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Frontiers in bioengineering and biotechnology2021; 8; 613621; doi: 10.3389/fbioe.2020.613621

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.

Abstract: Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor β1 concentrations were increased in the concentrates compared with whole blood or serum ( < 0.05), while white blood cells were reduced ( < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing ( < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies ( < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.
Copyright © 2021 Hagen, Lehmann, Aurich, Bauer, Melzer, Moellerberndt, Patané, Schnabel and Burk.
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Publication Date: 2021-01-21 PubMed ID: 33553119PubMed Central: PMC7859354DOI: 10.3389/fbioe.2020.613621Google 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 study focuses on developing a scalable production process of equine Platelet Lysate (ePL) and testing its efficiency in the culture of equine Multipotent Mesenchymal Stromal Cells (MSC). The aim is to provide an effective alternative to the current widely-used Fetal Bovine Serum (FBS) for the cultivation of equine MSC.

Collection and Preparation of Samples

  • Blood samples were collected from 20 healthy horses, and upon testing for pathogen contamination, 19 were included in the study.
  • Growth factors and platelets were collected from these samples after being concentrated using a buffy coat method.
  • This method also managed to reduce the content of white blood cells in the samples.
  • The concentrated platelets were then lysed through freeze/thaw cycles, eliminating the cells but maintaining the growth factor concentrations.
  • A negative correlation was observed between donor age and concentrations of platelets and growth factors.

Testing of Equine Platelet Lysate in MSC Culture

  • All lysates obtained were combined, culminating into the production of an ePL which was then used as a medium supplement in MSC culture, in comparison with FBS.
  • Adipose-derived MSCs from four unrelated horses were used for this experiment.
  • MSC proliferated well in both 10% FBS and 10% ePL, demonstrating the effectiveness of ePL as a growth medium.
  • However, lower concentrations of ePL (5% or 2.5%) led to inconsistent proliferation or complete loss of proliferation. Substantial differences were noted in generation times and confluences.

Analysis of MSC Expression and Differentiation

  • MSC in all culture media expressed CD90, CD44, and CD29 surface antigens while the detection of CD73 and CD105 was low.
  • Adipogenic and osteogenic differentiation of the MSC led to similar results irrespective of the culture media used.

Conclusion and Future Research

  • The buffy coat method showed usefulness in producing horse platelet concentrate which can be used in large-scale production.
  • This ePL also supports the expansion of MSC similarly to FBS, when used at identical concentrations (of 10%).
  • The study hence proposes that future research should focus on further researching equine MSC functionality in culture with ePL in an effort to establish ePL as an established alternative to FBS in cultivating equine MSC.

Cite This Article

APA
Hagen A, Lehmann H, Aurich S, Bauer N, Melzer M, Moellerberndt J, Patané V, Schnabel CL, Burk J. (2021). Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture. Front Bioeng Biotechnol, 8, 613621. https://doi.org/10.3389/fbioe.2020.613621

Publication

Frontiers in bioengineering and biotechnology
ISSN: 2296-4185
NlmUniqueID: 101632513
Country: Switzerland
Language: English
Volume: 8
Pages: 613621
PII: 613621

Researcher Affiliations

Hagen, A
  • Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany.
Lehmann, H
  • Department of Veterinary Clinical Sciences, Small Animal Clinic, Justus-Liebig-University Giessen, Giessen, Germany.
Aurich, S
  • Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig-University Giessen, Giessen, Germany.
Bauer, N
  • Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, Giessen, Germany.
Melzer, M
  • Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany.
Moellerberndt, J
  • Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany.
Patané, V
  • Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, Giessen, Germany.
Schnabel, C L
  • Faculty of Veterinary Medicine, Institute of Immunology, Leipzig University, Leipzig, Germany.
Burk, J
  • Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany.

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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