TITAN Project: Microfluidic and Sensing Tools for Immunotherapy †

: The TITAN project aims to improve immunotherapy, targeting the efficiency of methods to obtain genetically engineered T cells. Immunotherapy has achieved great success in clinical trials, but it is currently very expensive in terms of time required for analysis, reagents, and samples. TITAN aims to the continuous sampling of critical quality attributes, in order to quickly recognize deviations from the desired range and take appropriate corrective actions on process parameters for an optimal outcome. To achieve its aims, TITAN is currently developing microfluidic and sensing tools for the accurate and efficient real-time monitoring of the T cells amplification process.


Introduction
Immunotherapy with genetically engineered T cells has achieved some spectacular success in clinical trials and has obtained marketing approval.A key need is the widespread availability of small-scale bioreactors providing in-process monitoring.TITAN aims to achieve continuous sampling of critical quality attributes, to quickly recognize deviations from the desired range and take appropriate corrective actions [1].Parameters to be verified and related tools include bacterial contamination [2]; counting of cells by microfluidic and electrical detection; ratio of live/dead cells to be identified by gold nanoparticles on a capacitive sensor; cytokines production identified by electrochemical methods; T cell function tests through the production of spheroids into droplet microfluidic devices; and the electrochemical detection of metal nanoparticles (NPs) through anodic stripping voltammetry.

Materials and Methods
For each of the developed strategies for sensing and microfluidics, different technologies have been addressed, having the common purpose of using as low a volume of sample as possible, since it would originate from patient-derived tissues.In particular, we developed the following topics: 1.
Bacterial contamination check.

qPCR for Contamination Check on a POC Platform
An innovative protocol for qPCR working with a very low volume of raw sample (0.25 µL) and avoiding the step of nucleic acid extraction was optimized for a point-of-care (POC) instrument (scheme depicted in Figure 1).The optimized extraction-free protocol in minimal analysis volume, combined with the possibility to perform the qPCR assay on a portable device, demonstrates the possibility to avoid the time-consuming bacterial cell culturing.The achieved amplification of the Gram+ Staphylococcus aureus was performed in a 5 µL total volume of reaction, reaching the limit of detection of 1 CFU/mL.
protocol in minimal analysis volume, combined with the possibility to perform the qP assay on a portable device, demonstrates the possibility to avoid the time-consumi bacterial cell culturing.The achieved amplification of the Gram+ Staphylococcus aureus w performed in a 5 µL total volume of reaction, reaching the limit of detection of 1 CFU/m

CAR-T Cells Efficiency Tests
Lymphocytes spilled out from bioreactor will be focused and counted through simulated and optimized spiral channel (Figure 2A,B), which will help in the single c identification.Detection of Interleukins IL6 and IL10 is performed down to 2 ng/mL immobilizing antibodies on the surface of microfabricated gold electrodes (Figure 2C), b this limit can be improved by nanostructuring the surface of electrodes.Interleukins a cytokine detection will be integrated into functional assays to evaluate the efficiency CAR-T cells in killing tumor cells.To improve reproducibility and standardization of t assays, a microfluidic method to produce spheroids of the same dimensions was improv based on computational simulation and fabrication of a droplet generator working with phase emulsion of Matrigel-containing cells and oil (Figure 2D,E).To complete t functional test for CAR-T, tumor cells are loaded with gold NPs and their release af action of activated lymphocytes is detected through anodic stripping voltammetry.

CAR-T Cells Efficiency Tests
Lymphocytes spilled out from bioreactor will be focused and counted through a simulated and optimized spiral channel (Figure 2A,B), which will help in the single cell identification.Detection of Interleukins IL6 and IL10 is performed down to 2 ng/mL by immobilizing antibodies on the surface of microfabricated gold electrodes (Figure 2C), but this limit can be improved by nanostructuring the surface of electrodes.Interleukins and cytokine detection will be integrated into functional assays to evaluate the efficiency of CAR-T cells in killing tumor cells.To improve reproducibility and standardization of the assays, a microfluidic method to produce spheroids of the same dimensions was improved, based on computational simulation and fabrication of a droplet generator working with 2-phase emulsion of Matrigel-containing cells and oil (Figure 2D,E).To complete the functional test for CAR-T, tumor cells are loaded with gold NPs and their release after action of activated lymphocytes is detected through anodic stripping voltammetry.

Figure 1 .
Figure 1.Sketch of the qPCR assay procedure.

Figure 1 .
Figure 1.Sketch of the qPCR assay procedure.

Figure 2 .
Figure 2. (A,B) Spiral channel for cell focusing.Red cycle highlight the position of cells in th channel (C) Interleukin detection by DPV.(D) Simulated device for droplets generation wit emulsion of oil (green) in water (blu); (E) plastic device realized for droplet generation (red) i water.