When it comes to sorting cells, it is common practice to use fluorescent or magnetic tags to label certain cellular features. For a long time, that was the only practical way to separate cells and you had to hope that the labels you used did not interfere with subsequent analysis and testing. Given many innovations in the area of cell analysis and sorting, there is now a multitude of different ways of analyzing and sorting cells. Ways that do not necessitate the addition of often costly cellular labels to allow one to see what’s going on at the cellular level.
As you may be aware, cell sorting is now common practice in many research labs and hospitals. It seems that much of the future of precision and personalised medicine itself will rely on specifically isolated cell populations. This is of great potential benefit to improved medicines and treatments; however, there are some critical bottlenecks stopping the global adoption of many modern medical treatments that rely on cell sorting.
CAR T-cell therapy
Take CAR T-cell therapy for example - the reprogramming of a patient’s own white blood cells to target and attack their cancer. This therapy is one of the best up-and-coming examples in medicine where cell analysis and sorting is essential. It is currently a treatment for some cancer patients who have run out of other options. Some clinical trials with this type of treatment have proved to be a great success for specific cancers. Going forward, it is very plausible that CAR T-cell therapy could be used to treat many types of cancer that are currently difficult to suppress (1).
In CAR T-cell therapy, a sample of the patient’s blood is taken and immune cells called lymphocytes are subsequently isolated from it - via cell sorting. These isolated cells are then shipped off to a central bio-manufacturing plant where they are further sorted to isolate T-cells (2).
At the manufacturing plant, the T-cells are genetically engineered to provide them with a new receptor. A receptor that enables the T-cells to better target and kill cancer cells. Sounds easy enough but unfortunately, despite substantial recent advancements in the area, the current methods of genetic modification still come with some drawbacks. Other methods are being investigated, but many of these won't make the cut and research and development is ongoing (1, 3, 4, 5).
This imperfect process means that a lot of precious time and money is dedicated to quality control (QC) of newly made CAR T-cells (making sure you have a pure population of healthy, transfected cells). Estimates put QC between 10-20% of overall manufacturing cost and it has sometimes been found to double the amount of time between patient blood sampling and final CAR T-cell formulation (6).
After all this, you can finally transfer the cells back into your patient and (hopefully) save their life. Cells take on average 3-4 weeks to come back from the central manufacturing plant. Whether or not a patient is healthy enough to survive this waiting period is an unfortunate part of the patient screening process. (7)
Quality Control
Why exactly is the quality control stage so expensive and time-consuming? One of the main reasons stems from viral vectors currently being the main method of transfecting T-cells with new DNA.
Viral vectors need an incubation time of several days and afterward, cells must be strictly monitored for the possibility of unwanted and potentially cancerous mutations (insertional oncogenesis), or the virus itself not being safe for use. Highly skilled individuals with exceptionally expensive equipment such as flow cytometers are currently necessary to assess successful transfections (1, 3, 4, 5).
It’s not hard to see that this is a significant bottleneck in the system and many people are currently working hard to develop better methods of transfection. The research is now showing that the process of electroporation will soon be replacing viral vectors as the main method of DNA transfection. It lacks many of the costs and risks involved with using a virus and works significantly faster.
Electroporation involves applying an electrical field to cells which stimulates the formation of micropores on the cell surface. Successfully forming these pores puts the cell into a permeabilized state and newly engineered DNA can then enter the cell.
Unfortunately, even electroporation has a low success rate. Many cells fail to permeabilize or die during the process leading to cell loss, impure populations, and low transfection rates. The cells produced need their own quality control to make a pure population.
Currently, flow cytometry is used to assess the success of T-cell transfection and it is one of many lengthy and expensive steps in the QC process (3). Let's take a look.
FACS & MACS
The current best method of sorting cells is FACS - Fluorescent Activated Cell Sorting. A FACS machine uses fluorescent cytometry to analyze and then sort cells into desired populations. There are equivalent machines that use the exact same process but utilize a magnetic label instead of a fluorescent one, these are known as MACS.
