Executive summaries
D1.1: Report on the 2D simulation of the biosensor
In this report a 2D model of the electric field is adopted as part of an effort to simulate the dielectrophoretic phenomenon in microwells, the basic structure used in the COCHISE project. An analytical formulation is derived for a preliminary estimation of the dielectrophoretic force and compared with numerical simulations. We find that the analytical formulation is robust and serves as a predictive tool in determining the DEP force before rigorous numerical simulations.
D1.2: Design of test structures
Several test structures have been developed to support the development of the fabrication process and to allow for the experimental demonstration of important milestones of the COCHISE technology. Basic design rules and relations among all the geometrical parameters have been created, in order to guarantee the proper functionality of the prototypes. Scaling rules are also defined for both geometrical and electrical parameters.
D1.3: Report on the 3D simulation of the biosensor
In this report a 3D simulation model of the electric field, based on the solution of the Laplace
problem is adopted as part of an effort to simulate the dielectrophoretic phenomenon in microwells,
the basic structure used in the Cochise project. Starting from the computation of the electric field
with numerical simulations in Comsol 3.3, different analytical formulations of the dielectrophoretic
force are implemented, in order to develop a precise and efficient simulation strategy.
D1.4: Design of the first biosensor
In order to support high-throughput operations and experiments as required by the COCHISE
project we designed the first biosensor as a matrix of micro-well. The biosensor implements 1536
micro-laboratories on board following the standard of a 1536-well microtiter. We give a set of basic
rules for proper sizing of the micro-laboratory. We illustrate the matrix interconnection scheme and
present the basic rules to implement the signal generation and processing hardware that controls
the biosensor. We describe the layout of the first biosensor in relation with the design of the
microfluidics. We present the platform based on the biosensor, including the fluidic carrier, a
microscope, a cell dispenser, cameras control and automated motion.
D1.5: Design of the improved biosensor
The second version of the biosensor introduces several optimizations on the micro-well design and
on the system interfaced to the biosensor. Main optimizations on the biosensor architecture include
an optimized electrodes design to allow for simplified fabrication process and for trapping and
detection of cells of interest for the overall application. In addition a funnel shape on top of each
microwell was designed to guarantee simplified delivery procedures. At system level, a new
package design was conceived, featuring a fluid carrier and electrical interfaces that easily support
the biosensor substitution. Dedicated control electronics and software was designed and
implemented to support electrodes activation on microwell arrays.
D1.6: Report on test of the final biosensor
A novel biosensor architecture was developed in the latest part of the project, which allowed to address most critical issues previously found. Latest activities were aimed at providing a proof of concept of the innovative approach, by fabricating the new biosensor and by validating it through experiments. The new architecture was successfully tested and it supported a simplified cell delivery method, through microchannels integrated in the platform, and an efficient recovery method, which operates in parallel on multiple microwells, transferring the processed biological sample to standard microtiter plates. Other activities were focused on impedance-based monitoring of cell delivery in microwells, on optical detection of cell lysis, on the validation of a cooling system developed for the first version of the biosensor and on the characterization of the previous delivery method, based on the use of a microdispenser.
D2.1: Software program for the cell microdispenser
The delivery D2.1 describes a user's manual and a programmer's guide for the LabVIEW software Tethys23.vi (version 2.3) programmed by CEA to drive the cell microdispenser.
D2.2: Report on the cell delivery procedure for homogeneous cell suspension
One goal of the workpackage WP2 is to improve and transfer the CellJet microdispenser developed
previously in the Biochips Laboratory in CEA. The CellJet dispenser will be used at the University of
Bologna to deposit cells towards the microwell platform. This document presents the procedure to
achieve a correct dispense of cells.
D2.3: Fabrication of a recovery instrument
This report details the architecture and components of a recovery instrument for the collection of
cells of interest from a microwell platform. The microwells developed within the workpackage WP3
of the Cochise project have dimensions in the 300 µm range. Moreover, the liquid contained by the
wells is ~10-20 nL, which is under the capacities of standard commercial instruments. The homemade micropipette, associated with dedicated equipment, developed within WP2 to fulfil the cell recovery task is described in this document.
D2.4: Software program for the cell extraction system
The delivery D2.4 describes a user's manual and a programmer's guide for the LabVIEW software
CellsRecovery.vi (version 1.0) programmed by CEA to drive the pipeting of selected cells for the
Cochise project.
