T-Cells, B-cells, and Other Immune Cells: Cell Sorting for Developing Immunotherapies

Immune cells are highly sought-after targets for understanding each cell type’s role in disease progression and suppression and even developing effective immunotherapies. Isolating immune cells of interest can aid in clinically-relevant cellular engineering efforts as well as academic explorations of the body’s natural defense system.

T-cells and B-cells are closely related immune cells that are often studied for their correlations with prognoses, evaluation of patients’ inflammatory responses, and explored for potential development of precision medicine. Similar in size, morphology, and function, the likeness of these two immune cells can make their identification and separation difficult. For applications such as studying the tumor microenvironment or cellular engineering, both isolation and preservation of immune cells (e.g., T-cells, B-cells, monocytes) is a critical step. Separating these cells often requires a label, which may negatively impact sample quality for subsequent analysis or processing. Currently, label-based methods, although adequate for separation, may be too harsh on the cells, resulting in decreased viability, increased cell stress, and even inducing changes to the morphology and behavior of the cells. The use of labels in the sample may also make the reintroduction of the engineered cells to a subject more difficult. For more niche applications, one might even run into the issue that the necessary labels are unavailable for the separation to even be performed using conventional methods! Ideally, there would be a method that could perform such separations without a label, handle fragile cells gently, and still offer high-enough sensitivity to perform enrichment of T-cells among similar B-cells and vice versa – that’s where the CytoR1^TM comes in!

The CytoR1 Platform can characterize cell types, even if their identity is completely unknown, by leveraging voltage and frequency to manipulate cells based on the cell’s own bioelectrical properties. When characterizing these cells, we see that T-cells and B-cells trap similarly at low voltages and low frequencies (Figure 1). This would suggest that T-cells and B-cells would behave the same at these voltages and frequencies, which is not particularly helpful for separation. There is, however, a window of voltage and frequency where T-cells (Jurkat) and B-cells (Raji) differ in their responses on the CytoR1. At 200 Vpp, T-cells trap at ~4.8MHz while B-cells trap at ~3.2MHz.

Figure 1. Jurkat (T-cells) and Raji (B-cells) cells have distinct average frequency responses at 200Vpp. This difference in response indicates possible enrichment of these cell types using the CytoR1.

Based on the characterization of the individual cell types, T-cells and B-cells can be separated without a label based on their electrical properties at 4MHz and 190Vpp (Figure 2). Using these parameters, the Jurkat cells trapped on the posts of the CytoChip while the Raji cells flowed to the outlet for collection. From a 40/60 (Jurkat:Raji) mixed sample, the CytoR1 Platform enriched for Raji cells with 80% purity (15000 cells) and Jurkat cells with 60% purity (25000 cells) into 10 μL samples.  Throughout this sort, the CytoR1 Platform simultaneously enriches for viable cells over non-viable cells, significantly improving the overall quality of the sample. Further optimization of parameters and workflows could improve enrichments to achieve even higher purities.

Figure 2. Based on their bioelectrical properties, the CytoR1 Platform can enrich for T-cells from B-cells and vice versa.

The CytoR1 offers the unique flexibility to modify both the frequency and the voltage across a large spectrum to finely tune the system towards achieving your desired sort. By tuning the electrical signal afforded by the CytoR1 Platform, users can characterize their sample and achieve a sensitive sort without specific or harmful labels. CytoRecovery hopes to soon expand their sorting of the tumor microenvironment to include even more cell types including monocytes.

The CytoR1 presents a practical, sensitive, and gentle approach to identifying and enriching for immune cells in a manner compatible with exploratory and clinical efforts. The CytoR1 Platform, with its tunable nature, can sort and recover a variety of cell types – even yours!