Tubing-Free Microfluidic Microtissue Culture System Featuring Gradual, in vivo-Like Substance Exposure Profiles

Abstract

In vitro screening methods for compound efficacy and toxicity to date mostly include cell or tissue exposure to preset constant compound concentrations over a defined testing period. Such concentration profiles, however, do not represent realistic in vivo situations after substance uptake. Absorption, distribution, metabolism and excretion of administered substances in an organism or human body entail gradually changing pharmacokinetic concentration profiles. As concentration profile dynamics can influence drug effects on the target tissues, it is important to be able to reproduce realistic concentration profiles in in vitro systems. We present a novel design that can be integrated in tubing-free, microfluidic culture chips. These chips are actuated by tilting so that gravity-driven flow and perfusion of culture chambers can be established between reservoirs at both ends of a microfluidic channel. The design enables the realization of in vivo-like substance exposure scenarios. Compound gradients are generated through an asymmetric Y-junction of channels with different hydrodynamic resistances. Six microtissues (MTs) can be cultured and exposed in compartments along the channel. Changes of the chip design or operation parameters enable to alter the dosing profile over a large range. Modulation of, e.g., the tilting angle, changes the slope of the dosing curves, so that concentration curves can be attained that resemble the pharmacokinetic characteristics of common substances in a human body. Human colorectal cancer (HCT 116) MTs were exposed to both, gradually decreasing and constant concentrations of Staurosporine. Measurements of apoptosis induction and viability after 5 h and 24 h showed different short- and long-term responses of the MTs to dynamic and linear dosing regimes.

Keywords: drug dosing; microfluidics; microtissues; pharmacokinetics; tilting chip.

Figure 1

Substance concentration profiles in in vitro experiments as opposed to in vivo. Cell culture models in vitro are exposed to constant concentrations or sudden substance concentration steps generated by pipetting, while in the human body, the interplay of absorption, distribution, metabolism and excretion (ADME) produces distinct, dynamic substance concentration profiles upon oral administration or injection of a substance.

Figure 2

Working principle of the design. (A) Reservoirs 1 and 2 are filled with substance-loaded and plain cell culture medium. (B) Upon tilting of the chip, unequal volumes of liquid flow through the differently sized channels. While the substance concentration is, with each tilting interval Δt, gradually enriched in reservoirs 2 and 3, the substance is diluted in reservoir 1, until a concentration equilibrium is reached.

Figure 3

Modulation of concentration profiles. (A) Flow in the chip is dependent on the tilting angle α and the resulting height difference Δh between the liquid levels in the reservoirs at the left and right side. (B) Two reservoirs that are connected to channels with unequal flow rates Q₁ and Q₂ feed into a Y-junction. The flow-rate difference can be generated by different channel widths or dimensions. A cell culture site is located between the Y-junction and a third reservoir, and can be subjected to dynamic substance concentration profiles. (C) The channel resistances R_S and R_M, the initially applied liquid volumes V_S and V_M, and the tilting angle α can be used to modulate the concentration profiles. (D) The ratio of resistances can be used to define the time, after which an equilibrium is reached (for constant V and α). (E) The ratio of input volumes in reservoirs 1 and 2 defines the concentration, at which an equilibrium is reached (for constant R and α). (F) The tilting angle α modulates the slope of the concentration profile (for constant R and V).

Figure 4

Integration of the asymmetric design into a microfluidic microtissue-culture chip. (A) Microfluidic channel layout with the asymmetric Y-junction, followed by a mixing structure and six microtissue (MT) compartments. Details are shown in (B) the asymmetric Y-junction, (C) the micromixer including herringbone structures in a meander-shaped channel, and (D) the top view of a MT compartment with protective barrier structures and (E) a side view of a MT compartment. (F) Each microfluidic chip has the size of a microscopy slide and includes a dosing channel with a Y-junction, next to a control channel for linear exposure profiles. (G) Photograph of the microfluidic chip the channels and reservoirs of which have been filled with a colored liquid (scale bar: 10 mm).

Figure 5

Modulation of the substance concentration profile. (A) Amaranth red dye was used to simulate and track substance concentrations in the chip over time. Changing the tilting angle α modulates the slope of the concentration curve (V_S/V_M = 1; R_S/R_M = 4; n = 3 chips). (B) Decreasing concentration of 5-fluorouracil (5-FU) in the main channel over time. Peak area and concentration measurements were done by means of ultra-performance liquid chromatography, coupled to mass spectrometry UPLC-MS (V_S/V_M = 0.66; R_S/R_M > 0.25; α = 20°; n = 1 chip).

Figure 6

Staurosporine treatment of human colorectal tumor (HCT 116) microtissues (TuMTs) in the microfluidic chip. (A) Experimental layout: A chip was used to expose six TuMTs per channel to a PK-like dosing profile or a constant dose. The initial maximum concentration (C_max) of the PK-like profile was chosen as the constant concentration for the second channel. The perfusion control and the static control MTs were cultured without any compound treatment in the chip and in a 96-well microtiter plate, respectively. Readouts were taken 5 and 24 h after treatment start. (B) Relative caspase 3/7 activity, normalized to those of the static control of the respective sampling time points (data represented as mean ± SD; n = 6–12 TuMTs). (C) Intracellular ATP concentration as a measure of viability (data represented as mean ± SD; n = 6–12 TuMTs). (D) Representative images of TuMTs on the chip and in the microtiter plate at 0 and 24 h (scale bar: 500 μm).

Figure 7

Realization of more complex dosing profiles. A typical concentration profile (Kwon, 2001) upon oral administration of a substance could be approximated by using a sequence of different tilting angles. A medium exchange to replace the substance-loaded medium in the reservoir connected to the narrow channel with plain medium was performed after ~20 min. Amaranth red dye was used as drug-surrogate.