An Overview of Single-Cell Isolation Techniques

Zeb, Qudsia; Shafiq, Sarfraz; Liu, Liangyu

doi:10.1016/b978-0-12-814919-5.00006-3

Cited by 27 publications

(29 citation statements)

References 110 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Micromanipulation is a commonly used method in the laboratory. However, its throughput is low and requires highly skilled professional training (16). FACS was introduced in 1969 by Leonard Herzenberg (17) and is of wide application with high throughput.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Laser induced isolation and cultivation of single microbial cells

Peng

Wang

Wang³

et al. 2020

Preprint

View full text Add to dashboard Cite

Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

show abstract

Section: Introductionmentioning

confidence: 99%

“…Similar with FACS, MACS depends on a magnetic force to isolate cells from the cell suspension in a magnetic field. Although MACS is more rapid and less expensive than FACS, its isolation accuracy is lower than FACS (16). LCM uses a focused laser to cut a cell from its surroundings.…”

Section: Introductionmentioning

confidence: 99%

Laser induced isolation and cultivation of single microbial cells

Peng

Wang

Wang³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Limiting dilution cloning is economical and may cause less cellular stress than FACS sorting, but severely limited to generate highly validated single-cell clones. Furthermore, limiting dilution even is introducing affinity-based selection steps and in general the method is still inferior in control [14].…”

Section: Resultsmentioning

confidence: 99%

“…Many other techniques are also being developed, including manual isolation, microfluidic based sorting mechanisms, magnetic-activated cell sorting, or panning. However, none of them can meet all practical demands [14]. Recently, single cell printing is emerging [18], which is based on an inject-like technology in which free-flying microdroplets that encapsulate cells are generated by a non-contact dispensing procedure.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, single-cell printing technology (SCP) is emerging from others [14,15] as a gentle, low-cost and highly controlled technology, applicable for a wide array of specific cell-cloning applications ranged from the 0.8 µm prokaryotes to 100 µm plant cells [16,17]. It is based on inject-jet-like technologies generating on-demand free-flying microdroplets encapsulating cells to deposit them on variable substrates using a non-contact dispensing process.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

Groß

Jeney

Halm

et al. 2020

Preprint

View full text Add to dashboard Cite

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30 \ % of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.

show abstract

Single‐cell RNA sequencing technologies and applications: A brief overview

Jovic

Liang

Zeng

et al. 2022

Clinical & Translational Med

402

204

View full text Add to dashboard Cite

Single‐cell RNA sequencing (scRNA‐seq) technology has become the state‐of‐the‐art approach for unravelling the heterogeneity and complexity of RNA transcripts within individual cells, as well as revealing the composition of different cell types and functions within highly organized tissues/organs/organisms. Since its first discovery in 2009, studies based on scRNA‐seq provide massive information across different fields making exciting new discoveries in better understanding the composition and interaction of cells within humans, model animals and plants. In this review, we provide a concise overview about the scRNA‐seq technology, experimental and computational procedures for transforming the biological and molecular processes into computational and statistical data. We also provide an explanation of the key technological steps in implementing the technology. We highlight a few examples on how scRNA‐seq can provide unique information for better understanding health and diseases. One important application of the scRNA‐seq technology is to build a better and high‐resolution catalogue of cells in all living organism, commonly known as atlas, which is key resource to better understand and provide a solution in treating diseases. While great promises have been demonstrated with the technology in all areas, we further highlight a few remaining challenges to be overcome and its great potentials in transforming current protocols in disease diagnosis and treatment.

show abstract

An Overview of Single-Cell Isolation Techniques

Cited by 27 publications

References 110 publications

Laser induced isolation and cultivation of single microbial cells

Laser induced isolation and cultivation of single microbial cells

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

Single‐cell RNA sequencing technologies and applications: A brief overview

Contact Info

Product

Resources

About