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Abstract
This study proposed an optimized histogel construction method for histological analysis by
applying lung cancer patient-derived organoids (PDOs) to the developed histo-pillar strip.
Previously, there is the cultured PDOs damage problem during the histogel construction due to
forced detachment of the Matrigel spots from the 96-well plate bottom. To address this issue, we
cultured PDO on the proposed Histo-pillar strips and then immersed them in 4%
paraformaldehyde fixation solution to self-isolate PDO without damage. The 4 µl patient-derived
cell (PDC)/Matrigel mixtures were dispensed on the surface of a U-shaped histo-pillar strip, and
the PDCs were aggregated by gravity and cultured into PDOs. Cultured PDOs were self-detached
by simply immersing them in a paraformaldehyde fixing solution without physical processing,
showing about two times higher cell recovery rate than conventional method. In addition, we
proposed a method for embedding PDOs under conditions where the histogel temperature was
maintained such that the histogel did not harden, thereby improving the problem of damaging the
histogel block in the conventional sandwich histogel construction method. We performed
histological and genotyping analyses using tumor tissues and PDOs from two patients with lung
adenocarcinoma. Therefore, the PDO culture and improved histogel block construction method
using the histo-pillar strip proposed in this study can be employed as useful tools for the
histological analysis of a limited number of PDCs.