A standardized cell culture is not easily obtained as biological systems do not always behave reproducibly, e.g. in particular primary cell systems such as patient derived materials may vary widely in proliferation behavior. In addition, every cell biologist will vouch for the fact that cell culture performance is highly user dependent and requires a certain degree of expertise to achieve consistency in cell cultures.

While not always easy to maintain, standardized cell culture is of great importance as the condition of a cell culture may have a significant influence on the downstream results obtained with the cultured cells. 

One way to obtain a more standardized cell culture is to use cells for any downstream assay at consistent confluency. However, also the determination of cell confluency is very user dependent. Often different users will judge the same cell culture to be between 40-70% confluent, which poses a huge biological variation into any downstream assay. The CytoSMART™ System overcomes this issue in offering a user-independent analysis of the cell confluency which is recorded in the CytoSMART™ Project Page (here referred to as “cell coverage”). In addition, automatic alerts can be set so that the researcher receives an email notification once the cell culture has reached a chosen confluency, thus offering a very simple yet effective tool to easily standardize cell culture.

 


  Video of Normal Human Epidermal Keratinocytes – Adult (192627) maintained in KGM-Gold™ Keratinocyte Growth Medium over 4 days and recorded with CytoSMART™ Lux 10X Device.

 

Effects of Different Cell Culture Confluencies on Transfection Efficiency

With 15 years of transfection experience using the Nucleofector™ Technology, Lonza’s R&D experts know, that the confluency of a cell culture can have a great effect on transfection efficiencies. This is one of the reasons why each Nucleofector™ Optimized Protocol contains a recommendation at which confluency cells should be harvested for transfection. In the example below Normal Human Epidermal Keratinocytes were transfected using the Nucleofector™ Technology at 40% and 70% confluency. As can be seen, the transfection efficiency with pmaxGFP™ shows a 20% difference in GFP expression. This variation in transfection efficiency may affect the results of any downstream assay significantly, e.g. with higher standard deviations of the assay and in consequence a possible need for more repeated experiments to obtain an assay result with acceptable CV. 

Note that when well-versed cell biology scientists in our labs were asked to determine the confluency of the cell culture we obtained quite varying results ranging from 50-70%. See also the images taken with the CytoSMART™ Lux 10X System after 40% and 70% confluency.

 

 
Images of Neonatal Human Epidermal Keratinocytes at 40 and 70% confluency taken with the CytoSMART™ Lux 10X System.

 

  Neonatal Human Epidermal Keratinocytes(192906) were cultured until they reached 40 and 70% confluency. Cells were transfected with pmaxGFP™ using the Nucleofector™ Technology under the same conditions. Transfection efficiencies were determined by flow cytometry.

 

Take the opportunity now and standardize your cell culture with the CytoSMART™ System and its automatic confluency alerts. Don’t hesitate and request a quote for your personal CytoSMART™ System.

 

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