Expressão imuno-histoquimica de proteínas relacionadas com a reabsorção óssea em carcinoma epidermoide de palato duro e língua

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Expressão imuno-histoquímica de metaloproteína em carcinoma epidermóide de lábio inferior e língua

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Expressão imuno-histoquímica da e-caderina e da ß-catenina em carcinoma epidermóide oral com e sem metástase nodal

The adhesion molecules E-cadherin and β-catenin have been studied as possible markers to distinguish carcinomas with and without metastatic potential. The objective of this research was to study the imunohistochemistry expression of the E-cadherin and β-catenin in oral squamous cell carcinoma (OSCC), aiming to contribute for the better understanding of the biological behavior of this lesion. The sample consisted of 30 cases of OSCC, being 15 of tongue and 15 of lower lip. The profile and intensity of labeling and semi quantitative analysis of the percentage of immunopositive tumoral cells in membrane for E-cadherin and β-catenin had been related with the anatomical localization of the lesion, the presence or not of nodal metastasis and the histological grade of malignancy in the invasive front area of the tumor. It was registered the presence or not of cytoplasmic and nuclear labeling of the β-catenin. The results had been submitted to the statistical analysis, being used the Mann-Whitney Test, the Fisher Test and the Spearman Correlation Coefficient (α=0, 05). The results showed that the expression in membrane for E-cadherin and β-catenin was, predominantly, the heterogeneous profile in the lower lip and tongue carcinomas, as well as in the cases with and without nodal metastasis. It was not observed significant statistical difference between expression profile and amount of immunopositive cells for E-cadherin, β-catenin and the anatomical localization of the lesion and for the presence or not of nodal metastasis. However, there was significant difference of the reduced expression of these proteins with the high score of malignancy. It was verified that the expression of the β-catenin in cytoplasm was present in 22 (73.33%) of the 30 analyzed cases, and 6 cases (20%) showed nuclear expression. The statistical analysis detected significant association between the expression of the β-catenin in the cytoplasm with the histological grade of malignancy, being this molecule more frequently present in the cytoplasm in the cases of high score of malignancy. It was concluded that the reduced immunoexpression of these proteins in membrane can be related with the lowest cellular differentiation, as well as with the pattern of invasion in nests and isolated cells, demonstrated in the cases of OSCC with high histological grade of malignancy

Expressão imuno-histoquímica das integrinas α2ß1, α3ß, e α5ß1, em carcinoma epidermóide de lábio inferior e língua

The unpredictable biologic behavior of the oral squamous cells carcinoma has determined extensive research on the evolution of such tumor. Due to the existing relation between the outer cell matrix and the tumor cells, the integrins have been used as markers in the predictive study of the cell behavior. This study aims to analyze immunohistochemically the expression of the integrin α2β1, α3β1, and α5β1 connections for the collagen, the laminin and the fibronectin respectively in 15 cases of squamous cells carcinoma from the lower lip and 15 from the tongue, with different scores of malignance grading. A predominantly diffuse, cytoplasm and granular immunological marking was observed in the majority of the analyzed cases. According to the marking intensity, integrin α2β1 appeared positive in 80% of the lip and in 93,3% of the tongue cases. The immunological reactivity of integrin α3β1 was classified as positive in 60% of both the tongue and lip cases. For this integrin, 20% and 33.3% of the tongue and lip cases, respectively, were negative. In relation to integrin α5β1 the intensity was classified as positive in 53,3% of the cases and strongly positive in 46,7% of those located in the lip. In the tongue carcinomas, the intensity was positive in 46,7% of the cases and strongly positive in 53,3%. The statistic analysis did not show any significant differences or correlation of expression between these integrins nor between the anatomical sites or between different scores of malignancy grading. The expressive immunological marking of the integrins, α2β1, α3β1, and α5β1 in the studied cases of squamous cell carcinomas leads us to think of a great participation of these proteins in oral carcinogenesis; however, our results do not allow us to correlate its expression as an indicator of variations in the biological behavior of this neoplasia

Estudo in vitro dos efeitos da BMP-2 e do seu antagonista Noggin sobre a proliferação e migração celulares em carcinoma epidermóide de língua

Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy in the oral cavity and reach a large number of individuals, has become an important public health problem. Studies have demonstrated changes in pathway components BMP in various types of cancers as prostate, colon, breast, gastric and OSCCs. Is the current knowledge that these proteins may exert pro-tumor effect in more advanced stages of neoplastic development coming to favor progression and invasion tumor. The inhibition of the signaling pathway BMP-2 through its antagonists, have shown positive results of antitumor activity and use of Noggin may be a novel therapeutic target for cancer. Given this evidence and the few studies with BMP-2, Noggin and OSCC, the objective of this research was to evaluate the effect of BMP-2 and its antagonist Noggin on proliferation and migration cell in line of cell cultures of human tongue squamous cell carcinoma (SCC25). The study was divided in three groups, a control group, where SCC25 cells suffered no treatment, a BMP-2 group, in which cells were treated with 100ng/ml of BMP-2 and a group of cells that were treated with 100ng/ml of Noggin. For the proliferation assay and cell cycle were established three time intervals (24, 48 and 72 hours). Proliferative activity was investigated by trypan blue and cell cycle analysis by staining with propidium iodide flow cytometry. The potential for migration / invasion of SCC25 cells was performing by a cell invasion assay using Matrigel in a 48-hour interval. The proliferation curve showed a higher proliferation in cells treated with BMP-2 in 72 hours (p < 0.05), and lower overgrowth and cell viability in Noggin group. Recombinant proteins favored a greater percentage of cells in cell cycle phase Go/G1 with a statistically significant difference in the interval of 24 hours (p < 0.05). BMP- 2 produced a greater invasion of cells studied as well as its antagonist Noggin inhibits invasion of cells (p < 0.05). Thus, these results indicate that BMP-2 promotes malignant phenotype, dues stimulates proliferation and invasion of SCC25 cells and, its antagonist Noggin may be an alternative treatment, due to inhibit the tumor progression

Expressão imuno-histoquímica das integrinas a2ß1, a3ß1 e a5ß1 em adenoma pleomórfico de glândula salivar menor e maior e carcinoma adenóide cístico

Pleomorphic adenoma and adenoid cystic carcinoma (ACC) consist benign and malignant neoplasm from salivary gland, respectively. These neoplasms share some characteristics, such as cellular origin and considerable production of extracellular matrix, however, with distinct biological behavior. The aim of the present study was to compare the expression of D2E1, D3E1 e D5E1 integrins in pleomorphic adenoma from minor and major salivary glands and ACCs. Furthermore, it was investigated possible differences in the expression of these integrins according to histological subtypes of ACC. Fourteen cases of pleomorphic adenoma from major salivary gland, fourteen cases from minor salivary gland and ten cases of ACC were selected. It was taken into consideration the presence or absence, localization and intensity of integrin immunoexpression. The cases of pleomorphic adenoma were grouped in order to compare the expression between the distinct neoplasms. It was observed a highly significant difference (p<0,0001) in relation to D2E1 integrin between the neoplasms since pleomorphic adenoma showed a pronounced immunostaining. It was not possible to perform statistical tests considering the D2E1 integrin expression; nevertheless, it could be observed a tendency of higher staining in pleomorphic adenoma. For comparative reasons the cases ACCs were divided in two groups: solid and tubular/cribriform. It was not detected significant differences in regard to D2E1 integrin; and statistical analysis could not be realized in relation to D3E1 and D5E integrin expression. However, it was also verified a tendency of absence or reduced expression in the solid subtype. It can be concluded that the reduced D2E1 integrin expression observed in CACs may be related to a lesser degree of cell differentiation in this neoplasm and the reduced D5E1 integrin expression can be associated with aggressive biological behavior. Moreover, the absence and/or reduced expression of the studied integrins in solid ACC suggests a role in pathogenesis and more aggressive biological behavior of this histological subtype

Caracterização comportamental endócrina das fases ontogenéticas de sagüi comum (Callithrix jacchus)

