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Topic:Cell Culture
Cell culture in horses involves the in vitro cultivation of equine cells under controlled conditions. This technique is employed to study various cellular processes, including growth, differentiation, and response to external stimuli, in an isolated environment. Equine cell cultures can be derived from various tissues, such as skin, muscle, or bone, and are used in a range of research applications, including genetic studies, drug testing, and disease modeling. These cultures provide a valuable platform for understanding cellular mechanisms and developing therapeutic strategies. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and findings related to cell culture in equine research.
Understanding Stallion Sperm Capacitation: Species-Specific Mechanisms, Cryocapacitation, and Implications for IVF. Until recently, no repeatable protocol for equine in vitro fertilization (IVF) had been established, despite decades of trying. Recent studies have demonstrated reproducible IVF outcomes under specific experimental conditions, bringing equine IVF into a new perspective. Prolonged incubation of stallion spermatozoa in a TALP based medium supplemented with penicillamine, hypotaurine and epinephrine (PHE) appears to be important factors of protocols that support sperm capacitation and markedly improved IVF outcomes. The sperm capacitation procedure has now been applied successfully using both fre...
Influence of Follicle Number and Reproductive Cyclicity on Mesenchymal Stromal Cell Yield Isolated From Equine Ovarian Follicular Aspirates. Follicular aspirates have emerged as an alternative source of mesenchymal stromal cells (MSC) in mares; however, factors influencing successful MSC isolation remain unclear. This study evaluated whether reproductive cyclicity and follicle count affect MSC recovery from ovum pick-up (OPU)-derived follicular aspirates. Twenty-eight OPU procedures were performed on 13 mares, and cells were cultured until passage 3. Successful MSC cultures were obtained in 15/28 procedures (53.6%), while 13 procedures (46.4%) yielded non-MSC populations. Neither reproductive status (cycling vs. anestrous) nor foll...
Effect of Uncoated Magnetic Nanoparticles on Stallion Sperm Parameters: Influence of Semen Quality. The aim of this study was to evaluate the effect of uncoated, non-functionalised magnetic nanoparticles (MNPs) on stallion sperm and to determine whether these effects depend on initial semen quality. Ten cooled ejaculates from 3 stallions were exposed to different MNP concentrations ([MNPs] = 0, 5, 25, and 50 mg/mL), agitated, and exposed to an external magnet to recover the unbound sperm fraction. Ejaculates were classified as high- or low-quality groups according to initial evaluation sperm quality index (HSQi and LSQi), and the effect of [MNPs] was evaluated within groups using GLM. ...
Differential Gene Expression of Mesenchymal Stromal Cell and Granulosa Cell Markers in Equine Follicular Aspirates. Equine follicular aspirates obtained during ovum pick-up contain a heterogeneous mixture of mesenchymal stromal cells (MSC) and granulosa cells (GC). This study evaluates how selective isolation and expansion of MSC decrease GC populations. Follicular aspirates-derived cells were cultured up to passage four (P4) following a previously validated protocol. While freshly isolated follicular aspirates cells (FAC) displayed heterogeneous morphologies, early passages (P3-P4) showed a uniform, spindle-shaped phenotype compatible with MSC. Gene expression analysis by RT-qPCR revealed a marked upregula...
A single-cell RNA-seq dataset characterizing cellular diversity in healthy equine skin. The skin serves as the primary barrier tissue in horses and is frequently affected by immune-mediated dermatological conditions, most notably insect bite hypersensitivity (IBH). Yet, the cellular composition and transcriptional landscape of normal equine skin have never been characterized at single-cell resolution. Here, we present a single-cell RNA sequencing dataset of healthy equine skin comprising 85,574 high-quality transcriptomes from two horses, with one skin biopsy collected from each horse, divided into two portions for independent processing via manual or automated tissue dissociatio...
