iPSCs: The Future of Modeling and Validation
Session Number
Project ID: BIO 02
Advisor(s)
Dr. Angel Alvarez; Northwestern University Feinberg School of Medicine
Discipline
Biology
Start Date
22-4-2020 9:10 AM
End Date
22-4-2020 9:25 AM
Abstract
Introduction: Induced pluripotent stem cells (iPSCs) are generated from adult cells that are reprogrammed into an embryonic stem cell state with the capacity to differentiate into somatic cells. These cells can be differentiated into tissue organoids - 3D cellular structures self-organized to mimic a functional organ - which can be used for disease modeling. In this study, we aim to optimize the production of organoids by first optimizing conditions of cellular isolation, measuring the effect of bioplastics on stem cell growth, and comparing organoids generated from a bioreactor to those produced by conventional suspension cultures. We hypothesize the processing of blood prior to reprogramming will affect reprogramming success, that the material and sterilization process for bioreactor components will influence stem cell growth, and a spinning bioreactor will produce superior organoids to those generated using suspension culture. Methodology: Peripheral blood mononuclear cells (PBMCs) were isolated from patient blood and held at 37º Celsius or ice for four hours to mimic transport conditions. Moreover, we also determined the effects of varying blood volume on PBMC isolation using CPT tubes. Additionally, we tested the toxicity of two different 3D-printed plastics that were sterilized using ethanol or autoclaving by allowing them to leech into stem cell media. Then, iPSC colonies were allowed to expand on these plastic treated mediums. We also tested both autoclaving and using ethanol to determine which was the best method of sterilizing the plastics. This is due to 3d printed plastics being the main structural component of the bioreactor. Unfortunately, we did not have enough time to grow suspended-cultured iPSC organoids or produce organoids using a bioreactor.
Results: It was concluded that transporting the blood at 37º C was more viable than on ice. The PBMCs survived better under neutral temperatures as opposed to cold temperatures due to the physiological changes created by the extreme cold temperature.
Conclusion: Using iPSCs in disease modeling has become more prevalent in the recent past. Thus, determining which method to generate 3d organoids is of great significance to this field. Though we have not been able to prove or disprove our hypothesis, we were able to define important prerequisites to generating iPSCs from PBMCs and bioreactors. The continuation of this study will ultimately answer the question of which method, bioreactor or suspended culture, should be used when creating iPSC-based organoids.
iPSCs: The Future of Modeling and Validation
Introduction: Induced pluripotent stem cells (iPSCs) are generated from adult cells that are reprogrammed into an embryonic stem cell state with the capacity to differentiate into somatic cells. These cells can be differentiated into tissue organoids - 3D cellular structures self-organized to mimic a functional organ - which can be used for disease modeling. In this study, we aim to optimize the production of organoids by first optimizing conditions of cellular isolation, measuring the effect of bioplastics on stem cell growth, and comparing organoids generated from a bioreactor to those produced by conventional suspension cultures. We hypothesize the processing of blood prior to reprogramming will affect reprogramming success, that the material and sterilization process for bioreactor components will influence stem cell growth, and a spinning bioreactor will produce superior organoids to those generated using suspension culture. Methodology: Peripheral blood mononuclear cells (PBMCs) were isolated from patient blood and held at 37º Celsius or ice for four hours to mimic transport conditions. Moreover, we also determined the effects of varying blood volume on PBMC isolation using CPT tubes. Additionally, we tested the toxicity of two different 3D-printed plastics that were sterilized using ethanol or autoclaving by allowing them to leech into stem cell media. Then, iPSC colonies were allowed to expand on these plastic treated mediums. We also tested both autoclaving and using ethanol to determine which was the best method of sterilizing the plastics. This is due to 3d printed plastics being the main structural component of the bioreactor. Unfortunately, we did not have enough time to grow suspended-cultured iPSC organoids or produce organoids using a bioreactor.
Results: It was concluded that transporting the blood at 37º C was more viable than on ice. The PBMCs survived better under neutral temperatures as opposed to cold temperatures due to the physiological changes created by the extreme cold temperature.
Conclusion: Using iPSCs in disease modeling has become more prevalent in the recent past. Thus, determining which method to generate 3d organoids is of great significance to this field. Though we have not been able to prove or disprove our hypothesis, we were able to define important prerequisites to generating iPSCs from PBMCs and bioreactors. The continuation of this study will ultimately answer the question of which method, bioreactor or suspended culture, should be used when creating iPSC-based organoids.