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.
Ultracytochemical study of multiple dose effect of monastral blue uptake by equine pulmonary intravascular macrophages (PIMs). The pulmonary intravascular macrophages (PIMs) of the ponies possess all the characteristics of a fully differentiated resident macrophage, which forms adhesive plaques with the capillary-endothelial cells. In addition, it has a unique surface coat which does not conform to the concept of a carbohydrate rich membrane bound glycocalyx generally associated with macrophages. We studied the responses of these cells especially in the context of its globular surface coat to multiple doses of MB intravenously: 0.2 ml/kg body copper tracer substance. The ponies were treated with three doses of MB intr...
Babesia equi erythrocytic stage continuously cultured in an enriched medium. Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 1...
Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli. Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers. The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer. Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX. The possible relationship of this observation to recombinant expression of other heme proteins is discussed.
Detection of equine arteritis virus following amplification of structural and nonstructural viral genes by reverse transcription-PCR. A reverse transcription (RT)-PCR assay was developed for the detection of equine arteritis virus (EAV) in cell culture supernatant and in horse semen. Four different sets of oligonucleotide primers complementary to sequences located in the 3' end of the polymerase gene (open reading frame [ORF] 1b) and to sequences representing the entire ORFs 3, 4, and 7, which encode for nonstructural (ORFs 3 and 4) or viral nucleocapsid (ORF 7) proteins, were compared for their abilities to amplify the targeted EAV sequences by the RT-PCR procedure. The sensitivities of the RT-PCR for amplification of EAV s...
Phenotype and biological activity of neonatal equine chondrocytes cultured in a three-dimensional fibrin matrix. Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 x 10(5), 1 x 10(6), and 5 x 10(6) chondrocytes/cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, f...
Fine structure of equine oocytes matured in vitro for 15 hours. Transmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microscopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 +/- 0.2 degree C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero-time oocytes contained few cortical granules, and...
Secretagogue-induced [14C]aminopyrine uptake in isolated equine parietal cells. Equine oxyntic mucosal cells were obtained by sequential exposure to pronase and collagenase. Acid production by parietal cells was assessed by uptake of [14C]aminopyrine (AP), a weak base that accumulates in intracellular acidic spaces. Incubation for various times revealed a maximal AP uptake at 10 minutes for histamine and carbachol. Similar secretagogue responses were observed for parietal cells from the mucosal cell preparation or after enrichment by elutriation. Histamine and isobutyl-methylxanthine (IBMX) stimulated AP uptake with a dose-dependent response and maximal effective concentr...
Culture of equine embryos in media containing egg yolk, mare’s milk and saline: Preliminary results. A medium containing egg yolk, mare's milk and/or modified PBS was used to culture Day-8 to 8.5 equine blastocysts. Twenty-one variants of the medium containing different concentrations of the 3 components were prepared. Embryos were recovered nonsurgically and placed into the media at 37 degrees C for 24 h. A total of 45 embryos was cultured; of these 7 died in culture and 13 showed inadequate development at the onset, while 25 continued to grow in the media. It was established that embryos grew best in media containing 20 to 60% yolk, 20 to 60% mare's milk and/or 20 to 60% PBS. It was found e...
Effect of heparin on capacitation/acrosome reaction of equine sperm. The onset of sperm capacitation/acrosome reaction was evaluated using heparin. Equine semen was incubated at 38 degrees C for 4.5 h in culture medium with and without 10 micrograms/mL heparin and with and without 0.1 microM of Ca2+ ionophore. Sperm acrosome reaction was detected using chlortetracycline fluorescence (CTC) and transmission electron microscopy (TEM). The CTC assay provided staining patterns that corresponded with the capacitation/acrosome reaction in other mammalian species (man, mouse, guinea pig). The percentages of incapacitated sperm (PUC), capacitated acrosome-intact sperm (...
Investigation of stimulus-secretion coupling in equine sweat gland epithelia using cell culture techniques. When sweat glands isolated from samples of horse skin were explanted and cultured under favourable conditions, they could exhibit cellular outgrowth. This growth could be maintained for 2-4 weeks and these primary cultures were then disaggregated and the resultant cell suspensions used to initiate epithelial cell lines. Secretion from intact equine sweat glands is regulated by beta 2-adrenoceptors and appears to be mediated by cyclic AMP, but there is evidence that calcium may also play a role. Adrenaline could increase the cyclic AMP content of the cultured cells and this response was mediate...
In vitro interaction between oviduct epithelial and equine sperm. Coculture of stallion sperm with monolayers of equine oviductal epithelial cells (OEC) was evaluated. Monolayers were obtained from frozen-thawed OEC. Live sperm attached to the OEC in vitro, whereas sperm killed by heat treatment or glutaraldehyde fixation did not. Sperm attached to OEC showed flagellar motion for 4 d in vitro, during which time they gradually became released. Scanning electron-micrographs showed an intimate association between the sperm and OEC. Incubation of sperm for 4 h with either control, heparinized or OEC-conditioned medium (Tyrode's albumin lactate phosphate) resulte...
Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Reliable capacitation of equine spermatozoa has been a major obstacle in the development of equine in vitro fertilization. Experiments were done to compare in vitro capacitation of equine spermatozoa by use of heparin/caffeine, calcium ionophore, uterine tube epithelial cell (UTEC)-conditioned medium, and direct culturing of spermatozoa with UTEC (coculturing). Capacitation-like changes, as determined by chlortetracycline membrane staining patterns, developed with UTEC-conditioned medium and coculturing, equivalent to that with calcium ionophore. Both of these treatments induced more (P < 0.05...
