Chair: Doug Auld, Ph.D. (Novartis)
Select-to-Screen: Proximity-Driven In-Gel Encoded Library Synthesis and Screening –
Brian Paegel, Ph.D. (University of California Irvine)
Encoded library technologies typically involve affinity selection to identify hits, which identifies binding but not necessarily function. Transfer of encoded libraries to solid-phase enables activity-based hit generation using high-throughput screening assays. This has been accomplished for DNA-encoded libraries, but not other encoded library formats. This lecture will discuss the first steps toward enabling activity-based mRNA display library screening. DNA-functionalized magnetic microspheres are used to template via bulk emulsification the formation of uniform hydrogel particles. The DNA templates are transcribed and the product mRNA is captured locally in the gel via hybridization. The captured mRNA is then translated and puromycin capture results in polyvalent, monoclonal display of the translated peptide (100 nM peptide in gel by HiBiT quantitation). The beads are analyzed by FACS and enrichments of > 50-fold were observed in a model screen. The ultra-miniaturized gel particle library format scalably transfers output from selection to focused libraries for functional screening.
A Tale About Shoaling Bio-inspired Materials: Enabling High-Throughput Automated 3D Cell Culture –
Amir Nasajpour, Ph.D. (Twister Bioscience)
Entropic Biosciences, Inc. has designed a proprietary scaffold-free technology to grow high-throughput, automated, self-standing 3D cellular aggregates. We have used this platform technology to generate spheroids from various cancer cell types. This enables precision medicine and testing potential chemotherapeutics against 3D models derived from individual patients’ cancer and other verticles, including cellular agriculture and tissue engineering applications.
A Microfluidic DNA-encoded Library Approach to Enable Phenotypic Screening for Small Molecule
Direct Degraders of Proteins – Anastasia Velentza (Plexium)
Patient-derived primary cells offer valuable clinical information and hold immense potential for personalized medicine and disease diagnosis. Transgenic modification of these patient cells for ex vivo expansion may provide crucial molecular insights into diseases that the patient is suffering from and foster the development of novel therapeutic targeting strategies. However, existing transfection approaches for genetic cargo delivery to primary cells suffer from drawbacks such as cell viability and transfection rates, significantly limiting applications in clinical settings. The lack of established routines to assess the functionality of primary cells isolated from patient samples hinders the clinical integration of personalized therapy approaches.
To overcome these challenges, we present an innovative platform tailored for clinical sample testing designed to process patient-derived cells, and sequentially transfect genetic constructs. This entire procedure is seamlessly integrated onto a microfluidic chip. The platform utilizes vortex cell purification technology [1,2] to isolate primary cells based on biophysical size-based markers, ensuring the preservation of patient cells’ phenotype and integrity throughout the isolation process. Within primary cell trapping reservoirs on the microfluidic device, patterned microscale electrodes generate localized electric fields sufficient to electroporate trapped cells for controlled delivery of genetic cargo using low voltages (VPP < 40V) compared to bulk electroporation systems. The platform features 144 cell-trapping reservoirs, with 3.6-fold enhancement in the throughput over our previous prototype [3], enabling rapid sample processing. The underlying micro-circuitry is designed to minimize chamber-to-chamber voltage drop variations and counteract electrical resistance to support electroporation via AC square waves with tunable electroporation parameters, generating electric field strengths of up to 1.5 kV/cm. By formulating an in-house electroporation buffer, designed to enhance cell viability and achieve optimal cell membrane states during electroporation, the platform successfully delivers a wide range of transgenes. This includes robust expression of fluorescent reporter and chromosomal stability constructs, showcasing its versatility as a gene delivery tool. With these capabilities, it becomes an excellent choice for studying pathological molecular pathways and beyond.
In conclusion, our device offers transformative potential in healthcare settings, empowering both researchers and clinicians with a powerful tool to understand the complexities of diseases specific to each patient. Transgenic modification of patient-derived primary cells can directly address the challenges stemming from inter-patient variations in therapy response, promising to pave the way for more effective, patient-tailored interventions.
