Comparative cytotoxic characteristics of the interferon-inducing yeast RNA-tilorone complex]

Cytotoxicity of the yeast RNA-tilorona molecular complex (MC) with interferonogenic properties and its influence on the DNA replicative synthesis were studied in experiments with human lymphocytes and 3 cell lines. It was shown that the MC doses of 25, 100 and 250 micrograms/ml were absolutely nontoxic for all the cell lines. The main parameters of the MC toxicity based on the cell viability were calculated. The parameters were found to correlate in the order of their magnitude with those relating to interferonogens of the polynucleotide nature. Within the dose ranges of 10 to 100 micrograms/ml the MC had a stimulating effect on replicative processes in the cells. It was concluded that the use of the MC as an inductor in large-scale manufacture of human and animal interferons of type 1 was promising.     

Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Osteogenin inhibits proliferation and stimulates differentiation in mouse osteoblast-like cells (MC3T3-E1)

Osteogenin, a novel bone differentiation factor, was recently purified and characterized. We examined its effect on the proliferation and differentiation of MC3T3-E1 osteoblast-like cells. Cell proliferation was inhibited the first 48 h after addition of osteogenin, and this effect was independent of serum. Osteogenin did not influence the cell morphology. Alkaline phosphatase promptly increased in a dose and time-dependent manner and appeared to be specific. Treatment with TGF-beta 1 resulted in inhibition of alkaline phosphatase activity, and was reversed by osteogenin within 48 h. Cell cultures treated with osteogenin for 72 h after confluence became responsive to parathyroid hormone. Synthesis of collagenous proteins was stimulated by osteogenin. The present results demonstrate a significant influence of osteogenin on the differentiation of osteogenic phenotype in MC3T3-E1 cells in vitro.     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Monte Carlo simulation of gold nanoparticles for X-ray enhancement application

BackgroundGold nanoparticles (Au NPs) are regarded as potential agents that enhance the radiosensitivity of tumor cells for theranostic applications. To elucidate the biological mechanisms of radiation dose enhancement effects of Au NPs as well as DNA damage attributable to the inclusion of Au NPs, Monte Carlo (MC) simulations have been deployed in a number of studies.Scope of ReviewThis review paper concisely collates and reviews the information reported in the simulation research in terms of MC simulation of radiosensitization and dose enhancement effects caused by the inclusion of Au NPs in tumor cells, simulation mechanisms, benefits and limitations.Major conclusionsIn this review, we first explore the recent advances in MC simulation on Au NPs radiosensitization. The MC methods, physical dose enhancement and enhanced chemical and biological effects is discussed, followed by some results regarding the prediction of dose enhancement. We then review Multi-scale MC simulations of Au NP-induced DNA damages for X-ray irradiation. Moreover, we explain and look at Multi-scale MC simulations of Au NP-induced DNA damages for X-ray irradiation.General significanceUsing advanced chemical module-implemented MC simulations, there is a need to assess the radiation-induced chemical radicals that contribute to the dose-enhancing and biological effects of multiple Au NPs.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

Artifact-free CT images for electron beam therapy using a patient-specific non metallic shield

Patient’s CT images taken with metallic shields for radiotherapy suffer from artifacts. Furthermore, the treatment planning system (TPS) has a limitation on accurate dose calculations for high density materials. In this study, a Monte Carlo (MC)-based method was developed to accurately evaluate the dosimetric effect of the metallic shield. Two patients with a commercial tungsten shield of lens and two patients with a custom-made lead shield of lip were chosen to produce their non-metallic dummy shields using 3D scanner and printer. With these dummy shields, we generated artifact-free CT images. The maximum CT number allowed in TPS was assigned to metallic shields. MC simulations with real material information were carried out. In addition, clinically relevant dose-volumetric parameters were calculated for the comparison between MC and TPS. Relative dosimetry was performed using radiochromic films. The dose reductions below metallic structures were shown on MC dose distributions, but not evident on TPS dose distributions. The differences in dose-volumetric parameters of PTV between TPS and MC for eye shield cases were not clearly shown. However, the mean dose of lens from TPS and MC was different. The MC results were in superior agreement with measured data in relative dosimetry. The lens dose could be overestimated by TPS. The differences in dose-volumetric parameters of PTV between TPS and MC were generally larger in lip cases than in eye cases. The developed method is useful in predicting the realistic dose distributions around the organs blocked by the metallic shields.     

