolation.

Methodology

• Thirteen mares underwent a total of 28 ovum pick-up (OPU) procedures to collect follicular aspirates.
• The aspirated cells were cultured until passage 3 to assess the presence and viability of MSC populations.
• Researchers recorded whether cultures successfully yielded MSCs or resulted in non-MSC cell populations.
• They compared MSC culture success relative to reproductive status of the mare (cycling or anestrous) and the follicle count at the time of OPU.
• Cell counts at passage 0 (initial cell culture stage) were analyzed to evaluate their relationship to culture outcomes.
• Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value of initial cell counts for culture failure.

Key Findings

• Successful MSC cultures were obtained in 15 out of 28 procedures, representing about 53.6% success rate.
• The remaining 46.4% of procedures resulted in non-MSC populations, indicating culture failure for MSC isolation.
• The reproductive status of mares (whether cycling or anestrous) did not significantly influence MSC culture success.
• The number of follicles present at aspiration also did not affect the likelihood of successful MSC isolation.
• Interestingly, cultures that failed to yield MSCs had a significantly higher initial (passage 0) cell count compared to successful cultures (p < 0.001).
• The ROC analysis showed that a high initial cell count (≥700,000 cells) was an excellent predictor of culture failure, with an area under the curve (AUC) of 0.923.

Interpretation and Implications

• Ovarian follicular aspirates obtained by OPU are a consistent and viable source of MSCs in mares.
• The reproductive cycle phase and number of follicles do not need to be considered critically when planning MSC isolation from follicular aspirates.
• The finding that high initial cell counts predict culture failure is notable and somewhat counterintuitive, as more cells are often assumed to be beneficial.
• This suggests that a high number of contaminating or non-MSC cells at the start may inhibit MSC culture growth or that the cell population is not enriched for MSCs at higher counts.
• Practically, measuring the initial cell count at passage 0 can serve as a quick screening tool to forecast whether MSC culture will succeed or fail, potentially saving time and resources.

Conclusion

• The study establishes that MSC isolation from equine follicular aspirates is generally reliable and unaffected by follicle number or mare reproductive cyclicity.
• A high initial cell count after aspiration is a strong early indicator of likely MSC culture failure.
• This information can optimize MSC production workflows by allowing early identification and exclusion of samples unlikely to yield MSCs, improving efficiency in regenerative medicine applications for horses.