These methods involve adding a fluorescent or magnetic label to an antibody you know will stick to only the cells you want to isolate. This antibody linked label can be added to your cell population, and after a short incubation time, it will bind to the cells. The tagged cell population can then be put into the FACS machine. The machine will run the cells through a sorter which detects cells displaying your label and repels them away from the unwanted cells to create an isolated population.
Excellent, you have isolated T-cells. But they still have labels. Some labels will naturally dissociate from the cells and can be washed away. Some labels need another reagent to elute them before they can be washed off. Some labels just don’t come off. After all of this, you have to hope that the combination of reagents and labels used has not damaged your precious cells (8).
Aside from the time and effort it takes, FACS and equivalent machines are extremely expensive. To add to the cost, they require a specifically trained individual to operate them. Then think about all the human error that’s possible during these handling steps, we’ve now got quality control for our quality control. Fortunately, there are other developments in the pipeline that may just be the solution to some of these problems.
Impedance Cytometry
What if we could dramatically increase the purity of our CAR T-cell population within the transfection process itself rather than during the weeks of quality control afterward?
Cellix’s impedance technology is the first-in-field technology that can specifically sort for cells that become permeabilized during electroporation. A unique function that will directly impact the transfection process and the amount of QC required after.
Cellix’s impedance cytometry works by running an electrical current across a microfluidic channel. As cells are pumped through this channel, each individual cell interferes with the flow of electrical current. The interference can be measured on the receiving electrode as electrical impedance and it is always different depending on the electrical properties of the cell.
An impedance cytometer can, therefore, derive the physical properties of a cell from its unique electrical properties. These include:
Size
Morphology
Cell cytoplasm conductivity
Organelle impedance
Cellular DNA content
Cell membrane integrity.
When a cell’s membrane is permeabilized, its electrical properties change significantly. An impedance cytometer can detect this consistently, without the need for cell preparation and without damaging cells.
Impedance cytometry is not as specific as fluorescent or magnetic cytometry. You cannot search for specific proteins or other molecules within the cell; however, you can find the characteristics you need for cell transfection with much less effort. The robustness of the components also means that the technology can be scaled down to a much smaller size and can be multiplexed to a much higher throughput.
So now, without the requirement for multiple labeling steps, impedance cytometry can be harnessed to analyze and sort cells in-line with no need for preparation or human intervention. CAR T-cell QC is about to become a lot more manageable.
Most Importantly
We’re looking at the bigger picture here. Not only are impedance based cytometry instruments much smaller and more portable than current flow cytometers but simply eliminating the need for labeling, and harnessing QC more strategically, greatly reduces space, reagents, and staffing required.
When you first thought about label-free cell sorting and analysis, you may have expected that the main benefit was in removing the possibility of damaging cells that need to go back into the human body and facilitate normal life.
We here at Cellix know that it means so much more than that. As it stands, there are still some substantial manufacturing issues that need to be overcome to allow these groundbreaking therapies to be made available to those who need them and at a reasonable price.
Using the strategic benefits of label-free technology may be one of the necessary methods to cutting down time, reagents, highly skilled labour and overall cost. An essential step on the path of making gene therapy and other life-saving treatments accessible to everyone.
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References:
1
https://www.stemcell.com/media/files/wallchart/WA27041Production_of_Chimeric_Antigen_Receptor_T_cells.pdf
2
https://www.ncbi.nlm.nih.gov/pubmed/28344995
3
https://www.liebertpub.com/doi/pdf/10.1089/hum.2017.254
4
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909095/
5
https://www.ncbi.nlm.nih.gov/pubmed/29462761
6
https://www.sagentia.com/files/2018/07/Quality-control-testing-in-CAR-T-cell-manufacture.pdf
7
https://www.youtube.com/watch?v=RpD69Z-Xco4&t=450s
8
https://www.ncbi.nlm.nih.gov/pubmed/26767547