In this article, Cell Microsystems describes the challenges in the development of monoclonal cell lines, and what technologies are available to overcome them.
Sponsored Content by Cell MicrosystemsJul 12 2023Reviewed by Louis Castel Single-cell cloning involves generating a cell line from a single initial cell isolated from a heterogeneous population, typically obtained through transfections, transductions, or primary cells derived from tissue or biopsy samples.1
Pursuing more targeted therapies for diverse disease conditions has spurred advancements in single-cell workflows, particularly in cloning, which has become an indispensable necessity in biopharmaceutical research and product development.Current methods Limiting dilution and single-cell dispensing Two commonly utilized techniques for isolating single cells are limiting dilution and single-cell dispensing, occasionally assisted by fluorescence cell sorting.
Although retrieving individual cells from diluted cell suspensions may appear straightforward, employing manual pipetting or automated robotic platforms often proves challenging when obtaining an adequate number of desired monoclonal clones. Benchtop cell sorting systems equipped with multiple lasers and colors enhance the precision and speed of the sorting and selection process.18 However, these advancements necessitate significant gating requirements and setup for each population to be sorted, ensuring the retrieval of the maximum number of viable clones.19
This metabolic transition reduces reductive biosynthetic reactions involving NADPH. Cells undergoing this transition activate apoptotic genes or halt S-phase synthesis. Consequently, diminished outgrowth is frequently observed in single-cell progenitors initiated through FACS, and achieving the necessary outgrowth yields often takes longer.20
The systems employed for these methods of single-cell clone generation comprise multiple platforms, components, and steps. The subsequent outgrowth of the isolated single cells necessitates separate procedures, requiring additional equipment, resources, and space, which incurs additional costs beyond the initial platform investment, which can be substantial, ranging from several hundreds of thousands to millions of dollars.
With a saturated market offering a variety of platforms, researchers are faced with the challenge of navigating and selecting the most suitable and cost-effective system for their specific needs. Figure 3. CellRaft Technology Combines the Power of Flask-like Culture Conditions and Single-Cell Separation to Produce High Viability Cells, Colonies, and Organoids. Image Credit: Cell Microsystems
Consequently, the physical isolation of single cells is achieved without perturbation, ensuring the absence of biochemical or physiological changes. The CellRaft AIR System offers capabilities for individual imaging and analysis of cells using bright field or fluorescent imaging modalities. The absence of microfluidic separation or perturbation in the CellRaft Array ensures the viability and vitality of single cells, allowing them to develop into healthy clones.
At the heart of the CellRaft AIR System lies the CellRaft Cytometry software, which serves as its "brains." This software facilitates the selection, scanning, and imaging of thousands of CellRafts, enabling the automated identification and isolation of desired clones. . Workflow In the CellRaft AIR System workflow, cells are plated on the CellRaft Array and loaded into the system platform. The cells are imaged using three-channel fluorescence and bright field microscopy, and sorting is performed based on user-defined thresholds, filtering, and gating .
Automating all steps provides real-time imaging, identification, and isolation of cells and small colonies for outgrowth in 96-well plates . This accelerates the timeline for obtaining results and reduces costs and contamination risks. Figure 5. Timeline of Raji cell colony growth as an example of direct proof of track and trace functions. Image Credit: Cell Microsystems
It provides notable advantages over other systems, eliminating the need for additional space, equipment, and manual labor while ensuring cost-saving, viability, and high outgrowth rates.
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