366
J . T . D I N G L E A N D M . W E B B
If the identity of the mucopolysaccharide that is produced by the
Tissue Culture system is known, it is of course relatively simple to
estimate one of the constituents of the polysaccharide, for instance,
the uronic acid or the hexosamine. This has been done by a number
of authors; for example, Prince and Castor (1961) estimated mucopolysaccharides in serum-containing culture media from measurements
of the uronic acid content after dialysis of the used medium (10 ml),
at 4° and p H 5-8, and elimination of protein by heat coagulation.
Recoveries of added chondroitin sulphate of from 62 to 85 % and of
hyaluronic acid of from 73 to 100% were obtained by this method.
It is apparent from Table I that the production of hyaluronic acid
in Tissue Culture has been widely studied. One reason for this is the
availability of a simple and relatively specific test for the presence of
this non-sulphated mucopolysaccharide. Highly polymerized hyaluronic
acid, when present in solution together with protein, forms a so-called
u m u c i n clot" when acidified with dilute acetic acid. This mucin
clot only forms when the molecular weight is in excess of 1-2 million.
Precipitation of hyaluronic acid as a mucin clot is dependent not only
upon pH, but also upon the ionic strength; it does not occur when the
latter exceeds about 0-22 (Pigman, Hawkins, Gramling, Rizvi and
Holley, 1960). The clot may be washed, dried and weighed and this
method can be shown to be quite reproducible. Alternatively, a rough
estimate of hyaluronic acid content can be obtained by progressive
dilution of the culture fluid until a mucin clot no longer forms on
acidification. When this point is reached the hyaluronic acid concentration is about 30 ftg/ml (Grossfeld, 1961). The production of the
mucin clot, which Pigman et al. (1960) found to be a combination of
negatively charged hyaluronic acid and positively charged protein, can
be used in combination with hyaluronidases from various sources, both
for the identification of hyaluronic acid, and as a method to estimate
the production of the mucopolysaccharides. For example, Grossfeld,
Meyer and Godman (1955) and Daniel, Dingle and Lucy (1961) have
used this procedure to follow the production of hyaluronic acid by
cells and tissues in culture. A complication in the estimation of the
production of hyaluronic acid in Tissue Culture systems has been
reported, however, by Grossfeld (1958) who showed that the tissue
culture medium may contain active hyaluronidases. Certainly, it has
been mentioned frequently that media which contain embryo extract
and give a mucin clot reaction when initially prepared, lose this property when incubated at 37° for 3 days or more (e.g. Gaines, 1960).
Another useful method for the estimation of total mucopolysaccharides is the procedure developed by Meyer and Rapport (1952)
and Gaines (1960) from the initial studies of Kass and Seastone (1944)
J . T . D I N G L E A N D M . W E B B
If the identity of the mucopolysaccharide that is produced by the
Tissue Culture system is known, it is of course relatively simple to
estimate one of the constituents of the polysaccharide, for instance,
the uronic acid or the hexosamine. This has been done by a number
of authors; for example, Prince and Castor (1961) estimated mucopolysaccharides in serum-containing culture media from measurements
of the uronic acid content after dialysis of the used medium (10 ml),
at 4° and p H 5-8, and elimination of protein by heat coagulation.
Recoveries of added chondroitin sulphate of from 62 to 85 % and of
hyaluronic acid of from 73 to 100% were obtained by this method.
It is apparent from Table I that the production of hyaluronic acid
in Tissue Culture has been widely studied. One reason for this is the
availability of a simple and relatively specific test for the presence of
this non-sulphated mucopolysaccharide. Highly polymerized hyaluronic
acid, when present in solution together with protein, forms a so-called
u m u c i n clot" when acidified with dilute acetic acid. This mucin
clot only forms when the molecular weight is in excess of 1-2 million.
Precipitation of hyaluronic acid as a mucin clot is dependent not only
upon pH, but also upon the ionic strength; it does not occur when the
latter exceeds about 0-22 (Pigman, Hawkins, Gramling, Rizvi and
Holley, 1960). The clot may be washed, dried and weighed and this
method can be shown to be quite reproducible. Alternatively, a rough
estimate of hyaluronic acid content can be obtained by progressive
dilution of the culture fluid until a mucin clot no longer forms on
acidification. When this point is reached the hyaluronic acid concentration is about 30 ftg/ml (Grossfeld, 1961). The production of the
mucin clot, which Pigman et al. (1960) found to be a combination of
negatively charged hyaluronic acid and positively charged protein, can
be used in combination with hyaluronidases from various sources, both
for the identification of hyaluronic acid, and as a method to estimate
the production of the mucopolysaccharides. For example, Grossfeld,
Meyer and Godman (1955) and Daniel, Dingle and Lucy (1961) have
used this procedure to follow the production of hyaluronic acid by
cells and tissues in culture. A complication in the estimation of the
production of hyaluronic acid in Tissue Culture systems has been
reported, however, by Grossfeld (1958) who showed that the tissue
culture medium may contain active hyaluronidases. Certainly, it has
been mentioned frequently that media which contain embryo extract
and give a mucin clot reaction when initially prepared, lose this property when incubated at 37° for 3 days or more (e.g. Gaines, 1960).
Another useful method for the estimation of total mucopolysaccharides is the procedure developed by Meyer and Rapport (1952)
and Gaines (1960) from the initial studies of Kass and Seastone (1944)