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3T3 cells were established after a temporary dip in reproductive ability (top); once established they entered a resting state at a low cell density (bottom).% K7 p) m% Z4 I( s4 P
# S" E( u; P y- b$ u# K' |: B1 OGREEN
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In this mess of uncontrolled growth it was difficult or impossible to pick out cells that had been transformed after infection with oncogenic viruses, thus slowing the study of this transformation process. Todaro and Green (1963) took a different approach. "I wanted to do an experiment that somebody else could reproduce," says Green. He and Todaro controlled both inoculation density and frequency of transfer so that cell density would be strictly regulated. This was unlike the culturing techniques used by most researchers. "They would just transfer whenever they liked〞it was haphazard," says Green. "It's the way most people still do it."2 _5 C7 S7 i( s9 }4 \- c" ~ u" h
& K7 V1 c2 t$ KEstablishment of cell lines was thought to be a rare event, but with their strict protocol Todaro and Green succeeded in deriving stable cell lines in 9 of 11 attempts. Cells inoculated at higher density also grew optimally at higher density, but the most interesting line was one that was inoculated at a lower density of 3 x 105 cells per plate and transferred every three days. These 3T3 cells grew slowly at first, then recovered and grew stably to reach a reversible resting state. This state was reached at a much lower cell density than for other cell lines, and uniquely, these cells never grew over each other and did not show a reduction in size.& s2 B* E% M& |
! ?- p5 V ?: f T( N4 [9 M% h6 n* UThese unexpected and unique properties prompted a reassessment, still in progress today, of what steps are necessary for both the establishment and transformation processes. As Todaro and Green wrote in their paper, "the malignant properties of many established lines may be the result of the selective processes usually operating in cell culture and not related to the process of establishment per se.") r* h" b+ Z- {+ B( s! g& ]
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As Todaro's single semester turned into a postdoc of several years he found that 3T3 cells were an excellent target for transforming viruses (Todaro et al., 1964), as the growing, transformed cells could easily be picked out from the background of resting cells. 3T3 cells were similarly useful when cellular oncogenes were discovered, and Green also developed them as a model for adipose differentiation (originally dismissed as lipid accumulation secondary to cell death). They became a widely used feeder cell for the culturing of other cell types; Rheinwald and Green (1975) used them in this role to derive the first human keratinocyte cultures, which led to treatments for burn patients. 3T3 cells, in their many guises, have now been cited in over 25,000 publications.' P% s- A$ b& S5 U
/ }' \" h( P5 ~Todaro, G.J., and H. Green. 1963. J. Cell Biol. 17:299–313.
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Todaro, G.J., et al. 1964. Proc. Natl. Acad. Sci. USA. 51:66–73.
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5 I& q" \4 V/ `0 j( r* x1 SRheinwald, J.G., and H. Green. 1975. Cell. 6:331–343.(Cell lines were nothing new in 1961, whe) |
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发表于 2009-3-6 09:05
发表于 2015-6-6 11:41
