Single-cell cloning is the process of isolating an individual cell from a diverse population and subsequently enabling it to proliferate to establish a uniform community of cells. The population of cells derived from single-cell clones are genetically indistinguishable, facilitating gene and protein investigations, biopharmaceutical manufacturing and drug discovery applications.

Single-cell cloning is a crucial method for generating monoclonal cell lines used in research and drug production. It involves dilution cloning and deterministic methods that use single-cell images to ensure monoclonality. In biotechnology, single-cell cloning plays a vital role in recombinant protein production from mammalian cells, allowing the expression of high levels of the desired protein. Moreover, single-cell cloning proves invaluable in drug discovery and screening, identification of drug targets, investigation of drug resistance mechanisms and development of personalized treatment strategies.

Single-cell cloning is predicated on building a genetically homogeneous population to minimize gene expression variables. The genetic homogeneity is also a regulatory requirement for producing uniform active pharmaceutical ingredients like recombinant proteins or monoclonal antibodies.

Methods of Single-Cell Cloning and Isolation

Single-cell cloning by limiting dilution is an established technique to derive a monoclonal population from a pool of cells. This method relies on the Poisson distribution to rapidly and conveniently isolate individual cells from diluted cell suspensions using hand-pipettes or pipetting robots. However, limiting dilution is inefficient, leading to wells containing either zero or more than one cell .

Microfluidics-based methods for single-cell cloning employ microfabricated devices that allow precise manipulation and isolation of single cells, enabling researchers to establish clonal cell lines with high genetic fidelity. Microfluidic platforms enable the parallel processing and analysis of many cells. These methods require small volumes of reagents and samples, making them suitable for working with limited or precious cell samples. They are also compatible with current workflows as single cells can be easily transferred from the microfluidic device to standard culture plates for further expansion using conventional micropipettes and microscopes.

Laser-assisted cell microdissection (LACM) method is used to isolate single cells from heterogeneous environments like cytological preparations. It enables the differentiation of normal and morphologically abnormal cells as distinct populations from complex mixtures, allowing precise investigation of subcellular profiles. The biomolecules extracted from dissected cells, including RNA, DNA, and protein, can be utilized in various downstream applications such as gene expression studies using polymerase chain reaction, proteomic analysis, etc. The method utilizes either ultraviolet (UV) cutting systems or infrared (IR) capture systems in combination with light microscopy and laser technology.

Manual cell picking in single-cell sorting and cloning is also used to isolate individual cells. It utilizes a combination of an inverted microscope and micropipettes that can be moved via motorized mechanical stages. This allows for precise positioning of the pipette for the isolation of single cells. Each isolated cell can be observed and photographed under the microscope, ensuring unbiased selection. Manual cell picking is particularly valuable for isolating live cultured cells or embryo cells. These isolated cells are then transferred to separate culture vessels, allowing them to grow and generate cell lines for further study or application.

Techniques for Single-Cell Identification and Selection

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Challenges Associated with Single-Cell Cloning

Applications of Single-Cell Cloning

Validation and Confirmation of Clonality

Genotyping and sequencing can validate clonality by analyzing the genetic landscape to determine if the cells share identical genotypes. Techniques like PCR or RFLP analysis can detect specific markers or mutations unique to the clonal population. Functional assays and phenotypic analysis determine cell clonality by assessing functional characteristics capable of identifying aberrant phenotypes from normal ones.

Scaling Up Single Cell Clones

Subcloning and expansion strategies involve isolating individual cells from the clonal population and further propagating them to generate multiple subclones, each originating from a single cell to study variations and ensure clonal stability.

Moving single clones from multi-well plates to fed-batch cultures in flasks and bioreactors is a crucial step in biopharmaceutical production. A single clone is selected based on desired characteristics and transferred to a larger vessel for expansion.

Quality control and monitoring are essential to ensure the uniformity and consistency of clonal populations. Complying with regulatory guidelines requires production cell lines to originate from a monoclonal progenitor and be thoroughly assessed for growth rate, productivity and final product quality.

Cryopreservation and long-term storage methods are employed to preserve clonal populations by freezing them in liquid nitrogen or at ultra-low temperatures. This process is referred to as cell banking.

FAQs

Why is single-cell cloning important in biological research?

Single-cell cloning is crucial in biological research as it enables the study of an individual cell’s unique characteristics and behavior, providing valuable insights into the influence of genetics on cellular processes.

What are the advantages of single-cell cloning over other methods?

Single-cell cloning offers the ability to study individual cells with precision and resolution, providing insights into cellular heterogeneity and unique characteristics not achievable through bulk methods.

How does limiting dilution work in single-cell cloning?

Limiting dilution in single-cell cloning involves diluting a cell suspension to a point where each well receives only one single cell, ensuring clonal growth and isolation of individual cells.

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