Estimates Of Total Cell Numbers Research Articles

Novel stereological method for estimation of cell counts in 3D collagen scaffolds

Current methods for assessing cell proliferation in 3D scaffolds rely on changes in metabolic activity or total DNA, however, direct quantification of cell number in 3D scaffolds remains a challenge. To address this issue, we developed an unbiased stereology approach that uses systematic-random sampling and thin focal-plane optical sectioning of the scaffolds followed by estimation of total cell number (StereoCount). This approach was validated against an indirect method for measuring the total DNA (DNA content); and the Bürker counting chamber, the current reference method for quantifying cell number. We assessed the total cell number for cell seeding density (cells per unit volume) across four values and compared the methods in terms of accuracy, ease-of-use and time demands. The accuracy of StereoCount markedly outperformed the DNA content for cases with ~ 10,000 and ~ 125,000 cells/scaffold. For cases with ~ 250,000 and ~ 375,000 cells/scaffold both StereoCount and DNA content showed lower accuracy than the Bürker but did not differ from each other. In terms of ease-of-use, there was a strong advantage for the StereoCount due to output in terms of absolute cell numbers along with the possibility for an overview of cell distribution and future use of automation for high throughput analysis. Taking together, the StereoCount method is an efficient approach for direct cell quantification in 3D collagen scaffolds. Its major benefit is that automated StereoCount could accelerate research using 3D scaffolds focused on drug discovery for a wide variety of human diseases.

A Metric Fractionator for Estimation of Total Cell Number in Paraffin-Embedded Flat or Hollow Organs.

The physical fractionator is a convenient and practical solution for estimation of total cell number in a regulatory toxicology setting because it is insensitive to shrinkage allowing for paraffin processing/embedding and does not require measurement of the reference or organ volume. The principle involves sampling a known fraction of an organ in one or more steps and counting the total number of cells present in the final sample, physical disector section pairs. The total cell number in the organ is estimated by multiplying the cell count in the final fraction by the inverse of the sampling fraction(s). The key feature of the design is that tissue shrinkage due to paraffin processing occurs before the organ is uniformly sampled. Another requirement is that thermal expansion or contraction is avoided during the preparation of disector sections from the individual embedded subsamples, which ensures that the disector sections represent a known constant fraction. This vertical physical fractionator with subsampling is a simple and fast estimator to obtain precise and robust estimates of total cell number in large flat or hollow organs that do not prolong routine necropsy procedures. It is compatible with paraffin processing, avoids exhaustive sectioning, and allows for the collection of routine histopathology sections.

UV-irradiated parylene surfaces for proliferation and differentiation of PC-12 cells.

PC-12 cells originate from neuroblastic cells, which have an ability to differentiate into neuronlike cells. In this work, the purpose was to estimate the influence of microenvironments on cell attachment and neuritogenesis capacity of PC-12 cells on parylene-N and parylene-C films with and without ultraviolet (UV) light treatment. The estimate of total cell number after incubation for 72h, the ratio of adherent to suspended cells, counting of neurite outgrowths on parylene-N or parylene-C films after UV exposure suggested that these films were suitable for proliferation as well as differentiation of PC-12 cells. The differences in surface properties of parylene-N and parylene-C films with and without UV exposure were analyzed by contact angle measurement, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy. According to these analyses, introduction of oxygen-related chemical functional groups was presumed to result in increased hydrophilicity and efficiency of protein immobilization on parylene-N and parylene-C films after UV treatment. According to fluorescent staining, western blotting, and cell cycle analysis, UV-treated parylene-C and parylene-N films appear to effectively facilitate simultaneous proliferation and differentiation of PC-12 cells with neurite outgrowth.

How many TCR clonotypes does a body maintain?

We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude.

Viable Cells Density and its Relationship with the Vascularization of the Endplate in Lumbar Discs

