Fetal Cats Research Articles

Morphology of individual axons in crossed corticorubral projections in developing cats and effects of partial denervation.

Ordered neuronal connections in mature brains are thought to be sculpted from initially diffuse projections by elimination of inappropriate projections and strengthening of appropriate ones. Although evidence suggests that neuronal activity plays a role in these processes, the mechanism behind the modification of neuronal connections remains obscure. To gain insight into the mechanisms of axonal elimination and projection strengthening, we examined the morphology of individual axons that were to be eliminated as well as the consequences of partial denervation. While corticorubral projections in adult cats are thought to be uncrossed, early in postnatal development and after early unilateral lesions to the sensorimotor cortex, however, a significant amount of crossed corticorubral projections occurs. We examined the morphology of individual corticorubral axons in fetal cats and kittens from embryonic day 59 to postnatal day 48 and those that had received early unilateral lesions to the cortex, by serial reconstruction of Phaseolus-vulgaris-leucoagglutinin- or biocytin-labeled axons. For about 2 weeks during pre- and postnatal development, crossed axons remained simple in morphology, with few branches. Thereafter, they showed an increase in branch number, but then began to show fewer branches again. Axons and their collaterals were found in nonrestricted areas of the red nucleus (RN) throughout the period of observation, indicating that axons can sit at an inappropriate target for weeks but fail to ramify. In contrast, crossed corticorubral axons in kittens with cortical lesions showed terminal-arbor-like structures in the RN region that are in mirror symmetry to topographically appropriate areas in the ipsilateral RN, although some showed simple morphology without arbors. These complicated forms of morphology of individual axons during development and after partial denervation may not be explained by a simple activity-dependent mechanism.

Few neocortial and thalamic morphological changes after a neonatal frontal cortical ablation contrast with the effects of a similar lesion in fetal cats

To further understand the neuroanatomical consequences following perinatal brain injury, quantitative morphometric analysis was performed on the brain of cats receiving a unilateral frontal cortical ablation between postnatal days (P) 9 and P 14 and intact control cats. In all cats, the volume of the neocortex and thalamus was measured bilaterally and that of the thalamic ventrobasal complex (VBc) was measured ipsilaterally. In addition, using stereology, the neuronal and glial (presumably) cell packing densities (CPD) and the total number of neurons and glial cells (TCN) were measured in the ipsilateral VBc. The neuronal and glial cell coss-sectional areas (CSA) were also measured in the ipsilateral VBc. The mean ipsilateral and contralateral neocortex volumes were similar between the two animal groups. There was a statistically significant 14% and 13% reduction in mean ipsilateral and contralateral thalamic volumes, respectively for the lesioned animals, while the VBc shrank by 16% relative to intact controls. The mean neuronal and glial CPD were similar between the two groups. The mean neuronal TCN was reduced by 10% in the neonatal-lesioned cats, while the mean glial TCN was reduced by 31% in the same animals, however neither value reached significance. Lastly, the mean CSA of neurons and glial cells showed a tendency to be smaller in the lesioned cats by 8% and 9%, respectively. These results: (a) indicate that the neonatal lesion caused only minor morphological brain alterations and this sharply contrasts with the marked changes previously reported in cats with a similar lesion sustained prenatally; (b) suggest that the enhanced behavioral recovery and/or sparing reported for the present cats compared to fetal-lesioned animals is at least partially due to the morphological sparing reported here; (c) together with previous findings in fetal cats, support the hypothesis that the morphological changes after a neonatal neocortical lesion are qualitatively different and may depend on different mechanisms as compared to those occurring after similar damage sustained prenatally.

