The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research, including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option and should reduce the impact of operation steps, such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve labor-saving monitoring of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplets. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single-cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 ± 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.

Avian influenza A virus H5N1 is a highly pathogenic and persistently a major threat to global health. Vaccines and antibodies targeting hemagglutinin (HA) protein are the primary management strategies for the epidemic virus. Although camelids possess unique immunological features, the immune response induced by specific antigens has not yet been thoroughly investigated. Herein, we immunized an alpaca with the HA antigen of the H5N1 virus and performed single-cell transcriptome profiling for analysis of longitudinal peripheral blood mononuclear cell (PBMCs) behavior using single-cell sequencing technology (scRNA-seq). We revealed multiple cellular immunities during the immunization. The monocytes continued to expand after immunization, while the plasma cells reached their peak three days after the second antigen stimulation. Both monocytes and B cells were stimulated by the HA antigen and produced cell-type-specific cytokines to participated in the immune response. To our knowledge, this is the first study to examine the HA-specific immunological dynamics of alpaca PBMCs at the single-cell level, which is beneficial for understanding the anti-viral immune system and facilitating the development of more potent vaccines and antibodies in camelid animals.

Tumor metastasis causes the death in most cancer patients. During metastasis, cell migrated from primary tumor was the very first step and study the tumor cell migration and interaction with somatic cells is therefore of great important. Development of in vitro technology that can quantitatively gain migration and cell-cell interaction data is there high desirable. Conventional methods for study tumor migration and cell-cell interaction are plagued by inaccessible to get quantitative data or complication of microfabrication process which brings into additional efforts rather than study the biological question itself. Herein, we developed a 3D printed “LEGO”-like device combined with a quantitative data gaining process which can be used for investigation into tumor cell migration process dynamically at single cell resolution and tumor-stromal interactions with high flexibility.