Introduction The intervertebral disc is avascular and remains unknown if it has the capacity to maintain viable cells inside over the years. The vascularization of the endplate has a direct influence on the nutrition to the disc cells. This study aimed to analyze the vascularization of the endplate and its relation to the density of viable cells in lumbar intervertebral discs of young and old adult humans. Materials and Methods A total of 28 lumbar intervertebral discs were used and separated into two groups: Group 1 ( n = 15; younger than 50 years; Pfirrmann ≤ 3) and Group 2 ( n = 13; elder or equal to 80 years, Pfirrmann ≥ 4). Vascularization of the endplates was analyzed using Stereo Microscope Discovery V.8. Cell viability was assessed by MTT method. The cell density was measured by PicoGreen technique using amount of DNA as an estimate of total cell number. Results The viable cells remained steady in relation to the total cell density. The inferior endplate presented the highest number of holes ( p < 0.05). The greater number of cells was found in the annulus fibrosus ( p < 0.05). There was a positive correlation ( p < 0.05) between the density of viable cells and vascular openings in the inferior endplate. Conclusion The inferior endplate has the main supply of blood vessels to nourish the intervertebral disc and has direct bearing on the number of viable cells. Acknowledgments This work was supported by INFAR/UNIFESP Confocal and Flow Cytometry Facility and by grants from the “Fundacao de Amparo à Pesquisa do Estado de São Paulo” (FAPESP: Grant number: 2009/54793–6). D.E.M. was supported by a PhD's fellowship grant from CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nível Superior).

Stereological estimation of total cell numbers in the human cerebral and cerebellar cortex.

Our knowledge of the relationship between brain structure and cognitive function is still limited. Human brains and individual cortical areas vary considerably in size and shape. Studies of brain cell numbers have historically been based on biased methods, which did not always result in correct estimates and were often very time-consuming. Within the last 20–30 years, it has become possible to rely on more advanced and unbiased methods. These methods have provided us with information about fetal brain development, differences in cell numbers between men and women, the effect of age on selected brain cell populations, and disease-related changes associated with a loss of function. In that this article concerns normal brain rather than brain disorders, it focuses on normal brain development in humans and age related changes in terms of cell numbers. For comparative purposes a few examples of neocortical neuron number in other mammals are also presented.

Inhibition of cell survival, viability and proliferation by dentin adhesives after direct and indirect exposure in vitro

The influence of dentin adhesive systems (Scotchbond Multi-Purpose, XP Bond, Xeno V, Clearfil Protect Bond, AdheSE) on cell survival, viability and proliferation was characterized after direct and indirect exposure using different cell culture techniques. The primers and cured bonding parts were directly exposed to cells using cell culture inserts, and complete materials were analyzed in a dentin barrier test. Cell responses were examined in 3T3 mouse fibroblasts after 24- and 72-h exposure periods by the estimation of total cell numbers (survival), apoptosis (viability) and cell proliferation. Cell numbers were effectively reduced by the primers of AdheSE, Protect Bond, and Scotchbond Multi-Purpose as well as XP bond after direct exposure in a cell culture insert test device. Likewise, Scotchbond Multi-Purpose primer induced a rate of apoptosis (93.9%) even higher than detected with Protect Bond primer (91.6%). Cell proliferation was entirely inhibited by primers and by Xp Bond as well. The Scotchbond Multi-Purpose was most cytotoxic in a dentin barrier test device after a 24-h indirect exposure. It also increased the percentage of cells in apoptosis to 15.4% compared to untreated controls. Unpolymerized primers of dentin adhesives were more cytotoxic than polymerized bonding counterparts. Moreover, total etch dentin adhesives were more cytotoxic than self-etch adhesives. When dentin adhesives are used in deep cavities without a protective dentin barrier the leachable hydrophobic and hydrophilic component of dentin adhesive systems can penetrate to the pulp and may induce cytotoxic responses in pulp tissues.

Design-based Stereology

In regulatory toxicology studies, qualitative histopathological evaluation is the reference standard for assessment of test article-related morphological changes. In certain cases, quantitative analysis may be required to detect more subtle morphological changes, such as small changes in cell number. When the detection of subtle test article-related morphological changes is critical to the decision-making process, sensitive quantitative methods are needed. Design-based stereology provides the tools for obtaining accurate, precise quantitative structural data from tissue sections. These tools have the sensitivity necessary to detect small changes by combining statistical sampling principles with geometric analysis of the tissue microstructure. It differs from other morphometric methods based on tissue section analysis by providing estimates that are statistically valid, truly three-dimensional, and referent to the entire organ. Further, because the precision of the stereological analysis procedure can be predicted, studies can be designed and powered to detect subtle, potentially toxicologically significant changes. Although stereological methods have not been widely applied in toxicologic pathology, recent advances have made it feasible to implement these methods in a regulatory toxicology setting, particularly methods for estimation of total cell number.

