Summary. 1. A method for freezing uncultured amniotic fluid cells is presented, which allows their use as pregnancy-age matched controls in prenatal diagnosis of metabolic diseases. 2. Amniotic fluid cells were successfully cultured after up to 7 days in transport, which makes prenatal diagnosis available to parents living a long way from specialized centers.

Zusammenjassung. 1. Eine Einfriermethode fiir nichtkultivierte Fruchtwasserzellen wird beschrieben, wodurch man diese wie Kontrollmuster anwenden kann bei pranataler Diagnostik von Stoffwechselkrankhciten. 2. Fruchtwasserzellen wurden erfolgreich kultiviert nach lang-zeitigem Transport (7 Tage); dieser Befund bringt eine pranatale Diagnostik im Bereich ent-fernt von spezialisierten Zentren lebender Eltern.

An increasing number of inborn errors of metabolism nan he detected in early pregnancy (Milunsky et al., 1970; Milunsky and Littlofield, 1972). Until now metabolic defects have been determined by biochemical analysis of cell homo-genates obtained from large numbers (millions) of cultured amniotic-fluid cells. As a consequence the period of time required for prenatal diagnosis in most in­stances has been very long (4—10 weeks) (Brady et al., 1971 ; Fratantoni et al., 1969; Epstein et al., 1972; Cox et al., 1970).

This period can be substantially reduced for a number of metabolic diseases by using microchemical techniques allowing the analysis of enzyme activities in clones of a few hundred cells (Galjaard et al., 1972). Knowledge of the variation in enzyme activity among heterozygotes for the disease concerned and also a proper selection of control material is of great importance for a reliable prenatal diagnosis, as there is increasing evidence that the "biochemical behavior" of cultured cells can be influenced by the duration of cell culture (Robbins et al., 1970), the phase in the growth curve (DeMara, 1964) and the conditions of cell culture (Ceccarini and Eagle, 1971; Lie et al., 1973). It therefore seems preferable to compare analytical data from the pregnancy at risk with those from control amniotic fluid samples at the same stage of pregnancy and grown in identical

conditions at the same time. This can only be realized when control amniotic fluid is available at any moment when a pregnancy at risk is to be monitored, which will rarely occur. In this communication the transport of amniotic fluid samples is discussed and a method is presented for long-term storage of these samples with preservation of cell viability.

Samples of amniotic fluid are centrifuged for 5 min at 80 X g and the cell pellets (0.2 — 1.4 x 10^e cells) are resuspended in 1 ml cell culture medium (Ham's F 10) supple­mented with 15% fetal calf serum, 4% dimethyl sulfoxyde (as cryoprotective agent) and antibiotics. Cell suspensions are transferred into ampoules which, after sealing, are frozen at a rate of 1 degC/min over liquid nitrogen vapor down to —70°C and subsequently stored under liquid nitrogen. To test the viability of the cells ampoules are taken after 7—22 days and rapidly thawed (under running tapwater). The cell suspensions are then washed by resuspending in 5 ml of serum-free medium, centrifuged for 5 min at 80 X g, and resuspended

in F 10 supplemented with 25% fetal calf serum and antibiotics. Aliquots (0.75 ml) of cell suspensions are carefully pipetted onto 22 x 22 mm coverslips in 35 mm Falcon Petri dishes. After 12—24 hrs for cell attachment additional medium is added. A fully humidified incubator -*Mth 5% CO₂ in the gaseous phase is used.

The results of culturing of the stored samples were compared with those of aliquots from the same sample cultured immediately (Table 1). No difference was observed in the time interval for the appearance of dividing cells or the type of proliferating amniotic fluid cells. It may be concluded that samples of uncultured amniotic fluid cells treated according to the method described can serve as reliable controls in prenatal diagnosis of metabolic disorders.

Two of the samples (Nos. 7 and 8, Table 1) had even been in transport for 7 days without detrimental effects on in-vitro cell growth afterwards. This finding is also illustrated by results of cell cultures of amniotic fluid samples from various periods of gestation air-mailed from the USA to Europe during different transfer times (Table 2). First mitoses were observed after 3—7 days and sufficient cells for karyotyping were obtained after 6—20 days of in-vitro growth. These results indicate that a transport time of up to 7 days does not affect the possibility for prenatal karyotyping.

The main conclusions from these results are:

1. Storage of unprocessed amniotic fluid cell samples is possible with preser­vation of cell viability, thus allowing the use of optimal control material in the prenatal analysis of a pregnancy at risk for a metabolic disease.

2. Even a few days of transportation have no detrimental effect on in-vitro cell growth, offering the possibility of prenatal diagnosis for parents at risk for a genetic disease living a long way from specialized centers.

Acknowledgement. This work was supported in part by a grant from Preventie Fonds.