Researchers have literally pushed the possibilities of genetic testing to the extreme: by using extremely fine needles, they have succeeded in draining fluid from individual cells and examining their gene activity without killing them in the process. The method therefore has considerable potential for research: sampled cells can be examined again later in order to record changes in activity under certain circumstances. The scientists have already illustrated this possibility using the example of immune and fat cells.
Like on a kind of computer hard drive, all body cells carry the entire genetic code of our organism. However, they differ in terms of the genetic “programs” running in them. Genes are active when the respective code is converted into messenger RNA. These information carriers then shape the respective characteristics of the specialized body cells of an organism. In addition, genes are switched on or off by certain stimuli, thus enabling flexible cell reactions. However, this system is also prone to failure: If something goes wrong in gene regulation, diseases can occur.
Researchers have therefore long been developing methods to record gene activity. Using so-called single-cell RNA sequencing, it was even possible to record the patterns in the smallest units. The messenger RNA molecules present in the cell fluid are deciphered and thus assigned to the respective active gene sequences. But until now, the methods for recording the transcriptome of single cells had a catch: "The cells to be examined had to be isolated, dissolved and thus killed," says Julia Vorholt from the Swiss Federal Institute of Technology in Zurich (ETH). As a result, it was not possible to study how the gene activity of a specific cell develops. "This restriction was previously considered unavoidable," says Vorholt.
Gently tapped for rehearsals
But with the so-called live-seq method, which she and her colleagues are presenting, the transcriptome can now be recorded without killing the cell being examined. This is possible by sampling it minimally invasively, as in a biopsy. The basis of the method is the FluidFM micro-injection system developed at the ETH, which can manipulate tiny amounts of liquid under a microscope. Microscopically small channels are used for this. Vorholt and her group have previously developed a cell extraction method from these "world's smallest hypodermic needles" that makes it possible to extract tiny amounts of liquid from individual cells without killing them. In the current study, they now show that full-fledged single-cell transcriptomes can be created from samples obtained in this way: they succeeded in reading the RNA from these tiny amounts of cell fluid.
To demonstrate the potential of their live-seq method, the team successfully analyzed the transcriptome of different cell types and states. According to the researchers, the mere fact that the analyzed cells do not die is an advantage: "You can continue to observe the sampled cells under the microscope - how they develop and behave," says Vorholt. In addition, they can be left in their physiological context. "The microenvironment and the cell-cell interactions then remain in place," says co-author Orane Guillaume-Gentil from ETH.
Courses of gene activity detectable
Most importantly, Live-seq can also show the activity of thousands of genes in a single cell over time through repeated measurements. "In this way, the single-cell analysis is changing from an end point to a temporal and spatial analysis method," says Vorholt. To demonstrate this, the researchers recorded the transcriptomes of individual immune cells before and after stimulation by specific substances. They also looked at gene activity in adipose stromal cells - a type of stem cell - before and after they differentiated into fat cells. They were able to successfully identify changes in the transcriptome.
It has already become fundamentally clear that the method can help to investigate new biomedically relevant questions. "With Live-seq, we can now investigate, for example, why certain cells differentiate and their sister cells do not, or why certain cells are resistant to a cancer drug and others are not," senior author Bart Deplancke Deplancke from the Swiss Federal Institute of Technology in Lausanne explains the potential of the new procedure.
Source: Swiss Federal Institute of Technology Lausanne ETH Zurich, specialist article: Nature, doi: 10.1038/s41586-022-05046-9