Effect Of Cell Number Research Articles

Structural design and tunable mechanical properties of novel corrugated 3D lattice metamaterials by geometric tailoring

In this work, a novel corrugated 3D lattice metamaterial with stable mechanical response and deformation behavior was constructed by geometric tailoring. Through quasi-static compression experiments and finite element simulations, the effect of cell number, scaling factor and gradient design methods are systematically studied. Using specific energy absorption (SEA), plateau stress and densification strain as energy absorption indicators, the effects of various factors on the energy absorption performance of corrugated lattice metamaterials were evaluated. The results demonstrate that the proposed corrugated lattice has stable mechanical response and ideal energy absorption performance due to the stable deformation behavior. In addition, the scaling of the corrugated lattice unit cells can trim the lattice mechanical indicators. Furthermore, the continuous interface is effective for realizing controllable mechanical response and tunable energy absorption performance of gradient lattice metamaterials.

Mechanical Properties of ZrO2 TPMS Structures Prepared by DLP 3D Printing

Compressive strength is one of the most important mechanical properties for cellular ceramics. But the dependence of compressive strength of highly porous cellular ceramic structures on porosity remains unclear due to the limit of available methods for making such strong structures based on specific structural designs. In this paper, the TPMS structures, namely P-cell, and neovius structure, were prepared based on the DLP 3D printing technology. Samples with various unit cells were fabricated with zirconia. The relative density of the sintered samples exceeds 99%. The effects of cell number on the compressive strength and deformation of the model were investigated. Samples with similar relative densities exhibited comparable mechanical property in aspect of compressive strength. It is very interesting that the cell number eventually only influence the total strain.

Abstract 4614: Establishment of a HER2 positive breast cancer bone metastasis model for validation of novel therapies

Abstract Breast cancers with overexpression of human epidermal growth factor receptor 2 (HER2+) have aggressive clinical behavior, and at advanced stages are associated with increased risk for developing metastases to distant organs including bones, brain and lungs. The aim of this study was to establish a systemic metastasis model for HER2+ breast cancer with a special interest in bone metastasis. The effects of cell number, estrogen supplementation and mouse strain on metastasis formation were studied. In the study, 5-6 weeks old athymic nude and Rag2 mice (n=10-13 per group) were used. Half of the mice received estrogen supplementation (E2-releasing rods 5µg/day) one week before inoculation of the cancer cells. The mice were inoculated intracardially with 1 or 5 x 105 luciferase-labelled triple-positive (ER, PR positive and HER2 overexpressing) human BT-474 breast cancer cells. The formation of metastases was followed by bioluminescence imaging (BLI) at inoculation and once a week for the duration of the study. At sacrifice, X-ray imaging was performed and the bones were collected for histological analysis. The formation of bone metastases was observed in all study groups, and bone metastases were dominant in the model. Based on BLI, bone metastases developed in nude mice earlier than in Rag2 mice. However, detection of metastases in Rag2 mice was challenging due to dark fur hindering the signal transmittance. The bone metastases appeared between 7 to 36 days after inoculation of the cancer cells in nude mice and between 20 to 43 days in Rag2 mice. The number of bone metastases was higher in nude mice. E2 supplement accelerated the development of bone metastases in both mouse strains. In nude mice with E2 supplement, bone metastases appeared between 7 to 14 days compared to between 20 to 36 days in mice without E2 supplement. In Rag2 mice with E2 supplement bone metastases formed between 20 to 28 days compared to between 36 to 43 days without E2 supplement. The use of higher cell number accelerated the development of bone metastases but had no major effect on their incidence in both mouse strains. Take rate of bone metastases was 90-100% in nude mice with E2 supplement compared to 30-50% without E2 supplement. In Rag2 mice, the take rate of bone metastases was 50% with E2 supplement. X-ray imaging showed estrogen induced bone growth and large tumor-induced osteolytic lesions in the hind limbs in both strains. Due to the extensive bone lesions and occasional fractures, the first mice were sacrificed 50 days after inoculation of the cancer cells. Both mouse strains occasionally developed metastases in soft tissues including brain and ovaries. In conclusion, a high rate of bone metastasis was achieved in athymic nude mice supplemented with E2. This model can be used to study the efficacy of anti-cancer, such as HER2-targeted, compounds on tumor growth at metastatic locations or on the prevention of metastasis formation. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli, Derek Grant. Establishment of a HER2 positive breast cancer bone metastasis model for validation of novel therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4614.

Rapid and High-Throughput Detection of Peripheral Blood Chromosome Aberrations in Radiation Workers.

