Relevant principal factors affecting the reproducibility of insect primary culture

Norichika Ogata,Kikuo Iwabuchi

doi:10.1007/s11626-017-0140-7

Abstract

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

Highlights

Tissue culture has been defined as the maintenance of isolated portions of multicellular organisms in artificial containers outside the individual for considerable periods of time (Murray and Kopech 1953)
We have assessed how interindividual variation affects the development of primary explant cultures by observing 126 fat body explant cultures dissected from six Allomyrina dichotoma larvae
To test whether a random effect was significant in the development of primary explant cultures, we quantitatively analyzed our observations of primary explant culture histories using a mixed model (Bates et al 2015)

Summary

Introduction

Tissue culture has been defined as the maintenance of isolated portions of multicellular organisms in artificial containers outside the individual for considerable periods of time (Murray and Kopech 1953) It was devised in the twentieth century (Harrison et al 1907; Carrel 1912) to research the behavior of animal cells without the effects of homeostasis and experimental stress present in in vivo experiments (Freshney 2005). The freshness of explants markedly affects the quality of cultured cells and explants of poor quality exhibit lower reproducibility (Mothersill et al 2001; Drobna et al 2004; Freshney 2005) These problems are obstructive to studying developmental biology and molecular biology in vitro. The following two models were constructed: (1) a model containing two random effects, donor individuals and culture replicates, and (2) a model containing only culture replicates; and the likelihoods of these models were compared

Methods

Results

Discussion

Conclusion