Cultivated seafood is a potential alternative protein source that can address the rising global food demand with exponentially rising human population growth. Cultivated seafood is made by growing animal cells in vitro using stem cells for edible food, eliminating the need to raise the entire animal. A crucial first step in developing cultivated seafood is creating a well-characterized cell line that can continuously grow and differentiate into desired cell types. Due to difficulties in determining optimal primary cell culture conditions, no continuous cell lines of food-relevant mollusks have been established so far. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the decontamination, cell dissociation, and culture conditions suited for mollusk adductor muscle cells. Oyster adductor (OAD) cells were obtained via tissue explant, mechanical and enzymatic digestion. The cells were routinely monitored using an inverted microscope for phase-contrast and fluorescence imaging. Culture vessels were coated with surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine to promote cell attachment. The tissue decontamination with Penicillin-Streptomycin (100 µg/mL), Amphotericin B (0.25 µg/ml), and algaecide solution (0.03%) was effective in controlling microbial growth. OAD cells grew best at lower nutrient levels in a one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. The tissue explant method resulted in the optimal cell dissociation from the three methods, and proceeding cultures had attached cells surviving for up to 10 days. All the plate coatings promoted cell attachment, but fibronectin provided optimal cell attachment of OAD cells. Fibroblast-like, neuron-like, epithelial-like, and rounded cells were observed. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in primary cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production. / Master of Science in Life Sciences / For sustainable seafood production, alternative sources of seafood proteins are essential in ensuring food security in the future. Cultivated seafood is an alternative protein source to address this rising food demand without the need to raise, farm, or slaughter animals. In developing cultivated seafood, self-renewing stem cells of the animal of interest are grown and made into edible products. A crucial first step in making cultivated seafood is understanding the growth conditions of the primary cells taken from animal tissue. Marine mollusk composes a significant part of seafood consumption, and developing cultured mollusks can address the growing food demand as a seafood alternative. However, there are many gaps in understanding the biological and physiological requirements of mollusk cells. No continuous, self-renewing mollusk cells of food-relevant species have yet been established. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the tissue decontamination, cell dissociation, and culture conditions suited for oyster adductor muscle (OAD) cells. OAD cells were obtained via three cell dissociation methods. Cell growth was routinely monitored using an inverted microscope. Cell-surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine were used to promote cell attachment. The tissue decontamination was effective with Penicillin-Streptomycin, Amphotericin B, and algaecide. OAD cells grew best at lower nutrient levels in the one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. Various cell morphologies were observed in the OAD cell cultures. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production.

Twenty two methionine-containing di- to octa-peptides were evaluated for their ability to serve as methionine sources to support protein accretion and cell proliferation in C₂C₁₂ myogenic, MAC-T mammary epithelial and ovine myogenic satellite cells. Factors in serum that may be involved in regulating peptide utilization was investigated using MAC-T cells. Growth of MAC-T cells was studied in the presence of methionine-containing dipeptides with 6% desalted adult animal serum from chickens, horses, humans, pigs or rabbits. Serumal peptidase activities on the twenty two methionine-containing peptides were examined in cell-free, methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% fetal bovine serum. The cell cultures were incubated for 72 h at 37°C in a humidified environment of 90% air : 10% CO₂ for C₂C₁₂ and ovine satellite cells or 95% air : 5% CO₂ for MAC-T cells. The basal medium contained methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% desalted animal serum or one of the following serumal factors: .4% bovine serum lipids, 1% chemically defined lipid concentrate, bovine insulin (1 ug/mL), or 3% low protein serum replacement (LPSR-1). Treatment media tested included basal medium or basal media supplemented with L-methionine or one of the methionine-containing peptides. Cell cultures incubated with the basal media for 72 h were characterized by decreased cell number and decreased protein content compared with initial cultures. All the methionine-containing peptides (with the exception of glycylmethionine and prolylmethionine for C₂C₁₂ cells), regardles of chain length, were able to support protein accretion with responses ranging from 29 to 123% of that of free L-methionine. The DNA contents of ovine satellite cell cultures indicated that cell proliferation occurred in the presence of all the methionine-containing peptides with responses ranging from 45 to 144% of the L-methionine response. Bovine insulin and lipids were not effective in promoting peptide utilization by MAC-T cells. However, the LPSR-1 facilitated the utilization of methionine-containing peptides in C₂C₁₂ and MAC-T cells. In the cell-free, methionine-free Dulbecco’s modified Eagle’s medium, peptidases could release all the methionine residues from the tetra- to octapeptides during 24 h of incubation and 42 to 70% of the methionine residues from the di- and tripeptides tested. The results demonstrated that cultured animal cells possess the ability to utilize methionine-containing peptides as methionine sources for protein accretion and cell proliferation, but serumal peptidases are at least partially responsible for the observed responses. / Ph. D.

Irreversible electroporation (IRE) is a non-thermal ablation technique that allows for eradication of unresectable tumors in a minimally invasive procedure. While IRE will preferentially kill larger cells over smaller ones, it does not discriminate between cells with larger and small nuclei. Given that one of the hallmarks of cancer cell morphology is larger, more abundant nuclei, our team set out to explore the possibility of preferentially targeting this physical and geometrical characteristic. / Master of Science

The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs), pericytes, and astrocytic endfeet, which regulate the transport of molecules into and out of the brain. BMECs possess intrinsic barrier properties that limit the passage of approximately 98% of small molecules into the brain in healthy individuals. However, in some brain diseases, the BBB undergoes structural and functional alterations, which can contribute to disease progression. In this study, we aimed to investigate the BBB by exploring the effects of endothelial cell stretching and the optimal dimensionality of stretching to enhance endothelium barrier tightness in Chapter 2. Subsequently, we developed an endothelium gradient stretching device to further examine the stretching effect in Chapter 3. Additionally, we investigated the promotion of endothelium tightness through the use of electrospun fibers, wherein we controlled the pore size. Based on these findings, we designed and fabricated an organ chip model that incorporates mechanical stretching, microfluidic techniques, electrospun fibers, and hydrogel extracellular matrix (ECM). The results of permeability testing demonstrated that this chip significantly improved the tightness of microvascular selective transport ability and has the potential to be used in drug sorting for central nervous system (CNS) diseases.

In order to perform more robust and physiologically relevant research, the Microphysiological Systems laboratory required a method of culturing cells where the level of oxygen could be adjusted. To fulfill this need, I designed and manufactured a hypoxic cell culture incubator. I employed a Quality Function Deployment to understand the customer requirements and convert them into engineering specifications. Based on these specifications, I created various conceptual designs. I evaluated the conceptual designs as compared to existing products and decided to move forward with the control system separated from a small, standard chamber. I developed the circuitry schematics for the temperature control loop, incorporating three heating pads and a temperature sensor, and the gas concentration control loop, incorporating three solenoids and two gas concentration sensors for oxygen and carbon dioxide. I designed the chamber in SolidWorks and 3D printed it. Finally, I performed vigorous validation testing of the device’s ability to control the temperature, the concentration of carbon dioxide, and the concentration of oxygen. The incubator was able to maintain the temperature within 0.5 degrees Celsius of a setpoint, the concentration of carbon dioxide within 0.24% of a setpoint, and the concentration of oxygen within 0.05% of a setpoint. These results surpassed the determined requirements.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.