Stereological estimation of total cell numbers in the young human utricular macula

Conclusion: There is no change in the total cell population and hair cell:supporting cell ratio in the human utricular macula from gestational week 16 and onwards, whereas the lower hair cell:supporting cell ratio and lower total number of cells in the youngest specimens indicate that the utricle is still differentiating and adding new cells at the 10th to 12th gestational week. Objectives: Archival temporal bones were investigated to quantify cell numbers in the utricular macula in fetuses and children. Methods: The age of the subjects ranged from gestational week 10 to 15 years. The optical fractionator was used to estimate the total number of cells in the utricular macula. Results: The total cell number was found to be 143 000 in subjects older than gestational week 16. The number of hair cells and supporting cells did not change between the 16th gestational week and 15 years and was 36 000 and 107 000, respectively. In the youngest specimen (10th and 12th gestational week) there was a statistically significant lower total number of cells (62 000) and a lower hair cell:supporting cell ratio, resulting in a mean number of 13 000 hair cells and 49 000 supporting cells.

158APOPTOSIS IN IN VITRO PRODUCED BOVINE EMBRYOS ACCORDING TO DEVELOPMENTAL KINETICS

Apoptosis has been previously reported in embryos during late pre-implantation development. Fast-developing embryos are known to present higher developmental competence. The aim of the present work was to evaluate the quality of in vitro-produced bovine embryos with fast (8-cells at 48 hours post-insemination (hpi) and slow (8-cells at 90hpi) cleavage and study the correlation of this phenotype with programmed cell death occurrences. Embryos were produced from immature oocytes obtained from slaughtered cow ovaries, after maturation and fertilization, presumed zygotes were cultured in CR2 medium with 10% FCS, together with granulosa cells under 5% CO2 atmosphere. The number of nuclei in the inner cell mass and trophectoderm (ICM/TE), as well as the number of nuclei with fragmented DNA, were estimated by applying differential staining and TUNEL, respectively; data were analyzed by ANOVA (JMP—SAS Institute). To test the expression of apoptosis regulating genes, a pool of fifty 8-cell embryos from each group (fast and slow) were collected. After RNA extraction and reverse transcriptase reaction, cDNA was amplified with Bax and Bcl2 primers, individually. Results indicated, as expected, higher quality in fast-cleaving embryos, estimated by the number of ICM nuclei (20.8±1.4 and 15.6±2.1—P≤0.05); however, the number of TE didn’t show significant differences (54.9±2.4 and 53.2±3.8); the same was observed for total cell number (75.7±2.8 and 68.8±4.4). The frequency of blastocyst TUNEL-positive nuclei as an estimate of total cell number was significantly larger in the slow group when compared to the rapid development group (19.0±2.5% and 8.5±1.4%, respectively, P≤0.05). The greater proportion of morphologic abnormal nuclei in both groups was located in the ICM, and may explain the lower number of ICM nuclei in slow developing embryos. Hence, embryos of slow development show TUNEL-positive blastomeres at the 8-cell stage, but no fragmented nuclei were observed in embryos at 48hpi. Bax and Bcl2 cDNA amplification showed that both mRNAs were constitutively present at the 8-cell stage in both groups. It can be concluded that in vitro-produced bovine blastocysts, with slow development to the 8-cell stage, present lower quality compared with fast development homologues, estimated by mean number of ICM nuclei, as well as nuclei fragmentation in blastomeres (TUNEL-positive). There is a difference in fragmented nuclei proportion between both groups at the 8-cell stage, but this result may be biased by the numbers of hours in culture. It was possible to demonstrate the presence of mRNA for pro (Bax) and anti-apoptotic (Bcl2) genes in slow- and fast-developing embryos at the 8-cell stage, and the future determination of the ratio between these two transcripts may allow the evaluation of the participation of pre-transcriptional regulation of these genes on the induction of DNA fragmentation. Financial support: Grant 99/12351-3 FAPESP São Paulo, Brazil.

Estimation of absolute microglial cell numbers in mouse fascia dentata using unbiased and efficient stereological cell counting principles