D2.5: Evaluation of electrical signatures for NK, CTL and tumor cells
This work is dedicated to investigate the existence of electrical signatures indicative of cells used in
the COCHISE project. The first phase of this work was to demonstrate feasibility of electrical
detection of flowing cells using HeLa cells, an immortal cell line derived from cervical cancer cells
which is very common in cell biology laboratories. Then, an immortalized line of T lymphocyte cells,
Jurkat cells, was used as a reference cell type for lymphocytes. Results using Jurkat cells was
extrapolated to Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) lymphocytes whose handling
is more difficult as they require specific medium conditions for culture. Finally, electrical detection of
K562 cells was successfully performed. K562 is a human immortalized myelogenous leukaemia cell
line, i.e. a cell line involved in the cancer of blood and bone marrow. K562 cells are the cells of
reference for tumor cells within the COCHISE project. In addition to HeLa cells, they were used to
demonstrate the possibility to differentiate tumor cells and lymphocytes by differential impedance
spectroscopy according to their size. The size difference between lymphocytes and tumor cells can
thus be an electrical signature to distinguish these cells in the millisecond range when they are still
flowing through a microfluidic channel.
D2.6: Report on the cell recovery procedure
This report details the architecture and components of a recovery instrument for the collection of
cells of interest from a microwell platform. The microwells developed within the workpackage WP3
of the Cochise project have dimensions in the 70-300 µm range. Moreover, the liquid contained by
the wells is ~10-20 nL, which is under the capacities of standard commercial instruments. The
home-made micropipette, associated with dedicated equipment, developed within WP2 to fulfil the
cell recovery task is described in this document.
This delivery presents the main functionalities and principles for the LabVIEW software
CellsRecovery.vi (version 1.0) programmed by CEA to drive the pipeting of selected cells for the
Cochise project.
D2.7: Report on the cell delivery procedure
for heterogeneous cell suspension
This work is dedicated to present three technical approaches investigated to dispense cells into the
microwells of the COCHISE biosensor: the CellJet microdispenser, the MicroFab dispenser, and
micropipette handling. As technical problems appeared during the project with the CellJet
dispenser, more efforts were applied on the commercial jetting device from MicroFab Technologies
as an alternative for cell deposition. Statistical dispense of single cells with this system was
demonstrated. Integration of the MicroFab dispenser within the Cochise platform included
mechanical parts, motorized stages, software, and development of an operational loading
procedure. Moreover, micropipette handling also appears as a promising technique for depositing
cells into the microwells of the biosensor. Viability of cells deposited by the MicroFab dispenser and
by the micropipette was proved. Finally, some inspection procedures of the presence of cell(s) in
the microwells are presented.
D3.1: Test structures
This report resumes the process evaluation through test structures. Different approaches are evaluated to combine together PCB-standard dielectrics (e.g. epoxy, Pyralux, Polyimide) and novel metals (e.g. Platinum, Gold and Aluminum). Here Aluminum (Al) is in the focus because of its low price, biocompatibility, wide range of available foil thicknesses and good electrical conductivity. In this document the basic steps are described to combine Al with dielectric materials creating a PCB-stack and the structuring/drilling of these materials. Furthermore, surface treatments with thiols and fluorinated acrylates were inspected to allow designing the hydrophilicity/hydrophobicity microfluidics networks required for the COCHISE Project.
D3.2: Process Concept & Material Selection
In D3.2 a process is fixed for the realization of Aluminum based PCB technology using Polyimide
and Pyralux as dielectric layers. Drilling and structuring by laser and Aluminum etching features
were added to the Al-PCB process and verified with demonstrators for WP 1. Furthermore, the
surface modifications for all relevant surfaces allow the microfluidics networks necessary to
manage biological samples in the COCHISE platform.
Biocompatibility tests had been set up together with the University of Ferrara. Possible and typical
packaging materials had been selected for testing. Tests with lymphoblastoid cell-line (LCL) and
cytotoxic T-lymphocytes (CTLs) show good biocompatibility results for selected materials.
A second process flow based on glass and powder blasting technology has been set up and will be
evaluated in a demonstrator.
D3.3: First Biosensor
In D3.3 first biosensors are shown and described. Manufacturing technology and materials used are in principle already explained in D3.2.
Therefore, only a brief overview about the technology used for sensor realization is given.