Development of bovine embryos matured and fertilized in vitro in media containing human leukemia inhibitory factor

To evaluate neural stem/progenitor cell (NPC) transplantation therapy in cat models of neurodegenerative diseases, we have isolated, expanded and characterized feline NPCs (fNPCs) from normal fetal cat brain. Feline NPCs responsive to both human epidermal growth factor (hEGF) and human fibroblast growth factor 2 (hFGF2) proliferated as neurospheres, which were able to differentiate to neurons and glial cells. The analysis of growth factors indicated that both hEGF and hFGF2 were required for proliferation of fNPCs. In contrast to the effect on human NPCs, human leukemia inhibitory factor (hLIF) enhanced differentiation of fNPCs. Expanded fNPCs were injected into the brains of normal adult cats. Immunohistochemical analysis showed that the majority of transplanted cells were located adjacent to the injection site and some fNPCs differentiated into neurons. The survival of transplanted fNPCs over time was monitored using non-invasive bioluminescent imaging technology. This study provided the first evidence of allotransplantation of fNPCs into feline CNS. Cats have heterogeneous genetic backgrounds and possess neurological diseases that closely resemble analogous human diseases. The characterization of fNPCs and exploration of non-invasive bioluminescent imaging to track transplanted cells in this study will allow evaluation of NPC transplantation therapy using feline models of human neurological diseases.

Segregation of cerebrorubral and cerebellorubral synaptic inputs on rubrospinal neurons of fetal cats as demonstrated by intracellular recording

Cerebrorubral and cerebellorubral inputs are localized to distal dendrites and somata of red nucleus neurons in adult cats, respectively. To examine if this segregation is established early in development, we performed intracellular recording from rubrospinal neurons of fetal cats aged from embryonic day 58 to 65. Stimulation of the contralateral cerebellar nuclei evoked excitatory postsynaptic potentials (EPSPs). EPSPs were also induced by stimulation of the ipsilateral pericruciate cortex but they were much slower in time course and smaller in amplitude compared to cerebellar ones. We suggest that cerebrorubral and cerebellorubral synapses are segregated on soma-dendritic membrane of rubrospinal neurons early in development.

Neuraminic acid specific lectins as markers of early cortical plate neurons

Early cortical plate and subplate cells in the developing neocortex of many animal species and humans contain one specific plasma protein, fetuin. Fetuin is heavily glycosylated and it is possible that due to the large amount of sugars, this molecule may play a part in cellular recognition during brain development.Cellular and extracellular carbohydrates in the developing brain of the sheep were studied histochemically using a battery of fluorescein-labelled lectins. Two neuraminic acid specific lectins, Sambucus nigra and Maackia amurensis, labelled consistently the fetuin-positive cells as demonstrated by double labelling with lectins and antifetuin antibodies. Brain sections from other species, known to contain fetuin-positive cells (fetal cow, postnatal tammar wallaby) showed a similar lectin staining pattern to that of the sheep fetus. Additionally, sections from species thought to contain fetuin in their developing brains that failed to cross-react with available antifetuin antibodies (postnatal Monodelphis, fetal cat) also demonstrated lectin-positive staining in the same neuronal cell population.Thus, neuraminic acid is a common and well conserved terminal carbohydrate in cortical plate and subplate neurons of the developing brain. Neuraminic-specific lectins are useful markers for these neurons in addition to the more traditional use of immunocytochemical methods in studies of formation of the neocortex.

Chapter 3 Responses of isolated cat retinal ganglion cells to injected currents during development

Publisher Summary This chapter explains the response properties of dissociated ganglion cells to constant current stimulation. The whole-cell patch clamp recording technique offers a powerful tool for assessing the membrane properties of developing neurons. This method to record from acutely dissociated retinal ganglion cells obtained from postnatal and prenatal cats, in order to characterize the development of spiking properties in these neurons. The long-term goal of this work is to relate the functional development of retinal ganglion cells to the structural refinements that are known to occur in these neurons during ontogeny. It has been found recently that somatostatin-containing amacrine cells and ganglion cells, which at maturity are preferentially distributed in the inferior, are widespread in the fetal cat retina. It is conceivable that developing ganglion cells are particularly sensitive to certain transiently expressed neuromodulators, and that this contributes to the correlated discharges that have been noted in the developing retina.

