Desarrollo y fabricación de un filtro microestructurado para células tumorales circulantes
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Comisión Nacional de Energía Atómica. Instituto de Tecnología Sabato
Resumen
El cáncer es a día de hoy la principal causa de muerte en el mundo. La detección temprana
del cáncer implica una mayor probabilidad de respuesta al tratamiento y supervivencia; y el
aislamiento de células tumorales circulantes de la sangre surge como una posible vía de diagnóstico
temprano en lo que se denomina biopsia líquida. En este trabajo se estudió el desarrollo, la fabricación
y la evaluación de desempeño de un dispositivo de filtrado a través de una membrana microporosa
delgada biocompatible, que permitiría llevar a cabo el enriquecimiento, la clasificación y la
cuantificación de células tumorales circulantes a partir de sus propiedades físicas diferenciales,
brindando así una herramienta de diagnóstico temprano de diferentes tipos de cáncer y de monitoreo
de la progresión de la enfermedad. Para ello, se evaluaron tres rutas de microfabricación de
membranas microporosas: (1) depósito electroquímico de cobre y recubrimiento de titanio por
evaporación térmica; (2) depósito electroquímico de níquel; y (3) fotolitografía. Su caracterización
se hizo a través de técnicas tales como microscopía óptica y electrónica, espectroscopia por energía
dispersiva y perfilometría óptica. Se estudió la citocompatibilidad de los materiales utilizados y se
efectuó una caracterización funcional inicial del dispositivo.
El recubrimiento de membranas de cobre con titanio no ha sido completamente satisfactorio,
obstruyendo parcialmente los poros y presentando una pobre adherencia. Aun así, permitió la
utilización de las membranas en ensayos de filtrado. La fabricación por depósito electroquímico de
níquel en las condiciones utilizadas produjo membranas frágiles y no manipulables, principalmente
por la presencia de altas tensiones residuales en el depósito. La fabricación por fotolitografía brindó
un resultado prometedor, pudiendo obtenerse membranas enteras con poros definidos. Sin embargo,
dado el bajo espesor de resina utilizado, dichas membranas resultaron no ser manipulables. De los
ensayos de citocompatibilidad se obtuvieron resultados poco concluyentes, con altos márgenes de
error relativo. No pudo establecerse un orden de mérito de citocompatibilidad de los diferentes
materiales, aunque se detectó una aparente tendencia del aluminio a disminuir la viabilidad celular, a
diferencia de los demás materiales ensayados, que mantuvieron una viabilidad celular de orden
comparable al ensayo control. Los ensayos de caracterización funcional del dispositivo encontraron
como principal obstáculo la imposibilidad de disgregar completamente los cúmulos celulares. La
adición de un quelante de cationes calcio resultó perjudicial, pues se observó la destrucción total de
las células al filtrar en estas condiciones. En cuanto a la velocidad de deformación, una primera
aproximación parece indicar que el caudal óptimo de filtrado se encuentra acotado entre 90 y 190
μL/min. Por último, se presentan dos alternativas de rediseño de la unidad filtrante, de modo de
obtener una cámara de filtrado biocompatible. Todo esto indicaría que se han obtenido avances
significativos en el logro del objetivo planteado en este trabajo.
Cancer is today the leading cause of death in the world. Early detection of cancer implies a greater probability of response to treatment and survival; and the isolation of circulating tumor cells from the blood emerges as a possible way of early diagnosis in what is called liquid biopsy. In this work, the development, manufacture, and performance evaluation of a filtering device through a biocompatible thin microporous membrane was studied, which would allow the enrichment, classification, and quantification of circulating tumor cells from their differential physical properties, thus providing a tool for early diagnosis of different types of cancer and monitoring the progression of the disease. To do so, three microfabrication routes of microporous membranes were evaluated: (1) copper electroplating and titanium coating by thermal evaporation; (2) nickel electroplating; and (3) photolithography. Its characterization was made through techniques such as optical and electronic microscopy, dispersive energy spectroscopy and optical profilometry. The cytocompatibility of the materials used was studied and an initial functional characterization of the device was carried out. The coating of copper membranes with titanium has not been completely satisfactory, partially clogging the pores and presenting poor adhesion. Even so, it allowed the use of the membranes in filtration tests. Nickel electroplating manufacturing under the conditions used produced fragile and non-manipulable membranes, mainly due to the presence of high residual stresses in the deposit. Fabrication by photolithography provided a promising result, being able to obtain whole membranes with defined pores. However, given the low thickness of resist used, these membranes turned out to be non-manipulable. The use of an alternative resist to make thicker membranes was not successful. Inconclusive results were obtained from the cytocompatibility assays, with high relative error margins. An order of merit of cytocompatibility of the different materials could not be established, although an apparent tendency of aluminum to decrease cell viability was detected, unlike the other materials tested, which maintained a cell viability of comparable order to the control assay. The functional characterization tests of the device found the impossibility of completely disaggregating cell clusters as the main obstacle. The addition of a calcium cation binder was detrimental since the total destruction of the cells was observed when filtering under these conditions. As regards the strain rate, a first approximation seems to indicate that the optimal flow rate is between 90 and 190 μL/min. Finally, two alternatives to redesign the filter unit are presented, in order to obtain a biocompatible filter chamber. All of this would indicate that significant progress has been made in achieving the objective set out in this work.
Cancer is today the leading cause of death in the world. Early detection of cancer implies a greater probability of response to treatment and survival; and the isolation of circulating tumor cells from the blood emerges as a possible way of early diagnosis in what is called liquid biopsy. In this work, the development, manufacture, and performance evaluation of a filtering device through a biocompatible thin microporous membrane was studied, which would allow the enrichment, classification, and quantification of circulating tumor cells from their differential physical properties, thus providing a tool for early diagnosis of different types of cancer and monitoring the progression of the disease. To do so, three microfabrication routes of microporous membranes were evaluated: (1) copper electroplating and titanium coating by thermal evaporation; (2) nickel electroplating; and (3) photolithography. Its characterization was made through techniques such as optical and electronic microscopy, dispersive energy spectroscopy and optical profilometry. The cytocompatibility of the materials used was studied and an initial functional characterization of the device was carried out. The coating of copper membranes with titanium has not been completely satisfactory, partially clogging the pores and presenting poor adhesion. Even so, it allowed the use of the membranes in filtration tests. Nickel electroplating manufacturing under the conditions used produced fragile and non-manipulable membranes, mainly due to the presence of high residual stresses in the deposit. Fabrication by photolithography provided a promising result, being able to obtain whole membranes with defined pores. However, given the low thickness of resist used, these membranes turned out to be non-manipulable. The use of an alternative resist to make thicker membranes was not successful. Inconclusive results were obtained from the cytocompatibility assays, with high relative error margins. An order of merit of cytocompatibility of the different materials could not be established, although an apparent tendency of aluminum to decrease cell viability was detected, unlike the other materials tested, which maintained a cell viability of comparable order to the control assay. The functional characterization tests of the device found the impossibility of completely disaggregating cell clusters as the main obstacle. The addition of a calcium cation binder was detrimental since the total destruction of the cells was observed when filtering under these conditions. As regards the strain rate, a first approximation seems to indicate that the optimal flow rate is between 90 and 190 μL/min. Finally, two alternatives to redesign the filter unit are presented, in order to obtain a biocompatible filter chamber. All of this would indicate that significant progress has been made in achieving the objective set out in this work.