How are individual cells cloned?
Let’s talk about the criteria for successful cloning and one of the modern technologies for cell separation based on impedance
Single cell cloning is important in stem cell biology, cancer research and biotechnology. Major applications include cell line development, monoclonal antibody production, single cell multiomics, and the characterization of rare cytotypes such as circulating tumor cells.
There are two main steps in the single cell cloning process: cell division and subsequent growing them on a medium. In the case of cell cultures of multicellular organisms, it is not easy to clone individual cells correctly, because they will not grow on standard media that are easy to grow, for example, bacteria. And the dissociation of individual cells and the creation of pure clonal cell lines are generally long-standing problems in this area. The inefficiency of cell cloning represents a major obstacle to the standardization and optimization of gene editing in stem cells for basic and translational research.
Criteria for optimal cloning technology
The cloning process takes a lot of time and resources and involves sequential steps. It is critical that heterogeneous cell populations be segregated according to cell type and that individual cells can be isolated and analyzed.
The ideal single cell cloning technology must meet strict criteria:
It provides isolation of single cells with high efficiency and avoids isolating multiple cells, doublets, and aggregates.
She happens to be disposableto avoid cross-contamination of cells when moving from one experiment to another.
The procedure must be sterileto prevent bacterial and viral contamination.
Technology ensures conservation viability and functionality cells in such a way that the cells can either be analyzed using the most advanced methods of cell biology and omics analysis, or transplanted.
Proof of clonality is recorded in the mode real time in accordance with regulatory requirements.
Technology is simple, cost effective and compatible with standard workflows.
Several technologies are available for isolating single cells, some of which are commercially available. For example, a printer scp singlecell (Cytena, Germany) and cellenONE (Cellenion, France) based on optical detection for single cell dosing, or ICell813 (Takara Bio, Japan) and Rhapsody14 (BD), which allow the capture of single cells in nanoscale microcells.
Cell separation method based on impedance
Let us consider in more detail one of the modern single-stage methods for separating single cells for subsequent cloning using a convenient pipette (MullerSwitzerland), which allows universal isolation of viable single cells together with unambiguous evidence of their clonality.
The device is a portable pipette for placement impedance analyzerconnected to a computer, and micropump in combination with a sterile disposable sensitive tip that acts as a cell counter. The system recognizes and registers every single “particle” that passes through the tip opening. A single sharp peak on the impedance graph is a sign that we have a single cell in front of us, peaks of small amplitude indicate cell fragments, while multiple peaks result from doublets, aggregates.
This technology enables the efficient and traceable isolation of living cells, stem cells and cancer stem cells, which can then be individually cultured and transplanted.
The one-step cell isolation method using pipettes and an impedance sensor enables traceable isolation of live single cells and guarantees their clonality. The method is considered uncomplicated, effective and compatible with standard procedures. It produces cells that can be analyzed, transplanted, and used in cell population management in biotechnology, stem cells, and cancer research. We advise you to take a closer look at the technology.
In the next article, we will look at another popular cloning technology − microfluidic chips.
The article was prepared by Ilya Gridnev especially for Blastim