For the sensor realization Aluminum based PCB technology using Polyimide [PI] and Pyralux [Py] as dielectric layers was developed.Drilling and structuring by laser and Aluminum etching features were added to the Al-PCB process. Furthermore, the surface modifications for all relevant surfaces allow the microfluidics networks necessary to manage biological samples in the COCHISE platform. The technology was and still is developed by realizing sensor prototypes and is thus very closely adapted to later manufacturing. D3.3 shows two different types of cell levitation sensors with different geometrical aspects.
D3.4: Improved Biosensor
In D3.4 the improved biosensor is shown and described. Manufacturing technology and materials
used are in principle already explained in D3.2 and D3.3. Therefore, only a brief overview about the
technology used for sensor realization is given.
For the sensor realization Aluminum based PCB technology using Polyimide [PI] and Pyralux [Py]
as dielectric layers was developed. Drilling and structuring by laser and Aluminum etching features
were added to the Al-PCB process. Furthermore, the surface modifications for all relevant surfaces
allow the microfluidics networks necessary to manage biological samples in the COCHISE platform.
The funnel is realized in a thick Polyimide layer by drilling and therewith well matched to the PCB
manufacturing process flow. The feasibility of in inner layer connection by electroless Ni
metallization was also shown.
D3.5: Final process flow
In D3.5 the manufacturing process flow of the biosensor is described in detail. Principles of the
manufacturing technology and materials used are already explained in D3.2 and D3.3.
For the sensor realization Aluminum based PCB technology using Polyimide [PI] and Pyralux [Py]
as dielectric layers was developed. Drilling and structuring by laser and Aluminum etching features
were added to the Al-PCB process. Furthermore, the surface modifications for all relevant surfaces
allow the microfluidics networks necessary to manage biological samples in the COCHISE platform.
The funnel is realized in a thick Polyimide layer by drilling and therewith well matched to the PCB
manufacturing process flow. The feasibility of in inner layer connection by electroless Ni
metallization as well as the opening of inner Al layers is also shown.
D3.6: Demonstrator of biosensor
For the realization of a dielectrophoresis enhanced microwell device a technology based on standard PCB technology has been developed. As materials Aluminum, Polyimide and Pyralux have been selected with focus on biocompatibility and process compatibility. For these materials a process flow consisting of lamination, Al structuring by wet etching, microwell and via formation by laser drilling, funnel drilling and via metallization was fixed. Surface treatments have been evaluated for hydrophobic and hydrophilic modification. The technology was developed along demonstrators for general proof of concept. Different test structures have been developed especially for investigation of optimized cell delivery. Therefore funnels, pools and a microfluidic glass layer on top of the device have been realized. For the overall system a fluidic carrier for nutrient solution supply and cooling functionality has been developed and adapted for the different sensor demonstrators.
Also the technology up scaling to the final biosensor with 1536 wells for detection of cell-to-cell interactions has been successfully demonstrated.
D4.1: Protocols for handling single cells and in vitro expansion
Several cells/cell lines were employed to generate protocols for (a) culturing single cells; (b)
demonstrating their ability to grow and express biological parameters; (c) demonstrating the
maintenance of their phenotype after expansion. The cell lines employed were: (a) human cytotoxic
T lymphocytes; (b) human target lymphoblastoid cells; (c) the NKL natural killer cell line; (d) the .221
and .221-G1 cell lines and (e) two murine lymphoma cell lines (RMA and RMA-S) differentially
susceptible to in vivo NK-mediated lysis.
Due to the fact that COCHISE prototypes are still not available, the cloning of single cells were
performed by limiting dilutions in 96-wells plates. For the development of assays to detect biological
activity of low numbers of cells, the Bio-plex instrument was employed. For determinations of the
biological activity of in vitro expanded cells, several biological assays were employed, including CTL
mediated cytolysis (in vitro) and NK-mediated cytolysis (in vitro and in vivo).
D4.2: Protocols for handling single cells and in vitro expansion
We determined the effects of materials on biological functions of two cell lines growing in suspension
(the human chronic myelogenous K562 cells and the LCL leukemic cell lines) and one cell line growing
attached to the culture plates (the cystic fibrosis IB3-1 cell line). We analyzed the following parameters:
cell proliferation of all the cell lines studied, erythroid differentiation of K562 cells, CTL-mediated lysis of
LCL cells, release of cytokines/chemokines by IB3-1 cells. Due to the fact that COCHISE prototypes are
still in a phase of design and fabrication, this information is of great interest. We verified the possibility to
follow lysis of single cells by analyzing the possible increase of non specific lysis of cells targets of NK
cells and CTL after continuous or pulsed contact with the materials under study. We verified with
additional experiments with respect to D4.1 the possibility to follow CTL-mediated lysis of LCL after
pulse of the cells with calcein.
