Application of a combinatorial approach in clonogenic assay for plant extract screening

Aim. To develop and theoretically validate a combinatorial clonogenic assay design for optimizing the screening of plant extracts and to quantitatively evaluate its efficiency compared with the conventional approach.

Materials and methods. A theoretical model of a clonogenic assay was developed for a library of 10 plant extracts using two prostate cancer cell lines (DU145 and LNCaP) with three biological replicates. The conventional individual screening approach was compared with a hierarchical combinatorial design based on sequential pooling of extracts followed by deconvolution of positive pools. The required number of experimental stages, culture plates, and the overall efficiency of both approaches were calculated.

Results and discussion. The conventional approach required 60 culture plates to evaluate 10 plant extracts, whereas the proposed hierarchical combinatorial design reduced this number to 36 plates, corresponding to a 40% reduction in experimental workload. The efficiency of the proposed method increased with library size, reaching an 86% reduction in the number of experimental units when screening 100 samples. The proposed strategy decreases reagent consumption, shortens experimental time, and increases screening efficiency while maintaining the number of replicates and statistical reliability. The method is particularly effective for identifying the most active extract but requires subsequent validation when multiple active compounds or synergistic interactions are expected.

Conclusions. The hierarchical combinatorial design is an effective approach for optimizing clonogenic assays by substantially reducing the number of experimental procedures, time, and material costs while preserving the reliability of the obtained results. This strategy can be applied as a primary high-throughput screening tool for plant extracts and other biologically active compounds before detailed validation studies.

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