Influence of selenium on mercuric chloride cellular uptake and toxicity indicating protection: studies on cultured K-562 cells

Selenium and mercuric chloride (MC) interactions regarding cellular uptake and selenium protection on MC toxicity have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 microM) or selenomethionine (10 or 50 microM) together with MC (35 or 50 microM). Both treatments with selenite showed an increase of mercury uptake with increased selenium dose. In the pretreated or simultaneously treated selenite and 35 microM MC combinations, no inhibition of growth was seen, whereas all 50-microM MC combinations were toxic to the cells. A selenite-dependent protection was obtained for both exposure protocols when considering the cellular uptake of mercury. The cells died when the accumulation on d 4 reached more than about 0.8 x 10(-15) mol/cell of mercury, whereas they survived up to twofold more mercury uptake when exposed to selenite. Selenomethionine gave, with a few exceptions, similar effects as selenite on MC uptake and toxicity.     

Monte Carlo calculations of radiotherapy dose in “homogeneous” anatomy

Given the substantial literature on the use of Monte Carlo (MC) simulations to verify treatment planning system (TPS) calculations of radiotherapy dose in heterogeneous regions, such as head and neck and lung, this study investigated the potential value of running MC simulations of radiotherapy treatments of nominally homogeneous pelvic anatomy. A pre-existing in-house MC job submission and analysis system, built around BEAMnrc and DOSXYZnrc, was used to evaluate the dosimetric accuracy of a sample of 12 pelvic volumetric arc therapy (VMAT) treatments, planned using the Varian Eclipse TPS, where dose was calculated with both the Analytical Anisotropic Algorithm (AAA) and the Acuros (AXB) algorithm. In-house TADA (Treatment And Dose Assessor) software was used to evaluate treatment plan complexity, in terms of the small aperture score (SAS), modulation index (MI) and a novel exposed leaf score (ELS/ELA). Results showed that the TPS generally achieved closer agreement with the MC dose distribution when treatments were planned for smaller (single-organ) targets rather than larger targets that included nodes or metastases. Analysis of these MC results with reference to the complexity metrics indicated that while AXB was useful for reducing dosimetric uncertainties associated with density heterogeneity, the residual TPS dose calculation uncertainties resulted from treatment plan complexity and TPS model simplicity. The results of this study demonstrate the value of using MC methods to recalculate and check the dose calculations provided by commercial radiotherapy TPSs, even when the treated anatomy is assumed to be comparatively homogeneous, such as in the pelvic region.     

Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.     

Topography and morphometry of intestinal mast cells in children with Hirschsprung's disease

Mast cells (MC) are source of many biological active compounds like cytokines, arachidonic acid derivates, proteoglicanes, prostaglandins, proteases, free oxygen radials, NGF, PAF and many more. The role of MC in pathogenesis of Hirschsprung's disease (HD) is not clear. Substances produced by MC may exert an important effect on embryology, growth, differentiation and regeneration of intestinal nervous system. Additionally, MC products modulate inflammation processes thus influencing on the clinical course of HD. Present study was established to evaluate the morphologic MC examination as a support of making diagnosis in HD. The MC topography and morphometry were evaluated in specimens collected from aganglionic colon of patients with diagnosed HD. The results were compared with corresponding data from normally innervated colon of patients suffering from constipation, and normal colon of children not presenting defecation problems. MC were visualized using indirect immunohistochemical method LSAB with mouse antibody against human tryptase. The MC visualized in submucosa and muscular layer in Hirschsprung's disease were significantly larger in comparison with control group (p<0.05). Also the number of MC/mm2 in mucosa and lamina propria in HD was significantly elevated (p<0.05). However, the MC density in submucosa was also higher but it was not high statistically significant. In muscular layer and in serosa density of MC/mm2 was not statistically significant. In the intestinal wall MC in aganglionic segment in Hirschsprung's disease are significantly activated comparing with normally innervated colon segments taken from the patients from other groups. This may confirm the role of MC both in pathogenesis of HD and in the reparation processes of bowel nervous system.     

