Isolation, propagation, and cryopreservation of equine articular chondrocytes.
Abstract: Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.
Publication Date: 1992-12-01 PubMed ID: 1476323
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research focused on the isolation, propagation, and cryopreservation of horse cartilage cells (chondrocytes). It found that these chondrocytes could be harvested, grown in labs, preserved through freezing, and remain viable for periods of up to 9 months, potentially serving useful for cartilage regenerative therapies and pharmaceutical testing.
Chondrocyte Isolation
- The researchers collected the articular (relating to joint) cartilage cells known as chondrocytes, from horses of different ages. These cells were isolated from culture by digesting them in a collagenase solution.
- They found that cartilage from late-term unborn and newborn foals had more chondrocytes compared to older foals (3 to 12 months old).
Chondrocyte Propagation
- To cultivate the chondrocytes, they used a specific growth medium, Ham’s F-12, supplemented with fetal bovine serum, ascorbic acid, alpha-ketoglutarate, and L-glutamine. This medium was buffered with HEPES, and antibiotics (penicillin and streptomycin) were added for microorganism control.
- They observed that in primary monolayer (single layer) cultures of freshly isolated chondrocytes, population doubling (cell division) time was approximately six days. However, after up to the fifth passage, a shift in the shape and nature of the cells (dedifferentiation) towards more fibroblast-like (the cell that produces collagen and other fibers in connective tissues) was observed. This change was faster at lower cell-plating densities.
Chondrocyte Cryopreservation
- Chondrocytes were preserved by freezing, using a cryoprotectant, dimethyl sulfoxide. They were stored at -196 degrees Celsius for up to 9 months.
- The preserved cells retained their viability, but there was a decrease from 86% viability at 3 weeks to 31% at 12 weeks in newborn foal cells. However, cells from older foals and young adult horses showed better viability than newborn cells.
- These preserved cells could be thawed and used immediately or kept in lab cultures either in monolayer or 3-dimensional structure.
Application of the Study
- The researchers suggested that the cultivated and stored chondrocytes could be used for cartilage resurfacing experiments, a process where damaged cartilage in a joint is replaced.
- Additionally, these cultivated cells could be used for in vitro (outside the body) testing of potential pharmaceutical drugs on cartilage cells before starting any animal or human testing.
Cite This Article
APA
Nixon AJ, Lust G, Vernier-Singer M.
(1992).
Isolation, propagation, and cryopreservation of equine articular chondrocytes.
Am J Vet Res, 53(12), 2364-2370.
Publication
Researcher Affiliations
- Department of Clinical Sciences College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
MeSH Terms
- Animals
- Cartilage, Articular / cytology
- Cell Division
- Cell Separation / veterinary
- Cells, Cultured
- Cryopreservation / veterinary
- Horses
Grant Funding
- AR 35664 / NIAMS NIH HHS
Citations
This article has been cited 18 times.- Ciamillo SA, Pownder SL, Potter HG, Stefanovski D, Nixon AJ, Ortved KF. Correlation of Arthroscopic Grading and Optical Coherence Tomography as Markers of Early Repair and Predictors of Later Healing Evident on MRI and Histomorphometric Assessment of Cartilage Defects Implanted with Chondrocytes Overexpressing IGF-I.. Cartilage 2023 Jun;14(2):210-219.
- Kim AY, Duerr FM, Phillips JN, Samulski RJ, Grieger JC, Goodrich LR. Serotype-specific transduction of canine joint tissue explants and cultured monolayers by self-complementary adeno-associated viral vectors.. Gene Ther 2023 Apr;30(3-4):398-404.
- Linardi RL, Dodson ME, Moss KL, King WJ, Ortved KF. The Effect of Autologous Protein Solution on the Inflammatory Cascade in Stimulated Equine Chondrocytes.. Front Vet Sci 2019;6:64.
- Gilbertie JM, Long JM, Schubert AG, Berglund AK, Schaer TP, Schnabel LV. Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators.. Front Vet Sci 2018;5:150.
- Cassano JM, Schnabel LV, Goodale MB, Fortier LA. Inflammatory licensed equine MSCs are chondroprotective and exhibit enhanced immunomodulation in an inflammatory environment.. Stem Cell Res Ther 2018 Apr 3;9(1):82.
- Morgan R, Keen J, Halligan D, O'Callaghan A, Andrew R, Livingstone D, Abernethie A, Maltese G, Walker B, Hadoke P. Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways.. PLoS One 2018;13(2):e0192746.
- Reesink HL, Sutton RM, Shurer CR, Peterson RP, Tan JS, Su J, Paszek MJ, Nixon AJ. Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility.. Stem Cell Res Ther 2017 Nov 2;8(1):243.
- Ortved KF, Austin BS, Scimeca MS, Nixon AJ. RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.. Arthritis 2016;2016:3484961.
- Wilson B, Novakofski KD, Donocoff RS, Liang YX, Fortier LA. Telomerase Activity in Articular Chondrocytes Is Lost after Puberty.. Cartilage 2014 Oct;5(4):215-20.
- Ortved KF, Begum L, Mohammed HO, Nixon AJ. Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model.. Mol Ther 2015 Feb;23(2):363-73.
- Hemphill DD, McIlwraith CW, Samulski RJ, Goodrich LR. Adeno-associated viral vectors show serotype specific transduction of equine joint tissue explants and cultured monolayers.. Sci Rep 2014 Jul 29;4:5861.
- Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, Gray SJ, Samulski RJ. Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis.. Mol Ther Nucleic Acids 2013 Feb 5;2(2):e70.
- Goodrich LR, Choi VW, Carbone BA, McIlwraith CW, Samulski RJ. Ex vivo serotype-specific transduction of equine joint tissue by self-complementary adeno-associated viral vectors.. Hum Gene Ther 2009 Dec;20(12):1697-702.
- Feng G, Wan Y, Balian G, Laurencin CT, Li X. Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells.. Growth Factors 2008 Jun;26(3):132-42.
- Fortier LA, Miller BJ. Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes.. J Orthop Res 2006 Aug;24(8):1765-72.
- Akens MK, Hurtig MB. Influence of species and anatomical location on chondrocyte expansion.. BMC Musculoskelet Disord 2005 May 17;6:23.
- Burton-Wurster N, Gendelman R, Chen H, Gu DN, Tetreault JW, Lust G, Schwarzbauer JE, MacLeod JN. The cartilage-specific (V+C)- fibronectin isoform exists primarily in homodimeric and monomeric configurations.. Biochem J 1999 Aug 1;341 ( Pt 3)(Pt 3):555-61.
- Girdler NM. The role of mandibular condylar cartilage in articular cartilage repair.. Ann R Coll Surg Engl 1997 Jan;79(1):28-37.
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