The use of animal models in biomedical research is ever increasing. Models that use primates might also have advantages in terms of low maintenance costs and availability of biological knowledge, thereby favoring their use in different experimental protocols. Many current stress studies use animal models at different developmental stages since biological response differs during ontogeny. The aims of this study were to perform a detailed characterization of the developmental stages of common marmosets (Callithrix jacchus), a very important animal model used in biomedical research. Ten subjects, 6 females and 4 males, were followed from birth to initial adult age (16 months). Behavioral and fecal collection for measurement of adrenal (cortisol) and sex (progesterone, estradiol and androgens) hormones took place twice a week during the first month of life and once a week for the remainder of the study. Behavior was observed for 30 minutes in the morning (0700-09:00h) and afternoon (12:00-14:00h). Behavioral profile showed changes during ontogeny, characterizing the 4 developmental stages and the respective phases proposed by Leão et al (2009).. Differentiation of developmental stages was considered using the onset, end, change and stabilization of the behavioral profile parental care (weaning and carrying), ingestion (solid food), affiliation (social grooming) and autogrooming, agonism (scent marking and piloerection) and play behavior and endocrine profile. Infant weaning and carrying terminated within the infantile stage and the peak of solid food ingestion was recorded in the infantile III phase. Receiving grooming was recorded earlier than grooming performed by the infant and autogrooming. The first episode of scent marking was recorded in the 4th week and it was the least variable behavior, in terms of its onset, which, in almost all animals, was between the 5th and 7th week of life. Solitary play and play with the twin started around the 7th week and play with other members of the group started 8 weeks later. Sex hormone secretion started to differ from basal levels between the 21st and 23rd week of life, in males and females, suggesting that puberty occurs simultaneously in both sexes. Basal cortisol, even at an early age, was higher in females than in males. However, cortisol was not correlated with the juvenile stage, as expected, since this stage corresponds to the transition between infancy and adult age and most behaviors are intensified by this time. The behavioral and endocrine profile of subadult animals did not differ from that of the adults. These results provide more detailed parameters for the developmental process of C. jacchus and open new perspectives for the use of experimental approaches focused on the intermediate ontogenetic phases of this species

Aspectos anatômicos do olho e neuroquímicos da retina do mocó (Kerodon rupestris)

The visual system is an important link between the animal and the environment, com profound influences on the habits and lifestyle in various habitats. Adaptive mechanismsto the temporal niche are present in the visual system of many vertebrates, involving changins in ocular dimensios and design, retinal cell distribution and organization of neurochemical circuits related to the retinal resolution or sensitivity. The sensory system of the eye is represented by the retina, whose organization is responsible by receipty, initial analysis, and transmission of the information to the brain. The knowledge of the position of the eyes in the head and the distribution of retinal cells allow to identify adaptive aspects of each species to its visual field, which is characteristic to the ecological niche it occupies. In this research, we study eye anatomical characteristics and retina neurochemical features of the rock cavy (Kerodon rupestris), a tipical Brazilian rodent from the suborder Hystricomorpha, family Caviidae. The rock cavy has lateral eyes well constitute bony orbit and well differentiated extrinsic muscle. The study of the descriptive and morphometric anatomy of the showed mean values of axial diameter 10.7±0,5mm and equatorial diameter 11.6±0.7mm. The pupil is slit shaped and the lens has mean axial diameter 5.4±0.03 mm, corresponding to ~45% of the axial diameter of the eye. The posterior nodal distance and the retinal magnification factor were estimated at 6.74 mm e 118 μm/grau, respectively. Flat mounts were processed for Nissl stain, and the topographic distribution of ganglion cells showed a moderate visual band, just below the optic disc, with higher density in the ventral retina. Retinal vertical sections and flat mounts were processed for immunohistochemistry to visualize tyrosine hydroxilase (TH) and thus two types of TH+ cells were detected. Type 1 cells had strong TH-immunoreactivity, the body cell varied from 120.047 to 269.373 μm2 stratifying in the sublamina 1 of the IPL. Type 2 cells were weakly TH-imunoreactive, had cell body located mostly in the IPL, varying from 54.848 to 177.142 μm2, constituting ~10% of the TH+ cells. Both cell types exhibited similar topographic distribution with higher density found in a horizontal band along of the naso-temporal axis in the dorsal retina. The total population of dopaminergic cells was 2,156±469,4 cells, occupying an average area of 198,164 μm2. The presence of cones and rods was detected by immunohistochemistry in vertical sections and flat mounts. S cones density is around 10 times smaller than L cones, with different degree of spatial organization. Other retinal neuronal populations of the rock cavy were also detected in vertical sections with specific markers. Comparative analysis of the anatomical characteristics of the rock cavy eye 12 suggest that it was designed to acquire higher sensitivity to light, at expense of image sharpness, compatible with a vision at mesopic conditions. Additionally, the distribution of the 2 subtypes of dopaminergic cells in a naso-temporal band in the dorsal retina seems suitable to a gain in sensitivity, coherent with an animal with predominantly crepuscular activity pattern

