3D cel culture poster2
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Introduction/Abstract Materials and Methods Results Conclusions and Future Directions Results continued Mesenchymal Stem Cells (MSC) are multipotent cells that are capable of differentiating into several different cell types including osteoblasts, chrondroblasts, and adipocytes. They also have the ability to exert effects on other cell types by excreting immunomodulatory, anti-angiogenic, anti-inflammatory, pro-cardiovasculogenic, and pro-arteriogenic factors. These cells are primarily present in the bone marrow but can also be found in the blood, placenta, amniotic fluid, heart, skeletal muscle, and adipose tissue in both adults and fetuses. Due to these characteristics, there has been much interest in understanding the therapeutic potential of these cells in areas such as tissue repair and regeneration of bone, cartilage and heart muscle as well as tissue transplantation and treatment of autoimmune diseases. As such, it is important to understand how these cells behave in an environment that mimics the environment of native tissues such as a 3D extracellular matrix. In this study, we looked at the proliferation and migration of mouse mesenchymal stem cells in a 3D matrix of fibrinogen and thrombin and compared it to a non-coated 2D control. Proliferation and migration was observed over five days by staining the cells with Live/Dead stain, imaging using a fluorescent microscope, and measuring the amount of the Live stain fluorescence with a fluorescent plate reader. Preliminary results indicate that migration is affected by the 3D cell matrix with cells beginning to migrate towards one another by Day 3 and beginning to migrate to different planes within the matrix by day 5. There is also an increased proliferation seen in the 3D culture conditions compared to the 2D culture. These results will be useful in accessing the difference between mouse models and human models of cell migration and proliferation within a 3D matrix. 300 µL fibrin/ thrombin gel cultures were created with human fibrinogen (Sigma) solubilized in 0.85% sodium chloride at 37°C and human thrombin (Sigma) reconstituted in 1 mL of sterile dH 2 O. Mouse Mesenchymal Stem Cells (Invitrogen) were mixed with fibrin at 1,000,000 cells/mL (50,000 cells/well) in 24-well plates, a cell density found to maximize proliferation (cell concentrations were calculated using measurements obtained from the T4 Cellometer [Nexcelom]) and thrombin was added immediately following. Final fibrinogen concentration was 5 mg/mL. Final thrombin concentrations was 2.5 IU/mL. Non-coated control wells were also measured. 3D fibrin constructs were incubated at 37°C with 10% serum-containing DMEM/F-12 media. Measurements were taken one, three and five days after seeding, where day one samples were processed twenty four hours after fibrin construct formation. Mouse MSC proliferation was evaluated by labeling cells with Live/Dead Stain (Invitrogen), Imaging the cells using an Olympus IX-71 INVERTED MICROSCOPE and Qcapture pro software as well as reading average fluorescence intensity (494 nm ex / 517 nm em) over each well using a Molecular Devices F5 plate reader. FilterMax F5 Multi-mode Plate Reader Software (Molecular Devices) provided raw fluorescent read data. Excel software was used to evaluate raw data produced from the plate reader and produce graphical results including standard deviations. Day 1 and Day 3 2D and 3D Images The fluorescent microscope images of the mouse MSC cells indicated that the cells begin to migrate toward one another by day 3. By Day 5 the cells form what look like spherical colonies that have migrated to a higher plane within the 3D matrix. This could be due to the higher proliferation that is observed in the 3D cell culture conditions when compared to the 2D cell culture conditions. The cells are attempting to migrate higher within the matrix where there is more room to grow. This observation is confirmed in Figure 4. The higher levels of Live Cell fluorescence correlate to higher levels of proliferation seen in 3D versus 2D culture conditions. Future directions to take this research could involve varying the concentrations of Fibrinogen and Thrombin to determine the affects on migration and proliferation. It would also be interesting to let the experiment go longer (eight or ten days) to see if a greater number of spherical colonies form than what was observed at five days. Also it would be useful to compare mouse MSC to human MSC to determine if they behave the same way in 3D culture. Finally we would like to look at mouse embryonic stem cells in a similar experiment to determine if these cells behave in a similar way or if the MSC cells are migrating this way because they are more differentiated. It would also be interesting to see if the 3D matrix causes the mouse ES cells to differentiate and if this differentiation could be manipulated by changing the 3D culture conditions. Day 5 2D and 3D Images Assesment of Mouse Mesenchymal Stem Cell Migration and Proliferation in 3D Fibrin Constructs Melissa McCoy, Instructional Support Technician for MS Biotechnology Program, Suharika Thotakura, Mathew Cheney and William Ferguson • Chunnian Zhou, Ph. D, BIO 510 Instructor, Bill Tawil, Ph. D, BME 501 Instructor • Figure 1A Day 1 image of 2D mouse MSC cells- 50, 000 cells were seeded and grown overnight, stained with Live/Dead and imaged at 10x magnification Thrombin 2.5 