Perfused multiwell plate for 3D liver tissue engineering

Abstract

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.

Fig. 1

Photographs of a perfused multiwell with an array of 12 bioreactors. The size of the assembled multiwell plate is ~127.8 × 85.5 × 34 mm. The top view (a) includes inserted photographs of a bioreactor and a scaffold. The size of the white bar in the scaffold photograph is 300 µm. The bottom view (b) of a partially docked perfused multiwell shows the built-in connectors and pneumatic lines distributing positive and negative air pressure to individual valves and pump chambers.

Fig. 2

A schematic cross-section of a bioreactor (section A-A’ from Fig. 1a). Maintenance and post-seeding flow directions are indicated by arrows (1) and (2), respectively. Diameter of the reactor and reservoir well is 15 mm. Centers of the wells are 20 mm apart. Diameter of the scaffold is 14.9 mm. When there are 3 mL of culture medium in a bioreactor (a typical total volume), top of the scaffold is under ~7 mm of fluid. The valves, the pump chamber, and other features are not drawn to scale.

Fig. 3

A simulation of oxygen transport. The control volume is marked by a dashed line. Oxygen probes are sketched as black rectangles in the middle of the reactor and reservoir wells. The depth of culture medium in the channel was 1 mm corresponding to total volume of ~3 mL. The flow rate was 0.25 mL min⁻¹

Fig. 4

Measured concentration of dissolved oxygen in reservoir and reactor wells seeded with rat hepatocytes: (a) As a function of time post seeding with a flow rate of 0.25 mL min⁻¹ and a sampling rate of 0.2 measurements per minute. (b) As a function of flow rate (immediately following the time course measurement) with a sampling rate of 1 measurement per minute.

Fig. 5

Measured and modeled inlet and outlet oxygen concentrations as a function of flow for two depths of cell culture medium in the channel. The figure also shows modeled oxygen concentration at the oxygen probe location.

Fig. 6

(a) Cell viability assay of rat liver cells cultured for seven days in the perfused multiwell. The image shows multiple channels in a scaffold. Live cells are stained green with calcein AM while dead cells are stained red with ethidium homodimer-1. The areas occupied by the cells are bright green and areas without cells appear dark green or black. (b) Immunostaining of rat liver cells cultured in the perfused multiwell 7 days post seeding demonstrating retention of the hepatocyte-specific functional marker albumin (green). Staining was preformed with rat albumin antibody. This image shows a single channel with its boundary highlighted by a white dash line. (c) Image of rat hepatocytes and sinusoidal endothelial cells cultured in the perfused multiwell plate day 13 post seeding. Nuclei are stained blue with DRAQ 5, non-parenchymal cells express green fluorescent protein (GFP), and the functional marker for sinusoidal endothelial cells (SE-1) is stained red.