122
C H A R I T Y W A Y M O U T H
strains could be cultivated in a protein-containing medium for a period
and returned to the synthetic medium without an adaptive crisis. Other
strains fail to grow on return to a biological medium.
Holmes (1959) was able to grow Chang's human liver epithelial cells
in a semi-synthetic modification (Medium A) of Parker's medium
858, after a period of adaptation similar to that described by McQuilkin
et al. (1957). The serum concentration was rapidly reduced from 20°/o,
by replacing half of the medium with fresh defined medium, three
times a week. Difco Bactopeptone at 0*1 °/o in the medium reduced the
period of adaptation to 90 days. Addition of peptone to cells after
adaptation still stimulated growth, and these adapted cells were used
as test objects for studying fractions obtained from the peptone by
curtain electrophoresis. One heat-stable, dialysable fraction was
discovered with the capacity, when included at 0-1 °/o in the defined
medium, to give a cell population in 10 days double that produced
with the whole peptone at 0-1 % . Holmes and Wolfe (1961) fractionated
bovine serum in the same manner, and tested the fractions on Chang's
strain of liver cells and on HeLa cells, both grown in Medium A, or
the modification Medium A 2 , of 88 components. Perhaps the most
interesting supplements in Holmes' media A and A2 are ^-oestradiol,
included at 0-1 mg/100 ml, and cholic acid, at 20 mg/100 ml. Protein
components with ability to promote cell attachment, cell clumping
and cell multiplications were separated from the serum, and it was
observed that salmine sulphate at an appropriate concentration
(1 mg/100 ml) promoted excellent spreading of HeLa cells on glass,
in a manner similar to the albumin fraction from serum which had this
effect.
A human skin-cell strain, originally isolated in N C T C 109 + 3 0 %
serum and grown for some months in N C T C 109+10°/o serum, was
adapted by Bakken, Evans, Earle and Stevenson (1961) by gradually
reducing the serum concentration from 10, through 5, 1, 0-5 and
0-125°/o to zero. The period of withdrawal of serum was characterized by failure of the cells to adhere to the glass, and by cell
clumping. Adaptation to the point where islands of cells attached to
the glass took about 6 months. Better adhesion to the glass was promoted
by an improved glassware-cleaning procedure. After about 2 years,
most of the cells adhered to the glass. This strain was further adapted
to suspension culture in N C T C 109+0-45°/o of 4000 CPS Methocel
(Dow). Bakken et al. comment upon, without being able to explain,
the long period of adaptation required for human cells, both in their
experience with skin cells, and in the case of human liver cells (Holmes,
1959). This contrasts with the ready establishment of monkey kidney
cells in synthetic medium (Evans, Kerr, McQuilkin, Earle and Hull,
C H A R I T Y W A Y M O U T H
strains could be cultivated in a protein-containing medium for a period
and returned to the synthetic medium without an adaptive crisis. Other
strains fail to grow on return to a biological medium.
Holmes (1959) was able to grow Chang's human liver epithelial cells
in a semi-synthetic modification (Medium A) of Parker's medium
858, after a period of adaptation similar to that described by McQuilkin
et al. (1957). The serum concentration was rapidly reduced from 20°/o,
by replacing half of the medium with fresh defined medium, three
times a week. Difco Bactopeptone at 0*1 °/o in the medium reduced the
period of adaptation to 90 days. Addition of peptone to cells after
adaptation still stimulated growth, and these adapted cells were used
as test objects for studying fractions obtained from the peptone by
curtain electrophoresis. One heat-stable, dialysable fraction was
discovered with the capacity, when included at 0-1 °/o in the defined
medium, to give a cell population in 10 days double that produced
with the whole peptone at 0-1 % . Holmes and Wolfe (1961) fractionated
bovine serum in the same manner, and tested the fractions on Chang's
strain of liver cells and on HeLa cells, both grown in Medium A, or
the modification Medium A 2 , of 88 components. Perhaps the most
interesting supplements in Holmes' media A and A2 are ^-oestradiol,
included at 0-1 mg/100 ml, and cholic acid, at 20 mg/100 ml. Protein
components with ability to promote cell attachment, cell clumping
and cell multiplications were separated from the serum, and it was
observed that salmine sulphate at an appropriate concentration
(1 mg/100 ml) promoted excellent spreading of HeLa cells on glass,
in a manner similar to the albumin fraction from serum which had this
effect.
A human skin-cell strain, originally isolated in N C T C 109 + 3 0 %
serum and grown for some months in N C T C 109+10°/o serum, was
adapted by Bakken, Evans, Earle and Stevenson (1961) by gradually
reducing the serum concentration from 10, through 5, 1, 0-5 and
0-125°/o to zero. The period of withdrawal of serum was characterized by failure of the cells to adhere to the glass, and by cell
clumping. Adaptation to the point where islands of cells attached to
the glass took about 6 months. Better adhesion to the glass was promoted
by an improved glassware-cleaning procedure. After about 2 years,
most of the cells adhered to the glass. This strain was further adapted
to suspension culture in N C T C 109+0-45°/o of 4000 CPS Methocel
(Dow). Bakken et al. comment upon, without being able to explain,
the long period of adaptation required for human cells, both in their
experience with skin cells, and in the case of human liver cells (Holmes,
1959). This contrasts with the ready establishment of monkey kidney
cells in synthetic medium (Evans, Kerr, McQuilkin, Earle and Hull,