Cell sorting in a Petri dish controlled by computer vision

Abstract

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrate that both fluorescent and unlabeled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50–70 micrometers. Survival rate with a low number of 3T3 mouse fibroblasts and NE-4C neuroectodermal mouse stem cells was 66±12% and 88±16%, respectively. Purity of sorted cultures and rate of survival using NE-4C/NE-GFP-4C co-cultures were 95±2% and 62±7%, respectively. Hydrodynamic simulations confirmed the experimental sorting efficiency and a cell damage risk similar to that of normal FACS.

Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E‐selectin expression

“…The possible role of E-selectin in this static model of dPLB-985 binding to HUVECs is further supported by the inhibition of adhesion by soluble E-selectin. To assess the adhesion force between rMASP-1-treated HUVECs and dPLB-985 cells, we utilized the recently introduced computer controlled micropipette method [34,35]. Previously, it was applied to measure the adherence of macrophages and dendritic cells to fibrinogen, and to poly-L-Lys-graft-PEG.…”

“…We used a computer controlled micropipette method to quantify the adhesion force between dPLB-985 cells and rMASP-1-treated HUVECs [34,35]. HUVECs were induced with 2 µM of rMASP-1, 300 nM thrombin for 6 hours, or 10 ng/mL TNFalpha for 24 hours or left untreated.…”

“…A computer controlled micropipette (CellSorter), mounted onto an inverted fluorescence microscope (Zeiss Axio Observer A1) [34,35], was used to quantify the adhesion force between the variously pre-treated HUVECs and dPLB-985 cells. In brief, a confluent layer of HUVECs was cultured in a 35-mm tissue culture plastic Petri dish (Greiner) for one day.…”

Automated single cell sorting and deposition in submicroliter drops

“…We have reported that a micropipette controlled by computer vision allows automated single cell manipulations and sorting on a microscope. 24 A similar robot for the automated breeding of single cells has been recently developed. 25 This integrated instrument applies microwell arrays to immobilize cells on the surface for subsequent sorting.…”

“…Efficiency of sorting after optimizing experimental parameters is not expected to strongly depend on cell size or shape. 32 We did not investigate cell viability in this study as we carried out exhaustive viability experiment in our previous report 24 using the same system without automated cell deposition. We argue that automated cell deposition is not expected to affect cell viability when using the same vacuum and pressure parameters as in case of the manual deposition.…”