The initial client statement provided to the team:
“Design, develop, and characterize a 3D in vitro model of skeletal muscle ingrowth”
In order to accomplish this goal, the design team would need to successfully develop a new assay or improve the existing 3D assays in order to effectively model cell confluence, migration, and proliferation onto the scaffold. It was important that the team identified and prioritized design objectives and constraints, which would be iterated as the stakeholders’ needs were understood in depth. A final set of objectives and constraints were formed to assist the team
in moving forward with a comprehensive project strategy and testing mechanisms throughout the design phase.
3.3 Objectives
A list of objectives was created based on the initial client statement. Client and user interviews, along with team assessments, were used to then tailor the objectives to the wants and needs of the present stakeholders and determine success (Appendix B-D).
The initial objectives determined by the team were evaluated once client and user interviews provided detailed information regarding goals of the project. The team decided to focus on developing a new model system that would be compatible with 3D models, and then test this assay using a 3D microthread scaffold. Once the design iteration process began, the objectives and sub-objectives were further investigated for importance to the assay design. Figure 10 shows a hierarchical structure of the project objectives and their relevance to the design.
Figure 10. Project objectives hierarchical tree
The five high-level objectives specify that the assay will be Easy to Use, Reproducible, it will Interface with 3D scaffolds (e.g. fibrin microthreads), Support Cellular
Characterization, and be Cost Efficient. These objectives are defined in Table 1 and the sub- objectives are defined in Tables 2 through 6.
Table 1. Main objective definitions
Objective Definition
Easy to Use Model must be easy to assemble, easy to handle and intuitive to use
Reproducible Must produce results that are consistent across replicates and multiple users
Interface With 3D Scaffolds
Model needs to be biocompatible and conducive to C2C12 replication and migration onto the scaffold
Support Cellular
Characterization Must be able to obtain quantifiable results and be designed to allow the user to test multiple replicates at once and meet size constraints
Cost Efficient Model needs to be productive relative to the cost of building and using it
Table 2. Sub-objective definitions for Ease of Use
Sub-Objective: Ease of Use Definition
Minimal Preparation Time Model assembly and testing setup must be intuitive and efficient for user
“Off the Shelf” Design Components are prefabricated and minimal assembly is required
Easy to Handle Model must be easy to work with during setup, testing, and imaging
Easy Data Collection The user must be able to stain and image cells and analyze relevant throughput using available techniques
Easy to Clean Assay can be sterilized using 70% Ethanol Limited Monitoring
Table 3. Sub-objective definitions for Reproducible Sub-Objective: Reproducible Definition Consistent Assay Properties
Model must be able to show similar levels of cell outgrowth and confluence between replicates and between multiple tests Standardization Between
Users Model must be designed to minimize variability between users Precision of
Measurements
Little variance between cellular outgrowth distance measurements of same conditions (consistent) Accuracy of
Measurements
Cellular confluence predictive of in vivo response (shows cellular alignment, comparable leading cell outgrowth, formation of ECM around thread-gel interface)
in vivo Predictability Data provides benchmarks to recognize success and failure once the scaffold is tested in vivo
Table 4. Sub-objective definitions for Interface with 3D Scaffolds
Sub-Objective: Interface
with 3D Scaffolds Definition Support Microthread
Scaffolds The model must be designed to test fibrin microthreads as the primary focus Biocompatible Material Materials used for the model must be compatible with C2C12
cells and non-cytotoxic
Sustained Outgrowth The model must be able to sustain cells in order for proliferation to occur
Table 5. Sub-objective definitions for Supports Cellular Characterization
Sub-Objective: Supports Cellular Characterization
Definition
Quantify Cell Migration Model must support cell migration onto the microthread scaffold
Quantify Cell
Proliferation Model must be able to distinguish if outgrowth is caused by cell division or cell migration Quantify Cell Confluence Model must be able to quantify cell confluence around the
circumference of the microthread
Observe Cell Alignment Model must support the cell alignment required for functional muscle tissue regrowth
Ease of Data Collection Data collection must be efficient using microscopes and technology provided
Maximize Data Collection Rate
Efficient and intuitive set up leads to more assays being analyzed and more conditions being tested
Table 6. Sub-objective definitions for Cost-Efficient
Sub-Objective: Cost- Efficient
Definition Materials Purchased
Sustainably
The model must be made of materials which are available to the lab over time and affordable to purchase. Required consumables and disposable components must also be inexpensive.
Equipment is
Inexpensive or Provided
Model must be compatible with technologies already present in the lab and any additional accessories must be inexpensive to purchase
Minimize Use of Reagents
Lower the cost of materials / amount of materials used
3.4 Constraints
In order to complete the MQP project requirements, certain criteria must be met. These criteria were identified as constraints because they had the potential to limit the ability for continuation of the project. Table 7 defines the constraints.
Table 7. Project Constraints
Constraint Definition Financials Budget of $1,000
Time Project must be completed by April 19th, 2019 Resources Materials must be available or attainable for the team Materials Must not be cytotoxic or harm users
Sterility Must be able to be sterilized by lab standards or fabricated in a sterile environment.
The constraints provided the team with working limitations that must be met to design the assay. WPI provided a working budget of $1,000 and the team had until April 19th, 2019
(Project Presentation Day) to complete the MQP. It is important that the materials used for the project be available to the team and the users within the Pins lab. Additionally, the materials used to produce the assay must not be cytotoxic or harmful to users of the model system. Finally, the team needed to ensure that the assay could be sterilized by laboratory standards.
3.4.1 Design Dimension Constraints
The design of the developed assay must also fit physical constraints. Each singular model system must fit into a 34.8 mm diameter well. The container in which PDMS is cured must fit into a vacuum chamber, measuring 25 cm in diameter. Additionally, the PDMS has to be cured
at 60°C for at least one hour. The container in which the PDMS is cured must not be cytotoxic or leach toxins into the PDMS.