Prolyl-Hydroxyproline Promotes Structural Matrix Organization in 3D Equine Tendon Proper and Peritenon Post-Injury Model. Tendinopathy of the superficial digital flexor tendon affects horse health and performance, with slow healing and high re-injury rates due to the formation of mechanically weaker scar tissue. Tendons are rich in Type I collagen and may benefit from supplementation with collagen-derived dipeptide prolyl hydroxyproline (H-Pro-Hyp-OH) which improves collagen regeneration and reduces scarring in murine injury models. However, its effects on equine tenocytes in the tendon proper (TP) and peritenon (PERI) under stress are unknown. Three-dimensional fibrin-based TP and PERI constructs were used to ev...
Promises and Perspectives of Mesenchymal Stromal Cells in Companion and Farm Animals. Mesenchymal stromal cells (MSCs) are heterogeneous, multipotent cell populations that have become one of the most extensively investigated cell types in veterinary regenerative medicine. They can be isolated from a wide range of adult and perinatal tissues and have attracted considerable interest because of their immunomodulatory properties, broad secretory activity, and potential to promote tissue repair. However, increasing evidence indicates that MSCs should not be regarded as a uniform therapeutic product, as their biological behaviour is influenced by tissue source, donor characteristics,...
Ortholog-specific Spatial Expression of Vanins in the Respiratory Tract of Horses. Equine asthma (EA) is the most prevalent chronic respiratory disease in horses, but molecules thought to be relevant in its pathogenesis are not yet fully understood. Recently, increased Vanin-1 (VNN1) levels were found in bronchoalveolar lavage fluid from horses with severe EA. Human VNN1 is known as a pantetheinase, which is associated with respiratory diseases such as asthma, but the equine vanins (eVNNs) are uncharacterized. We identified the domain architecture of eVNN in silico and analyzed its tissue expression in healthy horses using RT-PCR (=5) and in situ hybridization (=3). Recombin...
Selective oncolytic activity of newcastle disease virus in equine sarcoid primary cells: a preclinical investigation. Equine sarcoids are the most common skin tumors in horses and are characterized by local invasiveness, high recurrence rates, and inconsistent responses to current therapies. Oncolytic virotherapy represents a promising alternative approach that exploits defects in antiviral signaling pathways in tumor cells. Newcastle disease virus (NDV), an avian paramyxovirus that is nonpathogenic in mammals, has demonstrated tumor-selective activity in several species; however, its effects on equine sarcoids have not been previously investigated. In this study, primary cell cultures derived from equine sar...
Equine herpesvirus myeloencephalopathy-associated mutations in equine herpesvirus 1 DNA polymerase confer the ability to replicate at elevated temperatures. Equine herpesvirus myeloencephalopathy (EHM), caused by equine herpesvirus type 1 (EHV-1), poses a major threat to the equine industry because of its devastating impact on animal welfare and athletic performance. A single-nucleotide polymorphism (SNP), G2254, in ORF30 (UL30), which encodes the viral DNA polymerase, has been used as a marker of neuropathogenic EHV-1 strains, although its contribution to EHM pathogenesis remains controversial. In addition, other ORF30 SNPs have been reported in association with EHM, but their functional significance is unclear. Clinical observations indicate tha...
Alpha-2-Macroglobulin-Enriched Plasma Modulates Inflammatory and Proteolytic Pathways in Experimental Equine Musculoskeletal Models. Joint inflammation and extracellular matrix degradation are major contributors to the development of equine osteoarthritis and reduced athletic performance. Alpha-2-macroglobulin is a naturally occurring broad-spectrum inhibitor of proteases and inflammatory mediators that has emerged as a promising orthobiologic therapy. This study investigated the anti-inflammatory and matrix-protective effects of alpha-2-macroglobulin-enriched plasma in complementary in vitro and ex vivo equine models. Primary equine chondrocytes and synoviocytes isolated from healthy horses were stimulated with interleukin...
