In situ patterned micro 3D liver constructs for parallel toxicology testing in a fluidic device

Abstract

3D tissue models are increasingly being implemented for drug and toxicology testing. However, the creation of tissue-engineered constructs for this purpose often relies on complex biofabrication techniques that are time consuming, expensive, and difficult to scale up. Here, we describe a strategy for realizing multiple tissue constructs in a parallel microfluidic platform using an approach that is simple and can be easily scaled for high-throughput formats. Liver cells mixed with a UV-crosslinkable hydrogel solution are introduced into parallel channels of a sealed microfluidic device and photopatterned to produce stable tissue constructs in situ. The remaining uncrosslinked material is washed away, leaving the structures in place. By using a hydrogel that specifically mimics the properties of the natural extracellular matrix, we closely emulate native tissue, resulting in constructs that remain stable and functional in the device during a 7-day culture time course under recirculating media flow. As proof of principle for toxicology analysis, we expose the constructs to ethyl alcohol (0-500 mM) and show that the cell viability and the secretion of urea and albumin decrease with increasing alcohol exposure, while markers for cell damage increase.

Figure 1

(a) Schematic of the assembled device, showing four discrete chambers, each addressable by an inlet and outlet. (b) Workflow for construct formation in the fluidic device. All fluidic channels (i) are filled with a mixture of HEPG2 cells and the HA/PEGDA precursor (light red, ii). A printed transparency photomask (grey) is employed to define constructs (iii). Following UV exposure, cross-linked constructs (dashed lines) are formed in the channels (iv) and the remaining solution is replaced, first with clean PBS and then with the desired ethanol mixture (v). Epifluorescence imaging (vi) confirms construct formation.

Figure 2

(a) Schematic representation of ethanol toxicity measurement. Media containing increasing concentrations of ethanol are flowed continuously through the parallel chambers of the fluidic device using a computer-controlled peristaltic pump. (b)–(e) L/D analysis on day 7 of constructs exposed to the indicated ethanol (EtOH) concentration. Each image represents a 3D reconstruction of a 150 μm Z-stack of confocal images taken at the conclusion of the experiment (day 7). Green fluorescence indicates calcein AM-stained live cells and red fluorescence indicates ethidium homodimer-1-stained dead cells; Scale bar −250 μm. The asterisk-indicated white region of the disk in each panel indicates the region of the organoid represented by the image. (f) Quantified cell viability percentages of constructs on day 7 at each ethanol concentration. (Significance: *p < 0.05 between all group-to-group comparisons.)

Figure 3

Assessment of (a) human serum albumin, (b) urea concentrations, and (c) alpha glutathione-S-transferase in the presence of 0 mM (black circles), 50 mM (red squares), 100 mM (blue upward triangles), and 500 mM (green downward triangles) ethanol. Significance in (a), (b) * p < 0.05 between 0 mM and all other conditions. Significance in (c) * p < 0.05 between 0 mM and 500 mM; ** p < 0.05 between 0 mM and 100 mM; *** p < 0.05 between 50 mM and 500 mM, and between 100 mM and 500 mM; **** p < 0.05 between 50 mM an 100 mM.

Figure 4

Confocal micrographs of small, Alexa Fluor 488 labeled hydrogel constructs fabricated in situ in a fluidic chamber. Photomask diameters are (a) 500 μm, (b) 250 μm, and (c) 100 μm. All scale bars represent 400 μm.