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Introduction
Perfused multiwell plate is an array of bioreactors integrated into a multiwell plate format. It fosters long-term maintenance of 3D tissue under constant microperfusion, mimicking in several important ways the complexity and 3D microenvironment of liver. It is assumed that the better a cell culture system mimicks the complexity and microenvironment of a tissue or an organ, the more useful and broader information it can provide.
The bioreactors have open wells, built-in pneumatically-actuated micropumps, and the perfused multiwell plate employes a docking design that facilitates handling and integration into conventional incubation and assays tools. The concept is scalable to a plate with a higher number of wells, such as a 96-well plate. The higher throughput capacity of the perfused 3D multiwell is beneficial for conducting assays for liver toxicology and metabolism. Perfused multiwell plate can be used to model hepatic disorders, cancer, and other human diseases. Although it was designed primarily for the culture of liver cells, it can be used for perfusion culture of other highly metabolically active cell types such as kidney, heart, or brain cells. More information about the perfused multiwell plate can be found in a journal article. [pdf]
Standard multiwell tissue culture plate format
• cells cultured as monolayers under static conditions
Photo: Karel Domansky
Perfused multiwell plate
• cells cultured as perfused 3D microtissue units in channels
(3D tissue contained in a channel of a scaffold represents a 3D microtissue unit)
Design of the perfused multiwell plate
The perfused multiwell is an array of 12 open well bioreactors, each consisting of a reactor well, a reservoir well, and an integrated micropump. The reactor well contains an extracellular matrix-coated scaffold where cells self-assemble into an array of 3D microtissue units. Each well accommodates 400 000 – 600 000 cells. The reservoir well holds cell culture medium that is continually circulated between the two wells and through the scaffold by a diaphragm micropump. Similar to static multiwell plates, all bioreactors are fluidically isolated and covered with a single lid. Thus, there is no cross-talk between the bioreactors on the plate. Single lid simplifies adding and removing cell culture medium to and from the wells.
Design: Karel Domansky with Walker Inman. Photo: Karel Domansky
Ridges around the outside of the plate and around each bioreactor reduce the risk of airborne contamination. The open well design is conducive to manual or automated cell seeding and fluid handling, and the center-to-center spacing between bioreactors (18 mm) is set to allow use of multichannel pipettors with alternating tips omitted.
Note: To better see the details, the lid was removed from the plate. Photo: Karel Domansky
Micropumps are actuated in parallel from pneumatic inputs connected to the perfused multiwell through a dock. Docks are kept in standard CO2 incubators for cell culture and in sterile hoods for seeding. Pneumatic lines run from the docks to small electronic controllers outside the sterile environment. No electronics are contained in the perfused multiwell. The docking design makes handling a perfused multiwell plate similar to handling a standard tissue culture plate.
Note: For illustrating purposes, the plate in the photograph is not fully engaged in the docking station.
Photo: Karel Domansky
Tissue perfusion
Perfusion of the tissue in the scaffolds by cell culture medium is done by diaphragm micropumps embedded in the perfused plate. The micropumps are actuated pneumatically. Sequencing and timing of the pneumatic signals and thus the flow rate and flow direction is achieved by an eletropneumatic controller. The controller contains electronic circuitry and solenoid valves. The pneumatic signals are transmitted from the controller through three pneumatic lines and a docking station to the perfused multiwell plate where they are distributed to all pumps on the plate connected in parallel. Pneumatic actuation permits to keep the electronic timing equipment outside the incubator (a harsh environment for electronic equipment) and connect the perfused multiwell plate kept inside the incubator with three pneumatic lines (see the green, yellow, and orange tubing in the photograph below).
Note: The plate is covered with a polystyrene lid that provides sterility barrier.
Design: Karel Domansky with Walker Inman. Photo: Karel Domansky
Scaffolds for cell attachment and tissue formation
The scaffold provides the 3D physical support for cell attachment and tissue formation. The main factors that play a role in choosing a particular scaffold geometry include tissue morphogenesis considerations and oxygen transport limitations. A variety of materials and microfabrication techniques can be used to manufacture the scaffolds ranging from soft materials such as hydrogels to hard amorphous thermoplastics such as polystyrene. In the photographs below, scaffolds were made from polystyrene by micromechanical milling and from silicon by deep reactive ion etching technique. Typically, scaffolds are coated with collagen, an extracellular matrix protein, to enhance cell adhesion.