units/ml Figure 2A Day 3 image of 2D mouse MSC cells- on day three cells were stained with Live/Dead and imaged at 10x magnification. Figure 3A Day 5- image of 2D mouse MSC cells- on day five cells were stained with Live/Dead and imaged at 10x magnification. Figure 1B Day 1 image of 3D mouse MSC cells- 50, 000 cells were seeded in 5 mg/ml fibrinogen and 2.5 units/ml thrombin 3D matrix. The cells were grown overnight, stained with Live/Dead and imaged at 10x magnification Figure 2B Day 3 image of 3D mouse MSC cells- on day three cells were stained with Live/Dead and imaged at 10x magnification. Figure 3B Day 5 3D image of mouse MSC cells- on day five cells were stained with Live/Dead and imaged at 10x magnification. Figure 3C Day 5 image of mouse MSC cells- same field of view as figure 3B but in brightfield at 40x magnification Figure 4 Live cell fluorescence intensity correlate to proliferation levels of mouse MSC cells in 2D and 3D culture conditions. Cells were stained with Live/Dead and fluorescent levels of the Live stain were quantified using Molecular Devices F5 plate reader. Samples were run in quadruplicated, averaged and standard deviations calculated to construct error bars. References/Acknowledgments 1. Haison Duong, M.Sc., Benjamin Wu, D.D.S., Ph.D., and Bill Tawil, Ph.D. “Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity”. TISSUE ENGINEERING: Part A Volume 15, Number 7, 2009. 2. Sese, N., Cole, M., Tawil, B. “Proliferation of HEKs and Co- cultured HEKs and HDFs in 3D Fibrin Constructs.” Tissue Engineering 2011; 17:429-437 3. Vaananen, H. Kalervo (2005) “Mesenchymal Stem Cells”. Annals of Medicine, 37:7, 469-479. This research was conducted as part of the development of the BME 501 course for the Master’s Biotechnology Program at CSUCI with the support of instructors Dr. Bill Tawil and Dr. Chunnian Zhou. BME 501 is a class offered in the MS Biotech Program under the Biomedical Engineering Emphasis, students interested in taking BME 501, please contact Dr. Ching-Hua Wang.
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Transcript of 3D cel culture poster2
Introduction/Abstract
Materials and Methods
Results
Conclusions and Future Directions
Results continued
Mesenchymal Stem Cells (MSC) are multipotent cells that are capable of differentiating into several different cell types including osteoblasts, chrondroblasts, and adipocytes. They also have the ability to exert effects on other cell types by excreting immunomodulatory, anti-angiogenic, anti-inflammatory, pro-cardiovasculogenic, and pro-arteriogenic factors. These cells are primarily present in the bone marrow but can also be found in the blood, placenta, amniotic fluid, heart, skeletal muscle, and adipose tissue in both adults and fetuses. Due to these characteristics, there has been much interest in understanding the therapeutic potential of these cells in areas such as tissue repair and regeneration of bone, cartilage and heart muscle as well as tissue transplantation and treatment of autoimmune diseases. As such, it is important to understand how these cells behave in an environment that mimics the environment of native tissues such as a 3D extracellular matrix.
In this study, we looked at the proliferation and migration of mouse mesenchymal stem cells in a 3D matrix of fibrinogen and thrombin and compared it to a non-coated 2D control. Proliferation and migration was observed over five days by staining the cells with Live/Dead stain, imaging using a fluorescent microscope, and measuring the amount of the Live stain fluorescence with a fluorescent plate reader. Preliminary results indicate that migration is affected by the 3D cell matrix with cells beginning to migrate towards one another by Day 3 and beginning to migrate to different planes within the matrix by day 5. There is also an increased proliferation seen in the 3D culture conditions compared to the 2D culture. These results will be useful in accessing the difference between mouse models and human models of cell migration and proliferation within a 3D matrix.
300 µL fibrin/ thrombin gel cultures were created with human fibrinogen (Sigma) solubilized in 0.85% sodium chloride at 37°C and human thrombin (Sigma) reconstituted in 1 mL of sterile dH2O. Mouse Mesenchymal Stem Cells (Invitrogen) were mixed with fibrin at 1,000,000 cells/mL (50,000 cells/well) in 24-well plates, a cell density found to maximize proliferation (cell concentrations were calculated using measurements obtained from the T4 Cellometer [Nexcelom]) and thrombin was added immediately following. Final fibrinogen concentration was 5 mg/mL. Final thrombin concentrations was 2.5 IU/mL. Non-coated control wells were also measured. 3D fibrin constructs were incubated at 37°C with 10% serum-containing DMEM/F-12 media. Measurements were taken one, three and five days after seeding, where day one samples were processed twenty four hours after fibrin construct formation. Mouse MSC proliferation was evaluated by labeling cells with Live/Dead Stain (Invitrogen), Imaging the cells using an Olympus IX-71 INVERTED MICROSCOPE and Qcapture pro software as well as reading average fluorescence intensity (494 nm ex / 517 nm em) over each well using a Molecular Devices F5 plate reader. FilterMax F5 Multi-mode Plate Reader Software (Molecular Devices) provided raw fluorescent read data. Excel software was used to evaluate raw data produced from the plate reader and produce graphical results including standard deviations.
