RESUMO
BACKGROUND: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. RESULTS: The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing--thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing--thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. CONCLUSIONS: The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing--thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.
Assuntos
Sobrevivência Celular , Criopreservação/métodos , Medicina Regenerativa , Congelamento/efeitos adversos , Humanos , Células U937RESUMO
BACKGROUND: The cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells. RESULTS: In the present study, U937 cells underwent the freezing and thawing cycle in the i3C device. Then a panel of vital tests was performed, including the number of dead cells (PI staining), apoptotic rate (Annexin V staining), mitochondrial membrane potential (TMRM staining), cytoplasm membrane integrity and intracellular metabolism (FDA staining), as well as post-thawing cell proliferation assays. Cells that underwent the freezing - thawing cycle in i3C devices exhibited the same functional activity as control cells. Moreover, the combination of the multi-parametric analysis at a single cell resolution and the optical and biological features of the device enable an accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells. CONCLUSIONS: The means and methodologies described here enable the freezing and thawing of spatially identifiable cells, as well as the efficient detection of viable, specific, highly biologically active cells for future applications.
Assuntos
Criopreservação/métodos , Anexina A5/metabolismo , Apoptose , Proliferação de Células , Sobrevivência Celular , Criopreservação/instrumentação , Congelamento , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Células U937RESUMO
The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.
Assuntos
Neoplasias da Mama/patologia , Linfonodos/patologia , Idoso , Neoplasias da Mama/metabolismo , Feminino , Humanos , Metástase Linfática , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-IdadeRESUMO
BACKGROUND: One of the major clinical problems in breast cancer detection is the relatively high incidence of occult lymph node metastases undetectable by standard procedures. Since the ascertainment of breast cancer stage determines the following treatment, such a "hypo-diagnosis" leads to inadequate therapy, and hence is detrimental for the outcome and survival of the patients. The purpose of our study was to investigate functional metabolic characteristics of living cells derived from metastatic and tumor-free lymph nodes of breast cancer (BC) patients. METHODS: Our methodology is based on the ability of living cells to hydrolyze fluorescein diacetate (FDA) by intracellular esterases and on the association of FDA hydrolysis rates with a specific cell status, both in physiological and pathological conditions. RESULTS: The present study demonstrates a significant difference in the ability to utilize FDA by lymph node cells derived from metastatic and tumor-free lymph nodes in general average, as well as in the metastatic and tumor-free lymph nodes of individual patients. Cells from metastatic lymph nodes had a higher capacity for FDA hydrolysis, and increased this activity after additional activation by autologous tumor tissue (tt). The association between increased FDA hydrolysis rate and activated T lymphocytes and antigen-presenting cells (APC) was shown. CONCLUSION: The results of the present study may contribute to predicting the risk of involvement of seemingly "tumor-free" axillary lymph nodes in occult metastatic processes, and to reducing false-negative results of axillary examination.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Esterases/metabolismo , Linfonodos/enzimologia , Idoso , Células Apresentadoras de Antígenos/enzimologia , Axila , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/secundário , Carcinoma Intraductal não Infiltrante/enzimologia , Carcinoma Intraductal não Infiltrante/secundário , Carcinoma Lobular/enzimologia , Carcinoma Lobular/secundário , Feminino , Fluoresceínas/metabolismo , Humanos , Linfonodos/patologia , Metástase Linfática , Ativação Linfocitária , Pessoa de Meia-Idade , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Linfócitos T/enzimologiaRESUMO
The importance of research involving non-adherent cell lines, primary cells and blood cells is generally undisputed. However, the task of investigating the complexity and heterogeneity of these cells calls for their long-run monitoring at a single-cell resolution. Such a capability is currently unavailable without having to use disruptive cell tethering. The present Cell Retainer (CR) concept enables high-content correlative multi-parametric measurements, from the functional to molecular level, of the same living individual non-adherent cells within a population. Thereby, despite extensive long-term bio-manipulations, the cells preserve their identity without tethering. Several exemplary experiments, using a microscope-slide-based version of the CR, are presented, which could not be performed by other state of the art methods.
