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Direct concentration, viability and phenotype measurement of stem cell

Direct concentration, viability and phenotype measurement of stem cell using a novel imaging cytometry method

Yan, haibo1*, Bodo Kohring2

1. Shanghai RuiYu Biotech, Shanghai, P.R. of China,

2. RuiYu Biotech Co., Ltd. Bachstrasse 16 D-32139 Spenge, 

Abstract: Mesenchymal stem cells are a subset of pluripotent stem cells which can be isolated from the mesoderm. With their self-replication renewal and multi-direction differentiation characteristics they possess a high potential for various therapies in medicine. Mesenchymal stem cells have a unique immune phenotype and immune regulation ability. Therefore, mesenchymal stem cells are already widely used in stem cell transplantations, tissue engineering and organ transplantation. And Beyond these applications, they are used as an ideal tool in tissue engineering as seeder cells in a series of basic and clinical research experiments. Up to now, there is not a widely accepted method and standard for the quality control of mesenchymal stem cells. The Countstar FL can monitor the concentration, viability and phenotype characteristics (and their changes) during the production and differentiation of these stem cells. The Countstar FL has also the advantage in obtaining additional morphological information, provided by the permanent brightfield and fluorescence based image recordings during the whole process of cell quality monitoring. The Countstar FL offers a fast, sophisticated and reliable method for the quality control of stem cells.

 

Materials and methods:

Adipose derived mesenchymal stem cells (AdMSCs) gifted by Professor Nianmin Qi, AO/PI staining solution (Shanghai RuiYu, CF002). Antibody: CD29, CD34, CD45, CD56, CD73, CD105, HLADR (BD Company).

AdMSCs were cultured in 37, 5% CO2 humidified incubator. Digest with trypsin before use.

CD marker staining procedure was followed as the manual of antibody.

CD marker detection with Countstar FL:

1. A signal-color application procedure was created by setting PE channel to image PE fluorescence.

2. 3 fields were captured from each chamber.

3. After imaging and initial analysis were complete, the threshold (log gate) setting for positive and negative transfection was set by FCS software.

 

Quality control of stem cell

The following Figure (Figure 1) shows the procedure of stem cell therapy. 


Figure 1: The procedure for stem cell therapy

  

Results:

Determining the concentration, viability, diameter and aggregation of AdMSCs.

The viability of AdMSCs were determined by AO/PI, A dual-color application procedure was created by setting Green channel and Red channel to image AO and PI fluorescence, plus a bright field. Example images were shown in Figure 2.


Figure 2. Images of prior to transport and after transportation of AdMSCs. A. Prior to transport; representative image shown. B. After transportation; representative image shown.

 The viability of AdMSCs were changed drastic after transportation when compared with prior to transport. The viability of prior to transport was 92%, but it reduced to 71% after transportation. The result was shown in Figure 3.


Figure 3. The viability results of AdMSCs (Prior to transport and after transportation)

 

The diameter and aggregation were also determined by Countstar FL. The diameter of AdMSCs were changed drastic after transportation when compared with prior to transport. The diameter of prior to transport was 19µm, but it increased to 21µm after transportation. The aggregation of prior to transport was 20%, but it increased to 25% after transportation. From the images which captured by Countstar FL, the phenotype of AdMSCs were changed drastic after transportation. The results were shown in Figure 4.


Figure 4: The diameter and aggregation results. A: The representative images of AdMSCs, the phenotype of AdMSCs were changed drastic after transportation. B: The aggregation of prior to transport was 20%, but it increased to 25% after transportation. C: The diameter of prior to transport was 19µm, but it increased to 21µm after transportation.

 

 

Determine the immunophenotype of AdMSCs by Countstar FL

The immunophenotype of AdMSCs were determined by Countstar FL, AdMSCs were incubated with different antibody respectively (CD29, CD34, CD45, CD56, CD73, CD105, HLADR). A signal-color application procedure was created by setting Green channel to image PE fluorescence, plus a bright field. Bright field picture reference segmentation was applied as a mask to sample the PE fluorescence signal. The results of CD105 were shown (Figure 5).


Figure 5: CD105 results of AdMSCs were determined by Countstar FL. A: Quantitative analysis of the positive percentage of CD105 in different samples by FCS express 5 plus software. B: High quality images supply additional morphological information. C: Validated results by thumbnails of each single cell, the FCS software tools divided the cells into different groups according to their varying protein expression.

 

Other antibodies results were shown in Fig 6


Figure 6:  A: A representative image of ASCs with typical spindle-shaped morphology. Captured by OLYMPUS microscope. Original magnification, (10x). B: Adipogenic differentiation of ASCs is evidenced by Ruthenium Red staining showing areas of mineralization. Captured by OLYMPUS microscope. Original magnification (10x). C: Countstar® FL characterization of ASCs.


Summary:

The Countstar FL can monitor the concentration, viability and phenotype characteristics (and their changes) during the production and differentiation of these stem cells. FCS express supplies the function to review every signal cell, validation the data through the image. The user can also have confidence to carry out next experiments based on Countstar FL results. The Countstar FL offers a fast, sophisticated and reliable method for the quality control of stem cells.