Scanning Microscopy


A method commonly used to measure the ability of cells to repair potentially lethal damage (PLD) is to compare immediate plating (IP) and delayed plating (DP) survival. Lower cell survival under IP conditions relative to that after DP conditions has been interpreted to indicate a higher ability of cells to repair potentially lethal damage (PLO) under DP conditions. However, this IP radiosensitization has not been observed in several cell lines and tumor models. IP conditions involve treatment of cells with trypsin and plating them into fresh growth medium. We have investigated the possibility that radiosensitization under IP conditions may be related to both the cell-shape and the nutrient concentration in growth medium (GM, MEM+15% serum). This idea predicts that the IP and DP survival of spheroids will show a response similar to the IP survival of cells in monolayers and that the IP and DP survival of crowded monolayer cells in high densities will be the same. Chinese hamster V79 cells grown in monolayers (spread cells) and spheroids (clumps of round cells) were used. The IP survival was lower than the DP survival for spread log phase monolayer cells but not for round log phase cells in spheroids. Radiosensitization of cells by fresh (as opposed to spent) growth medium was absent for high density plateau phase cells in monolayers at or above 2x106 cells/ml. However, PLO repair could be demonstrated in spheroid cells and in high density plateau phase cultures by exposing cells to hyperthermia or hypertonic saline.

Comparison of immediate plating versus delayed plating survival detects PLO repair only in well spread low density monolayer cells, but not in round spheroid cells nor in dense monolayer cells at > 107 cells/25 cm2 flask/5 ml medium. The absence of a difference between IP and DP cell survival does not mean that PLO repair is absent. Incorrect prediction of tumor response to radiotherapy can occur when PLO repair capacity is assayed as a ratio of DP/IP survival. More than one method must be used to measure the capacity of cells to repair their PLO.

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