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1.
Integr Biol (Camb) ; 7(11): 1396-401, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26235126

ABSTRACT

Apoptosis is characterised by many cellular events, but the standard Annexin-V assay identifies two; the transfer of the phospholipid phosphatidylserine (PS) from inner to outer leaflets of the plasma membrane, acting as an "eat me" signal to macrophages, and the permeabilisation of the plasma membrane. In this paper we compare the results from the Annexin-V assay with electrophysiology data obtained in parallel using dielectrophoresis, which highlights two changes in cell electrophysiology; a change in cytoplasmic conductivity which correlates with PS expression, and a membrane conductance spike that correlates with permeabilisation. Combining results from both methods shows a strong inverse relationship between conductivity and PS externalisation. One mechanism which may explain this correlation is related to intracellular Ca(2+), which is known to increase early in apoptosis. PS expression occurs when enzymes called scramblases swap external and internal phospholipids, and which are usually activated by Ca(2+), whilst the change in cytoplasmic conductivity may be due to K(+) efflux from intermediate conductance (IK) ion channels that are also activated by Ca(2+).


Subject(s)
Apoptosis , Electrophysiology/methods , Flow Cytometry/methods , Annexin A5/chemistry , Calcium/chemistry , Cell Membrane/metabolism , Cytoplasm/metabolism , Electrophoresis , Humans , Jurkat Cells , Phosphatidylserines/chemistry , Phospholipids/chemistry , Propidium/chemistry , Staurosporine/chemistry
2.
Analyst ; 140(15): 5198-204, 2015 Aug 07.
Article in English | MEDLINE | ID: mdl-26086875

ABSTRACT

Despite the accessibility of the oral cavity to clinical examination, delays in diagnosis of oral and oropharyngeal carcinoma (OOPC) are observed in a large majority of patients, with negative impact on prognosis. Diagnostic aids might help detection and improve early diagnosis, but there remains little robust evidence supporting the use of any particular diagnostic technology at the moment. The aim of the present feasibility first-in-human study was to evaluate the preliminary diagnostic validity of a novel technology platform based on dielectrophoresis (DEP). DEP does not require labeling with antibodies or stains and it is an ideal tool for rapid analysis of cell properties. Cells from OOPC/dysplasia tissue and healthy oral mucosa were collected from 57 study participants via minimally-invasive brush biopsies and tested with a prototype DEP platform using median membrane midpoint frequency as main analysis parameter. Results indicate that the current DEP platform can discriminate between brush biopsy samples from cancerous and healthy oral tissue with a diagnostic sensitivity of 81.6% and a specificity of 81.0%. The present ex vivo results support the potential application of DEP testing for identification of OOPC. This result indicates that DEP has the potential to be developed into a low-cost, rapid platform as an assistive tool for the early identification of oral cancer in primary care; given the rapid, minimally-invasive and non-expensive nature of the test, dielectric characterization represents a promising platform for cost-effective early cancer detection.


Subject(s)
Mouth Neoplasms/diagnosis , Mouth/pathology , Oropharyngeal Neoplasms/diagnosis , Oropharynx/pathology , Biopsy , Early Detection of Cancer/methods , Electrophoresis/methods , Humans , Mouth Mucosa/pathology , Mouth Neoplasms/pathology , Oropharyngeal Neoplasms/pathology
3.
J Tissue Eng Regen Med ; 9(2): 162-8, 2015 Feb.
Article in English | MEDLINE | ID: mdl-23225773

ABSTRACT

Dielectrophoresis (DEP) is a non-invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell-based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG-63 and SAOS-2) and an immunoselected enriched skeletal stem cell fraction (STRO-1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG-63, SAOS-2 and STRO-1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof-of-concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations.


Subject(s)
Bone and Bones/cytology , Electrophoresis/methods , Stem Cells/cytology , Antigens, Surface/metabolism , Bone Marrow Cells/cytology , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Separation , Cytoplasm/metabolism , Humans , Light , Osteocytes/cytology , Osteosarcoma/pathology
4.
Anal Bioanal Chem ; 401(8): 2455-63, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21877186

ABSTRACT

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C(Eff)) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C(Eff) of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C (Eff) and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.


Subject(s)
Carcinoma, Squamous Cell/pathology , Keratinocytes/pathology , Lab-On-A-Chip Devices , Mouth Neoplasms/pathology , Cell Line, Tumor , Cells, Cultured , Electric Impedance , Electrophoresis/methods , Humans , Mouth Mucosa/pathology
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