Robotic fluidic coupling and interrogation of multiple vascularized organ chips

Abstract

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

Figure 1.

(a) Rendering of the Interrogator CAD model. The system is comprised of a 3-axis motion system, automated liquid handler, peristaltic pump, and custom microscope stage that allows for the continuous perfusion, linking and image analysis of organ-on-chip models. (b) Organ Chips are placed between inlet and outlet reservoirs in modular docks below the main fluid handling deck. Left to right: brain, BBB, skin, lung, heart, kidney, liver, gut. Top view of the instrument deck layout in CAD (c) and within the graphical user interface (d). Component positions are determined from CAD then used to build a virtual deck in the control software. (e) Rendering of the microscope module, showing a compact optical path microscope mounted on a 3-axis positioning stage that is integrated into the Interrogator for real-time imaging and inspection of Organ Chips. (f) Micrograph of a Gut Chip channel showing villus-like structures. Scale bar: 500 μm.

Figure 2.

Schematic of stage calibration. (a) A matrix of x,y,z coordinates is corrected by touching the four calibration points with a probe attached to the pipettor head. (b) The actual coordinates are then mapped to the theoretical coordinate matrix to generate a calibration matrix that is automatically used to adjust fluid handler stage motion. Initial positional errors (c) can be reduced below 0.5 mm (d). (e) Accuracy and precision of liquid handler used in Organ Chip linking studies was measured using a standard dilution routine of fluorescent dye. N=8. (f) Accuracy of pipettor calculated as Error = SD/Mean. Error bars are SE of error and correspond to the precision. N = 60. Automated generation of absorption/adsorption calibration data using blank Organ Chips and inulin-FITC tracer dye during infusion and elution phases. The inlet reservoirs (g) show the addition of fluorescent tracer for the first 24 h, while the outlet reservoirs (h) exhibit dye dilution due to perfusion through the Organ Chip device.

Figure 3.

Linking scheme of 8 organ Human Body-on Chips. (a) Human organs for the Human-Body-on-Chip (HuBoC), each with a representative Organ Chip photograph and sectional schematic. Clockwise, starting top left, BBB: lower channel brain microvascular endothelial cells, upper channel brain pericytes and cortical astrocytes. Brain: differentiated human primary neural stem cells forming networks of neurons and astrocytes. Lung: lower channel human umbilical cord vascular endothelial cells, upper channel, lung epithelial cells. Skin: lower channel containing dermal microvascular endothelial cells, upper channel containing keratinocytes and dermal fibroblasts. Kidney: lower channel kidney derived endothelial cells, upper channel containing proximal tubule epithelial cells. Gut: lower channel human umbilical cord vascular endothelial cells, upper channel containing villi-like structures of gut epithelial cells, Liver: lower channel containing liver sinusoidal endothelial cells, upper channel containing hepatocytes. Heart: lower channel human umbilical cord vascular endothelial cells, upper channel cardiomyocytes differentiated from cardiomyocytes. Scale bar: 5 mm. (b) A total of 8 vital organs – Gut, Liver, Heart, Kidney, Lung, Heart, Brain, Blood Brain Barrier (BBB)), and Skin – were joined through vascular endothelial channels in order to create the Body-on Chips. The system enables multiple sampling points and variety of linking possibilities.

Figure 4.

Automated Human Body on Chips linkage demonstrates maintenance of organ viability and function for 3 weeks. Immunofluorecence imaging of the HuBoC organs and organ-specific assessment throughout 3-week linkage: (a) Gut Chip endothelium (VE-Cadherin) and parenchyma (ZO1); (b) Gut Chip permeability values for Inulin-FITC; (c) Liver Chip endothelium (VE-Cadherin) and parenchyma (MRP2); (d) Liver Chip albumin production; (e) Kidney Chip endothelium (VE-Cadherin) and parenchyma (ZO-1); (f) Kidney Chip albumin reabsorption; (g) Lung Chip endothelium (VE-Cadherin) and parenchyma (ZO1); (h) Lung Chip dextran (3 kDa) permeability; (i) Heart Chip endothelium (VE-Cadherin) and parenchyma (α-Actinin); (j) Heart Chip LDH secretion; (k) Skin Chip endothelium (VE-Cadherin) and parenchyma (white Loricrin and purple Keratin 14); (l) Skin Chip cascade blue^® (596 Da) permeability; (m) BBB Chip endothelium (VE-Cadherin) and parenchyma (white pericytes, purple astrocytes (GFAP)); (n) BBB Chip LDH secretion; (o) Brain Chip parenchyma (green astrocytes (GFAP) and red neurons (β-III-Tubulin)); (p) Brain Chip Glutamine:Glutamate ratio. Scale bars: 100 μm. Data recorded at the given time points from two independent experiments; micrographs acquired from unlinked control chips following 3 weeks of culture. The IHC is representative of multiple Organ Chips and Chip regions. Data from two independent studies shown.

Figure 5.

Long-term analysis of inulin-FITC (2-5 kDa) tracer dye pharmacokinetics in an 8 organ system linked via vasculature and supported by computational PBPK modeling. Inulin-FITC was given as a bolus dose once weekly on the parenchymal side of the Gut Chip and linked as shown in Fig. 4. Experimental values (white bars, days after dose of the second week and blue bars, days after dose of the third week) for Inulin-FITC concentration throughout the linked Organ Chips match with the PK model predictions (red bars) over 21 days in both the endothelial and parenchymal channels. Data from two independent experiments. Read-outs below 0.005% are at the limit of detection.