Supplementation of IVM media with equine follicular fluid of large follicles increases blastocyst yield in equine ICSI programs. Despite advances in the use of intracytoplasmic sperm injection (ICSI) for the in vitro production of equine embryos, oocyte maturation and competence to sustain pre-implantation embryo development remain significant challenges. This study evaluated the supplementation of the in vitro maturation (IVM) medium with equine follicular fluid (eFF), obtained from young mares (2-15 years old) with oocyte-to-blastocyst conversion rates >22%, as a strategy to improve oocyte developmental competence. In the first experiment, IVM medium was supplemented with 10% eFF collected from ovarian follicles of di...
Isolation, Expansion and Characterization of Mesenchymal Stromal Cells from Fresh and Cryopreserved Umbilical Cord Tissues in Horse and Dog. Mesenchymal stromal cells (MSCs) derived from perinatal tissues represent a promising resource for regenerative medicine due to their proliferative, immunomodulatory, and differentiation properties. Among these tissues, the umbilical cord is considered an accessible and ethically acceptable source of MSCs. This study evaluated the feasibility of whole umbilical cord cryopreservation in canine and equine species and assessed the biological properties of MSCs isolated from cryopreserved tissues compared with fresh samples. Equine Wharton's jelly and canine umbilical cord tissues were processed e...
The equine mesenchymal stromal cell (MSC) secretome modulates neutrophils and monocyte-derived macrophages and extracellular vesicles (EVs) impact macrophage viability. Neutrophil chemotaxis and phagocytosis are critical for protection against bacteria, but can be compromised by methicillin-resistant Staphylococcus aureus (MRSA). MRSA can also circumvent macrophage surveillance by affecting polarization and reactive oxygen species (ROS) production. We previously demonstrated that the secretome of equine mesenchymal stromal cells (MSCs), comprised of all secreted bioactive factors and collected as conditioned medium (CM), reduces the growth of MRSA both in vitro and in vivo. This study aimed to determine if the equine MSC secretome has additional anti-MRSA pro...
The in vitro application of high-intensity laser therapy on equine platelet-rich plasma and autologous protein solution does not alter select cytokine and growth factor concentrations. To quantify the effects of high-intensity laser therapy (HILT) on the concentration of key immunomodulatory cytokines and growth factors in equine platelet-rich plasma (PRP) and autologous protein solution (APS). We hypothesized that HILT treatment of PRP and APS in vitro would increase the concentration of cytokines and growth factors compared to untreated controls. Unassigned: Whole blood was collected from 6 adult horses housed at the University of Pennsylvania in March 2025 and processed into PRP or APS using commercially available systems (Zoetis). Platelet-rich plasma and APS samples wer...
Histone deacetylase inhibition alters mRNA expression of cell cycle- and senescence-associated genes in primary equine chondrocytes. Cellular senescence is a biological state of a cell that triggers inflammation and gradually contributes to pathological conditions. Unlike programmed cell death, prolonged inhibition of cell division induces a metabolic state in senescent cells, in which they release pro-inflammatory factors that drive inflammation affecting the tissue microenvironment. Along with prolonged excessive mechanical stress, low-grade inflammation raised by senescent chondrocytes may contribute to articular cartilage defects, which are prevalent in ageing populations. Moreover, the use of chemotherapeutics used to ...
Clinically relevant intra-articular corticosteroid doses impair equine osteoclast function in vitro. To assess the effects of clinically relevant doses of commonly employed intra-articular corticosteroids-specifically, triamcinolone acetonide (TCA), methylprednisolone acetate (MPA), and combination of betamethasone acetate and betamethasone sodium phosphate (BM)-on equine osteoclasts. Methods: In vitro controlled laboratory study. Methods: Three donor mares (13-19 years). Methods: Following differentiation from bone marrow aspirates, osteoclasts were cultured alone (pure osteoclast cultures; n = 18 wells per group) or on equine bone slices (osteoclast-bone cultures; n = 6 wells per ...