Padrão de atividades e de interações sociais de animais juvenis em dois grupos selvagens de Callithrix jacchus

The juvenile period represents the developmental phase between weaning and sexual maturity. Weaning occurs when the youngster does not receive direct care from the caretakers anymore. Individuals in the species Callithrix jacchus live in groups composed by the reproductive pair and successive twin sets. Cooperative care is the rule. Infants are weaned early, and from then on, food is provided by the adults in the group. These animals present high levels of social interactions, through play, grooming and social contact. During infant age, the twin becomes the main partner. There are few studies about the juvenile period, especially on Callithrix gender. The objective of this study was describing the pattern of activities and social interactions of four sets (one single and three twin sets) during juvenile phase in two Callithrix jacchus groups. We used instantaneous and continuous focal sampling for juveniles and scan sampling for adults behavioral recordings. Juveniles presented the same behavioral pattern as the adults relating the activity budget, in particular, foraging along the months. The composition of the diet was the same as that of the adults. Food transfer ended along the juvenile period. Social play as much as grooming were important socializing activities for the juveniles. The young individuals in the group were the main partners in social play, specially the twin. Adults were the main partners in grooming interactions. Scent marking differed between twins in the male/female sets, the female presenting the highest levels of marking. The juveniles were independent from adults in foraging activity. Social interaction varied according to group composition, but in general, interacted more with the twin and with the youngsters (infants and subadults), except in grooming. Even presenting many similarities, juveniles showed some differences between genders, which indicates the differentiation in behavior towards reproductive strategies early in the juvenile period

Caracaterização do núcleo pré-geniculado do sagüi (Callithrix jacchus) :projeção retiniana, neuroquímica e atividade celular (expressão de FOS)

In rodents, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are the main components of the circadian system. The SCN is considerate the site of an endogenous biological clock because can to generate rhythm and to synchronize to the environmental cues (zeitgebers) and IGL has been related as one of the main areas that modulate the action of SCN. Both receive projections of ganglion cells of retina and this projection to SCN is called retinohypothalamic tract (RHT). Moreover, the IGL is connected with SCN through of geniculohypothalamic tract (GHT). In primates (include humans) was not still demonstrated the presence of a homologous structure to the IGL. It is believed that the pregeniculate nucleus (PGN) can be the answer, but nothing it was still proven. Trying to answer that question, the objective of our study is to do a comparative analysis among PGN and IGL through of techniques immunohystochemicals, neural tracers and FOS expression after dark pulses. For this, we used as experimental model a primate of the new world, the common marmoset (Callithrix jacchus). Ours results may contribute to the elucidation of this lacuna in the circadian system once that the IGL is responsible for the transmission of nonphotic information to SCN and participate in the integration between photic and nonphotic stimulus to adjust the function of the SCN. In this way to find a same structure in primates represent an important achieve in the understanding of the biological rhythms in those animals

Caracterização citoarquitetônica, neuroquímica e de aferência óptica do complexo parabraquial do sagui (Callithrix jacchus)

The parabrachial complex (PB) is an area of the brainstem responsible for the processing and transmission of essential physiologic information for the survival of the organisms. This region is subdivided in approximately nine subregions, considering morphology, cytoarchitectural and functional characteristic. Its neurons have an extensive network of connections with other regions of the nervous system. The objective in this work was to map the retinal projection to the PB and make a citoarchitectonic and neurochemical characterization of this region in the common marmoset (Callithrix jacchus), a primate of the New World. The retinal projections were mapped by anterograde transport of the choleric toxin subunit b (CTb). The citoarchitecture was described through the Nissl method, and the neurochemical characterization was made through immunohistochemical technique to the some neurotransmitters and neuroactives substances present in this neural center. In marmoset PB, in the coronal sections labeled by Nissl method, we found a similar pattern to that evidenced in other animal species. The immunoreactivity against CTb was verified in the PBMv in fibers/terminal, characterizing such as retinal innervations in this area. The immunohistochemical technique reveled that the PB contain cells, fibers and/or terminals immunoreactives to the neuronal nuclear protein, Choline acetyl transferase, nitric oxide synthase, serotonin, enkephalin, substance P, Calcium-binding proteins (calbindin, calretinin e parvalbumin), and glial fibrillary acidic protein. The histochemical technique reveled cells and fibers NADPH-diaphorase reactive. Each one of those substances presented a characteristic pattern of demarcation in PB, and some serve as specific markers of subregions

