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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.
Identification and characterisation of beta-adrenoceptors on intact equine peripheral blood lymphocytes with the radioligand (-)-[125I]-iodocyanopindolol. In this study, beta-adrenoceptors of intact equine lymphocytes were identified and subclassified by (-)-[125I]-iodocyanopindolol (ICYP) binding. ICYP binding to intact equine lymphocytes was rapid, saturable (maximal number of binding sites 320 +/- 20 ICYP binding sites/cell, n = 12) and of high affinity (KD value for ICYP 14.4 +/- 1.7 pmol/l, n = 12). Binding was stereospecific as shown by the 10 times greater potency of (-)-propranolol to inhibit binding than its (+)-isomer. Beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline >(-)-adrenaline >(-...
Organisation of the cytoskeleton during in vitro maturation of horse oocytes. Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry ...
Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix. This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish fou...
Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media. The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st pola...
Inflammatory mediators induce endothelium-dependent adherence of equine eosinophils to cultured endothelial cells. Accumulation of equine eosinophils at sites of parasite infestation or allergic inflammation depends upon their adherence to vascular endothelial cells and subsequent migration through the endothelium and extracellular matrix. This study has examined whether cytokines, which cause endothelial cell-dependent eosinophil adherence in other species, and histamine and substance P, which increase adherence of equine eosinophils to protein coated plastic, induce equine eosinophil adherence to cultured equine digital vein endothelial cell (EDVEC) monolayers. The EDVEC monolayers were stimulated with r...
The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability. Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis, a neurologic disease of horses. The present study was designed to test the hypothesis that pyrantel tartrate can kill S. neurona merozoites growing in equine dermal cell culture. Sarcocystis neurona merozoites were exposed to a range of concentrations of pyrantel tartrate or sodium tartrate ranging from 0.001 to 0.01 M. Merozoites were then placed onto equine dermal cell cultures and incubated for 2 weeks to check for viability. At 1 and 2 weeks after inoculation, plaque counts were compared between treatments an...
Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure. Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovu...
DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus. In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR....
Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modi...
Increasing culture time from 48 to 96 or 144 hours increases the proportions of equine cumulus oocyte complexes with negative or fragmented nucleus morphology. The objective was to test the hypothesis that increasing equine oocyte culture time from 48 to 96 or 144 h increases nucleus maturation of equine oocytes. The hypothesis was not supported because condensed chromatin-stage oocytes decreased (P<0.01) from 33/126 (26.2%) at 48 h or 34/95 (35.8%) at 96 h to 11/117 (9.4%) at 144 h, and polar body-stage oocytes decreased (P<0.01) from 65/126 (51.6%) at 48 h to 25/95 (26.3%) at 96 h and (P<0.01) to 1/117 (0.9%) at 144 h. Negative (non-staining) oocytes increased (P<0.01) from 16/126 (12.7%) at 48 h or 15/95 (15.8%) at 96 h to 39/117 (33.3...
Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture. To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. Methods: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. Methods: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment...
Characterization of the Oregon isolate of Neospora hughesi from a horse. Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical si...
Adherence of eosinophils from allergic and normal ponies to cultured equine endothelial cells. To compare adherence of stimulated and unstimulated eosinophils from allergic and normal ponies to cultured equine vascular endothelial cells (equine digital vein endothelial cells; EDVEC) and examine the effect of eosinophil-derived factor(s) on cell adherence. Methods: Eosinophil adherence to unstimulated EDVEC or EDVEC pretreated with IL-1beta or supernatants from stimulated eosinophils was measured. Supernatants were also assayed for TNFalpha and IL-1beta-like bioactivity. Results: Adherence of unstimulated and rhIL-5 (10 ng/ml)-stimulated eosinophils from allergic ponies to rhIL-1beta-tre...
Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture. An isolate of Sarcocystis neurona (SN7) was obtained from the spinal cord of a horse with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The organism divided by endopolygeny and completed at least one asexual cycle in cell cultures in 3 days. The parasite was maintained by subpassages in bovine monocytes for 10 months when it was found to be non-pathogenic to gamma interferon knockout (KO) mice. Revival of a low passage (10th passage) of the initial isolate stored in liquid nitrogen for 18 months retained its pathogenicity for KO mice. Mero...