Equine infectious anemia virus Mat protein serves as a Tat cofactor to facilitate viral transcription. Equine infectious anemia virus (EIAV) is a member of the genus of the family and serves as an important model for studying lentiviral pathogenesis. A novel EIAV-encoded protein, designated Mat, was previously identified in our laboratory, yet its biological functions remained unclear. Here, we demonstrate that disruption of Mat moderately reduced EIAV replication in equine monocyte-derived macrophages, and this defect was restored when Mat was supplied in . Further analyses revealed that Mat exhibits nuclear localization and enhances Tat-mediated transcriptional activation of the long termin...
Effects of equine sperm incubation under capacitation-supporting conditions on homologous and heterologous ICSI outcomes. Intracytoplasmic sperm injection (ICSI) in horses has become sufficiently efficient to be considered for clinical and commercial application; however, several factors influencing its efficiency remain poorly understood. This study evaluated whether incubating equine sperm under capacitating conditions induces capacitation-associated changes and affects embryo development following homologous equine ICSI, as well as oocyte activation, early cleavage, and 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5hmC) levels following heterologous ICSI using bovine oocytes. Equine spermatozoa were ...
Effects of IGF1 Knockdown and Overexpression on the Phenotype and Function of Equine Primary Skeletal Muscle Cells. Skeletal muscle serves as the direct executor of movement, and the proliferation and migration capacities of its constituent cells represent the key physiological foundation determining equine endurance and speed. Previous whole-transcriptome analysis has shown that Insulin-like Growth Factor 1 () is significantly upregulated in following racing and is involved in the PI3K-Akt signaling pathway; however, its specific regulatory mechanism in equine skeletal muscle cells remains unclear. This study utilized primary equine skeletal muscle cells as a model. By constructing an overexpression plas...
Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage Florida Clade 2). Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing...
Genes Associated With Fracture Risk in Thoroughbred Horses Have Novel Roles in Osteogenesis. Bone fractures in Thoroughbred racehorses are a major welfare problem. Genetic factors contribute to fracture risk. Cell models have previously identified 112 differentially expressed genes in bone-forming osteoblasts derived from horses at high and low genetic risk of fracture. However, 42 of these genes have no published role in bone. In this study, we identified novel roles for a subset of these genes in bone formation. Twenty-six of the 42 genes were expressed in Saos2 cells during basal culture and/or after 21 days of osteogenic culture. Five of these genes (ADSSL1, CABP1, ENO2, SPARCL1...
Humoral immune response to equine mesenchymal stem/stromal cells (MSCs): MHC compatibility has more influence than inflammatory priming or chondrogenic differentiation. Mesenchymal stem/stromal cells (MSCs) hold great potential for treating various conditions, but their effectiveness and safety in allogeneic therapies can be affected by the recipient's immune response, among other factors. Allogeneic MSC administration has been associated with immune recognition in different species, including horses. These animals are both veterinary patients and translational models, offering valuable insights into how allogeneic MSCs interact with the immune system in vivo. Factors such as inflammation, chondrogenic differentiation, and major histocompatibility complex (MH...
Systematic establishment of approaches to the detection of equine macrophage polarization and their application in pathogenic infection. Macrophage phenotypic adaptation critically regulates inflammatory balance during infection; however, progress in equine immunology has been limited by a lack of specific tools for standardized identification. To address this gap, we established a reliable, antibody-based detection method for characterizing equine macrophage M1-like and M2-like marker profiles. After initially validating canonical marker genes via qPCR, we developed specific monoclonal antibodies targeting the differentially expressed surface proteins CD80 (M1-like) and CD163 (M2-like). These novel antibodies enabled the creat...
Anti-Inflammatory and Metabolic Effects of Fresh Versus Freeze-Dried Platelet-Rich Plasma on Equine Osteoarthritis in an Ex Vivo Cartilage-Synovium Explant Co-Culture System: A Pilot Study. Equine osteoarthritis (OA) is a major cause of lameness and economic loss in horses. While platelet-rich plasma (PRP) has clinical potential, the biological effects of fresh PRP (F-PRP) and freeze-dried PRP (FD-PRP) remain insufficiently defined. This pilot study compared 25% and 50% F-PRP and FD-PRP in an interleukin-1β-induced equine cartilage-synovium explant co-culture model. PRP treatments reduced inflammatory responses, with significant downregulation of COX-2 and PGE2 expression, and 25% F-PRP showed the most consistent inhibition of nitric oxide production. PRP also significantly redu...
Derivation of Equine Mesenchymal Stem/Stromal Cells from Induced Pluripotent Stem Cells via the Neural Crest Pathway and Characterisation by Immunophenotype and Tri-Lineage Differentiation. Mesenchymal stem/stromal cells (MSCs) hold promise for treating different equine conditions but enter senescence during culture. Using induced pluripotent stem cells (iPSCs) to derive MSC-like cells (iMSCs) can increase cell availability and diminish the need for invasive and repeated tissue harvesting. While human iMSCs are intensively studied, research on equine iMSCs (eqiMSCs) is very limited and has focused on strategies for spontaneous differentiation to obtain these cells. The aim of this study was to obtain MSC-like cells from equine iPSCs (eqiPSCs) by directing their differentiation vi...
Protective effects of adiponectin receptor agonists against equine lamellar endoplasmic reticulum stress. Lamellar endoplasmic reticulum (ER) stress occurs in hyperinsulinemia-associated and sepsis-related laminitis. Adiponectin is associated with reduced laminitis risk and inhibits ER stress in other species. Objective: To induce ER stress in ex vivo equine lamellar cells using pharmacological agents. To determine whether co-treatment with adiponectin receptor agonists (ARAs) reduces ER stress. Methods: In vitro. Methods: Lamellar cells from n = 4-5 horses were treated with tunicamycin or thapsigargin (10-1000 nM) for 2-24 h. ER-stressed cells were co-treated with full-length adiponectin ...
Comparison of equine PBMC isolation using two protocols: Impact on key cellular parameters. There are diverse uses for equine peripheral blood mononuclear cells (PBMC) in research. Studies have shown that PBMC isolation protocols can affect immune parameters of diagnostic and research assays. We aimed to assess the impact of equine blood handling techniques on cell yield, viability, and proliferation under mitogen stimulation. Whole blood (WB) samples were collected from eight healthy adult horses into heparinized tubes for PBMC isolation by comparing method 1 (M1) where freshly collected WB was layered directly over a density gradient medium for centrifugation, followed by PBMC harv...
FGF2, LIF, and IGF1 supplementation during in vitro maturation or culture improves equine ICSI embryo development in an age-dependent manner. Ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI) has reshaped equine assisted reproduction, enabling embryo production from mares and stallions with constrained breeding opportunities; however, limited efficiency of in vitro maturation (IVM) and in vitro culture (IVC) continues to hinder embryo production. Growth factor supplementation with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination (FLI) has been shown to enhance oocyte and embryo developmental competence in several species. Objective: This...
Allogeneic Platelet-Rich Gel Supernatant Reprograms the Cytokine and Growth Factor Microenvironment in an Equine In Vitro Inflammatory Tendon System. Tendinopathy involves a dysregulated inflammatory microenvironment in which cytokines, growth factors (GF) and extracellular matrix components interact dynamically. Platelet-rich plasma (PRP) is widely used as a regenerative therapy, but its mechanisms of action in inflamed tendon remain unclear. This study evaluated whether platelet-rich gel supernatant (PRGS) reprograms the inflammatory-anabolic mediator network in an equine in vitro tendon explant system stimulated with lipopolysaccharide (LPS). Tendon explants were cultured under six experimental conditions, including non-stimulated contro...