There is a pressing need to establish automated solutions for the rapid, high-throughput, and automatic detection of chromosome aberrations (CAs) in the occupational health surveillance of large-scale radiation workers. Here, we described and verified the accuracy of a new measurement system based on the automatic scanning and analysis of dicentric chromosomes (DICs). The effects of cell number on DIC detection by automatic scanning and analysis were studied, and the distribution of DIC values per cell was calculated. In total, 1088 cases were detected by automatic DIC scanning and analysis in 26 663 radiation workers, and 73 cases were further confirmed by a technician, including 5 cases in which radiation exposure lead to harmful medical consequences. Our approach reduces the workload by 96% and increases the speed of assessment approximately 7-fold. Overall, this study validates the utility of a novel rapid and high-throughput CA detection procedure as a means of occupational health surveillance of large-scale radiation workers.

Enhancement of backscattering by a conducting cylinder coated with gradient metasurface

This paper proposes a highly effective method for enhancing the backscattering by a conducting cylinder that is coated with a gradient metasurface. The employed metasurface exhibits a phase gradient continuously varying along the circumferential direction of the cylinder so that in-phase retroreflection can be produced to enhance the backscattering. It is demonstrated that the cylinder coated with the proposed gradient metasurface can generate backscattering very close to that from a conducting plate with the same dimensions as the cylinder's cross-section perpendicular to the incident plane wave. Compared with a bare conducting cylinder, the backscattering is significantly enhanced by the gradient metasurface made of conducting strips printed on a grounded dielectric substrate. Effects of cell numbers along the cylinder axis, incident angle, and polarization of the incoming electromagnetic wave on the backscattering enhancement are examined and discussed. A good agreement between simulated and measured backscattering results validates the observations.

On design of multi-cell tubes under axial and oblique impact loads

Multi-cell tubes have been drawn increasing attention for their excellent energy-absorbing ability. However, the effect of cell number and oblique loads on crashing behaviors has seldom been studied to date. In this paper, a group of multi-cell tubes with different cell numbers were comprehensively investigated under both axial and oblique loads. The finite element models were first established and then validated by experimental tests. The simulation results showed that the increase in cell number can be beneficial to the energy absorption (EA) but detrimental due to increase in peak force (Fmax) under axial load. When the oblique loads were taken into account, the tubes could undergo global bending, which is an inefficient deformation mode. By applying complex proportional assessment (COPRAS) method, the 7×7 tube was selected as the best based on multi-criteria under multiple loading angles. Then the Kriging modeling technique and multiobjective particle optimization (MOPSO) algorithm were integrated to address the optimization problems, where EA and Fmax were taken as objectives and tube sizes as design variables. The results demonstrated that different loading angles have different requirements on cell allocation and optimizations of multiple load cases (MLC) can yield better solutions in a weighted average fashion, whereas the optimization for separate single load cases (SLC) could result in inferior performance under other load cases.

Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier

Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT), have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0×104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property-dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed.

XS-E가 아토피피부염이 유발된 NC/Nga Mice의 피부상태에 미치는 영향

This study reports significant improvement of atopic dermatitis condition as a result of experiment using Xanthium strumarium L. extract (XS-E) at the dorsal skin of induced atopic dermatitis Nc/Nga mice. Skin clinical score has decreased (2.75±0.85, *p<0.05), showing visible change of skin condition. IgE (***p<0.001) and IgG1 (2522.00±32.80, ***p<0.001) in plasma also decreased significantly. mRNA (gene expression) level increased (RQ=2.75±0.10, ***p<0.001) within skin tissue of CD4+CD25+Foxp3+ Treg cell that’s activated by XS-E dosage, thereby discovering that there is an effect of suppressing proliferation and viability of Th2 cell, eosinophils, mast cell and inflammatory cell. Upon examining cells permeated with H&E and toluidine blue staining technique, thickness of epidermis and mast cell’s permeation decreased, and the result of examining the distribution of CCR 3+ eosinophils within ALN showed that it’s level fell down to that of wild type (normal group, NC/Nga-WT). By such results, it is suggested that XS-E is highly effective on atopic dermatitis, and it is considered that continued quantitative research and case study of clinical research such as effect of cell number in individual tissues or change of total cell number are necessary.

Stimulation of Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells with Notochordal Cell-Secreted Factors

IntroductionIntervertebral disc (IVD) degeneration, a major cause for low back pain, is characterized by a decreasing number of nucleus pulposus cells (NPCs),1 which are then unable to maintain a h...

Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 10(5), 10(6), or 10(7) cells per disc. The MSC-transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC-transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 10(6) MSCs transplanted disc, while less viable cells were detected in 10(5) MSCs transplanted and more apoptotic cells in 10(7) MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration.

Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection-based real-time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large-scale screening studies. A live cell staining lipophilic tracer-1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine iodide (DiR)-is used for accurate imaging of wound closure in a simple 96-well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images are analyzed using Image J software and the migration index is calculated. Effect of cell number, time after scratch and software settings are analyzed. The method is validated by showing concentration- and time-dependent effects of cytochalasin-D on fibroblast migration. Using this assay, we quantitatively evaluate the role of the MAPK-activated protein kinases MK2 and MK3 in fibroblast migration. First, the migratory phenotype of MK2-deficient MEFs is analyzed in a retroviral rescue model. In addition, migration of MK2/3-double-deficient cells is determined and the ability of MK3 to rescue cell migration in MK2/3-double-deficient fibroblasts is demonstrated.

Effects of cell number on teratoma formation by human embryonic stem cells

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source, potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein and (Fluc-eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n=35) or skeletal muscle (n=35) injection of 1x102, 1x103, 1x104, 1x105, or 1x106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after this period were followed out to day 365 to confirm tumor absence. Significantly, in this study, a minimum of 1x105 ES cells in the myocardium and 1x104 cells in the skeletal muscle was observed to be requisite for teratoma development, suggesting that human ES cell number is a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic.

Effects of cell number and cell size on petiole length variation in a stoloniferous herb

In stoloniferous species, the length of petioles is of pivotal importance because it determines the position of leaf blades within the canopy. From a mechanistic perspective, two developmental processes, cell division and cell elongation, are responsible for the length of a given petiole. This study aimed at quantifying the relative contributions of cell division and cell elongation to genotypic and plastic variation in petiole length of the stoloniferous herb Trifolium repens. Thirty-four genotypes of T. repens were grown under high light conditions and simulated canopy shade. Cells were counted and their lengths measured on epidermal prints from fully grown petioles of leaves that had been initiated in the experimental light conditions. Cell number was the main trait explaining petiole length differences among genotypes grown under high light, while both cell number and length changed in response to shading. Our study revealed a strong negative correlation between shade-induced changes in cell number and cell length: genotypes that responded to shading by increasing cell numbers hardly changed in cell length, and vice versa. Our results suggest that genotypic and phenotypic variation in petiole length results from a complex interplay between the developmental processes of cell elongation and cell division.

Design and fabrication of heart muscle using scaffold‐based tissue engineering

Cardiac tissue engineering strategies are based on the development of functional models of heart muscle in vitro. Our research is focused on evaluating the feasibility of different tissue engineering platforms to support the formation of heart muscle. Our previous work was focused on developing three-dimensional (3D) models of heart muscle using self-organization strategies and biodegradable hydrogels. To build on this work, our current study describes a third tissue engineering platform using polymer-based scaffolding technology to engineer functional heart muscle in vitro. Porous scaffolds were fabricated by solubilizing chitosan in dilute glacial acetic acid, transferring the solution to a mold, freezing the mold at -80 degrees C followed by overnight lyophilization. The scaffolds were rehydrated in sodium hydroxide to neutralize the pH, sterilized in 70% ethanol and cellularized using primary cardiac myocytes. Several variables were studied: effect of polymer concentration and chitosan solution volume (i.e., scaffold thickness) on scaffold fabrication, effect of cell number and time in culture on active force generated by cardiomyocyte-seeded scaffolds and the effect of lysozyme on scaffold degradation. Histology (hematoxylin and eosin) and contractility (active, baseline and specific force, electrical pacing) were evaluated for the cellularized constructs under different conditions. We found that a polymer concentration in the range 1.0-2.5% (w/v) was most suitable for scaffold fabrication while a scaffold thickness of 200 microm was optimal for cardiac cell functionality. Direct injection of the cells on the scaffold did not result in contractile constructs due to low cell retention. Fibrin gel was required to retain the cells within the constructs and resulted in the formation of contractile constructs. We found that lower cell seeding densities, in the range of 1-2 million cells, resulted in the formation of contractile heart muscle, termed smart material integrated heart muscle (SMIHMs). Chitosan concentration of 1-2% (w/v) did not have a significant effect on the active twitch force of SMIHMs. We found that scaffold thickness was an important variable and only the thinnest scaffolds evaluated (200 microm) generated any measurable active twitch force upon electrical stimulation. The maximum active force for SMIHMs was found to be 439.5 microN while the maximum baseline force was found to be 2850 microN, obtained after 11 days in culture. Histological evaluation showed a fairly uniform cell distribution throughout the thickness of the scaffold. We found that lysozyme concentration had a profound effect on scaffold degradation with complete scaffold degradation being achieved in 2 h using a lysozyme concentration of 1 mg/mL. Slower degradation times (in the order of weeks) were achieved by decreasing the lysozyme concentration to 0.01 mg/mL. In this study, we provide a detailed description for the formation of contractile 3D heart muscle utilizing scaffold-based methods. We demonstrate the effect of several variables on the formation and culture of SMIHMs.

Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents

In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability – over 12 weeks – with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5×10 4 cells/cm 2 on epidermal tissue quality equalled that of the standard density (2×10 5 cells/cm 2). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte–fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.

P210 Effect of cell number on the quality of the repair tissue in Autologous Chondrocyte Transplantation (ACT) in an in-vivo model

P210 Effect of cell number on the quality of the repair tissue in Autologous Chondrocyte Transplantation (ACT) in an in-vivo model

A study on type II structures. Part II:dynamic behavior of a chain of pre-bent plates

From the energy-absorption point of view, in general a cellular material may be composed of a number of type I and type II cells. A comprehensive study of the mechanisms, by which these two types of cells undergo large deformation under impact loading , will help to deepen our understanding of the dynamic behavior of cellular materials. In this paper, special attention is paid to the dynamic response of a chain of type II cells under a projectile impact. Both the FE analysis and the equivalent mass–spring model proposed in part I of the paper are employed, and the results agree well with each other while the mass–spring model is much more efficient than the FE simulation. Three collapse modes of type II chain are identified, depending on the initial impact velocity. Furthermore, based on the equivalent mass–spring model, the local contact behavior between the striker and the first cell of the type II chain, and the effects of the associated parameters (e.g., the effects of cell number, cell wall inertia, mass ratio, input energy, etc.) are discussed in detail.

A study on type II structures. Part II:: dynamic behavior of a chain of pre-bent plates

From the energy-absorption point of view, in general a cellular material may be composed of a number of type I and type II cells. A comprehensive study of the mechanisms, by which these two types of cells undergo large deformation under impact loading, will help to deepen our understanding of the dynamic behavior of cellular materials. In this paper, special attention is paid to the dynamic response of a chain of type II cells under a projectile impact. Both the FE analysis and the equivalent mass–spring model proposed in part I of the paper are employed, and the results agree well with each other while the mass–spring model is much more efficient than the FE simulation. Three collapse modes of type II chain are identified, depending on the initial impact velocity. Furthermore, based on the equivalent mass–spring model, the local contact behavior between the striker and the first cell of the type II chain, and the effects of the associated parameters (e.g., the effects of cell number, cell wall inertia, mass ratio, input energy, etc.) are discussed in detail.

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25-34), intermediate (40-55), and high (60-81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50-83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40-55) or high (60-81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40-55 cells) and 41% versus 25% (60-81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning.

Factors affecting cell viability during bisection of bovine embryos

The aim of this study was to investigate the effects of temperature, cytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae and blastocysts were bisected using a metal blade. In Experiment 1, splitting of embryos in control medium (PBS + 10% fetal calf serum) was compared with splitting in the presence of 7.5 μl/ml cytochalasin B. In Experiment 2, the control medium was compared with medium supplemented with 200 mM sucrose. In Experiment 3, the control medium was compared with medium supplemented with sucrose and cytochalasin B. Cell viability was measured by staining nuclei of embryos with Hoechst 33258 and propidium iodide. Cells with nuclei exhibiting pink fluorescence were considered lysed, while blue fluorescence was considered an indication of viable cells. Cells disaggregated during splitting were classified as extruded cells. An effect of the developmental stage was observed in the pooled data from the control groups of the 3 experiments, with a higher proportion of viable cells in bisected morulae compared with bisected blastocysts (77.6 vs 70.0%; P = 0.003). However, as there was no effect of cell number (P = 0.85), the influence of the developmental stage can be contributed to morphological changes rather than to increase of cells associated with this change. In Experiment 1, the cytochalasin B-treated embryos contained a higher percentage of viable cells than the control embryos after removal of the developmental stage effect (P < 0.01). In Experiment 2, no effect on sucrose could be observed. In Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this difference was not significant. In Experiment 1, there was a correlation between viable cells and temperature during splitting (r = 0.42, P = 0.05; temperature range 8.1 °C to 15.6 °C). No correlation was found in any other group in any of the experiments, nor in the pooled data from the control groups in the 3 experiments.