Stereology offers a set of unbiased principles to obtain precise estimates of total cell numbers in a defined region. In terms of microglia, which in the traumatized and diseased CNS is an extremely dynamic cell population, the strength of stereology is that the resultant estimate is unaffected by shrinkage or expansion of the tissue. The optical fractionator technique is very efficient but requires relatively thick sections (e.g., > or =20 microm after coverslipping) and the unequivocal identification of labeled cells throughout the section thickness. We have adapted our protocol for Mac-1 immunohistochemical visualization of microglial cells in thick (70 microm) vibratome sections for stereological counting within the murine hippocampus, and we have compared the staining results with other selective microglial markers: the histochemical demonstration of nucleotide diphosphatase (NDPase) activity and the tomato lectin histochemistry. The protocol gives sections of high quality with a final mean section thickness of >20 microm (h=22.3 microm +/- 0.64 microm), and with excellent rendition of Mac-1+ microglia through the entire height of the section. The NDPase staining gives an excellent visualization of microglia, although with this thickness, the intensity of the staining is too high to distinguish single cells. Lectin histochemistry does not visualize microglia throughout the section and, accordingly, is not suited for the optical fractionator. The mean total number of Mac-1+ microglial cells in the unilateral dentate gyrus of the normal young adult male C57BL/6 mouse was estimated to be 12,300 (coefficient of variation (CV)=0.13) with a mean coefficient of error (CE) of 0.06. The perspective of estimating microglial cell numbers using stereology is to establish a solid basis for studying the dynamics of the microglial cell population in the developing and in the injured, diseased and normal adult CNS.

Stereological quantitation in cerebella from people with schizophrenia.

Behavioural and anatomical studies in schizophrenia have pointed to cerebellar involvement. To provide stereological estimations of volumes and cell number in the cerebella of people with schizophrenia and a control group using post-mortem material. Stereological methods were applied to cerebella taken from eight male patients with a DSM-III diagnosis of chronic schizophrenia with no neurological disorder (mean age 57.5 years) and ten male controls (mean age 56.2 years). The Cavalieri principle was used to provide estimates of volumes, the optical disector method to obtain estimates of the numerical density of Purkinje and granule cells, and a combination of the two to obtain estimates of total cell numbers in the cerebellum. The rotator method was applied to obtain estimates of mean Purkinje cell volume. No global structural difference in major volumes, cell numbers of Purkinje cell volume was found between the groups. The most frequently reported pathological finding in the cerebellum in schizophrenia is vermal atrophy, which was not found in this small group of heavily affected patients.

Model of retinal surface area and neuron distribution in the avian eye

Changes in retinal neuron distribution may reflect normal or pathological changes in retinal function. The quantitative study of retinal neurons provides a better understanding of anomalous mechanisms, such as those controlling ocular growth and refractive error development in experimental animals. We developed a method to facilitate the quantitative analysis of amacrine neuron populations in wholemount chick retinae, since the domestic chicken is used extensively as an animal model in myopia studies. This method involved automated cell counting from confocal microscopic images and mathematical estimation of total cell numbers based on image cell density and retinal surface areas. Cell densities and cell counts were obtained from immunohistochemically-labeled amacrine neuron populations, using derived formulae to calculate retinal surface area based on vitreous chamber depth, equatorial width, ora serrata diameter and scleral thickness. Normalized total cell counts in each eye were compared, rather than cell densities, since changes in eye growth can affect cell densities. We also compared neuron distribution in central versus peripheral portions of the retina. This is an alternative technique for retinal analysis that supplements traditional anatomical cell counting methods, allowing higher numbers of specimens to be rapidly analyzed.

Cryopreservation and storage of embryonic rat mesencephalic dopamine neurons for one year: comparison to fresh tissue in culture and neural grafts

Blocks of embryonic rat ventral mesencephalic tissue containing the developing A8–A10 dopamine (DA) cell groups were cryopreserved and stored for approximately 1 year, at which time this tissue was thawed, dissociated into a cell suspension, and compared to a similar preparation of fresh mesencephalic tissue for viability in tissue culture and neural grafts. Estimates of total cell number immediately prior to plating in culture indicated that cryopreserved tissue yields fewer cells, but when this reduced cell number is compensated for, and equal numbers of cells were plated in culture, approximately equal total numbers of neurons, as well as tyrosine hydroxylase (TH)-positive neurons, were present in cultures from cryopreserved and fresh tissue. Grafting of equal numbers of fresh and cryopreserved mesencephalic cells into the striatum of adult rats with large unilateral lesions of the nigrostriatal DA pathway tended to yield smaller grafts with fewer surviving TH-positive cells with less extensive neuronal processes when tissue was previously cryopreserved. However, grafts derived from freeze-stored tissue provided a similar timecourse and extent of behavioral recovery in amphetamine-induced rotational tests to that provided by fresh tissue grafts. Taken together, our findings indicate that while cryopreservation of mesencephalic tissue has its costs — reduced cell yield in cultures and grafts, and compromised morphology in grafts — sufficient numbers of cryopreserved neurons survive the grafting procedure to ameliorate behavioral signs of DA depletion in the lesioned rat model.

