Final Design

Cell Recovery System Prototype

DESCRIPTION

- Bioreactor -

Houses the cells throughout the thawing phase.

The final design for the bioreactor was based around using a 500 mL container with uniform diameter. With this container in mind, four ports were added to the cap. These four ports allowed for the feeding of media, extraction of dead stem cells, filtered ventilation, and extraction of live cells. Both removal ports consist of an internal hose which hangs to a desired length (this was acceptable for the dead cell removal as a technician determines at each stage how much liquid is removed) and in the case of the live cell extraction port, to a certain location. After the thawing process was completed, the bioreactor was placed on a tilting stage for live cell removal. This stage orients the bioreactor at an angle which allows for the natural settling of cells directly below the port. At this point, the vessel loading process begins; an operation which lay outside the scope of this project.

Bulkheads were used to establish the four ports required of the container. These fixtures were advantageous as they require minimal fabrication and increased the ease of replication. It was also necessary to design these ports to maintain sterility throughout the test, which required further precautionary measures to ensure that each port was sealed. These precautionary measures came in the form of both O-rings and sealants, ensuring a tight seal around each of the bulkheads. With regards to the sealants that was used, it is imperative that any product used is Class VI capacity and completely biocompatible. Subsequently, we tested two types of sealants: Loctite Nuva-Sil silicone light cure adhesive and Loctite Hysol Epoxy structural adhesive. Both sealants are rated to bond to Polypropylene very well and are of classification VI biocompatibility. It was also important to note that the bulkheads are molded using Natural Polypropylene, which is of class VI biocompatibility and the same material as the Nalgene container provided by ViaCyte.

- Stirring Stage -

The final design of the stirring stage consists of a motor driven crank that allows for an adjustable radius of rotation of 1.27 cm (0.5 in) to a maximum of 8.255 cm (3.25 in).

Linear guide rails, ball bearing carriages, and 25.4 x 25.4 x 0.635 cm (10 x 10 x 0.25 in) precision ground aluminum plates are incorporated to distribute the weight of the bioreactor and provide stability during rotation.  Two linear guide rails are attached to the bottom plate on opposite edges, with the middle plate secured through the carriages on these rails.  Another set of guide rails are attached onto the middle plate on opposite edges than the bottom plate, with carriages securing the top plate of the table.  This allows for the plates to move in the X and Y direction, resulting in a circular motion as the crank base rotates.

The crank base is driven by a high torque stepper motor with a holding torque of 4800 g-cm and a low speed torque of 4280 g-cm.  A drive shaft with a tapped hole allows for secure screw fastening between the motor shaft and crank base as well as ease of disassembly.  The motor is attached to the bottom plate using custom made shafts.

To provide variable radii of rotation the crank has been designed with slots for the crank slide.  The crank slide is connected to the crank base with screws fastened underneath the crank.  Adjusting or removing the crank slide is simply achieved by loosening these screws on the collar mount.  The crank slide is attached to the top plate with a flanged bearing in order to rotate the top plate in a circular motion.

- Tilting Stage -

Allows for variability in set angle to guarantee removal of living cells.

The bottom plate was constructed of 6.35 mm (0.25 in) thick aluminum to provide a steady base. Two modified steel L brackets were fastened to the base using 0.25 in diameter socket head bolts; these are used to serve as the lower two hinge brackets that remain stationary at all times. Two slotted rings were attached to the lower hinge brackets to serve as varying angle settings. By means of the slots coupled with a locking pin (rated at 9,200 lbs), the stage has the ability to be set at 9 different angles (roughly 10 degrees apart) for fine tune adjustment by the lab technician. The lower hinge plates were coupled with upper steel counterparts (identical in dimensions) to form the hinges – utilizing a shoulder bolt as the hinge pin (rated at 70,000 psi). These upper hinge plates were then welded to the top plate, 3.18 mm (0.125 in). In order to keep the bioreactor properly set on the top plate throughout tilting, two resting pins were attached using 0.25 in screws. By this means, as the stage was tilted, the bioreactor came to rest against the two pins to prevent it from sliding off the apparatus. To ensure further stability, two L brackets were mounted on the internal side of the lower hinge brackets. This was done to account for possible instances of misuse/human error.

- Control System -

Controls pumps for extraction/insertion of media and stirring stage during the cell recovery process.

Considering the flexibility, low cost, and software support available from JR Kerr, it was decided that JR Kerr would provide the best components for the ViaCyte bioreactor control system. The concept for the bioreactor included a stepper motor for stirring and two servo motors for driving peristalsis pumps feeding and removing waste from the Bioreactor. Several equations were developed for this system that convert real world values into code the processor can execute. They include time keeping, converting user input into position, and velocity commands for the different motor controllers.

PERFORMANCE

The system prototype was tested for a duration of 24 hours, where the following operation process was conducted to simulate cell recovery.- Bioreactor - The goal of no cell adhesion was obtained.  The stirring stage induced enough shear into the container to prevent settling.  It was discovered that cells would clump around the feeding/extracting hoses, however this did not affect any process of the system.- Stirring Stage - The stirring stage was able to rotate at different speeds and radii of rotation.  At the maximum radius of rotation, 8.255 cm (3.25 in), the stirring stage remained within the incubator base dimensions of 46.26 x 46.26 cm (19 x 19 in).  The table was able to support a 9.0718 kg (20 lb) weight and rotate smoothly with no noticeable difficulties.- Tilting Stage - The optimum angle to extract live cells was found to be ~30 degrees. - Control System -

The controls were able to accurately remove the top layer of bioreactor fluid, refill the bioreactor with new fluid and power teh stirring stage at variable speeds.  The sensitivity of the control parameters was tested and adjusted to provide the highest level of accuracy.

The final assembly test proved that the system was able to maintain an environment that minimized the coagulation of cells in the bioreactor. Using magnetic beads to simulate the behavior of the stem cells, the system was able to prevent the beads from clumping together in the center of the bioreactor. The success of the system was due to the individual achievements for each component of the assembly.