Licht inzetten in microwell-systemen voor de isolatie van individuele cellen

Monoclonal antibodies (mAbs), valued for their high target-specificity, represent the largest class of biopharmaceuticals and are widely used in the treatment of diseases such as cancer, autoimmune disorders, and cardiovascular conditions. Fully human (FH) mAbs are particularly appealing because their human origin often reduces immunogenicity and increases clinical relevance, making them a key focus for therapeutic development. A promising route to generate FH-mAbs is direct cloning from human B cells, though a major challenge remains the identification and isolation of the specific B cells of interest. These target cells can be identified based on either membrane-bound Abs (present on the cell surface) or Abs secreted by the cells into their surroundings. Miniaturized microfluidic platforms hold great promise for advancing the identification and isolation of antigen (Ag)-specific Ab-expressing cells. However, current technologies face several limitations, including inefficient or cumbersome off-chip cell retrieval, limited multiplexing capabilities for detecting secreted Abs, and insufficient adaptability to varying experimental demands (e.g., well shape and dimensions, or molecules used for secretion detection). To overcome these challenges, this project focused on developing innovative microwell-based microfluidic platforms, that allow for the interrogation of cells to determine whether they express disease-relevant Abs, and to retrieve selected cells in a high-precision, single-cell manner. This microwell-based approach pursued two key objectives: (i) the development of light-based strategies for advanced single-cell screening, and (ii) the implementation of light-based methods for precise and efficient cell retrieval. These efforts led to the development of two complementary platforms: one that uses optical tweezers to retrieve cells identified by their membrane-bound or secreted Abs, and another that relies on a photodegradable hydrogel (HG) to retrieve cells, selected based on membrane-bound Abs. The optical tweezer-based platform (FLUIDOT) was first developed to identify target cells via membrane-bound Abs and enable their precise single-cell retrieval off-chip. Starting from blood samples of convalescent COVID19-patients, this system facilitated the discovery of FH-mAbs binding the receptor-binding domain (RBD) of SARS-CoV-2 and exhibit in vitro virus-neutralizing activity. Next, the platform was adapted to also enable the identification of cells based on multiplex monitoring of their secreted Abs. This required transitioning from OSTE+ to PDMS microwells, which allowed for high‑resolution spatial photopatterning of disease‑relevant Ags directly on the microwell walls. Binding of secreted Abs to these Ags resulted in spatially resolved fluorescent signals that indicate Ag‑specificity. This setup successfully identified SARS‑CoV‑2‑specific hybridomas in singleplex, duplex, and triplex formats, while also demonstrating the platform's ability for time-resolved screening of secreted Abs. To further enhance retrieval throughput and reduce the need for expert handling, we developed a second platform based on photodegradable HGs. The envisioned three-layer microfluidic device features microwells with bottoms composed of a light-sensitive Fe(III)-alginate HG. Upon identification of target cells, localized light exposure degrades the HG beneath the selected cells, allowing them to fall into a retrieval channel beneath the wells and flow toward the outlet for downstream analysis. In support of this system, we first characterized the HG's stiffness and photodegradation behavior by varying Fe³⁺ concentration, alginate concentration and alginate type. We then integrated an optimized HG formulation, balancing structural stability with fast photodegradation, into a working microfluidic prototype. As proof of concept, the device was used to retrieve fluorescent beads via localized HG degradation. While this platform requires further optimization (e.g., integrating microwells, tailored to fit single cells), it demonstrates a promising direction toward automated, scalable, and user-independent cell retrieval strategies. In addition to its application in FH-mAb discovery, the presented platforms offers broader potential for other applications in the context of single-cell secretome analysis. Its strengths lie in its design flexibility, ranging from microwell geometry to retrieval strategy and the types of capture molecules used for secretion detection. These innovations not only accelerate the discovery of therapeutic Abs but also lay the groundwork for future applications in single-cell analysis.