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Frontiers in veterinary science2021; 8; 759828; doi: 10.3389/fvets.2021.759828

Effects of Production Method and Repeated Freeze Thaw Cycles on Cytokine Concentrations and Microbial Contamination in Equine Autologous Conditioned Serum.

Abstract: Autologous conditioned serum (ACS) is a common intra-articular treatment for osteoarthritis in horses. The objective of this study was to investigate the influence of ACS preparation method on product contamination and concentrations of relevant cytokines and the influence of multiple freeze/thaw cycles. Blood was obtained from 10 healthy Thoroughbred horses and processed in parallel using a commercial and a non-commercial method to obtain ACS. Fluorescent microsphere immunoassay (FMIA) analysis was performed to quantify Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations in ACS obtained by both production methods. Effect of 3, 4 and 5 freeze/thaw cycles on concentrations of IL-1Ra, IL-10, IL-1β and TNF-α were assessed against baseline samples (2 cycles) in commercial ACS products. Standard aerobic and anaerobic culture methods were applied to both ACS products. Mixed effect one-way analyses of variance (ANOVA) were used to compare the two ACS production method for each cytokine. Repeated measures, mixed effect ANOVA were used to assess the effect of freeze/thaw on cytokine concentrations. Significance was set at < 0.05. There was no difference in cytokine concentration between production methods (IL-1Ra = 0.067, IL-1β = 0.752, IL-10 = 0.211 and TNF-α = 0.25). Microbial growth was only observed in two samples obtained using the commercial production method. When compared to baseline, IL-1Ra concentration was decreased following the 5th freeze/thaw cycle ( < 0.001). These results suggest that the concentration of important cytokines are not influenced by ACS production method. When storing ACS samples for future use, freeze/thaw cycles associated with standard clinical practice are unlikely to influence cytokine concentrations. However, the lack of outcome measures associated with 1 or 2 freeze/thaw cycles represents a limitation of this study.
Copyright © 2021 Hale, Hughes, Hall and Labens.
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Publication Date: 2021-11-25 PubMed ID: 34901249PubMed Central: PMC8656450DOI: 10.3389/fvets.2021.759828Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research focused on the impact of various preparation methods and repeated freeze-thaw cycles on the concentration of certain cytokines and the chance of contamination in equine autologous conditioned serum (ACS). It revealed that all tested factors didn’t have any noteworthy influence on the levels of these cytokines (natural biochemical substances) and contamination only occurred in a couple of instances when a commercial production method was used.

Methodology of the Study

  • Blood was acquired from 10 healthy Thoroughbred horses and processed evenly using both a commercial and a non-commercial method to obtain ACS.
  • An analysis was then carried out using a Fluorescent Microsphere Immunoassay (FMIA) to measure the concentrations of certain types of cytokines: Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) in ACS obtained by both production methods.
  • The effect of 3, 4 and 5 freeze/thaw cycles on concentrations of those cytokines were also assessed against baseline samples (2 cycles) in ACS products produced commercially.
  • Standard aerobic and anaerobic culture methods were applied to both ACS products.
  • Mixed effect one-way analyses of variance (ANOVA) were used to compare the two ACS production methods for each cytokine, alongside repeated measures and mixed effect ANOVA to assess the effect of freeze/thaw on cytokine concentrations.

Results of the Study

  • No significant difference in cytokine concentration were found between different production methods, with the results for IL-1Ra, IL-1β, IL-10 and TNF-α showing no statistical significance.
  • Microbes were only found in two samples obtained through the commercial production method.
  • When compared to the baseline, only the IL-1Ra concentration was noticed to decrease after the 5th freeze/thaw cycle, indicating that frequent freeze-thaw processes might affect particular cytokines stability.
  • Consequently, the study concluded that the concentration of key cytokines was unaffected by the ACS production method used.

Implications and Limitations

  • Storing ACS samples for future use with freeze/thaw cycles usually involved in standard clinical practice is not expected to affect cytokine concentrations.
  • The study limitations include an absence of outcome measures related to 1 or 2 freeze/thaw cycles, suggesting that the research may not fully cover the potential effects of less repeated freeze/thaw cycles on cytokine stability or microbial growth in the sample.

Cite This Article

APA
Hale J, Hughes K, Hall S, Labens R. (2021). Effects of Production Method and Repeated Freeze Thaw Cycles on Cytokine Concentrations and Microbial Contamination in Equine Autologous Conditioned Serum. Front Vet Sci, 8, 759828. https://doi.org/10.3389/fvets.2021.759828

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 8
Pages: 759828
PII: 759828

Researcher Affiliations

Hale, Josephine
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.
Hughes, Kristopher
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.
Hall, Sarah
  • Animal & Veterinary Sciences Department, Scotland's Rural College, Edinburgh, United Kingdom.
Labens, Raphael
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Citations

This article has been cited 3 times.
  1. Bowlby CM, Purmessur D, Durgam SS. Equine peripheral blood CD14(+) monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation. Vet Immunol Immunopathol 2023 Jan;255:110534.
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  2. Roth SP, Liso G, Brehm W, Wagner B, Schnabel CL, Troillet A. Selected cytokine and chemokine concentrations in equine autologous conditioned serum are similar under defined and practically relevant storage conditions. Front Vet Sci 2025;12:1588240.
    doi: 10.3389/fvets.2025.1588240pubmed: 40496923google scholar: lookup
  3. Yim KHW, Al Hrout A, Chahwan R. Intercellular Epigenomic Signalling via Extracellular Vesicles During B Cell Maturation. J Extracell Vesicles 2025 Jan;14(1):e70040.
    doi: 10.1002/jev2.70040pubmed: 39868437google scholar: lookup
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