D4.3: Protocol allowing the isolation of single T cells
We set up a method to manually micromanipulate individual resting or activated human T
lymphocytes and to subsequently analyze the level of expression of a few selected genes on these
single cells.
D4.4: Protocol for isolation of single lytic tetramer-positive T cells
We set up a method to label, sort, activate and micromanipulate single human CD8 T lymphocytes
of a defined specificity, in such a way that the expression of a few genes can then be analyzed in
each individual cell.
D4.5: Protocols allowing isolation and in vitro expansion of single NK cells
The analysis of the effects of biomaterial on biological functions has been performed on the
following experimental systems: (a) rat ippocampal cells differentiating in neurons and astrocytes;
(b) human IB3-1 tracheal cells producing a variety of interleukins and cytokines when treated with
TNF-alpha; (b) K562 cells induced to erythroid differentiation and production of hemoglobin by
mithramycin and (c) CTL mediated LCL-lysis. We have conclusively and reproducibly analysed all
the materials using long-term exposures, supporting the conclusion that among the 24 materials
tested (7 employed in surface treatments, 14 dielectrics/adhesives, and 5 metals) some display
strong effects of biological functions.
Several cells/cell lines were employed to validate the COCHISE Sensor platform: (a) human
cytotoxic T lymphocytes; (b) human target lymphoblastoid cells; (c) two Natural Killer cell lines, NKL
and NK-YST; (d) the 221 and 221-G1 cell lines and (e) two murine lymphoma cell lines (RMA and
RMA-S) differentially susceptible to in vivo NK-mediated lysis.
D5.1: Report on dissemination strategies
The COCHISE project is planning dissemination of the project results and a set of activities aimed at raising the level of public awareness about the perspectives of biosensor technology in oncology therapy. Dissemination strategy includes the following main points: definition of the objectives, identification of the target groups of the dissemination activity, identifications of a set of media to perform it, definition and scheduling of the dissemination activity in a plan.
D5.2: Preliminary dissemination report
The COCHISE project provided dissemination of the project results and a set of activities aimed at raising
the level of public awareness about the perspectives of biosensor technology in oncology therapy.
Dissemination strategy, as described in our previous deliverable D5.1, includes the following main points:
definition of the objectives, identification of the target groups of the dissemination activity, identifications of
a set of media to perform it, definition and scheduling of the dissemination activity in a plan.
D5.3: Final dissemination report
The COCHISE project provided dissemination of the project results and a set of activities aimed at
raising the level of public awareness about the perspectives of biosensor technology in oncology
therapy. Dissemination strategy, as described in our previous deliverables D5.1 and D5.2, has included
the following main points: definition of the objectives, identification of the target groups of the
dissemination activity, identification of a set of media to perform it, definition and scheduling of the
dissemination activity in a plan.
In the present final report the dissemination events carried out in the period M19-M36 of the COCHISE
project are listed and described in detail.
D5.4: Preliminary exploitation plan
The COCHISE consortium is planning exploitation of the project results. The exploitation strategy includes
the following main points:
- Development of a detailed exploitation plan for the use of the knowledge produced in the frame of
the COCHISE project;
- Protection of the knowledge produced in the frame of the COCHISE project;
- Assessment of the expected socio-economic impact of the knowledge and technology generated
in the frame of the COCHISE project;
- Conducting feasibility studies for the creation of one or more spin-off companies that could exploit
the results of the COCHISE project;
- Carrying out take-up activities to promote the early or broad applications of the state of the art
technologies.
The current deliverable report provides an overview of the COCHISE exploitation plan and strategy at midterm
in the project.
D5.5: Final exploitation plan
The present Deliverable reports a description of the exploitation plans of the COCHISE partners which
identified real opportunities deriving from the activities and the outcomes of the project. The partners
describe here possible applications of the technology developed within the COCHISE project as well as
possible interests of external companies and institutions in the technology discovered and developed by
each partner.