Synteny mapping in the horse using horse-mouse heterohybridomas. In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.
Development to blastocysts of one- to two-cell equine embryos after coculture with uterine tubal epithelial cells. Development of 1- to 2-cell in vivo fertilized equine embryos cultured with or without uterine tubal epithelial cells (UTEC) was studied. One- to 2-cell embryos (n = 26) were collected surgically from the uterine tubes of pony mares 1 day after ovulation. Four- to 8-cell embryos (n = 9) were collected 2 days after ovulation. Presumptive zygotes and 2-cell embryos were cultured with (n = 17) or without (n = 9) UTEC, and all 4- to 8-cell embryos were cocultured with UTEC as positive controls. Uterine tubal epithelial cells were used as cell suspensions within 2 weeks after initiation of cultures...
Isolation of an inhibitor of tumor necrosis factor-alpha-mediated cytotoxicity liberated from chemotaxin-stimulated equine white blood cell populations. Objectives of this investigation were to extract and isolate protein fractions inhibitory to the cytotoxic properties of tumor necrosis factor-alpha (TNF-alpha). In this context, mixed populations of WBC were harvested from equine blood and were stimulated with a combination of a synthetic chemotactic peptide and a calcium ionophore. Several methods were subsequently applied for the initial preparation of cell-free crude protein extracts, including fractional precipitation with gradient concentrations of ammonium sulfate and preparative-scale isoelectric focusing. In addition, protein fraction...
Culture confirmation of the carrier status of Babesia caballi-infected horses. Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.
Analysis of multiple mRNAs from pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse reveals a novel protein, Ttm, derived from the carboxy terminus of the EIAV transmembrane protein. Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regula...
In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. The chromatin configuration of resting horse oocytes and the time course of in vitro oocyte maturation was characterized using a fluorescent, DNA-specific label. Oocytes were classified as having either compact (CP) or expanded (EX) cumuli at the time of collection. Centrifugation of oocytes was effective in allowing visualization of the germinal vesicle. Two main chromatin configurations were found in oocytes known to have a germinal vesicle: condensed chromatin (CC), in which the chromatin formed a dense mass surrounding the nucleolus; and fluorescing nucleus (FN), in which the entire nucleu...
Biology and neurobiology of Borna disease viruses (BDV), defined by antibodies, neutralizability and their pathogenic potential. Borna disease viruses (BDV) isolated from more than 20 naturally infected horses, 2 sheep and a possible feline isolate were included in these studies. Most of these wild-type viruses were grown in rabbit cells. Specifically rabbit-adapted viruses establish persistent infection in immortalized cell lines of various animal species. Brain-, tissue culture-, and cell-free released viruses could all be neutralized with antibodies from naturally and experimentally infected animals (horse; hamster, rat, rabbit, mouse, and chicken), with highest titres in birds. Splenectomized rabbits, which were sub...
Isolation, propagation, and cryopreservation of equine articular chondrocytes. 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 buffere...
Animal immunodeficiency viruses. Feline immunodeficiency virus (FIV) has morphological, physical and biochemical characteristics similar to human immunodeficiency virus (HIV), the cause of AIDS in man. However, it is antigenically and genetically distinct from HIV; an antigenic relatedness with equine infectious anaemia virus has been demonstrated. FIV has been molecularly cloned and sequenced. Diagnostic tests are commercially available and attempts at preparing inactivated, subunit and molecularly engineered vaccines are being made in different laboratories. During FIV infection a transient primary illness can be recognized...
The activity of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) against equine herpesvirus-1 (EHV-1) in cell cultures, mice and horses. The activity of the nucleotide analogue, (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC), against equine herpesvirus-1 (EHV-1) was tested in cell culture, mice and foals. The ED50 for plaque reduction was found to be 0.07 and 0.03 microgram/ml in RK-13 and EEL cells respectively. In mice, a single administration of HPMPC (20 mg/kg, s.c.) was very effective at reducing clinical signs and virus replication if given on the day before intranasal inoculation with EHV-1. Treatment on the day of infection or day 1 p.i. was less effective, but still significantly reduced clinical sign...
In vitro production of specific antibody by equine peripheral blood mononuclear cells using tetanus toxoid as a recall antigen. Anti-tetanus toxoid (TT) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells (PBMC) were measured by an indirect enzyme-linked immunoabsorbent assay (ELISA). Optimal anti-TT Ig production occurred at concentrations of stimulating, purified TT of between 0.001 and 0.1 micrograms ml-1, which varied depending on the cell concentration. Optimal anti-TT Ig production was most consistently produced when the cell concentration was 5 x 10(6) ml-1. At this cell concentration maximal anti-TT Ig was induced using 0.1 micrograms ml-1 TT. At a cell c...
Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7. A sandwich enzyme-linked immunsorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the n...
The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas. Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the b...
Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares. In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) develo...
Interleukin-1 stimulation of equine articular cells. Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiment...
Isolation and culture of glandular epithelial and stromal cells from the endometrium of mares. Glandular epithelial and stromal cells were isolated from the endometrium of mares by collagenase digestion and were incubated on plastic for 7-9 days until the cells formed confluent monolayers. The cells differed in morphology: epithelial cells appeared polyhedral and stromal cells were spindle like. The monolayers were incubated in the presence and absence of oxytocin. Medium was removed from wells after 2, 8 and 24 h of incubation. Concentrations of prostaglandin F (PGF) in the medium increased significantly during this time. Glandular epithelial cells produced significantly more PGF than ...
Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002. The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF a...
Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells. The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibi...