RESUMO
The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
Assuntos
Barreira Hematoencefálica/metabolismo , Fenômenos Mecânicos , Técnicas Analíticas Microfluídicas/instrumentação , Fenômenos Biomecânicos , Barreira Hematoencefálica/citologia , Linhagem Celular , Impedância Elétrica , Células Endoteliais/metabolismo , Humanos , Microscopia ConfocalRESUMO
The gold standard of semen analysis is still an manual method, which is time-consuming, labour intensive and needs thorough quality control. Microfluidics can also offer advantages for this application. Therefore a first step in the development of a microfluidic chip has been made, which enables the man the semen analysis at home. In this article recent efforts to determine the concentration and motility using a microfluidic chip are summarized.
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Vascular endothelial cells form the inner lining of all blood vessels and play a central role in vessel physiology and disease. Endothelial cells are highly responsive to the mechanical stimulus of fluid shear stress that is exerted by blood flowing over their surface. In this study, the immediate micromechanical response of endothelial cells to physiological shear stress was characterized by tracking of ballistically injected, sub-micron, fluorescent particles. It was found that the mean squared displacement (MSD) of the particles decreases by a factor 1.5 within 10 min after the onset of shear stress. This decrease in particle motion is transient, since the MSD returns to control values within 15-30 min after the onset of shear. The immediate micromechanical stiffening is dependent on activation of the vascular endothelial growth factor receptor (VEGFR)-2, because inhibition of the receptor abrogates the micromechanical response. This work shows that the cytoskeleton is actively involved in the acute, functional response of endothelial cells to shear stress.
Assuntos
Células Endoteliais/metabolismo , Microesferas , Movimento (Física) , Estresse Mecânico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Actinas/metabolismo , Forma Celular , Células Cultivadas , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Hemorreologia , Humanos , Injeções , Microfluídica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Porous tubular poly(trimethylene carbonate) (PTMC) scaffolds for vascular tissue engineering, with an inner diameter of 3 mm and a wall thickness of 1 mm, were prepared by means of dip-coating and subsequent leaching of NaCl particles. The scaffolds, with an average pore size of 110 µm and a porosity of 85%, showed a smooth muscle cell (SMC) seeding efficiency of only 10%. To increase the efficiency of cell seeding, these scaffolds were coated with a microporous PTMC outer layer with a thickness of 0.1-0.4 mm, an average pore size of 28 µm, and a porosity of 65%. Coating of the scaffolds with the microporous outer layer did not influence the inner pore structure or the mechanical properties of the scaffolds to a significant extent. The intrinsic permeability of the scaffolds decreased from 60 × 10(-10) m(2) to approximately 5 × 10(-10) m(2) after coating with the microporous outer layer. The latter value is still relatively high indicating that these scaffolds may facilitate sufficient diffusion of nutrients and waste products during cell culturing. The efficiency of SMC seeding determined after 24 h cell adhesion in the scaffolds increased from less than 10% to 43% after coating with the microporous outer layer. The cells were homogeneously distributed in the scaffolds and cell numbers increased 60% during culturing for 7 days under stationary conditions. It is concluded that coating of porous tubular PTMC scaffolds with a microporous PTMC outer layer facilitates effective cell seeding in these scaffolds.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Dioxanos/química , Miócitos de Músculo Liso/metabolismo , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/metabolismo , Células Cultivadas , Humanos , Teste de Materiais , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Porosidade , Ovinos , Suínos , Resistência à Tração , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
The physiology of vascular endothelial cells is strongly affected by fluid shear stress on their surface. In this study, a microfluidic assay was employed to analyze the alignment of actin filaments in endothelial cells in response to shear stress. When cells were cultured in microfluidic channels and subjected to shear stress, the alignment of filaments in the channel direction was significantly higher than in static cultures. By adding inhibitory drugs, the roles of several signaling proteins in the process of alignment were determined. Thus, it is shown how microfluidic technology can be employed to provide a mechanistic insight into cell physiology.
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Acceptance of microfluidic technology in everyday laboratory practice by biologists is still low. One of the reasons for this is that the technology combines poorly with standard cell biological and biochemical analysis tools. Flow cytometry is an example of a conventional analytical tool that is considered to be incompatible with microfluidic technology and its inherent small sample sizes. In this study, it is shown that properly designed microfluidic devices contain cell populations that are large enough to be analyzed by flow cytometry. To illustrate this, the uptake of fluorescent human low-density lipoprotein (LDL) by human endothelial cells that were cultured in a microfluidic channel was analyzed. It was found that the uptake of LDL by the cells increased linearly over time. Moreover, the uptake decreased when cells were pretreated with fluid shear stress inside the microfluidic devices. This study shows that microfluidic technology can be combined with conventional flow cytometry, while retaining the advantages of working with microfluidics such as low reagent use and dynamic cell culture conditions. This approach of combining microfluidic technology with conventional laboratory tools may contribute to greater acceptance of microfluidic devices in biological research.
Assuntos
Células Endoteliais/metabolismo , Citometria de Fluxo/métodos , Lipoproteínas LDL/metabolismo , Células Endoteliais/citologia , Humanos , Técnicas Analíticas Microfluídicas , Estresse MecânicoRESUMO
Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M(n) = 4.37 x 10(5)) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50-70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 microm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.
Assuntos
Dioxanos/química , Elasticidade , Maleabilidade , Polímeros/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Elasticidade/efeitos dos fármacos , Elasticidade/efeitos da radiação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Raios gama , Géis , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos da radiação , Maleabilidade/efeitos dos fármacos , Maleabilidade/efeitos da radiação , Porosidade/efeitos dos fármacos , Porosidade/efeitos da radiação , Ovinos , Sus scrofa , Resistência à Tração/efeitos dos fármacos , Resistência à Tração/efeitos da radiaçãoRESUMO
Vascular cell biology is an area of research with great biomedical relevance. Vascular dysfunction is involved in major diseases such as atherosclerosis, diabetes, and cancer. However, when studying vascular cell biology in the laboratory, it is difficult to mimic the dynamic, three-dimensional microenvironment that is found in vivo. Microfluidic technology offers unique possibilities to overcome this difficulty. In this review, an overview of the recent applications of microfluidic technology in the field of vascular biological research will be given. Examples of how microfluidics can be used to generate shear stresses, growth factor gradients, cocultures, and migration assays will be provided. The use of microfluidic devices in studying three-dimensional models of vascular tissue will be discussed. It is concluded that microfluidic technology offers great possibilities to systematically study vascular cell biology with setups that more closely mimic the in vivo situation than those that are generated with conventional methods.
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Vasos Sanguíneos/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Animais , Pesquisa Biomédica/instrumentação , Pesquisa Biomédica/métodos , Vasos Sanguíneos/citologia , Humanos , Doenças Vasculares/patologia , Doenças Vasculares/fisiopatologiaRESUMO
Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm(2) for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.
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OBJECTIVES: Fulvestrant is an estrogen receptor (ER) antagonist that binds, blocks and degrades the estrogen receptor and is currently used in adjuvant treatment in postmenopausal women with ER-positive breast cancer as an alternative for tamoxifen. As an antagonist, it may induce or aggravate climacteric symptoms. In order to alleviate these symptoms, one could consider hormone therapy. The objective of this study was to analyze the effect of fulvestrant alone or in combination with different steroids in human breast cancer cells in vitro, and to demonstrate whether these steroids will compromise the efficacy of fulvestrant in ER-positive breast cancer cells. METHODS: We performed experiments in vitro with various hormone therapy preparations (estradiol (E2), dihydrodydrogesterone (DHD) and tibolone) at a concentration of 10(-6) mol/l alone or combined with fulvestrant in different breast cancer cell lines, ER-positive and ER-negative. After an incubation of 144 h, proliferation and apoptosis were measured. The first was measured by quantification of the expression of cyclin D1 mRNA, the latter by the Nicoletti fragmentation assay. RESULTS: This in vitro study revealed clear differences in results when various hormone therapy preparations, alone or combined with fulvestrant, are added to ER-positive and ER-negative breast cancer cell lines. CONCLUSIONS: Our study demonstrated that fulvestrant, an ER antagonist used in the treatment of ER-positive breast cancer, combined with E2 and DHD or in combination with tibolone, is not compromised in its efficacy in inducing apoptosis in ER-positive breast cancer cell lines in vitro.
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Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Didrogesterona/análogos & derivados , Didrogesterona/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Estrogênios/farmacologia , Feminino , Fulvestranto , Terapia de Reposição Hormonal , Humanos , Técnicas In Vitro , Norpregnenos/farmacologia , Progestinas/farmacologia , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Heat-induced apoptosis proceeds via mitochondria by permeabilization of the outer mitochondrial membrane (MOMP), resulting in the release of cytochrome c. This essential step is mediated by Bcl-2 family proteins, such as Bax. Recently, caspase-2 was assigned a prominent role in regulating Bax. Therefore, we studied the initiation of heat-induced apoptosis by monitoring Bcl-2 family members and the release of cytochrome c with or without caspase-2 inhibition. Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1) were exposed to heat treatment and/or X-radiation. Expression and localization of Bax and Bcl-2 proteins was investigated by flow cytometry (FCM) and confocal microscopy respectively. Cytochrome c release was measured with FCM as evidence for MOMP. In addition, the role of caspase-2 in heat- and radiation-induced apoptosis was assessed using the specific caspase-2 inhibitor zVDVAD-fmk. Here we present evidence that heat treatment, and not irradiation, increases intracellular Bax protein expression and subsequently stimulates MOMP, resulting in the release of cytochrome c. Furthermore, by selective blocking of caspase-2 using zVDVAD-fmk less Bax was expressed and subsequently a significant decrease in cytochrome c release was observed. In conclusion, heat treatment of hematopoietic cells does require caspase-2 activation for the initiation of Bax-mediated MOMP.
Assuntos
Caspase 2/metabolismo , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Proteína X Associada a bcl-2/fisiologia , Apoptose , Linhagem Celular Tumoral , Citometria de Fluxo , Temperatura Alta , Humanos , Microscopia Confocal , Raios XRESUMO
PURPOSE: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis). MATERIALS AND METHODS: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5 degrees C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion. RESULTS: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines. CONCLUSION: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.
Assuntos
Apoptose/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Genes p53/efeitos da radiação , Proteínas de Choque Térmico HSP70/efeitos da radiação , Sistema Hematopoético/efeitos da radiação , Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes p53/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Sistema Hematopoético/citologia , Sistema Hematopoético/metabolismo , Temperatura Alta , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Raios XRESUMO
Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.
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Proliferação de Células/efeitos dos fármacos , Difosfonatos/farmacologia , Alendronato/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ácido Clodrônico/farmacologia , Ciclina D1/genética , Fragmentação do DNA/efeitos dos fármacos , Ácido Etidrônico/análogos & derivados , Ácido Etidrônico/farmacologia , Feminino , Citometria de Fluxo , Humanos , Ácido Ibandrônico , Imidazóis/farmacologia , Pamidronato , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Risedrônico , Ácido ZoledrônicoRESUMO
PURPOSE: The aim of the study was to investigate the molecular mechanisms involved in apoptosis of human promyelocytic cells (HL60) induced by hyperthermia and to compare this to radiation-induced apoptosis as a reference model. MATERIALS AND METHODS: Apoptosis of HL60 cells was induced by heat-treatment (430C during 1 h) or by gamma-radiation (8 Gy) and followed at increasing time periods after treatment with Annexin V binding to phosphatidylserine (PS). The transition of the mitochondrial membrane potential (delta psim) was estimated by the extent of mitochondrial JC-1 uptake. Bcl-2 and Bax protein expression levels were monitored using fluorescent-labelled antibodies. Caspase activation was studied using a fluorochrome-labelled pan-caspase inhibitor (FLICA), which also allowed one to study the kinetics of the apoptotic cascade. RESULTS: After heat-treatment or irradiation of HL60 cells, a decreased delta psim as well as PS membrane expression were detectable after 8 h. Bcl-2 and Bax protein expression levels were decreased and increased, respectively, 1 h after heat-treatment or irradiation. The apoptotic rate of HL60 cells, as measured by the FLICA binding, was faster with heat-treatment as compared to gamma-irradiation. Addition of a pan-caspase inhibitor prevented PS externalization after heat-treatment but not after irradiation. The presence of a pan-caspase inhibitor did not influence the decrease of delta psim both after heat-treatment and gamma-irradiation. However, the addition of the specific caspase-2 inhibitor zVDVAD-fmk prevented the mitochondrial breakdown after heat-treatment. Inhibition of caspase-2 had no effect on the gamma-irradiation induced apoptosis. CONCLUSION: These results suggest that the commitment to apoptosis in HL60 cells after heat-treatment is started by mitochondrial membrane transition involving the Bcl-2 family members and is mainly executed in a caspase-dependent pathway. The results suggest that caspase-2 plays a key role in the heat-induced apoptosis.
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Apoptose/fisiologia , Caspases/metabolismo , Raios gama , Hipertermia Induzida , Mitocôndrias/fisiologia , Anexina A5/farmacologia , Apoptose/efeitos da radiação , Proteínas de Ligação ao Cálcio/farmacologia , Células HL-60 , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/efeitos da radiaçãoRESUMO
Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (M(w)=8 x 10(6)) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (T(d)=79 degrees C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.
Assuntos
Colágeno/química , Elastina/química , Engenharia Tecidual , Animais , Bovinos , Colágeno/ultraestrutura , Elastina/ultraestrutura , Elétrons , Microscopia Eletrônica de Varredura , ViscosidadeRESUMO
Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.
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Prótese Vascular , Colágeno/uso terapêutico , Elastina/uso terapêutico , Engenharia Tecidual/métodos , Fenômenos Biomecânicos , Técnicas de Cultura de Células , Reagentes de Ligações Cruzadas , Liofilização , Humanos , Miócitos de Músculo Liso/citologia , Porosidade , SolventesRESUMO
BACKGROUND: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness). METHODS: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor-alpha/cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation. RESULTS: TNF-alpha treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed. CONCLUSIONS: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.