Monte Carlo as quality control tool of stereotactic body radiation therapy treatment plans

Purpose/objectiveThe objective of this study was to verify the accuracy of treatment plans of stereotactic body radiation therapy (SBRT) and to verify the feasibility of the use of Monte Carlo (MC) as quality control (QC) on a daily basis.Material/methodsUsing EGSnrc, a MC model of Agility™ linear accelerator was created. Various measurements (Percentage depth dose (PDD), Profiles and Output factors) were done for different fields sizes from 1x1 up to 40x40 (cm²). An iterative model optimization was performed to achieve adequate parameters of MC simulation. 40 SBRT patient’s dosimetry plans were calculated by Monaco™ 3.1.1. CT images, RT-STRUCT and RT-PLAN files from Monaco™ being used as input for Moderato MC code. Finally, dose volume histogram (DVH) and paired t-tests for each contour were used for dosimetry comparison of the Monaco™ and MC.ResultsValidation of MC model was successful, as <2% difference comparing to measurements for all field’s sizes. The main energy of electron source incident on the target was 5.8 MeV, and the full width at half maximum (FWHM) of Gaussian electron source were 0.09 and 0.2 (cm) in X and Y directions, respectively. For 40 treatment plan comparisons, the minimum absolute difference of mean dose of planning treatment planning (PTV) was 0.1% while the maximum was 6.3%. The minimum absolute difference of Max dose of PTV was 0.2% while the maximum was 8.1%.ConclusionSBRT treatment plans of Monaco agreed with MC results. It possible to use MC for treatment plans verifications as independent QC tool.     

Aberration induction by mitomycin C in early primary spermatocytes of mice

MC is well known to induce dominant lethal mutations in mouse spermatocytes. Tests were done to determine whether chromosomal aberrations could be identified in spermatocytes as being responsible for the dominant lethal effects. Male mice were treated with single doses of MC during DNA synthesis preceding meiosis and during early prophase of meiosis. Simultaneous labeling was performed to identify cells that were in S-phase during the time of treatment. Diakineses-metaphases I were analyzed for the occurrence of univalents, gaps, fragments and rearrangements. The frequencies of cells with aberrations increased with dose and time after treatment. Maximal values were obtained after 12 days, indicating that MC was most effective in cells undergoing DNA replication. 95% of these cells were labeled. The majority of aberrant cells contained one or more fragments. These cells will lead to dominant lethality of the zygotes after fertilization. Cells with rearrangements occurred 11 and 12 days after treatment. These cells can develop into sperm carrying a reciprocal translocation which would then give rise to semi-sterile progeny after fertilization. Further investigations are needed to study the transmission of rearrangements observed in primary spermatocytes.     

Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targeting properties of two metal cyclized alpha-MSH peptide analogues, (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH. Initially, the presence and density of the MC1 receptor were determined on a bank of human melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 +/- 0.68 %ID/g of (188)Re-(Arg(11))CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of (188)Re-(Arg(11))CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. (188)Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as (188)Re-(Arg(11))CCMSH. However, the kidney uptake value of (188)Re-CCMSH was two times higher than that of (188)Re-(Arg(11))CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover, the biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH highlight their potential as therapeutic agents for human melanoma.     

