<b>Developing Nanoparticle Sensor Technology for Real Time in Vitro Application</b>(Study on Human mesenchymal stem cells for Bone and Cartilage tissue)
Creators: name: AlSalhi, Dr. Sara Ibrahim
Publisher: name: figshare
Year: 2024
Subjects: classificationCode: 329999
subject: Other biomedical and clinical sciences not elsewhere classified
classificationCode: 400304
subject: Biomedical imaging
classificationCode: 400311
subject: Tissue engineering
classificationCode: 400310
subject: Rehabilitation engineering
classificationCode: 400399
subject: Biomedical engineering not elsewhere classified

description: This project aims to develop a novel fluorescently tagged nanoparticle that can monitor Oxygen, pH and Glucose levels within the in vitro environment which has multiple fluorescent wavelength band levels that might interfere with the quantification of cell viability and proliferation assays. The fluorescent optical sensors have gained particular features due to their high sensitivity, good photostability and ideal dimension for in vitro and in vivo applications, which play a vital role in regenerating tissue in biological applications. This project aims to understand the different concentrations of Nanosensors in 2D HMSC cultures and use the obtained information as a baseline for characterizing oxygen, pH, and glucose in a 3Dimesnional hydrogel system. Assays (MTT, Cy- Quant, LDH and Live dead assays) and differentiation assays which contain (Collagen-I, Collagen-II, Osteo Calcian, and ALP activity) were measured for 28 days at different data points at (3, 10, 17, 25 days).Contains reviews and topics that contributed to a comprehensive understanding of the achievement of the overall objective of the project such as Tissue Engineering and the importance of MSC in Tissue Engineering, Culture MSC in vitro in 2D and 3D, The Role of O2, pH, and glucose in stem cells behaviour, Nanosensors in monitoring O2, pH, Glucose, Working principle of Nanosensors (oxygen, pH, and glucose), Current research used to monitor and optimize oxygen, pH, and Glucose in 3D differentiation along with Notable Research Gaps.The experiment in this project was performed in four stages as shown in figure 7, ranging from synthesizing nanoparticle sensors (starting with oxygen sensors, pH sensors and finally glucose sensors), culturing Human mesenchymal stem cells, incubating HMSC stem cells in 96 well plates with the three different nanoparticle sensors (oxygen, pH, and glucose) at cell seeding density (2300 cells/ cm2), measuring the response in the HMSCs behaviour during the incubation period (28 days) through characterizing the viability assays Cy-Quant, LDH, and Live Dead, proliferation MTT at different time points 3,10,17 and 25 days and differentiation assays Collagen-I, collagen-II, Osteo Calcin, ALP Activity.MTT, Cy-Quant, LDH and Live dead assays were conducted to assess the proliferation and viability of Human mesenchymal stem cells in a 96-well plate in a 2D environment. HMSCs cultured with Nano (oxygen, pH, Glucose) sensor and assessed by differentiation assays Collagen-I, Collagen-II, Osteo Calcin, ALP activity.The result showed that the viability of HMSC cells in the oxygen concentration of 1mg/mL seriously affected the viability whereas the glucose sensors showed a negligible effect on proliferation and differentiation assays. Interestingly the pH sensors showed a higher staining of osteocalcin both in normal and osteo differentiation media indicating that there is a potential effect of amine-conjugated FITC dye in HMSC differentiation into osteo lineage. Further experiments with repeats are necessary to confirm the differentiation capacity. It is suggested to observe an in concentration of pH sensors in application to stem cell differentiation.Oxygen, pH and glucose nanoparticle-based sensors were synthesized and treated these sensors in the HMSC cellular environment in 96 well plates with two different concentrations 0.1 mg/mL and 1mg/mL. Lower concentrations (0.1mg/mL) of sensors show better viability for HMSC cells and can be used for monitoring oxygen, pH and glucose in vitro 3D environmental applications.