Kristy Chau – Independent Project

Kristy Chau – Independent Project

Class of 2017

Introduction to Topic

Exposure to mustard gas can cause severe burning to the eyes and depletion of the corneal endothelial layer. Long term effects include blindness or a loss of clarity. The destruction of the corneal endothelial layer is irreversible at the moment, due to the fact that the corneal endothelial cells (CECs) do not regenerate in-vivo. Currently, the only form of treatment is corneal transplants, however there is a low supply of eye donors. Fortunately, since the CECs do proliferate in-vitro, mass producing this kind of cell in the lab is possible. An alternative way must be developed to alleviate the worldwide shortage of donors.

Project Description

At USAMRICD, my project revolved around the eye and the lethal effects of mustard gas to the corneal endothelium. With the ultimate goal of alleviating the shortage of eye donors, the provided equipment and guidance of others allowed experimentation on corneal endothelial cells and human mesenchymal stem cells. The extraction of CECs from rabbit, pig, and mouse eyes, and hMSCs from human bone marrow were necessary to induce differentiation. In order to expose the two cells to see such a reaction, the construction of a co-culture system in which CECs and hMSCs grown in the same vessel can be separated by a membrane or barrier containing five micrometers. Through 3D printing, the porous membrane can be manufactured and held in place by a frame intended to promote the differentiation of hMSCs into a CEC phenotype. This morphological change can indicate if the use of hMSCs were effective in cellular therapy and repairing the corneal endothelium from damage and disease.

Experience Description

During the summer of 2016, I was given the opportunity to work alongside scientists at U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) in Edgewood, Maryland. I was selected through the Science Engineering Apprenticeship Program (SEAP) and was ecstatic to learn more about the Science Technology & Engineering (STEM) field as I have had much interest in medicine.

On the first day, one of my mentors informed me of the focus of my main project. My project was primarily about the human eye, a delicate organ incorporating multiple parts to produce vision. With the guide of my three mentors, Dr. Timothy Varney, Erik Eaton, and Robert Haggins, and a college intern, Zachary Murray, we were able to experiment ways in producing corneal endothelial cells and human mesenchymal stem cells. Our ultimate goal was to potentially help alleviate the worldwide shortage of eye donors. The process involved dissecting rabbit albino eyes, swine eyes, etc. to isolate the corneal endothelial cells, and grow hMSCs. Every other week, eyes were delivered to USAMRICD, in which I could extract the corneal endothelial cells and store them for later use. I incubated them for periods of time, in order to find the most suitable flask of cells that would be used for the differentiation. We utilized human mesenchymal stem cells (hMSCs) extracted from human bone marrow. We continued with the experiment by dissecting more eyes, feeding, and incubating the cells until there was an adequate amount to be used for the experiment. I was given multiple protocols to follow during the dissections, as I worked under their supervision.

During this process, I realized the importance of sterilization and impact of time on the growth of cells. Within weeks, the cells may have been too confluent to use, so utilizing my time wisely was essential to acquire the necessary amount of cells. Also, my mentors emphasized on sterilization, for all equipment had to be sprayed with alcohol to ensure cleanliness. All materials in use must have been placed in the fume hood. In addition to the equipment at school, I was able to use new materials, such as biopsy punches, flasks, Mr. Frosty, etc. Similar materials used in school include incubators, water baths, centrifuges, and micropipettes. Safety precautions were always taken, since we were required to wear personal protection equipment (PPE) at all times.

After obtaining the adequate amount of cells, we constructed a co-culture system in which CECs and hMSCs were grown in the same vessel separated by a membrane containing five micrometers. Through research and experimentation, I was able to understand the influence of stem cells on corneal endothelial cells. I would like to continue working on this project to discover the factor secreted by the hMSCs, for the results were unexpected, but useful. From this experience, I realized my passion for the medical field, especially optometry.

Innovation Description

The creation of a co-culture system, in which the two cells can be grown in the same vessel separated by a membrane, can promote the differentiation of hMSCs into a CEC phenotype. Ultimately, we want to see how the human mesenchymal stem cells will morph into hexagonal shapes, as a replica of the depleted corneal endothelial cells.

Using Alibre, SolidWorks, or some kind of computer aided design (CAD) to build the model, the barrier should be able to fit across an 8 well plate, in terms of width, length and height. Taking the measurement of one well in the 8 well plate, and then adding three more side by side to the first will make it easier to keep the measurements consistent. Within the measurement of one well, the gaps between the wells should also be taken into consideration when building the model. Once the wells are placed next to each other to make a row of four with gaps in between each of them, rectangular holes should be cut out in the middle of each. Through 3D printing, the membrane can be manufactured with Ninjaflex, a bendable material that is easier to manipulate than plastic. NinjaFlex allows for repeated movement and impact without wear as it has an abrasion resistance better than other materials, a constant diameter and a chemical resistant that proves for it to be the most suitable for the creation of the barrier. The use of NinjaFlex will produce a strong, flexible, and high quality print. Using Krazy Glue, sheets of nylon will cover the holes on one side of the barrier and be cut out under a sterile environment to reduce the chances of contamination. Most likely done so in a fume hood, any pair of scissor can be used to cut the rectangular sheets of nylon, as long as they are sterile. The nylon will prevent the CECs and hMSCs from being in contact yet allow them to share the same growth media. The second side of the barrier will be glued to the other side of the barrier with sheets of nylon and secured onto the plate with Silicon. The membrane will then be held in place by a frame intended to promote the differentiation of hMSCs into a CEC phenotype.

With previous attempts in accessing similar results, the barrier allows the growth media to be shared between the CECs and hMSCs, while keeping the two cells separate. This makes the feeding process a lot faster and easier than previous methods. Compared to the pharmaceutical products and magnetic fields to treat corneal endothelial degeneration, the use of human mesenchymal stem cells seems to be the most effective due to their similarity to corneal endothelial cells. Other methods in approaching the same problem are inserts. The barrier allows each cell type to be evaluated in the same conditions. Inserts create two different environments which the cells are adhering to separate bases. This process requires more time to remove and add the growth media for each individual insert, whereas the barrier reduces the time to feed both types of cells at the same time.

Project Topic

Introduction to Topic

Exposure to mustard gas can cause severe burning to the eyes and depletion of the corneal endothelial layer. Long term effects include blindness or a loss of clarity. The destruction of the corneal endothelial layer is irreversible at the moment, due to the fact that the corneal endothelial cells (CECs) do not regenerate in-vivo. Currently, the only form of treatment is corneal transplants, however there is a low supply of eye donors. Fortunately, since the CECs do proliferate in-vitro, mass producing this kind of cell in the lab is possible. An alternative way must be developed to alleviate the worldwide shortage of donors.

Project Overview

Project Description

At USAMRICD, my project revolved around the eye and the lethal effects of mustard gas to the corneal endothelium. With the ultimate goal of alleviating the shortage of eye donors, the provided equipment and guidance of others allowed experimentation on corneal endothelial cells and human mesenchymal stem cells. The extraction of CECs from rabbit, pig, and mouse eyes, and hMSCs from human bone marrow were necessary to induce differentiation. In order to expose the two cells to see such a reaction, the construction of a co-culture system in which CECs and hMSCs grown in the same vessel can be separated by a membrane or barrier containing five micrometers. Through 3D printing, the porous membrane can be manufactured and held in place by a frame intended to promote the differentiation of hMSCs into a CEC phenotype. This morphological change can indicate if the use of hMSCs were effective in cellular therapy and repairing the corneal endothelium from damage and disease.

Experience

Experience Description

During the summer of 2016, I was given the opportunity to work alongside scientists at U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) in Edgewood, Maryland. I was selected through the Science Engineering Apprenticeship Program (SEAP) and was ecstatic to learn more about the Science Technology & Engineering (STEM) field as I have had much interest in medicine.