Assuntos
Células Sanguíneas/citologia , Técnicas de Cultura de Células , Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Microfluídica/instrumentação , Células U937RESUMO
Optical monitoring of various tissue physiological and biochemical parameters in real-time represents a significant new approach and a tool for better clinical diagnosis. The Tissue Spectroscope (TiSpec), developed and applied in experimental and clinical situations, is the first medical device that enables the real-time monitoring of three parameters representing the vitality of the tissue. Tissue vitality, which is correlated to the oxygen balance in the tissue, is defined as the ratio between O(2) supply and O(2) demand. The TiSpec enables the monitoring of microcirculatory blood flow (O(2) supply), mitochondrial NADH redox state (O(2) balance), and tissue reflectance, which correlates to blood volume. We describe in detail the theoretical basis for the monitoring of the three parameters and the technological aspects of the TiSpec. The comparison between the TiSpec and the existing single parameter monitoring instruments shows a statistically significant correlation as evaluated in vitro as well as in various in vivo animal models. The results presented originated in a pilot study performed in vivo in experimental animals. Further research is needed to apply this technology clinically. The clinical applications of the TiSpec include two situations where the knowledge of tissue vitality can improve clinical practice. The major application is the monitoring of "nonvital" organs of the body [i.e., the skin, gastrointestinal (G-I) tract, urethra] in emergency situations, such as in the operating rooms and intensive care units. Also, the monitoring of specific (vital) organs, such as the brain or the heart, during surgical procedure is of practical importance.
Assuntos
Sobrevivência Celular/fisiologia , Microcirculação/fisiologia , Mitocôndrias/fisiologia , NAD/metabolismo , Consumo de Oxigênio/fisiologia , Análise Espectral/instrumentação , Sobrevivência de Tecidos/fisiologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Respiração Celular/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Gerbillinae , Intestinos/irrigação sanguínea , Intestinos/fisiologia , Masculino , Sistemas On-Line/instrumentação , Projetos Piloto , Ratos , Análise Espectral/métodosRESUMO
BACKGROUND: The involvement of mitochondria in pathological states, such as neurodegenerative diseases, sepsis, stroke, and cancer, are well documented. Monitoring of nicotinamide adenine dinucleotide (NADH) fluorescence in vivo as an intracellular oxygen indicator was established in 1950 to 1970 by Britton Chance and collaborators. We use a multiparametric monitoring system enabling assessment of tissue vitality. In order to use this technology in clinical practice, the commercial developed device, the CritiView (CRV), is tested in animal models as well as in patients. METHODS AND RESULTS: The new CRV enables the optical monitoring of four different parameters, representing the energy balance of various tissues in vivo. Mitochondrial NADH is measured by surface fluorometry/reflectometry. In addition, tissue microcirculatory blood flow, tissue reflectance and oxygenation are measured as well. The device is tested both in vitro and in vivo in a small animal model and in preliminary clinical trials in patients undergoing vascular or open heart surgery. In patients, the monitoring is started immediately after the insertion of a three-way Foley catheter (urine collection) to the patient and is stopped when the patient is discharged from the operating room. The results show that monitoring the urethral wall vitality provides information in correlation to the surgical procedure performed.