In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord

Magnetic resonance (MR) imaging was evaluated for its possible diagnostic application in determining the survival of fetal central nervous system tissue grafts in the injured spinal cord. Hemisection cavities were made at the T11-L1 level of eight adult female cats. Immediately thereafter, several pieces of tissue, either obtained from the fetal cat brain stem on embryonic Day 37 (E-37), from the fetal neocortex on E-37, or from the fetal spinal cord on E-23, were implanted into the cavities made in seven cats. The eighth cat served as a control for the effect of the lesion only. In another group of four animals, a static-load compression injury was made at the L-2 level. Seven weeks later, the lesion was resected in three cases and fragments of either fetal brain-stem or spinal cord tissue were introduced. A small cyst was observed in a fourth cat in the compression injury group and a suspension of dissociated E-23 brain-stem cells was injected into this region of cavitation without disturbing the surrounding leptomeninges. Five months to 2 years posttransplantation, MR imaging was performed with a 2.0-tesla VIS imaging spectrometer by acquiring multislice spin-echo images (TR 1000 msec, TE 30 msec) in both the transverse and sagittal planes. Collectively, these intermediate-weighted images revealed homogeneous, slightly hyperintense signals at the graft site relative to the neighboring host tissue in seven of the 11 graft recipients. Two of the remaining four cats exhibited signals from the graft site that were approximately isointense with the adjacent host spinal cord, and the final two cats and the lesion-only control presented with very hypointense transplant/resection regions. The hyperintense and isointense images were tentatively interpreted as representing viable graft tissue, whereas the hypointense transplant/resection sites were considered to be indicative of a lack of transplant survival or the absence of tissue in the lesion-only control animal. Postmortem gross inspection of fixed specimens and light microscopy verified the MR findings in the control animal in 10 of the 11 graft recipients by showing either transplants and/or cysts corresponding to the MR images obtained. In one cat in the hemisection group, histological analysis revealed a very small piece of graft tissue that was not detected on the MR images. Therefore, it is suggested that within certain spatial- and contrast-resolution limits, MR imaging can reliably detect the presence of transplanted neural tissue in both the hemisected and compression-injured spinal cord of living animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Conduction velocity of identified central neurons in fetal and neonatal cats

Conduction velocity of identified central neurons in fetal and neonatal cats

Mapping the early development of projections from the entorhinal cortex in the embryonic mouse using prenatal surgery techniques

The purpose of this work was to study the development of specific projections from the postero-lateral cortex during the third trimester of gestation in the mouse. To do this, we labeled undifferentiated lateral cortex with the fluorescent carbocyanine dye, Dil, in the embryonic day (E) 16 mouse embryo using exo utero surgical techniques (Muneoka, Wanek, and Bryant, 1986). Embryos were allowed to develop to term (postnatal day 0, P0) at which time the fiber patterns emanating from the marked regions were studied. Dye placement in the undifferentiated postero-ventral cortex produced labeled fibers in the hippocampal formation. A robust projection of the angular bundle into the CA1 region of the hippocampus was heavily labeled. In addition, in some animals, cortical tracts, such as the anterior commissure, corpus callosum, and a corticotectal tract, were labeled. These tracts have been described previously as scaffolding pathways in the fetal cat (McConnell, Ghosh, and Shatz, 1989), and other vertebrates (Wilson, Ross, Parrett, and Easter, 1990). Dye placement in adjacent, more anterior or dorsal areas showed strong labeling in cortical structures but no labeling in the hippocampal formation. These data indicate that, by birth, the temporal cortex is subdivided along the rostro-caudal axis as entorhinal cortex and perirhinal cortex, and along the dorso-ventral axis, as entorhinal cortex and neocortex. Also, these earliest connections are similar to adult connections in their specificity of target area selection. Therefore, these early, yet specific, connections may play a role int he formation of future connections during postnatal development.

