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Charles A Garcia, Pavel G Molchanov, Martín M Hernández Torre, Jorge E Moreno Cuevas, Sergey N Cherenkevich, Andrei A Denisov, Pavel M Bulai, Victor V Boksha, Christine Lopez-Yang, Raquel Cuevas Diaz; On-chip stem cell preparation system for retinal disease treatment. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):747.
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Stem cell therapy is an emerging field in ophthalmology. To improve outcomes, our goal is to increase the specificity and quantity of stem cells through sorting. Current FACS and MACS cell sorting methodologies are costly and have limited specificity (91-95% purity of the desired cell type) as well as a low yield. We have developed a new cell sorting technique, which increases the specificity of cell lineages and total number of cells.
The technique is system-on-chip designed for long-term monitoring of cell cultures. It is a layer of electro-optical sensors, designed for long-term measurements of electrical and optical characteristics of cultured cells, as well as for cell elimination by electroporation. The system does not require human intervention in its operations. The array structure of the system allows individual monitoring and stimulation of any cell. With the use of selective dye labels, the system allows sorting of cells against specific cell markers on the basis of fluorescent signals. The important result of the electrical cell stimulation arises when the stimulating field strength reaches a sufficiently high intensity. This evokes the electroporation of the cellular membrane located on a microelectrode. Electroporation of individual cells can be achieved by choosing specific values of duration and frequency of stimulation of each microelectrode. Thus, it becomes possible to electroporate selective individual cells even in a heterogeneous culture.
We have tested our system on different cell lines and stem cell types. It has shown effective cell selection (more than 95%) by applying dye labels and close to 100% selective elimination through electroporation. Up to 60 million cells can be obtained from bone marrow aspirate after 3-4 days of sorting with high homogeneity (more than 95%) of cell lineages. We have shown accelerated cell proliferation (up to 45%) under chronic electrical stimulation. On-chip cell sorting shows many advantages in comparison to FACS and MACS: higher viability (less cell damage via reduction of shear stress), single use disposables, and no contamination issues in an easy to use, compact device.
We have developed an economical, efficient, higher yielding apparatus for cell separation for planned clinical trials, such as hESC or autologous CD34+ cells for diabetic retinopathy.
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