A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds

Abstract

We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps-one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each-we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7).

Keywords: 3D printing; VeroWhite; bioreactor; live imaging; microfabricated scaffolds; perfusion culture; peristaltic pump.

FIG. 1.

Design of the bioreactor. Three parts of the bioreactor namely base, body, and cap. The bottom of the base has a countersink which matches the highest magnification objective lens and also has a seating for the cover-slip. The base has internal threads which match the external threads on the body. The body has two grooves on the bottom and top for seating the silicone O-ring. The media inlet is S-shaped so that the media enters the bioreactor at a height lower than that at which it exits. The external thread on the body matches the internal threads on the cap. The cap too has a groove for seating the silicone O-ring and a cylindrical protrusion which will be filled with polydimethylsiloxane (PDMS).

FIG. 2.

Assembling the bioreactor. A thin PDMS ring is placed on the seating made for the cover-slip on the base. A cover-slip is then placed on the PDMS ring. Further, a silicone O-ring is placed on top of the cover-slip. The body is then screwed on to the base with due care to make sure that the silicone O-ring sits in the groove meant for it on the bottom of the body. Another silicone O-ring is placed on top of the body and the cap is screwed on top with the silicone O-ring sandwiched between the grooves on both the body and the cap.

FIG. 3.

Initial bioreactor and peristaltic pump designs. (A) Initial bioreactor designs. Left: The design did not have a base and the cover-slip was just stuck to the body using vacuum grease. Center: A PDMS ring gasket was used for sealing by placing it on top of the cover-slip with matching protrusions and grooves on the base and body, respectively. Right: The cap was just screwed on top of the body without any silicone O-rings for sealing. (B) Initial peristaltic pump designs. Left: A modular peristaltic pump with inlet and outlet ports. The tube is connected between the inlet and outlet ports on the inside of the housing (not shown). The housing is bent because of the force exerted by the tube on the rotor. Center: Housing and rotor made out of metal to avoid the bending of the housing and wearing out of the rotor. The force exerted by the tube on to the rotor and the support force exerted by the housing on the motor is also shown. Right: Pins and cylinders used as rollers on the rotor. The wear on the pins due to the friction during operation of the pump is shown.

FIG. 4.

Design of the miniature peristaltic pump. The peristaltic pump has three main parts—housing, rotor, and casing. The housing holds the motor and has a seating for the ball bearing, which holds the rotor in place. The rotor has three needle bearings, which act as rollers. The needle bearings roll on pins that are held between two flanges. The flange which attaches to the motor shaft has a hole that matches the profile of the shaft. The rotor is held on its other end by another ball bearing that sits on the seatings of the rotor and the casing. The casing is secured on to the housing using four bolts.

FIG. 5.

Assembling the miniature peristaltic pump. A ball bearing is press fitted on to the seating on the housing. The tubings are then inserted through the two holes on the top of the housing. The rotor is then carefully press fitted into the ball bearing inside the housing such that the tubing is pressed between the rotor and the housing. Further the motor is fixed from behind the housing such that the motor shaft inserts into the flange on the rotor. Another ball bearing is press fitted to its seating on the casing and the casing is then fitted on the housing such that the rotor is held by the ball bearing. The casing is secured on the housing using four bolts.

FIG. 6.

Perfusion culture setup and the pump control circuit. (A) An assembly plate for securing the bioreactors, peristaltic pumps, media reservoirs, and the electric control circuit. A control circuit placed at the center draws power from an external source and drives four pumps. Tubes are routed from the reservoir to the pump and then to the bioreactor. The outlet of the bioreactor drains back into the reservoir. (B) The pump control circuit essentially consists of a potentiometer (P) across which several motors (M) are connected. Another resistor (R) is connected in series with the potentiometer to avoid shorting of the circuit case when the potentiometer is set to zero resistance. There is also a switch (S) in series with the resistor and the potentiometer for conveniently powering off the circuit.

FIG. 7.

Bioreactor and pump characterization. (A) Shear stress in the bioreactor—The total shear stress on the cells due to the flow of media from the peristaltic pump is shown. The maximum shear stress is around 0.29 mPa and occurs close to the inlet. (B) Pump flow characterization—Flow rate (mL/min) of the pump at zero pressure head for various voltages (in Volts; top) and at a constant voltage (3V) for various pressure heads (in kPa; bottom).

FIG. 8.

Viability of cells under perfusion in the bioreactor. Different bars represent different days of culture. Blue, day 2; red, day 4; green, day 6. The days are counted from the day when cells were seeded. Percentage of live cells in 24-well plate, 24-well plate coated with collagen, static and perfusion bioreactors are shown. Representative fluorescent images of each culture are shown above with the days increasing from top to bottom. Cells are stained using Calcein AM (live) and Ethidium Bromide (dead). All images were taken at 10× magnification.

FIG. 9.

Live imaging of NIH 3T3 fibroblasts growing on ring scaffolds.

FIG. 10.

Primary rat hepatocytes in the static bioreactor.

FIG. 11.

High-magnification time-lapse imaging of HuH7 cells incubated with live–dead assay mixture (Calcein AM–Ethidium Bromide).