Christophe Lautrette

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

Microwave biosensors for identifying cancer cell aggressiveness grade

This paper illustrates the potential of microwave frequencies for biological analysis. Once penetrating inside biological cells, microwaves can interact with their intracellular content and inform on their safe or malignant state. This work demonstrates that their cancer grade (i.e. aggressiveness level) can also be identified by this way. Hence, based on permittivity measurements on three colon cancer cell lines loading RF resonators, the presented results show significant differences of electromagnetic signature in the cancer grade of analyzed cells. This sensing method appears very promising to develop new powerful tools for early cancer diagnostic.

Tunable frequency resonant biosensors dedicated to dielectric permittivity analysis of biological cell cytoplasm

This paper presents an improved design of resonant biosensor, dedicated to dielectric analysis on biological cells at microwave frequencies. Such sensor uses the capability of microwaves to penetrate inside biological cells in order to interact with their intracellular content. Hence, individual dielectric properties of the cell cytoplasm can be known and then used as a signature of the cell pathological state (living or dead, malignant or safe...). In this paper is introduced a continuously tunable frequency sensor prototype, able to perform an accurate dielectric analysis over at least a 1GHz bandwidth while keeping enough sensitivity to detect and analyze a single cell. As a proof of concept, permittivity measurements have been led on calibrated size polystyrene beads: achieved results show good agreement with expected permittivity values. Finally experiments on Glioblastoma cells will be presented.

Sedimentation field flow fractionation monitoring of in vitro enrichment in cancer stem cells by specific serum-free culture medium.

The development of methods to enrich cell populations for cancer stem cells (CSC) is urgently needed to help understand tumor progression, therapeutic escape and to evaluate new drugs, in particular for colorectal cancer (CRC). In this work, we describe the in vitro use of OncoMiD for colon, a CRC-specific primary cell culture medium, to enrich CRC cell lines in CSC. Sedimentation field flow fractionation (SdFFF) was used to monitor the evolution of subpopulations composition. In these models, medium induced a loss of adherence properties associated with a balance between proliferation and apoptosis rates and, more important, an increased expression of relevant CSC markers, leading to specific SdFFF elution profile changes.

Oncogramme, a new individualized tumor response testing method: application to colon cancer

Colon cancer is the second leading cause of cancer-related death in industrialized countries. Many anti-cancer researches are consequently performed and individualized tumor response testing (ITRT) methods are now used to individualize patient chemotherapeutic administrations. Then, a new ITRT method, Oncogramme, was developed for colon cancer. Colon tumor fragments from different patients were dissociated and seeded in a defined culture medium. Cell preparation process as well as culture medium allowed high cell viability and a good primary culture success rate. After treatment of isolated tumoral cells by chemotherapeutics alone or in combination, cytotoxicity was determined by cell death assay allowing the Oncogramme establishment, which was validated by statistical analysis. Indeed, significant results were obtained such as different profile for each patient’s cells with various drugs, and variability between patient’s cells in the response to each drug. Procedure described here to obtain the Oncogramme is a new, fast and technically reliable ITRT method applied to colon cancer. For an individualized cancer treatment use, this test should be further validated by a phase I clinical trial.

High frequency microfluidic biosensors for intracellular dielectric spectroscopy

This paper deals with the development and characterization of a high frequency (HF) label-free microfluidic biosensor for the non-invasive analysis of cell intracellular properties. The presented microfluidic biosensor is based on a band pass filter architecture made of thick gold electrodes designed to ensure a high sensitivity to cells flowing in the microfluidic channel. In a first step, to prove the feasibility of the proposed approach, HF measurements have been successfully achieved on polystyrene beads. Then, combining HF measurements with dielectrophoresis forces, to trap cells in the sensitive area, it has been possible to characterize cell dielectric properties without any denaturation. We demonstrate here the proof of concept of using high frequency impedance spectroscopy to analyze single cells in a microfluidic environment.

Improved sedimentation field-flow fractionation separation channel for concentrated cellular elution

SdFFF is now commonly used for cell sorting. Nevertheless, as with many other separation methods, SdFFF Hyperlayer elution leads (1) to sample dilution resulting in cell loss which could restrict further use; and (2) to a high output flow rate impacting detector sensitivity and selectivity. In order to limit these problems, we proposed modifications of the SdFFF separation channel consisting both in downscaling and the insertion of an outlet stream splitter. This last system corresponded to a strip which divides the flow rate output into two parts, one containing concentrated cells in a reduced volume and flow rate, the other containing the excess mobile phase useless for further cell manipulation, detection and characterization. For the first time we have shown that splitter implementation and downscaling respected channel flowing and resulted in Hyperlayer elution of around 95% of cells in less than 50% of input flow rate. Improved cell sorting was demonstrated by enrichment (∼10 times) of cancer stem cells from WiDr cells with two times less quantity of injected cells.