On the first day, one of my mentors informed me of the focus of my main project. My project was primarily about the human eye, a delicate organ incorporating multiple parts to produce vision. With the guide of my three mentors, Dr. Timothy Varney, Erik Eaton, and Robert Haggins, and a college intern, Zachary Murray, we were able to experiment ways in producing corneal endothelial cells and human mesenchymal stem cells. Our ultimate goal was to potentially help alleviate the worldwide shortage of eye donors. The process involved dissecting rabbit albino eyes, swine eyes, etc. to isolate the corneal endothelial cells, and grow hMSCs. Every other week, eyes were delivered to USAMRICD, in which I could extract the corneal endothelial cells and store them for later use. I incubated them for periods of time, in order to find the most suitable flask of cells that would be used for the differentiation. We utilized human mesenchymal stem cells (hMSCs) extracted from human bone marrow. We continued with the experiment by dissecting more eyes, feeding, and incubating the cells until there was an adequate amount to be used for the experiment. I was given multiple protocols to follow during the dissections, as I worked under their supervision.

During this process, I realized the importance of sterilization and impact of time on the growth of cells. Within weeks, the cells may have been too confluent to use, so utilizing my time wisely was essential to acquire the necessary amount of cells. Also, my mentors emphasized on sterilization, for all equipment had to be sprayed with alcohol to ensure cleanliness. All materials in use must have been placed in the fume hood. In addition to the equipment at school, I was able to use new materials, such as biopsy punches, flasks, Mr. Frosty, etc. Similar materials used in school include incubators, water baths, centrifuges, and micropipettes. Safety precautions were always taken, since we were required to wear personal protection equipment (PPE) at all times.

After obtaining the adequate amount of cells, we constructed a co-culture system in which CECs and hMSCs were grown in the same vessel separated by a membrane containing five micrometers. Through research and experimentation, I was able to understand the influence of stem cells on corneal endothelial cells. I would like to continue working on this project to discover the factor secreted by the hMSCs, for the results were unexpected, but useful. From this experience, I realized my passion for the medical field, especially optometry.

Innovation

Innovation Description

The creation of a co-culture system, in which the two cells can be grown in the same vessel separated by a membrane, can promote the differentiation of hMSCs into a CEC phenotype. Ultimately, we want to see how the human mesenchymal stem cells will morph into hexagonal shapes, as a replica of the depleted corneal endothelial cells.

Using Alibre, SolidWorks, or some kind of computer aided design (CAD) to build the model, the barrier should be able to fit across an 8 well plate, in terms of width, length and height. Taking the measurement of one well in the 8 well plate, and then adding three more side by side to the first will make it easier to keep the measurements consistent. Within the measurement of one well, the gaps between the wells should also be taken into consideration when building the model. Once the wells are placed next to each other to make a row of four with gaps in between each of them, rectangular holes should be cut out in the middle of each. Through 3D printing, the membrane can be manufactured with Ninjaflex, a bendable material that is easier to manipulate than plastic. NinjaFlex allows for repeated movement and impact without wear as it has an abrasion resistance better than other materials, a constant diameter and a chemical resistant that proves for it to be the most suitable for the creation of the barrier. The use of NinjaFlex will produce a strong, flexible, and high quality print. Using Krazy Glue, sheets of nylon will cover the holes on one side of the barrier and be cut out under a sterile environment to reduce the chances of contamination. Most likely done so in a fume hood, any pair of scissor can be used to cut the rectangular sheets of nylon, as long as they are sterile. The nylon will prevent the CECs and hMSCs from being in contact yet allow them to share the same growth media. The second side of the barrier will be glued to the other side of the barrier with sheets of nylon and secured onto the plate with Silicon. The membrane will then be held in place by a frame intended to promote the differentiation of hMSCs into a CEC phenotype.

With previous attempts in accessing similar results, the barrier allows the growth media to be shared between the CECs and hMSCs, while keeping the two cells separate. This makes the feeding process a lot faster and easier than previous methods. Compared to the pharmaceutical products and magnetic fields to treat corneal endothelial degeneration, the use of human mesenchymal stem cells seems to be the most effective due to their similarity to corneal endothelial cells. Other methods in approaching the same problem are inserts. The barrier allows each cell type to be evaluated in the same conditions. Inserts create two different environments which the cells are adhering to separate bases. This process requires more time to remove and add the growth media for each individual insert, whereas the barrier reduces the time to feed both types of cells at the same time.

By | 2017-05-15T15:33:54+00:00 May 15th, 2017|Biomed Capstone Project 2017|0 Comments

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