Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.
Normal adult human dermal fibroblasts were cultured at 37 degrees C on a thermoresponsive substratum composed of poly-N-isopropyl acrylamide (PNIPAAm) and type I collagen. The confluent fibroblasts were forced to detach from the substratum as a monolayer cell sheet made of polygonal cells linked together with many microvilli by decreasing an ambient temperature from 37 degrees C to a temperature below the lower critical solution temperature of PNIPAAm (about 30 degrees C). The floating cell sheet shrank and condensed into an aggregate with gap and tight junctions within several hours and finally formed a spheroid by 2 days. Spheroids were covered with squamous cells and contained cuboidal cells inside. Cycloheximide and actinomycin D reversibly inhibited the spheroid formation. Extracellular matrix fibrils deposited among inner cells when the culture period extended over 7 days. A 28-day-old spheroid with a diameter of about 600 microns contained live cells even in the central region. Cellular metabolic activities such as glucose consumption, lactic acid production, and ATP content of spheroids were significantly lower than those of monolayers. The spheroid described in the present study seems to be a useful in vitro model of connective tissues.
Takezawa
T
T
Japan Research Center, W. R. Grace & Co.-Conn., Kanagawa.
Mori
Y
Y
Yonaha
T
T
Yoshizato
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005109:Extracellular Matrix / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007773:Lactates / Q000378:metabolism; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009940:Organoids; D013997:Time Factors
D007773:Lactates; D003609:Dactinomycin; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D005947:Glucose