The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.

Teresa M Desrochers,Erica P Kimmerling,Barbara E Ehrlich,Ivana Y Kuo,David L Kaplan,Wei-Chun Chin

doi:10.1371/journal.pone.0120097

Abstract

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.

Highlights

Mycoplasmas are a group of microorganisms that lack a rigid cell wall and their small size (0.3 to 0.8 μm) and the flexibility afforded by the lack of a cell wall allows these contaminating organisms to pass through most sterile filtration units utilized in conventional cell biology laboratories [1]
We have examined the growth of a group of pig kidney epithelial cells (LLC-PK1) cells with different gene knockdowns of two different calcium channels, an isoform of the intracellular calcium channel inositol triphosphate receptor type 3 (InsP3R3) or polycystin 2 (PC2) in both 2D and 3D cultures both before and after mycoplasma removal
To examine mycoplasma contamination in 2D culture, we used a group of mycoplasma infected LLC-PK1 cells that had been unmodified or modified with short hairpin RNA to knockdown two different calcium channel proteins

Summary

Introduction

Mycoplasmas are a group of microorganisms that lack a rigid cell wall and their small size (0.3 to 0.8 μm) and the flexibility afforded by the lack of a cell wall allows these contaminating organisms to pass through most sterile filtration units utilized in conventional cell biology laboratories [1]. We show for the first time that even though a mycoplasma contamination did not result in a proliferation defect or other notable cell culture defects in short-term 2D culture, the presence of the mycoplasma did have a significant impact upon 3D tissue morphology. Both the mycoplasma-positive and mycoplasma-negative cell lines were grown in 2D culture for 48 hours and their proliferation was measured by an MTT assay.

Results

Conclusion