Quantifying confluency to reduce MSC passage timing variation

Quantifying confluency to reduce MSC passage timing variation

Point

Quantification of the proliferative capacity of human somatic stem cells in culture provides an important index for the objective evaluation of cell state.

Overview

It is known that the proliferative capacity of human somatic stem cells , such as MSCs (mesenchymal stem cells), will decrease with increasing passage number to the point that cells eventually stop dividing. For this reason, it is useful to know the proliferative capacity of cells in culture over time.
In many cases, it is considered desirable to perform passaging at subconfluency, when about 70 to 80% of the culture vessel surface is occupied by cells. This judgment is normally made by visual observation, which varies among operators. Unbiased quantification of cell coverage area helps address these issues.

Current

Issue-1
Variability in passage  timing decisions.

Operators usually judge the degree of confluency by visual observation, a qualitative method that is further confounded by variabilities in judgment among multiple operators.

Issue-2
Reliable prediction of the degree of confluency is difficult

The proliferation rate of primary cells especially varies from lot to lot and will further vary with passage number. Therefore, it is difficult to predict when cells will reach the desired degree of confluency for passaging.

Solution

Non-invasive phase contrast imaging and image analysis allows for the quantification of MSC coverage area.Phase contrast imaging and image analysis of human somatic stem cells (such as MSCs ) allows for the automatic identification of regions occupied by cells, and quantification of the area covered.
Using the BioStation CT, a cell culture observation system with a built-in microscope and camera inside its incubator , and the BioStudio-T, a cell observation system that can be installed inside an incubator , phase contrast images can be captured over a long period without perturbing the culture environment. A growth curve can be generated from the time lapse acquisition data.Cell image analysis software CL-Quant add-on ModuleCell culture observation device BioStation CTCell observation device BioStudio-T

Measurement of the area occupied by MSCs through non-invasive phase contrast imaging and image analysis

Measurement of the area occupied by MSCs through non-invasive phase contrast imaging and image analysis (bone marrow-derived MSC UE7T-13 lines (JCRB1154))
Cell regions, including cell pseudopodia, can be detected even in an image with varying cell density
Left: phase contrast images, right: detection of area occupied by cells by image analysis
top: low density, bottom: mid density

Utilization scene

As an evaluation criterium for establishing cell culture processes and procedures

  • Validation of culture conditions
  • Understanding the upper limit of passage number and population doubling number, medium composition, passaging procedure, etc.
  • Detection of cells which proliferate abnormally due to transformation
  • Validation of culture environment (temperature, oxygen concentration, etc.)
  • Comparison of proliferation among cell lines

Cell image analysis software
CL-Quant add-on module

CL-Quant

CL-Quant Add-on Module is an add-on software for CL-Quant, specialized for individual cell's characteristics and analysis to make CL-Quant easier to use. With the easy 3 steps, cells can be evaluated efficiently even if you are new to image analysis.

See here for details

Cell culture observation device
BioStation CT

BioStation CT

Up to 30 culture vessels can be automatically observed by phase contrast/fluorescence in a stable culture environment of an incubator, reducing the burden on developers. Its excellent position repeatability expands the possibility of live cell imaging for a wide range of cell types.

See here for details

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Cell observation device
BioStudio-T

BioStudio-T

The BioStudio-T offers a unique imaging platform with a stationary sample surface and moving objective lens. This configuration allows observation of mechanically sensitive samples, such as stem cells, with minimal perturbation.

See here for details

Nikon will contribute to solving your cell culture issues with its image analysis
techniques and know-how on cell quality evaluation.

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