A Microfluidic Chip Architecture Enabling a Hypoxic Microenvironment and Nitric Oxide Delivery in Cell Culture

Abstract

A hypoxic (low oxygen level) microenvironment and nitric oxide paracrine signaling play important roles in the control of both biological and pathological cell responses. In this study, we present a microfluidic chip architecture for nitric oxide delivery under a hypoxic microenvironment in human embryonic kidney cells (HEK-293). The chip utilizes two separate, but interdigitated microfluidic channels. The hypoxic microenvironment was created by sodium sulfite as the oxygen scavenger in one of the channels. The nitric oxide microenvironment was created by sodium nitroprusside as the light-activated nitric oxide donor in the other channel. The solutions are separated from the cell culture by a 30 µm thick gas-permeable, but liquid-impermeable polydimethylsiloxane membrane. We show that the architecture is preliminarily feasible to define the gaseous microenvironment of a cell culture in the 100 µm and 1 mm length scales.

Keywords: cell culture; gasotransmitter; hypoxia; microenvironment; microfluidic chip; nitric oxide; oxygen depletion; sodium nitroprusside.

Figure 1

Microfluidic chip design. (a) The main chip type containing two separate, but interdigitated channels. The channels are 100 µm wide and the gap between two channels is 150 µm. The channel height is 40 µm. (b) A chip variant used for mm scale patterning of four different microenvironments: (1) hypoxia and nitric oxide, (2) only hypoxia, (3) only nitric oxide, and (4) neither treatment. The channels have two gap dimensions, 150 µm and 650 µm.

Figure 2

Light dependency of sodium nitroprusside (SNP) to degrade and release nitric oxide (NO). NO increased by time and the maximum release of NO in our study was observed from SNP under light after 3 h.

Figure 3

Time dependence of light-activated nitrite release from SNP in the microfluidic channels with or without concurrent hypoxia.

Figure 4

Microfluidic chip setup for cell experiments. PDMS, polydimethylsiloxane.

Figure 5

Fluorescence signal from hypoxia and nitric oxide in the cells. (a) Hypoxia in one channel; (b) hypoxia in two channels; (c) nitric oxide in one channel; (d) nitric oxide in two channels. The black and grey datasets are two independent experiments.

Figure 6

Simultaneous hypoxia and nitric oxide response from the cell culture. Simultaneous fluorescence signal from hypoxia and nitric oxide increased in the cells after 3 h of pumping O₂-depleted H₂O and SNP in meanders. The data were gathered from three independent experiments (n = 3) and error bars were measured from standard deviation.

Figure 7

Combined patterning of hypoxia and nitric oxide microenvironment. (a) Hypoxia signal in the hypoxia only experiment. (b) Optical image of the cells in the hypoxia only experiment. (c) Nitric oxide signal in the nitric oxide only experiment. (d) Optical image of the cells in the nitric oxide only experiment. (e) Hypoxia signal in the combined experiment. (f) Nitric oxide signal in the combined experiment. (g) Phase contrast image of the cells before the simultaneous hypoxia and nitric oxide experiment. (h) Phase contrast image of the cells after the simultaneous hypoxia and nitric oxide experiment (image magnification: 100×).

Figure 8

Simultaneous response to hypoxia and nitric oxide on a microchip. (a,c) Hypoxia and nitric oxide signals from cells simultaneously. (b,d) Spatial variation of hypoxia and nitric oxide cell responses on the microchip. The result shown in (a) is analyzed in (b) and the result shown in (c) is analyzed in (d).

Figure 9

Oxygen measurements of microchip on oxygen sensor foil. (a) Calibration of oxygen on oxygen sensor foil with standards: oxygen-depleted water (0% oxygen: black colour) and oxygen saturated water (100% oxygen: white colour). (b) Oxygen signal from pumping one channel with oxygen depleted water. (c) Oxygen signal from pumping two channels with oxygen depleted water.

Figure 10

Fluorescence signals from the four different microenvironments achieved by the second microchip design. (a) Simultaneous response to nitric oxide and hypoxia. (b) Only hypoxia cell response. (c) Only nitric oxide cell response. (d) Signal from control area without treatments (image magnification: 100×).