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1 3.051J/20.340J Lecture 13: Quantifying Cell Behavior 1. Cell Cultures a. Why use cell cultures? in vivo (living organisms) in vitro (glass, i.e., culture) Pros 1. native 3D environment 1. simplified model systems 2. all relevant signals present 2. study parameters independently 3. observe as function of time Cons 1. many variables – noisy data 1. unnatural 2D environment 2. animal rights concerns 2. may lack important signals b. Types of cell cultures Primary cultures: directly from living organism ☺ reflect function of real organism  eventually die off; #  some cell types poor dividers (nerve) Primary cells time Cell lines: derived from primary cells (not clones) ☺ altered genes ⇒ immortality • isolated spontaneous mutation • tumor cells • viral oncogene transfection # Cell lines time2 3.051J/20.340J 2. Assays of Cell Function  Adhesion  Migration  Proliferation  Differentiation (change in phenotype, ex. blood stem cells ⇒ leukocytes)  Secretion (protein production) a. Cell Adhesion Assays Importance: cell adhesion is necessary for many other cell functions— provides biophysical and biochemical stimulation  Sedimentation assay: 1. Cell type seeded onto surface of interest at given density (#/area) for a specified time in vitro 2. Surface is gently washed, and remaining cells counted, e.g., by optical microscopy or Coulter counter (cells detached, suspended & “count” by electrical resistance change thru narrow channel) % seeded adhered treated surface TCPS (control) 3.For testing cell resistance, serum-containing medium is a stricter test than cells seeded in protein-free solution. Note: plasma vs. serum plasma—liquid portion of blood (cells removed) serum—plasma with coagulants (e.g., FGN) removed3 3.051J/20.340J ⇒ Sedimentation assays give no info. on strength of adhesion.  Cell Spreading Assay 1. Cell type seeded onto surface of interest at given density (#/area) for a specified time 2. Measure projected surface area (optical microscopy) Closer in spirit, but still not a direct adhesion measurement Spreading indicates focal contact formation—metabolic activity  Centrifugation assay: (normal force) 1. Cell type seeded into 24-well plate with surface coating for a specified time 2. Plate is inverted in centrifuge and cells attached vs. applied force is measured4 3.051J/20.340J Normal Force Shear Force (centrifugation) (flow chamber) Shear Normal cells 100% remaining 50% F 0% 50% Applied Force Metric for comparison  Flow Chamber assay: (shear force) 1. Cells seeded into parallel plate chamber 2. Fluid flow velocity gradient results in shear force on cell-surface bonds – pop like a “seam”5 3.051J/20.340J b. Cell Migration Assays Importance: • tissue organization (embryonic) • immune and inflammatory response (chemotaxis of white blood cells) • angiogenesis—endothelial cell migration to form vasculature • wound healing—fibroblast migration to form connective tissue • tumor metastasis How do cells migrate? Actin polymerization in cytoskeleton ⇒ “lamellipod” extension Contractile force generated on actin filaments Force translated to substrate through focal contacts ⇒ net translation focal contact actin filament lamellipod Over short times: cell motion appears directional Over long times: cell motion is analogous to a diffusive process (in absence of chemical gradients or physical barriers)6 3.051J/20.340J Quantification approaches: 1. individual cell measurements 2. migration of cell populations  Individual Cell Measurements: Time-lapse videomicroscopy tracks cell motion (beneath fluid or gel) on a surface as a function of time elapsed. d 1. Measure distance traveled from starting pt. (d) as a function of time (t) 2. Fit data to model to obtain rms speed (S) and persistence time (P) (cell dependent) P: time before memory of initial direction is lost (typically min-hrs) S: measure of centroid displacement per time (typically 1-50 µm/h) /2 2 −tP )⎤()2 = nS ⎡Pt − P (1− ePersistent Random Walk Model: dt ⎣ ⎦ M # of dimensions (2) t 2()2 = 1 ∑ di ()where dt Mi=1 M = # of measurements Note: <d(t) > ≡ 0, t >>P Why?7 3.051J/20.340J M = # of cells if each time step used as a single data point d1(4∆t) d2(4∆t) Cell 1 path Cell 2 path Requires measuring many cell paths for good statistics (TEDIOUS!!) Alternately, we can use data from a single cell: Strategy (i): count each pt. on its trajectory as a “starting pt.” (all sampling points are equivalent) d2(4∆t) d1(4∆t) d3(4∆t) Strategy (ii): break migration path into M = N/j segments of j∆t steps (N=total # of sampling intervals ∆t) d1(4∆t) d2(4∆t) d3(4∆t)8 3.051J/20.340J Fitting to the Persistent Random Walk Model 2 /2 −tP )⎤()2 =nS ⎡Pt −P (1−edt ⎣ ⎦ For short times t<P: 2 axexp( −ax) ≈− +()−...1 ax 2 ()2 2 2dt = S t ()2 1/ 2 distance traveled = speed × time or dt = St (directional motion) ()2 2 2For long times t>P: dt = nS Pt − nS P 2 ()2 vs. t: ×103 10 Plotting dt()2dt m = nS2P (µm2) 0 slope = nS2P y-intercept = -nS2P2 time (min) ⇒ values of P & S b = -nS2P29 3.051J/20.340J Persistence time (P) Comparison of P & S (min) for different cell types migrating on same substrate… How do speeds relate to event sequence in the inflammatory response? Figure by MIT OCW. From D.A. Lauffenburger & J.J. Speed (S) (µ m/min) Linderman, Receptors: Models for Binding, Trafficking and Signaling, Oxford U. Press, 1993.()2 2For t >>P: dt = nS Pt ()2 1/ 2 distance traveled ~ time1/2 or dt = S nPt (diffusive motion) Analogous to the diffusion coefficient (D) for atoms and molecules, we can define a motility coefficient, µ , for cells: < > 1/22 ()2 = n µ t or dµ= SP / n ⇒ dt 2 2 = 4µ t for 2d Typical values: µ ~ 10-9-10-8 cm2/sec3.051J/20.340J 10 Chemotaxis If chemotactic or haptotactic (surface-bound signaling) agent is present, mean displacement <d(t)> is nonzero. We can quantify the degree of chemotacticity of a migration with the Chemotactic Index, CI: ⎧ t ⎫<d() >t −tP ⎪CI = ⎪⎨1−⎛⎞−1 ⎡1−e ⎤⎦⎬−1 ⎜⎟⎣ / P ⎭Lpath ⎪⎩⎝⎠ ⎪ Lwhere <d(t)> is the mean displacement up a concentration gradient, and path is the total cell path (contour) length. Concentration gradient of chemotactic agent in x-direction <d() >t For long times (t>>P): CI =< Lpath () > ( 0 < CI <1)t113.051J/20.340J 


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