Early dendritic outgrowth of primate retinal ganglion cells

The pattern of dendritic stratification of retinal ganglion cells in the fetal monkey (Macaca mulatta) was examined using horseradish peroxidase and retinal explants. Ganglion cells in the rhesus monkey are born between embryonic day (E) 30-70 (LaVail et al., 1983). At E60, E67, and E68, approximately 50% of all ganglion cells within the central 3.0 mm of the retina had dendritic arbors that were unistratified within the inner plexiform layer (IPL), while the remaining 50% had bistratified arbors. Unistratified cells had relatively flat arbors that ramified within a restricted portion of the IPL. In contrast, bistratified cells had one portion of the arbor that branched in the inner half of the IPL and a second portion that branched in the outer half of the IPL. Relatively few bistratified cells were encountered in the central 1.0 mm of the retina but were more numerous with increasing eccentricity. At E81, E90, and E110, the dendritic arbors of ganglion cells increased in both area and complexity, but occupied a relatively small percentage of the total depth of the IPL. The bistratified cells encountered at these fetal ages were typically located in the far retinal periphery. Between E125-E140, the dendritic arbors of individual ganglion cells increased in area and depth to occupy a greater proportion of the total IPL than at earlier fetal ages. These observations suggest that ganglion cells in the macaque undergo at least three stages of dendritic stratification: (1) an initial period of dendritic growth during which the cells have either unistratified or bistratified dendritic arbors; (2) a loss of the majority of bistratified cells through cell death or remodeling of the arbor; and (3) growth or expansion of the arbor to occupy a greater percentage of the total depth of the IPL. The first two stages are similar to recent observations in the fetal cat (Maslim & Stone, 1988) with the exception that dendritic development in the primate lacks an initial diffuse ingrowth to the IPL. Additionally, primate ganglion cells undergo a third stage of dendritic growth in late fetal development during which the arbor occupies a greater proportion of the depth of the IPL.

Animal models: Prenatal diagnosis of Chediak‐Higashi syndrome in the cat by evaluation of cultured chorionic cells

The autosomal recessive disease Chediak-Higashi syndrome (CHS) is a progressive and generally fatal disease of humans. The underlying genetic defect in CHS is unknown and prenatal diagnostic methods have not been applied to this disease. The purpose of this study was to determine if CHS chorionic cells expressed a characteristic of CHS--enlarged lysosomes--that would permit the prenatal diagnosis of the disease. Cats with CHS, which have been shown to be homologous with human CHS, were used as the model system in this study. Chorionic tissue samples were obtained from CHS and control cat fetuses and cultures of cells were established. Acid phosphatase was utilized as a marker of lysosomes and cultures of chorionic fibroblasts from CHS and control fetuses were stained histochemically for acid phosphatase. The diameter of the largest lysosomes in 150 cells of each fetus was determined. The mean (+/- SD) diameter (in microns) of the largest lysosomes of normal fetuses was 0.9 +/- 0.13 (range 0.5-7.0 microns), whereas the mean diameter of lysosomes in CHS chorionic cells was 3.9 +/- 0.65 microns (range 0.5-25 microns). These means were significantly different (P less than 0.0001). These data suggest that it should be possible to diagnose human CHS in the first trimester by chorionic villus sampling.

Synchronous Bursts of Action Potentials in Ganglion Cells of the Developing Mammalian Retina

The development of orderly connections in the mammalian visual system depends on action potentials in the optic nerve fibers, even before the retina receives visual input. In particular, it has been suggested that correlated firing of retinal ganglion cells in the same eye directs the segregation of their synaptic terminals into eye-specific layers within the lateral geniculate nucleus. Such correlations in electrical activity were found by simultaneous recording of the extracellular action potentials of up to 100 ganglion cells in the isolated retina of the newborn ferret and the fetal cat. These neurons fired spikes in nearly synchronous bursts lasting a few seconds and separated by 1 to 2 minutes of silence. Individual bursts consisted of a wave of excitation, several hundred micrometers wide, sweeping across the retina at about 100 micrometers per second. These concerted firing patterns have the appropriate spatial and temporal properties to guide the refinement of connections between the retina and the lateral geniculate nucleus.

Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenii)

A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of FLG in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and alpha 2HS glycoprotein in human fetal brain. The sequence of appearance of FLG-positive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of FLG in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed.

