Culture conditions found to minimize false positive diagnosis of lysosomal storage disorders.

The effect of culture conditions on the ultrastructure and enzyme activities of cultured skin fibroblast cells relevant to the diagnosis of lysosomal storage disorders are reported. The parameters examined were: pH of the culture media, type of media, increasing cell passage, and day of harvest. Ultrastructural changes were defined in terms of the number of lysosome-like inclusion bodies per cell according to a method devised in our laboratory and proven reliable in the detection of affected individuals. Our biochemical results included determination of enzyme activities of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase-lysosomal enzymes, arylsulfatase C, a microsomal marker, and 5' nucleotidase, a plasma membrane marker. Our results indicate that the cellular ultrastructure is more sensitive than enzyme activity to changes in culture conditions. The resulting ultrastructural "artifacts" observed under certain conditions were severe enough to result in a mistaken diagnosis. Due to certain difficulties we had previously encountered in heterozygote cultures (for lysosomal storage disorders) of amniotic cells, we decided to examine heterozygote cultures of skin fibroblasts. From these (preliminary) studies it seems that an elevation in the pH over the physiologic levels in the culture media may help to define between normal individuals and affected heterozygotes. On the basis of our results, we recommend that to minimize false positive ultrastructural results for the diagnosis of lysosomal storage disorders, cultures be grown in minimal essential medium, the pH of the medium carefully monitored to remain below 7.4, examining the cultures not later than cell Passage 8 and no later than Day 10 after subculture.