Análise imunoistoquímica da distribuição de Serotonina, transportador de serotonina e receptores de serotonina no hipotálamo do sagui (callithrix jacchus)

The hypothalamus is a diencephalic portion located around the third ventricle below the hypothalamic sulcus, limited by the optic chiasm, and by the mammillary bodies, acting as a center that integrates behavioral and homeostatic functions. Serotonin is a neurotransmitter produced in limited sites in the midbrain and brain stem, but is distributed throughout the central nervous system and has many functions, acting through specific receptors that are also distributed throughout the nervous system. Using immunohistochemical techniques, the aim of this study was to delineate the hypothalamic nuclei of the marmoset (Callithrix jacchus) and study the distribution of serotonin transporter and serotonin receptors in the hypothalamus of this species. We used the Nissl method to determine the cytoarchitecture of the hypothalamic nuclei, and immunohistochemistry to reveal the presence of NeuN as a method to determine the contours of the hypothalamic nuclei. As a result, we found serotonin containing fibers and terminals throughout the rostrocaudal extent of the hypothalamus, more concentrated in some nuclei, and even absent in some. Like serotonin, serotonin transporter was observed between pre-optic area and tuberal region of the hypothalamus, in densities and distribution similar to serotonin. The 5-HT1A and 5-HT1B receptors were found with minor differences among itselves regarding the disposition and intensity of staining.

Efeitos do substrato e da densidade populacional sobre as atividades comportamentais e níveis de homócitos em relação à densidade em Litopenaeus vannamei (BOONE, 1931)

Carciniculture in Brazil occupies world-wide prominence due to shrimp culture, and the state of Rio Grande do Norte has presented the best results in the culture of the Litopenaeus vannamei in the last decade. This species has been shown to adapt easily to different environments and is between the five most cultivated penaeids of the world. The ponds are usually constructed in areas close to water courses and estuaries. Stock density and substrate ponds can pollute environment, causing losses in the growth and survival of the shrimps, being considered stress factors. Shrimps in inadequate densities and substrates can result reduced productivity of the farm; and favor diseases. So, it is important to verify how these variables influence the development of the animals in the culture farms. Our objective was to study the influence of the type of substrate and the stock density on the behavior and haemocyte count of the L. vannamei. Individually marked juvenile shrimps were kept in aquaria with 30 L of seawater and continuous aeration, in 12L-12D photoperiod. They were observed through Ad libitum and focal sampling instantaneous methods during thirty days, five times per week, six times per day (8:00 to 18:00) in windows of 15 minutes every two hours. The marking of carapace permitted quantifying molting and the feeding was supplied three times a day. Two experiments were carried out: the first one tested animals in the three different substrates (fine sand, smaller rocks-SPP and biggest rocks-SGR) with 33 shrimp/m2. In the second one, the animals were tested in three stock densities (26, 52 and 66 shrimp/m2) in fine sand substrate. At the end of experiment, biometry (first and second ones) and haemocyte count (second one) were made. The behavior of the L. vannamei seems to have been influenced by substrate and stocking density. In low granulometry of the substrate; the exploratory behavior became more frequent and inactivity of the shrimps was reduced. Burrowing was registered in sand substrate, specially in the initial period of the day. Cleaning was gradually higher along the day, presenting the biggest levels as the dark phase approached. The ingestion of feeding was more frequent in low density, and the animals were bigger and heavier at the end of the experiment. In the fine sand condition, the animals presented better growth, probably associated with the burrowing. The molting was equivalent in all types of substrate, but it was more frequent in high densities. Mortality of the shrimps was more frequent in high densities, and cannibalism and diseases were also registered in that condition. The clinical signals were similar to the ones of infectious mionecrosis (IMNV), generally associated with environment and physical stress. The haemocyte count was low for the hematologic standards of the penaeid, which we attributed for greater dilution of haemolymph in the postmolting phase. Smaller shrimps presented lower levels of haemocytes in relation to the bigger animals, count was also low in 26 shrimp/m2 density. The study demonstrates that stocking density and the granulometry of the substrate can affect the welfare, the health and the behavior of the L. vannamei. The sand substrate and low stocking density can be important tools in the management systems of shrimp production

