A capillary based vacuum assisted instrument for rapid and efficient collection of individual live cells from adherent cultures for single cell and clonal analysis (LB56)

There is a high demand for a method permitting simple and rapid collection of individual cells from adherent cell cultures that may be used from simple selection of transfected cells to single cell analysis. Numerous protocols for single cell analyses have been developed, which include whole genome sequencing and expression analyses. Although current flow sorting technology permits collection of single cells, acquisition of cells directly from cell culture dishes based on their morphology, location or fluorescent label remains challenging. Recently we introduced a novel cell and tissue acquisition system (KuiqpicK) and demonstrated its application for brain tissue microdissection. Here, we examined KuiqpicK’s ability to efficiently collect individual live cells. Collection of cells was performed from various adherent cultures, including neuroblastoma SH‐SY5Y, Chinese hamster ovary (CHO), human melanoma MDA‐MB‐435 cell cultures and primary neural progenitor cells established from embryonic and adult mouse and rat brains. Single and multiple cell acquisitions were performed based on the cell morphology, location in the plate and fluorescent label (e.g. CellTracker probes). To test the viability of collected cells, Trypan blue exclusion test and single cell recultivation experiments were performed using CHO and SH‐SY5Y cells. Trypan blue assay demonstrated survival rates ranging from 80 to 95% (n>300) for all cells tested. The clonal expansion of collected single SH‐SY5Y and CHO cells was demonstrated within 6 and 25 days, respectively. In addition, applicability of the instrument for the collection of single cells grown in three‐dimensional (3D) culture system was shown using MDA‐MB‐435 cells. Moreover, isolation of RNA from collected cells exhibited high integrity and low degradation rates. Subsequent T7 based labeling procedure consistently produced high incorporation yields making it suitable for downstream sequencing and gene expression analyses. To summarize, our experimental data convincingly demonstrated that the capillary based vacuum assisted instrument, KuiqpicK is a convenient approach to collect single and multiple cells (e.g. colonies) from both adherent and 3D cell cultures, capable of providing cells with high survival rate and suitable for downstream functional studies. Grant Funding Source : Supported by NIMH 2R44MH091909