Future directions for this project would include various experiments to allow better understanding of cellular response to the ABS. Testing could occur in a series of studies that each isolate a different aspect of the design as follows:
Study 1: 2D vs 3D
The purpose of this first study is to see if there is a significant difference in cell viability between 2D and 3D culture exposure to ABS. The control group will consist of cells plated in the traditional 2D (flat) manner on top of the same collagen ECM that is used to make the 3D lumen in the new design. The cells will be exposed to stagnant ABS (no flow). The test group will consist of flowing ABS (at the same concentration used for the control group) through a 3D lumen seeded with cells for the same duration of time as the 2D cells are exposed to the ABS. After the set period of exposure, viability testing of the 2D and 3D cultures will be conducted to reveal any differences of viability as a result of geometric culturing methods (2D vs 3D). It is anticipated that cell viability will be greater in the 3D culture vs the 2D culture, as it is suspected that the stagnant exposure is what is most immediately toxic to cells
Study 2: Media vs ABS
The purpose of this second study is to observe the differences in viability between exposure to media vs exposure to ABS. The control group will consist of a cell-seeded lumen that will have a 50/50 composition of the growth media for each of the two cell types flowing through it. The test group will feature a cell seeded-lumen that will have ABS flowing through it (at the same flow rate as the media in the control group). After a set period of exposure, the viability of the media and ABS groups will be examined to determine if any differences in viability correlate with media vs ABS exposure. It is anticipated that greater cell viability will be observed in the media group than the ABS group. This study attempts to eliminate the influence of the 3D culture on cell viability and focuses on how cells respond to the media/ ABS.
Study 3: Increasing Concentrations of ABS
The purpose of this study is to see if the toxicity response observed in the cells (cell death) is related to the concentration of ABS or whether the presence of ABS at all is enough to induce cell death (this could have clinically relevant results). For the control group, cell-seeded lumens will have a 50/50 composition of the growth media for each of the two cell types flowing through it:
There will be about 3-4 control groups in which the concentration of media changes
Conc. A: media (0% water)
Conc. B: media + minimal concentration of water (10%-20%)
Conc. C: media+ moderate concentration of water (21-30%)
Conc. D: media+ higher concentration of water (31-40%)
The purpose of this control group is to ensure that any significant changes observed in cell death between ABS concentrations is not due to a lower concentration of media but due to the increasing toxic exposure from ABS. For the test groups:
There will be about 3-4 test groups in which the concentration of ABS changes:
Conc A: media+ water (no ABS)
Conc B: media + minimal concentration of ABS (10%-20%)
Conc C: media+ moderate concentration of ABS (21-30%)
Conc D: media+ higher concentration of ABS (31-40%)
After predetermined time of exposure, viability of each control and test group will be tested.
While it is anticipated that both groups will experience decreases in cell viability with each successive concentration group, it is expected that the rate of decrease in cell viability will be greater in the ABS group than the control group. The results of this study could be further augmented by additional data yielded from the collection and analysis of metabolites from the cells. If there is sufficient media volume and it is cycled through the system multiple times, metabolite analysis could provide meaningful insight into the status of cellular function in addition to cell viability during/ after exposure to ABS/ different concentrations of ABS.
Study 4: Increasing Time of Exposure
The purpose of this study is to observe cell response to ABS over greater periods of time (clinically relevant). The control groups will consist of cell-seeded lumens that will have a 50/50 composition of the growth media (at same concentration as the media in the ABS mixture, diluted with water) for each of the two cell types flowing through it with increasing periods of time that each will be run:
Duration 1, Duration 2, Duration 3, Duration 4
The test groups will feature cell seeded-lumens that will have ABS flowing through it (same concentration for all durations)
Duration 1, Duration 2, Duration 3, Duration 4
Viability tests will be conducted after each duration. It is expected that viability will decrease with increased time duration, or that it may saturate at one of the intermediate durations. It is also expected that the rate of decrease in cell viability will be greater in the ABS group than in the control media group.The results of this study could be further augmented by additional data yielded from the collection and analysis of metabolites from the cells. If there is sufficient media volume and it is cycled through the system multiple times, metabolite analysis could provide meaningful insight into the status of cellular function in addition to cell viability during/ after exposure to ABS at varying durations.
Study 5: Particle Velocity vs Set Flow Rate
A high speed camera can measure the velocity of particles in the flow over time to establish a relationship between the flow rate set on the pump and the actual observed velocity of particles in flow (that may be affected by the ability of the particles to deform and the resistance they may encounter). This would give more insight into the hemodynamics of the ABS in the blood vessel environment. It is anticipated that particle velocity will increase as the programmed flow rate increases, but a 1:1 correlation is not expected.