Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J....
-
Upload
laureen-nelson -
Category
Documents
-
view
214 -
download
2
Transcript of Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J....
![Page 1: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/1.jpg)
Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability
Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. FletcherBiophysical Journal, 2006
Sophie Wong20.309, MIT
November 20, 2008
![Page 2: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/2.jpg)
Background
• AFM used to quantify mechanical properties of biological material
• Current methods for measuring elasticity and viscoelasticity– Require indentation of cells adhered to substrate– Not feasible for non-adherent cells
• Increased stiffness of lymphocytes may be the cause of diabetes mellitus and leukemia
![Page 3: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/3.jpg)
Three Goals• Develop method for characterizing and
comparing deformability of leukemia cells
• Compare mechanical properties of –myeloid (HL60) and lymphoid (Jurkat) lines –normal neutrophils
• Compare Hertzian Mechanics Model vs. Liquid Droplet Model
![Page 4: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/4.jpg)
Developing the Method
• Used microfabricated wells to trap cells
50 µm 2 µm
• Array of 8 – 20 µm diameter wells
•Jurkat and HL60 cells trapped in 13.6 µm wells
•Neutrophils trapped in 10.8 µm wells
![Page 5: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/5.jpg)
Deflection-position curve
• Cantilever deflection small compared to indentation
• Piezo platform extended at constant rate = 1506 nm/s
• Deflect cantilever until ~ 800pN applied or cell indented 3 µm
HL60
![Page 6: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/6.jpg)
HL60s stiffer than Jurkat cells and neutrophils
• consistent with a model of leukostasis
• Stiffness contributes to vessel blockage
855 Pa
48 Pa 156 Pa
![Page 7: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/7.jpg)
Fitting the model• Models used to determine cell elasticity and
viscoelasticity• Hertzian Mechanics Model assumptions– Homogeneous, Isotropic, Linear, Elastic (HILE)– Material undergoes infinitesimally small strains
• Liquid Droplet Model assumptions– Internal contents are homogeneous viscous– Cortical tension constant around cell– Cortical shell deforms around tip during indentation– Radius of cell remains constant during indentation
![Page 8: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/8.jpg)
Hertzian Model fits better
• Gray line = data• Dash line = Hertzian mechanics model• Dotted line = Liquid droplet model
![Page 9: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/9.jpg)
Significance & Future Studies
• Deformability of leukemic cells plays important role in leukostasis
• Where does difference in cell stiffness originate? – Filament networks?– Cytoplasm?– Nuclear or cell membranes?
• Investigate other factors involved in leukostasis: adhesion, transmigration
![Page 10: Force Microscopy of Non-adherent Cells: A Comparison of Leukemia Cell Deformability Michael J. Rosenbluth, Wilbur A. Lam, and Daniel A. Fletcher Biophysical.](https://reader036.fdocuments.us/reader036/viewer/2022083006/56649f385503460f94c548f9/html5/thumbnails/10.jpg)
Questions?