Delayed grafting of fetal CNS tissue into chronic compression lesions of the adult cat spinal cord

This review summarizes a series of experiments involving transplants of embryonic feline CNS tissue into chronic compression lesions of the adult cat spinal cord. Fetal spinal cord (FSC), caudal brainstem (BSt), neocortex (NCx) or a combination of either FSC/NCx or FSC/BSt was transplanted as solid pieces or as a suspension of dissociated cells into the developed cystic cavities produced by static-load compression trauma 2-10 weeks prior to grafting. All cats were immunosuppressed with cyclosporin A and their locomotor function was assessed for 6-30 weeks. Following the period of evaluation, all recipients were perfused with fixative and tissue specimens, taken at the transplantation site, were processed for general histological and/or immunocytochemical analysis. Viable graft tissue was found in all animals with the exception of two cats which showed active rejection of their transplants. All of the viable intraspinal grafts were extensively vascularized and did not show any signs of imminent or on-going tissue rejection. Fetal cat CNS grafts showed an extended maturational phase in that features of immature neural tissue (e.g. a paucity of myelination) were still seen even 6-9 weeks after transplantation. By 20-30 weeks, FSC and BSt grafts had attained a more advanced stage of maturation. Transplants in these chronic lesions were extensively blended with both the gray and white matter of the host spinal cord and could be visualized by magnetic resonance imaging (MRI). MRI could also detect regions of cavitation at the graft-host interface, as well as within some transplants. While preliminary evidence from behavioral studies suggest that the FSC and BSt grafts may improve or spare locomotor function in some recipients, a more rigorous analysis of post-grafting locomotor function is required to determine conclusively the functionality of these transplants.

Chediak-Higashi syndrome in the cat: Prenatal diagnosis by evaluation of amniotic fluid cells

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease in humans, cats, and 8 other species. The homology of CHS in humans and cats has been demonstrated. Since human CHS is a progressive, serious, and eventually fatal disease, a method for prenatal diagnosis would be desirable. This study was designed to determine whether CHS could be diagnosed prenatally by examination of amniotic fluid cells. The amniotic fluid samples were obtained from CHS and control cat fetuses on the 45th day of gestation and cultures of cells were established. Because the underlying enzyme deficiency in CHS has not been identified, it was necessary to use a secondary manifestation of the syndrome in these studies. The secondary manifestation used was the characteristic enlargement of lysosomes associated with the disease. The lysosomes of these cells were stained by acid phosphatase histochemistry and the diameter of the largest lysosome in each cell was measured by light microscopy with a calibrated ocular micrometer. The diameters of the largest lysosomes in cells of normal fetuses ranged from 0.5 to 7.0 micron (means ranged from 0.9 to 1.8 micron), whereas the diameter of the largest lysosomes in the cells of CHS fetuses ranged from 0.5 to 30 microns (means ranged from 6.4 to 12.8 microns). The approximate t-test for independent samples with unequal variances disclosed that the largest acid phosphatase-positive lysosomes in amniotic fluid cells of CHS cat fetuses were significantly larger than the lysosomes in the cells of normal cat fetuses (P less than 0.0001). This information should, by extrapolation, provide the basis for the prenatal diagnosis of human CHS by amniocentesis.

Cardiac Mapping of Regional Glucose Utilization in Fetal Cats, Rabbits, and Chicks Using [14C]2‐Deoxyglucosea

Cardiac Mapping of Regional Glucose Utilization in Fetal Cats, Rabbits, and Chicks Using [¹⁴C]2‐Deoxyglucose^a

Comparison of the Size of the Vascular Compartment of the Carotid Body of the Fetal, Neonatal and Adult Cat

The carotid bodies from full-term fetal cats, 3- to 4-day-old neonates and adult cats were perfusion-fixed at normal arterial blood pressure with 3% phosphate-buffered glutaraldehyde. Serial 5-microns sections were cut and stained by the MSB method. Using an interactive image analysis system, determinations were made of the volumes of the carotid body and of its vascular and extravascular compartments. Compared to the fetus, the carotid body of the neonate increased in volume by 51% and by 286% in the adult cat. There was a proportional increase in the volumes of the vascular compartment and of the small vessels (5-12 microns diameter) in that compartment. The volume of the small vessels, expressed as a ratio of the total volume of the organ, remained constant in the three animal groups at 5-7%. The small vessel endothelial surface area, expressed as a ratio of the extravascular volume (which was assumed to consist largely of type 1 and type 2 cells), was the same in the neonate as in the full-term fetus. Thus, there were no apparent quantifiable morphological features of the carotid body and its vasculature which would account for the resetting of the hypoxic sensitivity of the organ from the fetal to the adult range within a few days of birth.