Assuntos
Fluxometria por Laser-Doppler/métodos , Mitocôndrias/fisiologia , Monitorização Intraoperatória/instrumentação , Monitorização Intraoperatória/métodos , Análise de Variância , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/cirurgia , Dióxido de Carbono/metabolismo , Procedimentos Cirúrgicos Cardíacos , Cuidados Críticos , Metabolismo Energético , Fluorometria , Gerbillinae , Humanos , Concentração de Íons de Hidrogênio , Modelos Lineares , Microcirculação , Mitocôndrias/química , Modelos Biológicos , NAD/química , NAD/metabolismo , Oxigênio/metabolismo , Ratos , Espectroscopia de Luz Próxima ao Infravermelho , Uretra/citologia , Uretra/fisiologiaRESUMO
Multicellular spheroid models have been recognized as superior to monolayer cell cultures in antitumor drug screening, but their commercial adaptation in the pharmaceutical industry has been delayed, primarily due to technological limitations. The current study presents a new spheroid culture platform that addresses these technical restrictions. The new culturing device is based on a multiwell plate equipped with a glass bottom patterned with an array of UV adhesive microchambers. Each microchamber is designed to accommodate a single spheroid. The system facilitates the simultaneous creation and culturing of a large number of spheroids, as well as screening their response to antitumor drugs. The volume of the spheroids is easily controlled by seeding density. The location of each spheroid is preserved in the same microchamber throughout its growth, treatment with soluble agents, and imaging. The growth ratio parameter, a non-intrusive size analysis of the same spheroid before and after exposure to drugs, was found to be a sensitive indicator for the reaction of MCF7 breast cancer spheroids to cytotoxic drugs. This feature helps reveal the heterogeneity within the spheroid population during the formation process and their drug response, and provides an opportunity to detect specific, highly active or drug-resistant spheroid sub-groups. The advantages of this spheroid-based system make it an efficient drug-screening tool that may be valuable to related fields of research and clinical applications.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Técnicas de Cultura de Células/instrumentação , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Polímeros/química , Esferoides Celulares/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Desenho de Equipamento , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Esferoides Celulares/citologiaRESUMO
Direct quantitative experimental investigations of the function of lymphocytes and other immune cells are challenging due to the cell mobility and the complexity of intercellular communications. In order to facilitate such investigations, an in vitro system is required that is noninvasive and provides kinetic data on cellular responses to challenges such as drug treatments. The present work reports the development of a disposable, inexpensive polymer-made device, the Polymer Live Cell Array (PLCA), for real-time, kinetic analysis of immune cells. The PLCA proved to be optically and biologically compatible, thus individual immune cells can be observed and treated independently without being tethered. The cells share a common space which facilitates cellular communications via secreted molecules or via direct intercellular interactions. These properties facilitate real-time, non-intrusive, repeated measurements of immune cells under multiple experimental treatments.
Assuntos
Linfócitos/citologia , Microscopia de Fluorescência/métodos , Polímeros/química , Análise Serial de Tecidos/instrumentação , Comunicação Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Desenho de Equipamento , Humanos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Lisossomos/ultraestrutura , Potencial da Membrana MitocondrialRESUMO
A high throughput Microtiter plate Cell Retainer (MCR) has been developed to enable, for the first time, high-content, time-dependent analysis of the same single non-adherent and non-anchored cells in a large cell population, while bio-manipulating the cells. The identity of each cell in the investigated population is secured, even during bio-manipulation, by cell retention in a specially designed concave microlens, acting as a picoliter well (PW). The MCR technique combines micro-optical features and microtiter plate methodology. The array of PWs serves as the bottom of a microtiter plate, fitted with a unique flow damper element. The latter enables rapid fluid exchange without dislodging the cells from their original PWs, thus maintaining the cells' identity. Loading cell suspensions and reagents into the MCR is performed by simple pouring, followed by gravitational sedimentation and settling of cells into the PWs. Cell viability and cell division within the MCR were shown to be similar to those obtained under similar conditions in a standard microtiter plate. The efficiency of single cell occupancy in the MCR exceeded 90%. No cell dislodging was observed when comparing images before and after bio-manipulations (rinsing, staining, etc.). The MCR permits the performance of kinetic measurements on an individual cell basis. Data acquisition is governed by software, controlling microscope performance, stage position and image acquisition and analysis. The PW's unique micro-optical features enable rapid, simultaneous signal analysis of each individual cell, bypassing lengthy image analysis.