Devices for the capture of rare cells from biological samples for diagnostic purposes

Abstract

The chance of surviving to a disease often depends on early diagnosis and effective therapy. In the field of early prenatal diagnosis, micromanipulation is a reliable technique for manual selection and isolation of rare fetal cells in maternal biological fluids for molecular or cytogenetic analysis. This technique allows obtaining pure cell populations for analysis, but it is expensive and time consuming, as it requires qualified and experienced staff and specific equipment [1]. This research aims at making the prenatal diagnosis more economical and reproducible in the hospital environment, by creating a device that allows selecting rare cells from biological samples in a semi-automated way. The device consists in electrospun nanofiber mats with surface functional groups conjugated to antibodies capable of selectively binding to the antigens present on the surface of target cells. Nanofiber mats were produced from polymer mixtures of Nylon 6.6 and Polyacrylic Acid (PAA) in a suitable solvent, with or without the addition of a third polymer, synthetized in house, that should prevent non-specific cell binding. The first phase of the work was devoted to the determination of the operating parameters for electrospinning in order to optimize the morphology of the mats, their mechanical resistance and handling characteristics. Bioconjugation protocols, based on EDC/NHS (1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride)/(Sulfo-(N- hydroxysulfosuccinimide) chemistry were developed to provide the electrospun mats with specific cell capture abilities. In order to facilitate the recognition of the antibody by the receptors expressed by the target cells, the functional groups of the mat were previously reacted to a linker that acts as a spacer arm. Antibodies labelled with fluorescent probe were used to be able to assess the success of the conjugation reactions by fluorimetry and spectrofluorimetry analyses. Protocols for cell capture tests on antibody-decorated mats were devised using different cell suspensions: fetal cells (CF), mesenchymal stem cells (MSC) and lymphocytes (WBCs, White Blood Cells). The results of the capture tests were obtained by observing the mats under the optical and electron microscopes (SEM). References [1] G. Makrydimas, G. Damiani., C. Jakil, V. Cigna., M. Orlandi, F. Picciotto, K. H. Nicolaides, 2020, Ultrasound in Obstetrics & Gynecology, 56(5), 672-677.