Identification of variables that optimize isolation and culture of multipotent mesenchymal stem cells from equine umbilical-cord blood.
Abstract: OBJECTIVE-To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics. SAMPLE POPULATION-UCB samples from 79 Thoroughbred and Quarter Horse mares. PROCEDURES-UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion. RESULTS-MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes. CONCLUSIONS AND CLINICAL RELEVANCE-Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.
Publication Date: 2009-12-03 PubMed ID: 19951125DOI: 10.2460/ajvr.70.12.1526Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article discusses a study aimed at optimizing the extraction and cultivation of mesenchymal stem cells (MSCs) from the umbilical cord blood (UCB) of horses. The study further explores the variables that boost successful MSC extraction and analyzes how shipping, processing, and cryopreservation affect the stem cells’ viability, recovery rates, and expansion kinetics.
Objective and Sample Population
- The main goal of the research was to identify the best methods and variables that improve the isolation and cultivation of MSCs from UCB.
- 79 samples of umbilical cord blood were analyzed in this study. The samples were collected from Thoroughbred and Quarter Horse mares.
Procedures and Analysis
- The UCB samples underwent processing to reduce volume and remove Red Blood Cells (RBCs).
- Nucleated cells (NCs) from the processed samples were then either cryopreserved (preserved by cooling to sub-zero temperatures) or cultured under different conditions to maximize the growth and proliferation of MSCs.
- Both donor and UCB-sample variables were analyzed to ascertain their impact on the success of MSC isolation and cultivation.
Results
- The research was successful in growing MSCs, capable of multilineage differentiation, from over 80% of the UCB samples.
- Automating UCB processing and controlling shipping temperatures were found to aid in maintaining sterility and standardizing RBC reduction and NC enrichment from UCB samples.
- The number of NCs after UCB sample processing was found to be the only variable that predicted successful MSC growth.
- The UCB-derived MSCs and NCs were successful in cryopreservation and thawing with no loss in cell recovery, viability, or MSC growth rate.
- Improvements in MSC isolation rate were observed with the use of fibronectin-coated culture plates and reducing incubator oxygen tension from 20% to 5%.
- Some UCB-derived MSC clones proliferated for more than 20 passages before senescence. The onset of senescence correlated with specific immunocytochemical changes.
Conclusions and Clinical Relevance
- The study concluded that equine UCB samples are a good source of readily obtainable, highly proliferative MSCs that could be stored and used therapeutically.
- This discovery has significant clinical implications as stem cells are an essential ingredient in regenerative medicine.
Cite This Article
APA
Schuh EM, Friedman MS, Carrade DD, Li J, Heeke D, Oyserman SM, Galuppo LD, Lara DJ, Walker NJ, Ferraro GL, Owens SD, Borjesson DL.
(2009).
Identification of variables that optimize isolation and culture of multipotent mesenchymal stem cells from equine umbilical-cord blood.
Am J Vet Res, 70(12), 1526-1535.
https://doi.org/10.2460/ajvr.70.12.1526 Publication
Researcher Affiliations
- Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA.
MeSH Terms
- Animals
- Cell Culture Techniques
- Cryopreservation
- Fetal Blood / cytology
- Horses / blood
- Mesenchymal Stem Cells / cytology
- Mesenchymal Stem Cells / physiology
- Multipotent Stem Cells / cytology
- Multipotent Stem Cells / physiology
Citations
This article has been cited 13 times.- Kulus M, Sibiak R, Stefańska K, Zdun M, Wieczorkiewicz M, Piotrowska-Kempisty H, Jaśkowski JM, Bukowska D, Ratajczak K, Zabel M, Mozdziak P, Kempisty B. Mesenchymal Stem/Stromal Cells Derived from Human and Animal Perinatal Tissues-Origins, Characteristics, Signaling Pathways, and Clinical Trials.. Cells 2021 Nov 23;10(12).
- Zhou Y, Liao J, Fang C, Mo C, Zhou G, Luo Y. One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells.. Bio Protoc 2018 Nov 20;8(22):e3080.
- Mocchi M, Dotti S, Bue MD, Villa R, Bari E, Perteghella S, Torre ML, Grolli S. Veterinary Regenerative Medicine for Musculoskeletal Disorders: Can Mesenchymal Stem/Stromal Cells and Their Secretome Be the New Frontier?. Cells 2020 Jun 11;9(6).
- Ayala-Cuellar AP, Kang JH, Jeung EB, Choi KC. Roles of Mesenchymal Stem Cells in Tissue Regeneration and Immunomodulation.. Biomol Ther (Seoul) 2019 Jan 1;27(1):25-33.
- Textor JA, Clark KC, Walker NJ, Aristizobal FA, Kol A, LeJeune SS, Bledsoe A, Davidyan A, Gray SN, Bohannon-Worsley LK, Woolard KD, Borjesson DL. Allogeneic Stem Cells Alter Gene Expression and Improve Healing of Distal Limb Wounds in Horses.. Stem Cells Transl Med 2018 Jan;7(1):98-108.
- Owens SD, Kol A, Walker NJ, Borjesson DL. Allogeneic Mesenchymal Stem Cell Treatment Induces Specific Alloantibodies in Horses.. Stem Cells Int 2016;2016:5830103.
- Tessier L, Bienzle D, Williams LB, Koch TG. Phenotypic and immunomodulatory properties of equine cord blood-derived mesenchymal stromal cells.. PLoS One 2015;10(4):e0122954.
- Villatoro AJ, Fernández V, Claros S, Rico-Llanos GA, Becerra J, Andrades JA. Use of adipose-derived mesenchymal stem cells in keratoconjunctivitis sicca in a canine model.. Biomed Res Int 2015;2015:527926.
- Mohanty N, Gulati BR, Kumar R, Gera S, Kumar S, Kumar P, Yadav PS. Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood.. Cytotechnology 2016 Aug;68(4):795-807.
- Mohanty N, Gulati BR, Kumar R, Gera S, Kumar P, Somasundaram RK, Kumar S. Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood.. In Vitro Cell Dev Biol Anim 2014 Jun;50(6):538-48.
- Wang X, Wang Y, Gou W, Lu Q, Peng J, Lu S. Role of mesenchymal stem cells in bone regeneration and fracture repair: a review.. Int Orthop 2013 Dec;37(12):2491-8.
- Carrade DD, Lame MW, Kent MS, Clark KC, Walker NJ, Borjesson DL. Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells().. Cell Med 2012;4(1):1-11.
- Kretlow JD, Spicer PP, Jansen JA, Vacanti CA, Kasper FK, Mikos AG. Uncultured marrow mononuclear cells delivered within fibrin glue hydrogels to porous scaffolds enhance bone regeneration within critical-sized rat cranial defects.. Tissue Eng Part A 2010 Dec;16(12):3555-68.
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