Chediak‐Higashi syndrome: Prenatal diagnosis by fetal blood examination in the feline model of the disease

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease of humans, mink, cattle, mice, killer whales, cats, and blue and silver foxes. The disease is characterized by incomplete oculocutaneous albinism, recurrent and severe pyogenic infections, a bleeding tendency secondary to a platelet storage pool deficiency, and enlarged granules in many types of cells. Humans with CHS usually die during childhood. It has been suggested that the prenatal diagnosis of CHS should be possible by the demonstration of enlarged granules in neutrophils of fetal blood. We tested this hypothesis using 20 cat fetuses obtained 18 days at prepartum. Two litters (6 fetuses) were from CHS to CHS matings and four litters (14 fetuses) were from CHS male to heterozygous female matings. Fetuses were identified as CHS or phenotypically normal by histologic examination of the size of melanin granules in the ciliary body and by the size of periodic acid-Schiff-positive granules in renal tubular epithelial cells. The diameter of the peroxidase-positive granules in neutrophils of the 15 CHS fetuses ranged from 0.3 to 3.0 microns whereas those of the five normal fetuses ranged from 0.3 to 1.0 micron. All 20 fetuses were correctly classified as CHS or phenotypically normal. These data indicate that examination of the size of fetal blood neutrophil granules can be used to diagnose CHS prenatally.

Neocortical connections in fetal cats

The major extrinsic projections to and from visual and auditory areas of cerebral cortex were examined in fetal cats between 46 and 60 days of gestation (E46–E60) using axonal transport of horseradish peroxidase either alone or in combination with tritiated proline. Projections to visual cortex from the dorsal lateral geniculate nucleus and lateral-posterior/pulvinar complex exist by E46, and those from the contralateral hemisphere, claustrum, putamen, and central lateral nucleus of the thalamus are present by E54–E56. In addition, cells in the medial geniculate nucleus project to auditory cortex by E55. At E54–E56 efferent cortical projections reach the contralateral hemisphere, claustrum, putamen, lateral-posterior/pulvinar complex and reticular nucleus of the thalamus. Cells in visual cortex also project to the dorsal and ventral lateral geniculate nuclei, pretectum, superior colliculus and pontine nuclei, and cells in auditory cortex project to the medial geniculate nucleus. Except for interhemispheric projections, all pathways demonstrated are ipsilateral, and projections linking cerebral cortex with claustrum, dorsal lateral geniculate nucleus and lateral-posterior/pulvinar complex are reciprocal. The reciprocal projections formed with the dorsal lateral geniculate nucleus, lateral-posterior/pulvinar complex and the claustrum show a greater degree of topological organization compared to the projections formed with the contralateral hemisphere and superior colliculus, which show little or no topological order. Therefore, the results of the present study show that the major extrinsic projections of the cat's visual and auditory cortical areas with subcortical structures are present by the eighth week of gestation, and that the origins and terminations of many of these projections are arranged topologically.

Cultivation techniques for animal coronaviruses: Emphasis on feline infectious peritonitis virus, canine coronavirus, transmissible gastroenteritis virus, and porcine hemagglutinating encephalomyelitis virus.

SummaryTechniques are described for the growth and characterization of some mammalian coronaviruses. Because of the fastidious nature of their growth requirements, most will replicate only in cells derived from the natural host or a closely related species. Fetal cat cells are used to grow FIPV, and porcine cells are used to grow TGEV and HEV. However, CCV will replicate in both feline and canine cells. Although all four of these viruses prefer to replicate in a cell in the stationary phase of growth, FIPV is able to replicate in an actively growing cell. Each virus causes a cytopathic effect in monolayer cell cultures under agar or media 18 to 72 h postinfection. Primary isolation of each virus from field specimens is difficult, although most can usually be isolated after 1 to 3 blind passages in the cell culture.