Growth on extracellular matrix surface
The culture surface has a definite influence on the rate of attachment and proliferation. To attach to the culture surface, AFCs must create their own microenvironment, consisting of glycoproteins, collagen, laminin, and fibronectin, among others (ECM proteins). Fetal bovine serum contains fibronectin and so does human AF at 15–18 weeks of gestation.643 If serum concentrations of less than 10 percent are used (such as in Chang‐type media supplements), the presence of AF in the culture setup might facilitate the coating of fibronectin on plastic surfaces. Chang and Jones607 reported optimal cloning and growth when cultures were initiated with equal parts of AF and growth factor‐supplemented medium (including 4 percent fetal bovine serum). Two other studies have shown that precoating the plastic surfaces with ECM improves both cloning and rate of growth of AFCs.644, 645 In both laboratories, ECM‐coated dishes were custom made from bovine corneal endothelial cells. The use of such precoated surfaces may be advantageous for cell attachment and cloning if suboptimal media have to be used. It appears impractical for routine use unless the laboratory is prepared to accept the extra expense involved in purchasing precoated dishes. A number of manufacturers offer such “biologic” plastic ware.
Reduction of oxygen supply
Other efforts directed at improving the cellular microenvironment take into account that atmospheric oxygen conditions are not optimal for most mammalian cells in culture. Brackertz et al.646 and Held and Sönnichsen647 demonstrated improved AFC growth under hypoxic conditions. If multigas incubators are opened frequently, however, their humidity tends to decrease, which could disadvantage cells growing in open dishes.
Testing and handling fetal bovine serum
The single most important factor in determining the speed and success of prenatal cytogenetic diagnosis is the quality of the growth medium and its supplements.557, 607 Since the traditional medium supplement, fetal bovine serum, represents a complex mixture of growth‐promoting substances, considerable effort has been directed toward formulating serum‐free media in mammalian cell culture.648 Human AFC culture has benefited greatly from the success of these efforts.649, 650 Fetal bovine serum or Chang‐type medium, which includes serum, requires proper storage and handling to preserve its effectiveness. Freeze–thaw cycles and exposure to light are particular problems.651
Defined growth factor supplements
The commercial versions of the growth factor‐supplemented media are based on the formulation provided by Chang et al.652, 653 The classic “Chang medium” included transferrin, selenium, insulin, tri‐iodothyronine, glucagon, fibroblast growth factor, hydrocortisone, testosterone, estradiol, and progesterone. These factors are added to a 1 : 1 mixture of Dulbecco's modified Eagles' medium (DMEM) and Ham's F12 medium, plus sodium bicarbonate, and small amounts of HEPES buffer and antibiotics. Chang et al. pointed out that their preferred basic medium mixture can be replaced by a number of other formulae (e.g. Ham's F10 or F12, Coon's modified Ham's F12, McCoy's 5A, RPMI 1640, DMEM, minimal essential medium and TC 199) without detriment. Chang and AmnioMAX media that differ in their buffering systems are available for use in closed or open cell culture systems. As with other aspects of the cell culture art and science, local preferences vary with respect to choice of specialized media, fetal or newborn bovine serum, and whether to mix these media with less costly media.609, 633, 634, 653, 654
It is assumed that the various peptides, hormones, and trace elements act synergistically on the recruitment of cells into the cycle and keep them from reverting to the G0 stage after completed division. A greater number of cells within a colony will therefore stay in the proliferative pool. The cycle time of individual cells, with the possible exception of the duration of the G1 phase, is not likely to change. Unless Claussen's micropipette method655 is used, a culture period of 5–7 days will thus remain the minimum time requirement for prenatal cytogenetic diagnosis employing AFC cultures. In our laboratory, we experimented with 12‐hour Colcemid exposure and very early harvests. We obtained a small number of metaphase cells after 3 and even 2 days in cell culture but the number of metaphases was insufficient for a complete analysis.
A drawback noted by some users of Chang and AmnioMAX media – other than the expense – is their limited shelf‐life. Lyophilized or other, more stable media supplements are offered by some manufacturers (e.g. Condimed, UltroSer) but cloning efficiency testing so far has failed to identify a commercial product that consistently yields higher cloning efficiencies than fresh lots of Chang media.604 Use of Chang‐type media may augment the incidence of chromosome breakage and chromosomal mosaicism in AFC cultures but rarely to the extent that the cytogenetic interpretation is compromised.656–659 This may in part result from the fact that Chang media can facilitate the growth of E‐type colonies and these colonies yield higher rates of random chromosome changes.588, 611 However, the advantages gained by the reduction of turnaround time and the substantial decrease of culture failures using Chang‐type media appear to outweigh the potential drawbacks of increased chromosomal breakage and pseudomosaicism.
Culture failure
For most laboratories today, reports are completed in 6–14 days with a mean of 7–11 days to the final report, and the rate of culture failure is below 1 percent, depending somewhat on the timing of the amniocentesis,660 and in many laboratories averages closer to 0.1–0.2 percent (van Dyke, unpublished data). There are multiple reasons for cell culture problems and outright failure.661 With the increased experience of the obstetrician and the universal use of high‐resolution ultrasound, maternal urine is now rarely received as an AF sample. Anecdotal evidence of some labs suggests that the risk of culture failure is higher in cases of fetal aneuploidy. In one published retrospective study, 56 (0.7 percent) of 7,872 AF samples did not grow.662 Twenty‐four of these were judged technically inadequate and 10 were from women whose fetuses had died. Of the remaining 32 cases, 4 had proven (determined by repeat amniocentesis) and 4 had possible (extrapolated from fetal phenotype) aneuploidy. This 25 percent rate of growth failure associated with proven or likely chromosomal aberrations was not confirmed in a similar study comprising 6,369 cases and a growth failure rate of 1.2 percent.663 A study of 14,615 cases identified a higher incidence of culture failure in advanced pregnancies with abnormal ultrasound findings but no association with aneuploidy.664 In addition to a baseline level of less than 1 percent unexplained culture failure (the standard set by the American College of Medical Genetics is 2 percent),623, 624, 665 a number of known hazards can interfere with cell growth.
Syringe toxicity and delayed transportation
One serious hazard is transmittal of AF in toxic syringes or tubes.666, 667 AF samples should not be transported in syringes; rather, the fluid should always be promptly transferred and transported in conical centrifuge tubes with plastic caps, spinal tap tubes or similar specimen transport containers. Rubber‐capped tubes and stoppered syringes should not be used as storage or transport containers for AF. Problems reported in the United States prompted one manufacturer to recommend minimizing both the time of AF in the syringe and contact with the stopper attached to the plunger rod.
Although it is advisable to deliver AF samples to the laboratory without delay, in our experience with AF specimens transported by courier and various delivery services, cell viability is maintained for