Photo: Karel Domansky
The scaffold is backed by a filter and a filter support. The filter captures cells in the scaffold immediately after seeding. There is also a filter in the reservoir well. The presence of filters in both wells prevents the cells from entering the valves and pumps.
Cells types cultured in perfused multiwell plate
Rat and human hepatocytes
Silicon scaffold microfabricated by deep reactive ion etching. Photo: Sharon Karackattu
Mouse hepatocytes (day 7)
Polystyrene scaffolds with 340-micrometer diameter microdrilled holes. Photo: Karel Domansky
3D model of the perfused multiwell plate
The pumps, valves, and fluidic capacitors are made by sandwiching a thin (~25 µm), highly flexible polyurethane membrane between the top (fluidic) and bottom (pneumatic) plates. The membrane also provides a partition between the sterile upper half (containing the cell cultures) and the non-sterile lower half (containing the pneumatics). The alignment between the plates is guided by two Dowel pins. Two flexing silicone membranes on the front face of the pneumatic plate move in tandem with the polyurethane mebrane in the valves and pumps. Thus, they indicate proper connections and function of the pneumatics.
Design: Karel Domansky with Walker Inman
3D model: Karel Domansky
A detailed exploded view of the components occupying each reactor well
Pumps, valves, and fluidic capacitors
An O-ring, a support scaffold, a filter, and a scaffold are placed into each reactor well, pressed together, and secured with a tightlly fitting retaining ring. A filter is dropped into each reservoir well and locked in place with a retaining ring (filter support is built-in in the bottom of the well). The pockets in sides of each well allow extration of the retaining rings and other components from the wells with tweezers.
An empty bioreactor (upper image) and a bioreactor loaded with components (lower image).
Perfusion of the tissue is performed by pneumatically driven micropumps integrated into the multiwell plate. Immediatelly after cell seeding, pumps circulate cell culture medium downward through the scaffold (direction 2). After cells attach to the scaffolds, flow direction is reversed to dislodge cell debris from the scaffold and the backing filter (direction 1). This is the long-term maintenance flow direction.
A cross-section of a bioreactor loaded with components.
The pumps feature a clamshell-like pump chamber and two active valves. The contoured shape of the pump chamber in combination with a thin, flexible membrane reduces actuation pressure requirements and leads to a constant stroke volume. A relatively large stroke volume allows for a high compression ratio and results in a pump that is seff-priming and bubble tolerant.
fluidic side
pneumatic side
3D model: Karel Domansky
Schematic cross-sections and top view photographs of the valves and pump chamber illustrating six phases of the pump cycle.
Photo: Karel Domansky with Walker Inman
A fluidic capacitor is used to convert pulsatile flow on the pump outlet into a nearly continuous flow through the tissue, thereby eliminating disruptive ocillatory movement of the cells in the channels during cell attachment.
Pumped volume plotted versus time during one pump cycle.
Performance of the fluidic capacitor is demonstrated by imaging flow in a capillary. The imaging was done at millisecond time increments with high-speed video camera Phantom V7.1 by Vision Research. The upper image show flows in a capillary that was attached directly to the outlet of a diaphragm micropump that generates a pulsatile flow. Then, the fluidic capacitor was inserted between the micropump outlet and the capillary (lower image). A dramatic flow smoothing effect of the fluidic capacitor can be observed.
Video: Karel Domansky and Walker Inman
More details about the pumps, valves, and fluidic capacitors can be found in a journal article. [pdf]
Incubator setup
During the cell culture, perfused multiwell plates are plugged into docking stations attached to the shelves of a standard incubator. Pneumatic lines connect the docking stations with solenoid valves. Switching the solenoid valves (and therefore managing the tissue perfusion patterns) is done by an electronic controller mounted on the incubator door.
Note: For a more explanatory depiction of the incubator setup, a photograph of the incubator interior is superimposed on the incubator door. Insert shows an enlarged photograph of the controller. A hair dryer on the microscope is used for defogging the lids on the perfused multiwell plate.
Photo: Karel Domansky
Additional resources:
1) Perfused multiwell plate instruction manual. [pdf]
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