A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures

Abstract

Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem-Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (V(b)=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, -5°C, -10°C, and -20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, -10, -20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10(-3) cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C-12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.

FIG. 1.

Schematic diagram showing the orientation of a microperfusion system positioned on a temperature-controlled cryostage relative to an objective lens of an inverted microscope. Also shown are cells adhering to a poly-D-Lysine-coated slide within a microperfusion channel with inlet and outlet ports.

FIG. 2.

The microperfusion system. (A) PDMS microperfusion channel situated on poly-D-lysine-coated slide (ie, microperfusion system). Arrow points to microperfusion channel. (B) The microperfusion system is located on a temperature-controlled cryostage. Arrow points to injection needle attached to tubing extending from an adapter on the inlet port. PDMS, polydimethylsiloxane.

FIG. 3.

Confocal images of a UT-7/TPO cell stained with a membrane dye, MINI67, that are immobilized on a poly-D-lysine-coated glass coverslip. (A) Top view (x–y plane). (B) Right side view (y–z plane).

FIG. 4.

Boyle Van't–Hoff relationships for UT-7/TPO cells at 37°C. The osmotically inactive cell volume (V_b) is determined to be 0.15 of initial volume.

FIG. 5.

(Left) UT-7/TPO cells immobilized on surface-treated substrate in a microchannel perfused with 3×PBS. (Right) Grayscale and binary images of the single cell in the cropped area (rectangle) at each time slide. PBS, phosphate-buffered saline.

FIG. 6.

Normalized cell volume change with respect to time when cells are perfused with 3×PBS at −20°C, 0°C, and 37°C.

FIG. 7.

Arrhenius plot of the UT-7/TPO cells showing the natural logarithms of the average water membrane permeability coefficient (Lp) versus the reciprocal of the absolute temperatures.

FIG. 8.

Normalized cell volume change with respect to time when cells are perfused with 10% DMSO+0.9% NaCl at 0°C, 22°C, and −20°C. DMSO, dimethyl sulfoxide.

FIG. 9.

Arrhenius plot of the UT-7/TPO cells showing the natural logarithms of the average DMSO membrane permeability coefficient (Ps) versus the reciprocal of the absolute temperature.