On the recovery of adhesiveness by trypsin-dissociated cells

Abstract

Summary.A sensitive method for assaying aggregation of dissociated cells has been developed which allows the determination of the mean number of cells per aggregate of a cell population. We have demonstrated that exposure of dissociated 6-or 7-day chick embryo neural retinal cells to trypsin in calcium-free solution renders them unable to aggregate for a half hour in stirred cell suspensions. Aggregation was noticeable first at 30 to 40 minutes and progressed to the formation of massive compact aggregates. Because the half-hour aggregation lag occurred both in the absence of serum and in medium reclaimed from aggregated preparations, the possibilities were excluded that it was due either to an inhibitor of aggregation in the serum, or was the time required for release into the medium of soluble aggregation-promoting materials emanating from the cells themselves. Cells dissociated by divalent cation withdrawal (Ca + +, Mg + +-free saline with EDTA) aggregated without a lag. The trypsin-induced lag does not appear to be the result of trypsin adsorbed to the surfaces of dissociated cells, as the lag is not abolished by addition of trypsin inhibitors to the aggregation medium. Microelectrophoresis of dissociated cells did not reveal changes in surface charge density during recovery from trypsinization. A variety of proteins and calcium ion, if present during trypsinization, protect the cells against the trypsin-induced aggregation lag. If the temperature was reduced from 37 to 6 ~ aggregation of fully adhesive cell populations came to a complete halt within 2 to 3 minutes. Aggregation resumed with a 5 to 10 minute delay when the temperature was returned to 37 ~ The rapidity of onset and reversal of inhibition of aggregation by low temperature treatment militates against the hypothesis that the low-temperature inhibition of aggregation acts by suppressing the synthesis of cell surface components necessary for adhesion. The abolition of the aggregation lag in trypsinized cells was also shown to be temperature-dependent; a 20-minute cold pulse administered in the middle of the lag period extended the length of the lag by exactly 20 minutes.Adhesiveness of cell surfaces is required so that cells can construct coherent tissues. A problem basic both to cell biology and to developmental

A correlation between membrane fluidity and the critical temperature for cell adhesion.

“…The correlation between the characteristic temperature for cell adhesion and membrane fluidity was not observed in the presence of Mg 2 § Since the two divalent cations, Ca 2+ and Mg 2 § are considered to act in qualitatively different ways in cell adhesion (27,29), it is probable that Mg 2+ is effective in cell adhesion in such a manner that is independent of the change in membrane fluidity at about 10~ SUMMARY BHK 21 cells can adhere to a protein-coated plastic dish in the presence of Ca 2+ at temperatures above 12~ However, they cannot adhere below 8~ The ESR spectrum of cells spin-labeled with a stearic acid label indicated that the membrane fluidity changed characteristically at 10~ 20~ and 300C. The critical temperature for cell adhesion coincided well with one of the characteristic temperatures for the membrane fluidity change.…”

“…The change in the physical state of the lipids may also affect the insertion (13) and distribution (3,4,8,20,23) of proteins in the membrane, and the activity of membrane-bound enzymes (31). These molecular events would also be relevant to cell adhesion (12,24,27,30).…”

Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth.

“…C ELL-CELL interactions are fundamental to all aspects of animal development, as well as to the functioning of the mature organism. Since the pioneering experiments of Steinberg et al (1973) and Takeichi (1977), it has become clear that a major role in these interactions is played by calcium-dependent adhesion systems. Calcium-dependent adhesion is accomplished principally through the actions of the cadherins, a family of cell surface glycoproteins (reviewed by Takeichi, 1988).…”

Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells

“…2c). Trypsin destroys the cell-to-cell adhesion molecules such as adhesion receptor, and enables cells a singlecell suspension state which makes it an effective reagent to detach adherent cells (Edwards et al 1975;Steinberg et al 1973). Recombinant trypsin is more suitable for the production of pharmaceutical proteins because it is non-toxic and free of virus contamination.…”

“…In addition, in the cell culture process trypsin is often used to detach cells, which can destroy the cellto-cell adhesion molecules (Edwards et al 1975;Steinberg et al 1973), thereby enabling cells to stay in single-cell suspension state. Nowadays, there are still some commercial trypsins derived from animal pancreas, which may be toxic and cause infectious contamination to cultured cells (Golden et al 2002).…”