Total numbers of various cell types in rat cerebellar cortex estimated using an unbiased stereological method

A new and efficient stereological method for estimating the total number of the different cell types in rat cerebellar cortex is presented. The cells have been subdivided into the following categories: Purkinje cells, granule cells, Golgi cells, glial cells in the granular layer, Bergmann glial cells and neurons and glial cells in the molecular layer. The method has been used to estimate the total number of some of the cell types in rats exposed to orally administered toluene at doses of 200, 400 and 800 mg/kg/day for 12 weeks compared with a control group. No statistically significant differences were found between the exposed and non-exposed animals. The described method can be used in a number of both biological and experimental studies. With the use of new stereological methods it is possible to get precise estimates of total cell numbers in a much shorter time than earlier required. This makes it possible to improve the reliability of the final result by increasing the number of cases processed.

Stereological quantitation of human brains from normal and schizophrenic individuals

The complex organization and high density of nerve cells in the human brain presents a challenge to the estimation of total cell numbers. The first unbiased counting method, the disector, was described in 1984 and has since made it possible to accurately count total neuron numbers in any region that can be defined, while excluding artifacts of earlier counting methods. The disector method has been applied to normal neocortex and to four subcortical brain regions from schizophrenics and controls. The total neuron number in neocortex in 26 normal individuals was estimated to be 25.10(9). A 40 and 50% reduction of total nerve cell number was found in the mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics, respectively. Furthermore, the Cavalieri principle has been used to estimate the volume of human cortex, white matter, central grey regions and the volume of the ventricular system in both controls and schizophrenics. As shown by these studies, the introduction of unbiased stereological methods and subsequent modifications in recent years have made it possible to estimate a number of parameters in the human brain without the biases included in most classical works. By comparison, earlier conventional counting methods have been relatively time consuming, mostly biased to a smaller or larger degree, and generally less precise. The application of these new neurostereological methods will undoubtedly provide greater confidence for future brain studies.

Effects of undernutrition during early life on granule cell numbers in the rat dentate gyrus

Undernutrition during early life is known to affect the morphology of the hippocampal formation. Recent advances in stereological techniques have made it possible to make relatively unbiased estimates of total cell numbers in well-defined brain regions. It was decided to use these methods to determine the effects of different levels of undernutrition during early postnatal life on the granule cells of the rat dentate gyrus. Male hooded Long Evans rats were undernourished between the 16th day of gestation and 30 postnatal days of age to two different levels. The daily food intake of level-1 and level-2 rats represented about 60 and 40%, respectively, of that eaten by well-fed, age-matched controls. Nutritional rehabilitation of the rats was commenced when they had reached 30 days of age by placing them on an ad libitum diet. Groups of control and experimental rats were killed at 70 and 212 days of age. The Cavalieri principle was used to determine the granule cell layer volume within the dentate gyrus, and the "dissector" method was used to determine numerical densities of these granule cells. These estimates were used to calculate the total numbers of granule cells. There were between 260,000 and 320,000 granule cells within the dentate gyrus of 70-day-old control and experimental rats. By 212 days of age, well-fed controls had an average of about 834,000 granule cells. The level-1 and level-2 previously undernourished rats had about 515,000 and 595,000 granule cells, respectively. Two-way analysis of variance procedures showed significant main effects of nutrition and age as well as a significant interaction between them.(ABSTRACT TRUNCATED AT 250 WORDS)

The cerebral cortex in congenital hydrocephalus in the H-Tx rat: a quantitative light microscopy study

Hydrocephalus in the H-Tx rat first develops in late gestation and causes death at 4-7 weeks. The effect of hydrocephalus on overall cortical dimensions and on five specific regions (frontal, sensory-motor, parietal, auditory and visual) has been studied by quantitative light microscopy at 10 and 30 days after birth. The lateral ventricle volumes in hydrocephalic rats were about 40 x larger than controls and increased fourfold between 10 and 30 days. Cortical volume was reduced by a small amount at 10 days but was larger in hydrocephalics at 30 days. Thinning of the cortical mantle was severe with disruption of the laminar structure, particularly in the auditory and visual regions, where it was already present at 10 days. The density of cortical cells (neurones and glia) was not altered in hydrocephalics at 10 days but was reduced in all regions at 30 days. Estimates of total cell number suggest that the lower density was not associated with an overall loss of cells. Capillary numerical density was not affected by the hydrocephalus at 10 days after birth but by 30 days it was significantly lower, particularly in the worst-affected posterior regions. The results show that the cerebral cortex is severely distorted and that in advanced hydrocephalus, although overall cell number is not affected, both cell density and capillary density are lower by up to 30%.