Evaluation of PENFAST – A fast Monte Carlo code for dose calculations in photon and electron radiotherapy treatment planning

The aim of the present study is to demonstrate the potential of accelerated dose calculations, using the fast Monte Carlo (MC) code referred to as PENFAST, rather than the conventional MC code PENELOPE, without losing accuracy in the computed dose. For this purpose, experimental measurements of dose distributions in homogeneous and inhomogeneous phantoms were compared with simulated results using both PENELOPE and PENFAST. The simulations and experiments were performed using a Saturne 43 linac operated at 12 MV (photons), and at 18 MeV (electrons). Pre-calculated phase space files (PSFs) were used as input data to both the PENELOPE and PENFAST dose simulations. Since depth–dose and dose profile comparisons between simulations and measurements in water were found to be in good agreement (within ±1% to 1 mm), the PSF calculation is considered to have been validated. In addition, measured dose distributions were compared to simulated results in a set of clinically relevant, inhomogeneous phantoms, consisting of lung and bone heterogeneities in a water tank. In general, the PENFAST results agree to within a 1% to 1 mm difference with those produced by PENELOPE, and to within a 2% to 2 mm difference with measured values. Our study thus provides a pre-clinical validation of the PENFAST code. It also demonstrates that PENFAST provides accurate results for both photon and electron beams, equivalent to those obtained with PENELOPE. CPU time comparisons between both MC codes show that PENFAST is generally about 9–21 times faster than PENELOPE.     

Betulin binds to melanocortin receptors and antagonizes alpha-melanocyte stimulating hormone induced cAMP generation in mouse melanoma cells

Betulin is a principal component of birch bark and is known to possess a broad range of biological activities, including antiinflammatory, antiviral and anticancer actions. The present study was carried out in vitro to clarify the influence of betulin on melanocortin (MC) receptor-ergic signalling by using COS-7 cells transfected with corresponding human MC receptor DNA. The results showed that betulin binds to the human melanocortin MC1, three to five receptors with selectivity to the MC1 subtype (K(i) value 1.022 +/- 0.115 microM). Betulin binds to the MC receptors with the following potency order-MC > MC3 > MC5 > MC4. Betulin itself does not stimulate cAMP generation, however, it slightly antagonizes alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in the mouse melanoma cell line B16-F1. As a water-insoluble substance, betulin was dissolved in DMSO therefore DMSO competition with the labelled ligand NDP-MSH for the binding to the MC receptors was tested in the identical experimental set-up. We found that DMSO competes for binding to all the MC receptor subtypes, at 20% concentration and above. Selectivity for one or another receptor subtype was not observed. We have demonstrated for the first time, the ability of the plant compound betulin to bind to the MC receptors. One may suggest MC receptor MC1 subtype as the essential target for the antimelanoma action of betulin and its structurally close molecules such as betulinic acid. Moreover, we have found a new non-peptide small molecule MC mimetic, that is betulin. Thus, we report a new chemical motif for the binding to the MC receptors that could be used as a template for the search of more selective MC mimetics.     

Evidence for a direct effect of growth hormone on osteoblasts

In order to determine whether growth hormone (GH) exerts a direct effect on osteoblasts, in vitro and in vivo immunocytological studies were carried out on newborn rat calvaria and a clonal osteoblast-like cell line (MC3T3-E1) isolated from newborn mouse calvaria. After exposure to human growth hormone (hGH) or 1,25 dihydroxyvitamin D3 (1,25(OH)₂D₃), a significant increase in alkaline phosphatase activity was observed in MC3T3-E1 cells. Simultaneous exposure of MC3T3-E1 cells to hGH and 10 nM 1,25(OH)₂D₃ showed a synergistic effect of the two hormones on this activity. The optimal dose of hGH was 0.1 nM. An immunocytological procedure was performed on ultrathin frozen sections from 7-day-old rat calvaria and MC3T3-E1 cells cultured with hGH. GH-like immunoreactivity was observed in both cases. In calvaria, endogenous GH-like immunoreactivity was localized at the same ultrastructural level (plasma membrane, cytoplasmic and nuclear matrices) as exogenous GH-like immunoreactivity in MC3T3-E1 cells. Following the initial step of binding to the plasma membrane, GH may be internalized in the cytoplasmic matrix and nucleus. In situ hybridization revealed the presence of mRNA coding for GH receptor in calvaria cells. The density of these receptors seemed to be lower in osteoblasts than in hepatocytes. In MC3T3-E1 cells, hGH induced a dose-dependent secretion of insulin-like growth factor 1. In conclusion, these results indicate that GH may act directly on osteoblasts.     

Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.