Influência da laserterapia na proliferação de células-tronco do ligamento periodontal humano

Low level laser irradiation (LLLI) has been used in Dentistry to promote wound healing and tissue regeneration. The literature shows a positive effect of LLLI on cell proliferation, but little is known about their effectiveness in promoting stem cells proliferation. The aim of this study was to evaluate the effect of LLLI on the proliferative rate of human periodontal ligament stem cells. Extracts of periodontal ligament were isolated from two third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were grown in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were irradiated with a InGaAlP-diode laser, using two different energy densities (0,5J/cm 2 - 16 seconds and 1,0J/cm² - 33 seconds), with wavelength of 660nm and output power of 30mW. A new irradiation, using the same parameters, was performed 48h after the first. A control group (non irradiated) was kept under the same experimental culture conditions. The Trypan blue exclusion test and the mitochondrial activity of the cells measured by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] essay were performed to assess the cell proliferation in the intervals of 0, 24, 48 e 72 h after irradiation. The data of cell counts were submitted to nonparametrical statistical tests (Kruskal-Wallis and Mann-Whitney), considering a confidence interval of 95%. DAPI (4 -6-Diamidino-2-phenylindole) staining of the cells was performed at 72h interval to evaluate possible nuclear morphological changes induced by LLLI. The results of this study show that the energy density of 1,0 J/cm² promoted greater cell proliferation compared to the other groups (control and 0,5 J/cm²) at intervals of 48 and 72h. The mitochondrial activity measured by MTT essay showed similar results to the Trypan blue cell counting test. The group irradiated with 1,0J/cm² exhibited a significantly higher MTT activity in the intervals of 48 and 72h, when compared to the group irradiated with 0,5J/cm². No nuclear morphological change was observed in the cells from the three groups studied. It is concluded that LLLI has stimulatory effects on the proliferation of human periodontal ligament stem cells. Therefore, the use of laser irradiation in this cell type may be important to promote future advances in periodontal regeneration

Atividade biológica de células-tronco da polpa de dentes decíduos humanos submetidas à criopreservação

Dental pulp stem cells have been widely investigated because of their ability to differentiate into both dental and non-dental cells, with potential use in therapies involving tissue engineering. The technique of cell cryopreservation represents a viable alternative for the conservation of these cells, since it stops reversibly, in a controlled manner, all of cell biological functions in an ultra low temperature. The present study aimed to evaluate, using in vitro experiments, the influence of a cryopreservation protocol on the biologic acti vity of stem cells from human exfoliated deciduous teeth (SHED). Cells obtained from the pulp of three deciduous teeth on end-stage exfoliation or with indicated extraction were expanded in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. At second subculture (P2), a group of cells were submitted to cryopreservation for 30 days in 10% DMSO diluted in fetal bovine serum, at -80º C, while the remind cells continued under normal conditions of cell culture. Cell proliferation was evaluated in both groups (not cryopreserved or cryopreserved) by Trypan blue stain essay at intervals of 24, 48 and 72h after plating. Cell cycle analysis of SHEDs submitted or not to the cryopreservation protocol was performed in the same intervals. Events related to cell death were studied by Annexyn V and PI expression under flow cytometry at the intervals of 24 and 72h. The presence of nuclear morphological changes was evaluated by DAPI staining at 72h interval. It was observed that both groups exhibited an upward cell proliferation curve, without considerable changes in cell viability throughout the experiment. The distribution of cell in the cell cycle phasis was consistent with cell proliferation in both groups. There were no nuclear morphological damages in the end range of the experiment. therefore, it is concluded that the proposed cryopreservation protocol is efficient for storing the studied cell type, allowing its use in future experimental studies