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

DesRochers TM, Kuo IY, Kimmerling EP, Ehrlich BE, Kaplan DL - PLoS ONE (2015)

Bottom Line: 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, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Cyst formation is improved in 3D tissues without mycoplasma contamination.Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.
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pone.0120097.g003: Cyst formation is improved in 3D tissues without mycoplasma contamination.Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.

Mentions: The morphology of multiple regions of numerous 3D tissues was examined after 2 weeks in culture by both hematoxylin and eosin (H&E) (Fig. 3) and whole mount staining (Fig. 4A). H&E staining provided an insight into the organization of the formed cysts including the alignment of cells within the cysts and the hollow structure formed by the cysts. The mycoplasma-positive tissues appeared diffuse and lacked a solid structure compared to the mycoplasma-negative; as examples, the results from LLC-PK1 control and InsP3R3 KD cells are depicted (Fig. 3). Whole mount imaging provided a view of the total number of cysts formed within the tissues (Fig. 4A), with fewer cysts being present in the mycoplasma-positive conditions (as expanded below).


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

DesRochers TM, Kuo IY, Kimmerling EP, Ehrlich BE, Kaplan DL - PLoS ONE (2015)

Cyst formation is improved in 3D tissues without mycoplasma contamination.Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368695&req=5

pone.0120097.g003: Cyst formation is improved in 3D tissues without mycoplasma contamination.Mycoplasma positive and mycoplasma-negative cells were grown in 3D tissues for 2 weeks at which time the tissues were fixed, sectioned, and stained for H&E. Note that the cysts are more diffuse in the mycoplasma-positive tissues. Scale bars = 200 μm.
Mentions: The morphology of multiple regions of numerous 3D tissues was examined after 2 weeks in culture by both hematoxylin and eosin (H&E) (Fig. 3) and whole mount staining (Fig. 4A). H&E staining provided an insight into the organization of the formed cysts including the alignment of cells within the cysts and the hollow structure formed by the cysts. The mycoplasma-positive tissues appeared diffuse and lacked a solid structure compared to the mycoplasma-negative; as examples, the results from LLC-PK1 control and InsP3R3 KD cells are depicted (Fig. 3). Whole mount imaging provided a view of the total number of cysts formed within the tissues (Fig. 4A), with fewer cysts being present in the mycoplasma-positive conditions (as expanded below).

Bottom Line: 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, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus