Cell death along single microfluidic channel after freeze-thaw treatments

Abstract

Cryotherapy is a prospective green method for malignant tumor treatment. At low temperature, the cell viability relates with the cooling rate, temperature threshold, freezing interface, as well as ice formation. In clinical applications, the growth of ice ball must reach a suitable size as cells could not be all killed at the ice periphery. The cell death ratio at the ice periphery is important for the control of the freezing destruction. The mechanisms of cryoinjury around the ice periphery need thorough understanding. In this paper, a primary freeze-thaw control was carried out in a cell culture microchip. A series of directional freezing processes and cell responses was tested and discussed. The temperature in the microchip was manipulated by a thermoelectric cooler. The necrotic and apoptotic cells under different cryotreatment (duration of the freezing process, freeze-thaw cycle, postculture, etc.) were stained and distinguished by propidium iodide and fluorescein isothiocyanate (FITC)-Annexin V. The location of the ice front was recorded and a cell death boundary which was different from the ice front was observed. By controlling the cooling process in a microfluidic channel, it is possible to recreate a sketch of biological effect during the process of simulated cryosurgery.

Figure 1

Schematic diagram of cooling system for one-dimensional freezing in a PDMS microchip. The cooler was a set of TECs. The cooling side of the bottom TEC was attached to the top surface of the PDMS chip and the cooling effect was conducted through the PDMS slab to the channel. The image on the right was a phase contrast picture of seeded CaSki cells in the microchannel.

Figure 2

The images of ice spreading in the microchannel: (a) The moving ice front (around 2000 μm from the beginning reservoir) where the cooling rate was high; (b) the stabilized ice front (around 4100 μm from the beginning reservoir).

Figure 3

Phase contrast and fluorescent images of cells (a) before and [(b) and (c)] after 15 min freezing (region located 1600–2000 μm from the beginning reservoir).

Figure 4

The mapping of (a) phase contrast images and (b) fluorescent images along the channel after freezing for 15 min and thawing for 5 min (cycle I, region located between 2000 and 4100 μm). Upstream cells were all dead so the images were not shown (within 2000 μm). The red dotted line represented the death boundary where dead cells only occurred in the upstream of the line. The gray dotted line was the stabilized ice front. The region between two lines was defined as zero-loss region as no cells were dead in this position. The arrows in (a) indicated the position of stabilized ice front by comparing with Fig. 2b.

Figure 5

The fluorescence images of the cells after incubation for different period after the freeze-thaw cycle I on the same chip. (a) The upstream (∼1300 μm away from death boundary) of the death boundary after 10 min incubation. (b) The same position as (a) after incubation for 120 min. The white arrow indicated the apoptotic cells. Marked cells in (a) were primarily apoptotic as only the membrane was fluorescently labeled. In (b) both nucleus and membrane were labeled for the same marked cells which illustrated that the late apoptosis occurred after incubation. (c) Near the death boundary after 10 min incubation. (d) Near the death boundary after 120 min incubation.

Figure 6

The overlay of fluorescence and phase contrast images of the cells for different cooling duration: (a) Duration 15 min for cycle II and (b) duration 40 min for cycle III.

Figure 7

Full view of cell viability along the whole channel (0–4100 μm) after cycle III.