Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids

Abstract

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Figure 1. Microfluidic cell culturing workflow.

(A) Layout of the microfluidic device, consisting of a 4-by-6 array of interconnected drops with a single inlet and single outlet. Liquid is loaded from the top and automatically distributed all over the network due to capillary forces. (B) Single-cell loading into standing drops by using FACS and spheroid loading by using pipets. (C) Culturing under controlled perfusion and a close-up of the outlet: state 1, larger drop = liquid removal; state 2, smaller drop = no liquid outflow. (D) Parallel cell and spheroid transfer from the hanging-drop network to a receiver plate.

Figure 2

(A) Bright-field picture of the hanging-drop chip, loaded with cellular spheroids, after turning it upside down and cell sedimentation. (B) Green-fluorescence micrographs of cells, sorted into the hanging drop chip, after turning it upside down and sedimentation. The resulting and intended cell numbers per drop are indicated. (The target drops are marked in black at the top of each image. Both scale bars are 200 μm).

Figure 3

(A) Picture of the hanging-drop chip filled with red food dye for showing the hanging drop network. (B) Close-up view of the outlet drop with the tubing tip placed at the liquid–air interface defining the drop height. (C) Measured drop height variation over 10 h at a flow-rate 5 μL/min. (D) Bright-field and fluorescence micrographs of eight spheroids at the beginning and after 3 days in culture (loaded drops are marked in black, scale bars are 200 μm). (E) Relative spheroid growth in perfusion mode and (F) ATP content at the start and after 3 days in a static, D3(s), and perfused chip culture, D3(p). (Medians are marked in black).

Figure 4

(A) Bright-field images of 300 fluorescent HCT-116 cells, sorted in a chess-board-like pattern into hanging drops, and of subsequent spheroid formation during 125 h (target drops are marked in black, scale bar is 200 μm, see Supplementary Movie 1 for full time-lapse sequence). (B) Fluorescence intensity increase of 8 spheroids derived from 300 sorted, fluorescent HCT-116 cells over time. (C) Fluorescence intensity of cell clusters after 125 h in culture in dependence of the initially sorted cell number (n = 2). (D) Fluorescence intensity development versus time for clusters consisting of low numbers of cells, and fluorescence micrographs at different time points (scale bar is 200 μm, contrast has been increased for better visibility).

Figure 5

(A) Phase contrast images (10×) of all 24 drops at day 1, after loading 50 granulocyte/macrophage progenitor (GMP) cells from bone marrow suspension into each drop using a FACS unit, and at day 5 after subsequent culturing in the hanging drops (target drops are schematically marked in black, scale bar is 200 μm). (B) Flow-cytometric characterization of retrieved cells after 5 days in culture. (C) Yellow fluorescence micrographs of the hanging-drop culture taken every day (scale bar is 200 μm, see Supplementary Movie 2 for full time-lapse sequence).

Figure 6

(A) Photograph of the harvesting platform including the hanging drop chip at the top and the receiver plate below. (B) Spheroids imaged after harvesting on the receiver plate. Gray shadows are optical artifacts in transmission-light microscopy mode, which are produced by the curved drop liquid–air interface. Initially loaded drops are marked in black in the top schematic. Scale bar is 200 μm.