LUCERO BIO: Smart Optofluidic Isolation of Spheroids for Early-Stage Drug Discovery

Project Information

LUCERO-BIO

Grant agreement ID: 101058056

DOI

10.3030/101058056

Project closed

EC signature date 26 October 2022

Start date 1 January 2023

End date 31 March 2026

Funded under

• The European Innovation Council (EIC)

Total cost

€ 1 298 712,50

EU contribution

€ 1 298 712,00

Coordinated by

Lucero AB
Sweden

Periodic Reporting for period 2 - LUCERO-BIO (LUCERO BIO: Smart Optofluidic Isolation of Spheroids for Early-Stage Drug Discovery)

Reporting period: 2024-01-01 to 2026-03-31

Summary of the context and overall objectives of the project

Lucero is working on a novel AI-assisted cell handling platform with the goal of enabling the scalable use of 3D culture in pharmaceutical drug discovery. Personalised medicine can address variations of drug efficacy and toxicity linked to different patient subgroups. Pharmaceutical companies need innovative technologies to predict the effects of drug candidates on patients before entering clinical trials. Preclinical drug procedures require that technologies be selective for the detection and isolation of patient-derived 3D cell cultures and scalable for high-throughput drug screening.

The objective of the EIC Transition project is to expand our prototype’s functionalities, adjust the design to automatically detect spheroid viability, and automate the isolation and dispensing of 3D cell culture to enhance the drug screening workflow.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Activities performed during the first 12 months focused on the transition from single-cell manipulation to sorting and isolating miniature hepatocyte spheroids.

Lucero performed phototoxicity and dragging speed studies and reported that optical tweezers are optimal in our system for sample sizes ranging between 20 micrometres (single cell) to 50 micrometres (small cell clusters). These studies accounted for key parameters such as viability and throughput.

To cover the full-size range and throughput requirements of our target customers, Lucero developed novel microfluidic designs that accommodate a broader size range. These designs include microfluidic sorting-dispensing features and on-chip reservoirs that result in a compact disposable that can host, sort, and dispense miniature spheroids.

Lucero implemented a new microfabrication method during the past year using UV resin 3D printing. This method allowed for continuous iterations and optimization of the microfluidic module for sorting-dispensing protocols in 384 well-plates.

The opening times of our high-speed microvalves together with the flow rates and pressure inside the microfluidic chip were calibrated to control the throughput and the dispensed volume. As a result, we can dispense individual spheroids in droplets of approximately 2 microlitres in the centre of each well.

Lucero has been working on a hydrogel-based solution to keep the spheroids in a known location within each well after dispensing. This will increase the flexibility for the end user because they can dispense the spheroids into their choice of commercially available well plate. Immobilizing the spheroids in a scaffold also makes them compatible with a wider range of downstream technologies for fast and efficient liquid handling.

Lucero updated the imaging configuration of the prototype by including two independent imaging paths. A top, low numerical aperture imaging path captures the real-time activity inside the microfluidic chip and works as an input to the AI sorting algorithm. A bottom imaging path monitors the dispensed sample inside the well plate.
Lucero’s machine-learning algorithm was trained to detect spheroids with experimental images of miniature hepatocyte spheroids. To make the object classification robust, other objects of interest for our microfluidic experiments, such as beads, bubbles, debris, and single cells were included in these experimental images. These images were all taken with our prototype and labelled by the Lucero team. Further, the performance of the algorithm was characterized through real-time detection and sorting of miniature hepatocyte spheroids in a realistic environment.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

3D cell cultures are promising alternatives to 2D as they show cell-cell interactions lacking in 2D. However, moving from 2D to 3D is a step up in complexity. Currently, 3D assays are too expensive to be used in early-stage drug development. Further, 3D assays are not yet reproducible enough for standard use. This is in part due to a lack of enabling technologies and standardized workflows needed to culture, handle, and analyse these new models.

Lucero aims to provide the pharmaceutical industry with the first standardized workflow for using miniature spheroids for chemistry optimization and metabolic profiling. To this end, we have:

1. AI qualification of miniature hepatocyte spheroids
Lucero has successfully developed an AI algorithm that can classify spheroids based on size and shape in real-time. This feature allows users to qualify spheroids before dispensing them into wells, which will help standardize assays using hepatocyte spheroids by ensuring that each data point they generate is relevant.

Lucero is working towards the goal of qualifying spheroids based on their viability, label-free. Further research must be done to understand and establish an industry-wide consensus on which morphological traits indicate viability and to understand how reliable and accurate the AI algorithm can be for this task.

By achieving this goal, Lucero’s value offering would extend far beyond the current state of the art. It would have a significant impact on the standardization of 3D assays across the industry while simultaneously preserving the vital biological function of the models by avoiding the use of labels.


3. Sorting and dispensing of single, miniature hepatocyte spheroids for chemistry optimization and metabolic profiling
Lucero has successfully demonstrated the ability to dispense single, miniature spheroids using our novel cell-handling platform. Further work must be done to increase the robustness, reproducibility, and automation of our system.

Further, in preparation for an on-site pilot project with our partners, our prototype must be packaged to ensure user safety and to reduce the instruments footprint in the lab.