Cell density determines epithelial migration in culture.

Abstract

ABSTRACr The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approximately 6.6 X 105 cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 days showed that the cells accelerated at a uniform rate of 0.24 gm-hr 2 and, by extrapolation, possessed an apparent initial velocity of 2.8 gsmhr at the time of wounding. The apparent initial velocity was considered to be the result of a brief (<10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 105 cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 ;&m'hr-2) Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. The use of epithelial cultures to study the wounding response exploits the simplicity of working with a monolayer of a single cell type. Although there are drawbacks in attempting to correlate migration on plastic or glass with an in vivo system, it is still possible to make comparisons (1). The following experiments were carried out with the dog kidney epithelial cell line (MDCK), which has the properties of a transporting epithelium (2). It will be shown that the migration of an epithelial sheet can be divided into two stages: first, a brief (<10 hr) rapid acceleration that is dependent on a threshold cell density, and second, movement at a slower constant rate of acceleration, which is considered to result from an aggregate of the individual movements of each migrating cell. MATERIALS The edge of the wound was marked by the razor at its initial placement.For the experiments begun at the maximal cell density, the times of wounding were staggered so that results at various time points could be collected at convenient hours. However, for those experiments carried out at intermediate cell densities, all of the dishes were wounded at the same time (i.e., over a period of 50 min for 45 dishes). During the experiments, the medium was changed daily. Two or three dishes were used for each time point and observations were made every 4 hr in the first 36 hr and every 6 hr up to 120 hr. Cells were fixed in 10% (vol/vol) buffered formalin and were stained with Harris-modified hematoxylin and Giemsa (both from Fischer Scientific, Fair Lawn, NJ).Distances of migration were measured with an ocular graticule by light microscopy. Fifty measurements for each ...

Collective cell migration patterns: Follow the leader

“…As was found in a previous study (9), the present work shows that the average width of the cell culture increases quadratically with time. The mechanism that drives this acceleration in the average velocity of the culture edge is not clear and may be related to the formation of leader cells and cell columns (Fig.…”

Collective migration of an epithelial monolayer in response to a model wound

“…Like other studies of classical wounds on epithelial monolayers (22,24), our study showed a nonlinear progression of the free edge. The acquired average velocity accelerated from 0 to 10 Ϯ 5 m⅐h Ϫ1 in typically 15 h. In ref.…”

“…At the same time, as already mentioned, the epithelia keep their integrity; the cell-cell contacts are maintained in particular by cadherins (21), although it has been observed that MDCK cells migrate actively within the monolayer by developing active ''cryptic'' lamellipodia under the other cells (20). Interestingly, although the onset of migration significantly depends on the initial cell density (22), cell division does not play an active role but rather appears to redensify the monolayer (20).…”

“…The acquired average velocity accelerated from 0 to 10 Ϯ 5 m⅐h Ϫ1 in typically 15 h. In ref. 22, the dynamic behavior of the mean progression ͗s͘ of the edge was empirically fitted by a parabolic law. Fitting our data with a power law ͗s͘ ϭ a ⅐ t n gave results compatible with this parabolic evolution because the average progression for various initial widths was well described by a common exponent n ϭ 1.8 Ϯ 0.4 (Fig.…”

Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion

“…In order for a cell to migrate, it first develops a polarized phenotype with a lamellipod at the front of the cell, whereby the migration front leads the movement, attaching to the substrate and propelling the rest of the cell forward. Cell migration can be reconstituted in vitro by treating cells with motogenic stimuli, such as hepatocyte growth factor (HGF, also called scatter factor) (3), or by mechanically wounding a monolayer of epithelial cells, such as Madin-Darby canine kidney (MDCK) cells, stimulating cell migration on each side of the wound toward the opposite side, thereby closing the gap (4).…”