Day 1 and Day 3 2D and 3D Images
The fluorescent microscope images of the mouse MSC cells indicated that the cells begin to migrate toward one another by day 3. By Day 5 the cells form what look like spherical colonies that have migrated to a higher plane within the 3D matrix. This could be due to the higher proliferation that is observed in the 3D cell culture conditions when compared to the 2D cell culture conditions. The cells are attempting to migrate higher within the matrix where there is more room to grow. This observation is confirmed in Figure 4. The higher levels of Live Cell fluorescence correlate to higher levels of proliferation seen in 3D versus 2D culture conditions. Future directions to take this research could involve varying the concentrations of Fibrinogen and Thrombin to determine the affects on migration and proliferation. It would also be interesting to let the experiment go longer (eight or ten days) to see if a greater number of spherical colonies form than what was observed at five days. Also it would be useful to compare mouse MSC to human MSC to determine if they behave the same way in 3D culture. Finally we would like to look at mouse embryonic stem cells in a similar experiment to determine if these cells behave in a similar way or if the MSC cells are migrating this way because they are more differentiated. It would also be interesting to see if the 3D matrix causes the mouse ES cells to differentiate and if this differentiation could be manipulated by changing the 3D culture conditions.
Day 5 2D and 3D Images
Assesment of Mouse Mesenchymal Stem Cell Migration and Proliferation in 3D Fibrin Constructs
Melissa McCoy, Instructional Support Technician for MS Biotechnology Program, Suharika Thotakura, Mathew Cheney and William Ferguson • Chunnian Zhou, Ph. D, BIO 510 Instructor, Bill Tawil, Ph. D, BME 501 Instructor
•
Figure 1A Day 1 image of 2D mouse MSC cells- 50, 000 cells were seeded and grown overnight, stained with Live/Dead and imaged at 10x magnification
Thrombin 2.5 units/ml
Figure 2A Day 3 image of 2D mouse MSC cells- on day three cells were stained with Live/Dead and imaged at 10x magnification.
Figure 3A Day 5- image of 2D mouse MSC cells- on day five cells were stained with Live/Dead and imaged at 10x magnification.
Figure 1B Day 1 image of 3D mouse MSC cells- 50, 000 cells were seeded in 5 mg/ml fibrinogen and 2.5 units/ml thrombin 3D matrix. The cells were grown overnight, stained with Live/Dead and imaged at 10x magnification
Figure 2B Day 3 image of 3D mouse MSC cells- on day three cells were stained with Live/Dead and imaged at 10x magnification.
Figure 3B Day 5 3D image of mouse MSC cells- on day five cells were stained with Live/Dead and imaged at 10x magnification.
Figure 3C Day 5 image of mouse MSC cells- same field of view as figure 3B but in brightfield at 40x magnification
Figure 4 Live cell fluorescence intensity correlate to proliferation levels of mouse MSC cells in 2D and 3D culture conditions. Cells were stained with Live/Dead and fluorescent levels of the Live stain were quantified using Molecular Devices F5 plate reader. Samples were run in quadruplicated, averaged and standard deviations calculated to construct error bars.
References/Acknowledgments1. Haison Duong, M.Sc., Benjamin Wu, D.D.S., Ph.D., and Bill Tawil, Ph.D. “Modulation of 3D Fibrin Matrix Stiffnessby Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity”. TISSUE ENGINEERING: Part A Volume 15, Number 7, 2009.
2. Sese, N., Cole, M., Tawil, B. “Proliferation of HEKs and Co-cultured HEKs and HDFs in 3D Fibrin Constructs.” Tissue Engineering 2011; 17:429-437
3. Vaananen, H. Kalervo (2005) “Mesenchymal Stem Cells”. Annals of Medicine, 37:7, 469-479.
This research was conducted as part of the development of the BME 501 course for the Master’s Biotechnology Program at CSUCI with the support of instructors Dr. Bill Tawil and Dr. Chunnian Zhou.
BME 501 is a class offered in the MS Biotech Program under the Biomedical Engineering Emphasis, students interested in taking BME 501, please contact Dr. Ching-Hua Wang.
