A stone farm: development of a method for simultaneous production of multiple calcium oxalate stones in vitro

Abstract

We have previously shown how individual calcium oxalate stones of about 1 cm can be grown in vitro. While this proved a design concept, it was severely limited as an experimental tool because of the time required to undertake comparative studies. Here we describe a development of this system in which six parallel pairs of stone generators are supplied with feed solutions generating a medium that is supersaturated with calcium oxalate. Twelve stones were grown simultaneously in aseptically prepared artificial urine over a period of 32 days from 100 mg to about 250 mg. Flow rates, pH and [Ca(2+)] were stable and reproducible over the course of the experiment. Sodium azide (0.02%) was included in the growth medium of six stones and caused a modest decrease in growth rate from 5.5 to 3.4 mg/day. The experimental design is such that this was readily detectable both visually and statistically ( p<0.001). This multiple stone growing system ("a stone farm") shows improved consistency and illustrates the statistical power of the technique. Azide has only a minor effect on the growth kinetics and can be used as an antibacterial agent in studies involving urinary macromolecules. The technique is suitable for practical and meaningful investigation of calcium oxalate stone formation in vitro.

Citrate inhibits growth of residual fragments in an in vitro model of calcium oxalate renal stones

“…As we are pumping about 240 mg of calcium per day past each stone only a small proportion of the precipitated or total calcium is being incorporated into the stone. Applying the same reasoning to oxalate consumption, about 4% of available oxalate was incorporated into the control (2 mmol/L citrate) stones grown in artificial urine (compared to approximately 5% reported previously [13]). …”

“…Our model of stone growth, while not itself immune from all criticism, is very different from all that has gone before. It is based on a recognized principle, mixed suspension mixed product removal [11], is statistically powerful and robust [13] and addresses some of the deficiencies of other in vitro studies [12].…”

“…Further refinement and expansion of the technique as a "stone farm" allows 12 stones to be grown simultaneously making controlled studies feasible. This is fully detailed by Chow et al [13]. Briefly, fragments of stones retrieved from a patient who had undergone percutaneous nephrolithotomy, were adjusted to a dry weight of 100 ± 2 mg with abrasive paper.…”

“…To date there has been no direct experimental evidence to link the processes behind crystallization and actual stone growth. We have established a new technique of growing macroscopic calcium oxalate stones in vitro, based on the mixed suspension mixed product removal system, [12,13]. This model system (a stone farm) is ideal to test more directly the effects of citrate on residual fragments.…”

Kidney Stone Models for In Vitro Lithotripsy Research: A Comprehensive Review

“…15 This technique was expanded to allow for simultaneous growth of calcium oxalate phantoms. 16 The selection of the appropriate stone model is the first step in designing an appropriate in vitro analysis of a lithotripter. Other considerations regarding the in vitro set-up for testing the lithotripter (wet versus dry environment, maintenance of pressure/contact on the stone etc.)…”