Direct agglutination test for the detection of antibodies to Sarcocystis neurona in experimentally infected animals. Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in the Americas. The apicomplexan protozoan most commonly associated with EPM is Sarcocystis neurona. A direct agglutination test (SAT) was developed to detect antibodies to S. neurona in experimentally infected animals. Merozoites of the SN6 strain of S. neurona collected from cell culture were used as antigen and 2-mercaptoethanol was added to the antigen suspension to destroy IgM antibodies when mixed with test sera. Mice fed sporocysts of S. speeri or S. falcatula-like sporocysts from opossums did not sero...
Ultrastructure of schizonts and merozoites of Sarcocystis neurona. The ultrastructure of Sarcocystis neurona schizonts and merozoites was studied in specimens derived from cell culture and from the brains of infected mice. Schizonts and merozoites were located in the host cell cytoplasm without a parasitophorous vacuole at any stage of development. Merozoites divided by endopolygeny. Fully formed merozoites had a pellicle, numerous polysomes and ribosomes, smooth and rough endoplasmic reticulum, 22 subpellicular microtubules, 9-16 dense granules, 25-75 micronemes, a plastid, a Golgi complex, 1-3 mitochondria, a conoid, 2 apical rings, 2 polar rings, 0-6 lipid...
Isolation and characterization of an equine foamy virus. Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%...
Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay. The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical ...
Expression and characterisation of equine interleukin 2 and interleukin 4. In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycos...
Characterization of cultured smooth muscle cells obtained from the palmar digital arteries of horses. To develop methods to isolate, culture, and characterize smooth muscle cells (SMC) from equine palmar digital arteries. Methods: Segments of the medial or lateral palmar digital arteries from the forelimbs of 6 horses. Methods: To obtain smooth muscle explants, arterial segments were incised longitudinally. The tunica intima was gently scraped from the underlying tunica media, and explants were obtained from the tunica media. Approximately 18 to 24 explants were obtained from each palmar digital arterial segment. A substrate-attached technique was used to initiate primary culture of SMCCulture...
The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses. To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). Methods: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effe...
In vitro model of equine muscle regeneration. Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their res...
Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro–the role of CRH pre-incubation. The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-mi...
Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. Methods: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. Methods: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated fro...
Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture. We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy obser...
Determination of the activity of ponazuril against Sarcocystis neurona in cell cultures. The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures. Ponazuril inhibited merozoite production by more that 90% in cultures of S. neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril. Ponazuril may have promise as a therapeutic agent in the treatment of S. neurona induced equine protozoal myeloencephalitis (EPM) in horses.
Neospora hughesi: experimental infections in mice, gerbils, and dogs. Neospora hughesi is a recently described cause of equine protozoal myeloencephalitis (EPM). A rodent model for pathogenicity would facilitate development of therapies to be used in horses. In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of N. hughesi isolated from a horse from AL, USA. Only the gamma-IFNKO mice developed severe clinical disease following infection with N. hughesi and died 19-25 days after infection and exhibited se...
A unique exocelom-like space during early pregnancy in the horse. The free allantois and allantochorion of conceptuses from 17 mares between 20 and 90 days of gestation were examined to determine the manner in which the associated mesodermal derivatives differentiated. It was found that a robust basement membrane developed under the allantoic endoderm, and that this basement membrane was partially isolated from the vascular layer of the allantois by a mesothelial layer and an exocelom-like space. The exocelom-like space persisted until approximately the stage of villous formation, and remnants of the space persisted over larger allantoic vessels even later. ...
Establishment and characterization of equine autonomic ganglion cell lines to enable direct testing of candidate toxins involved in equine dysautonomia (grass sickness). To enable direct testing of a range of potential toxins or pathogens that might be involved in grass sickness, equine thoracic sympathetic chain ganglion cell lines were established from primary cell cultures by retroviral-mediated transduction of the temperature-sensitive mutant of the establishment oncogene encoding SV40 large T antigen. Morphological and behavioral features, temperature dependence, and immunocytochemical characteristics of the cell lines were investigated. The majority of cells were noradrenergic neurons in which dopamine-beta-hydroxylase, the enzyme that catalyzes norepine...
