Cell culture is foundational to modern biotechnology, biomanufacturing, drug discovery, vaccine production, regenerative medicine, and genetic engineering. However, most established cell culture protocols, media formulations, and immortalized or pluripotent cell lines exist only for well-characterized "model" organisms (e.g., human, mouse, rat). For many vertebrate species, including those of emerging interest to defense, biosecurity, agricultural, conservation, and public-health stakeholders, no validated cell culture baseline exists.
Filling this gap is critical for gene-drive and genetic-biocontrol research. Gene drives, genetic elements engineered to propagate a trait through a target population faster than standard inheritance, hold significant promise for controlling disease vectors, suppressing invasive or pest species, and protecting agriculture, ecosystems, and public health. Yet engineering, optimizing, validating and characterizing the safety and efficacy of gene-drive constructs requires robust, species-specific cell lines for the target organism that can enable rapid in vitro assessment of drive activity (e.g., homing/conversion efficiency, off-target activity, and stability) that would otherwise require slow, whole-organism breeding studies.
Establishing cell culture for a novel, non-model species today is a slow and irreproducible process. It requires years of manual optimization across an enormous parameter space (media composition, growth factors, substrate, oxygen tension, temperature, passaging cadence, reprogramming-factor delivery, and differentiation cues), depends heavily on individual operator skill, and frequently fails to transfer between laboratories. This bottleneck limits the speed for standing up new biological capabilities, constrains the breadth and resilience of the bioeconomy, and directly impedes the responsible development of gene-drive and biocontrol countermeasures.
This Phase II SBIR topic seeks a generalizable platform that fundamentally changes this paradigm by combining (1) species-agnostic biological methods for deriving and maintaining cells from arbitrary vertebrate and invertebrate species, (2) autonomous, closed-loop automation that systematically explores culture parameter space and produces reproducible, transferable protocols and cell lines, and (3) the cellular substrate and engineering workflows needed to build and validate in vitro testbeds for gene-drive technologies in those cell lines.
Proposers must address Component 1 and Component 2 below, and are highly encouraged to address Component 3. A proposer may submit only one proposal to this topic.
Component 1: Cross-Species Universality (Species-Agnostic Cell Culture & iPSC Derivation)
Develop generalizable biological methods that allow viable, stable cell lines to be established from diverse, non-model vertebrate and invertebrate species without bespoke, multi-year, per-species protocol development.
Areas of interest include, but are not limited to:
• Universal or rapidly tunable media, substrate, and supplement formulations applicable across taxonomically diverse species (e.g., across multiple vertebrate classes such as mammals and reptiles).
• Species-agnostic strategies for primary cell isolation, immortalization, and/or induced pluripotency (iPSC reprogramming), including reprogramming-factor delivery and selection approaches that generalize across species.
• Methods for establishing, characterizing, and validating pluripotency, viability, genomic stability, and lineage differentiation potential in species lacking established reference reagents or markers.
• A principled, design-of-experiments (DOE) or model-driven framework for rapidly converging on viable culture conditions for a previously uncharacterized species.
• Cryopreservation, banking, and quality-control approaches that ensure cell line stability and reproducibility across time and locations.
Component 2: Autonomous, Closed-Loop Culture Automation
Develop automation that performs cell culture operations with minimal human intervention and that systematically and reproducibly drives protocol optimization for new species.
Areas of interest include, but are not limited to:
• Per-vessel or per-well environmental independence (e.g., independent control of temperature, gas composition, and media conditions) to enable many simultaneous, independent optimization experiments.
• Automated execution of routine and complex culture operations, including feeding, passaging, reprogramming, differentiation, imaging, and sampling.
• Integrated, automated imaging, data capture, and analysis (including machine-learning–enabled morphology/health assessment) feeding a closed-loop optimization engine that proposes and executes the next experimental conditions.
• Software, data architecture, and electronic protocol capture that make optimized protocols portable and reproducible across instruments and laboratories.
• Throughput and parallelization sufficient to compress new-species protocol development from years to weeks or months.
Component 3: Gene-Drive and Genetic-Biocontrol Enablement
Develop the cellular substrates, engineering workflows, and validation methods needed to characterize gene-drive and related genetic-biocontrol technologies in non-model target species.
Areas of interest include, but are not limited to:
• Establishment of species-specific cell lines (including germline-relevant or developmentally relevant cell types) suitable as a testbed for gene-drive construct development.
• In vitro assays to characterize gene-drive efficiency, homing/conversion rates, off-target effects, fitness costs, and genetic stability prior to any whole-organism work.
• Containment, reversibility, and safeguard strategies (e.g., molecular confinement, reversal/immunizing drives) evaluable at the cellular level.
Proposers addressing Component 3 must explicitly address responsible-research practices, applicable biosafety/biosecurity and regulatory considerations, and appropriate institutional oversight. All Component 3 work under this topic shall be limited to in vitro / cellular research; no environmental release, and no work resulting in a gene-drive-competent whole organism, is contemplated or permitted under this effort.[BR4.1]
Component Integration
Proposers should describe how species-agnostic biology, closed-loop automation, and (where applicable) gene-drive enablement combine into a single coherent platform - e.g., how the automation systematically searches biological parameter space to converge on a validated cell line for a new target species, and how that cell line then serves as a reproducible substrate for gene-drive engineering and in vitro validation.
