Guide to Determining Viability and Concentration of Stem Cells Grown on Microcarriers – AZoM

Table of Content

Introduction Precise Counting of Adipose-Derived Mesenchymal Stem Cells Counting and Viability of Hematopoietic Stem Cells Counting Cells Growing on Microcarriers Cell Count Determination in Spheroids

Stem cell research and the significant potentials in stem cell therapy show a great deal of promise in 21st century medical treatments. Since stem cells typically need specialized conditions in culturing, knowing the concentration and capability of stem cells can be challenging.

Precise techniques for the determination of cell concentration and viability are needed to confirm consistency and reproducibility in stem cell culture. A wide range of specialized assays satisfying the needs of dedicated stem cell laboratories are offered by NucleoCounter instruments.

For reproducibility in stem cell research and production, detailed determination of cell viability and concentration is very crucial. Enhanced protocols are included in the NucleoCounter instruments to establish the count and viability of adipose-derived mesenchymal stem cells (MSC), that develop on microcarriers, and hematopoietic stem cells (HSC) found in the peripheral blood mononuclear cell (PBMC) fraction directly from whole blood and cell count determinations in spheroids.

Figure 1. Counting mononuclear cells including adipose-derived MSC in the stromal vascular fraction. (A) The stromal vascular fraction contains beside MSC, preadipocytes, endothelial cells and leukocytes also micelles, liposomes and microvesicles. By the use of Solution 10 the cells and the artifacts will be dissolved and the membranes permeabilized allowing for staining of nuclei with DAPI. (B) Image cytometry of cells stained with DAPI shows only the nucleated cells. (C) The accompanying NucleoView software allows the user to verify that all cells have been counted correctly.

A great deal of interest is produced by MSC from the stromal vascular fraction since these multipotent stem cells can be easily accessed and can be refined in large numbers directly from adipose tissue. Apart from MSC, leukocytes, preadipocytes, and endothelial cells are found in the stromal vascular fraction, as well as micelles, liposomes, and microvesicles.

It is a challenge to establish the cell count and viability of these newly isolated mononuclear cells, since liposomes, microvesicles, and micelles can be easily mistaken for cells while manually counting or when using automated cell counters.

To solve this issue, the NucleoCounter series instruments, NC-250, NC-200, and NC-3000, count only nucleated cells, as illustrated in Figure 1. After a lysis buffer is added, cells and other artifacts are lysed and thus, only the free nuclei in suspension are left out for staining with DAPI and for counting with the NucleoCounter systems.

Figure 2. Cell count and viability determination of PBMC from whole blood using the Viability and Cell Count Blood Assay. (A) Blood samples contain MSC, red blood cells, platelets and leukocytes, including HSC. Solution 17 will lyse red blood cells to minimize the quenching effect of the hemoglobin and to ensure robust staining of PBMC with Acridine Orange and DAPI to detect the total and dead cells respectively. Platelets are not stained due to the lack of nuclei and the weak staining. (B) Image cytometry of a stained whole blood sample shows the total count (green) and the dead count (blue). (C) The accompanying NucleoView software allows the user to verify that all cells have been counted correctly.

Until now, allogenic or autologous stem cell transplantations for the treatment of blood or bone cancer such as leukemia or myeloma, HSCs have been often used. All blood cells are produced by these stem cells and their purification can be easily performed from different sources such as peripheral blood and umbilical cord blood from the PBMC fraction.

Automated cell counters operating according to bright field cannot adequately distinguish between red blood cells and PBMC, and since the patient material still comprises of red blood cells, it becomes hard to determine the cell count and viability of newly isolated PBMC.

The NucleoCounter instruments NC-250, NC-200, and NC-3000 will count only PBMC, and will not count red blood cells and platelets, as they are faintly stained. To manage a high concentration of red blood cells, the NucleoCounter instrument offers Viability and Cell Count Blood Assay for whole blood, as illustrated in Figure 2.

In this blood assay, samples are incubated in Solution 17 to lyse the red blood cells and to confirm intense staining of the PBMC. The basic Viability and Cell Count Assay can be used to process the purified HSC.

Figure 3. Cell count and viability determination of cells growing on microcarriers. (A) The addition of reagent A100 and B lyses the cells bringing the nuclei into suspension. The total number of cells will be stained with DAPI and can be detected by the NucleoCounter family instruments NC-200, NC-250 and NC-3000. Afterwards, the dead count will be determined. (B) Image cytometry of cells stained with DAPI shows only the nucleated cells. (C) The accompanying NucleoView software allows the user to verify that all cells have been counted correctly.

The increasing demand in stem cell therapy and stem cell research is further compelled by the growing need for mass productions of human cells. To overcome these issues, mass productions leverage microcarriers, which are support matrix used for growing adherent cells such as mesenchymal stem cells (MSC).

Establishing the viability and concentration of cells, which are grown on microcarriers, is a highly complicated process, where cells have to be detached with trypsin that generally takes a lot of time. NC-250, NC-3000, and NC-200 offer a special protocol to count and determine the viability of cells, which are grown on microcarriers and that too without the need for earlier detachment, as shown in Figure 3.

Cells are lysed using the Viability and Cell Count A100 and B Assay that brings the nuclei into suspension. DAPI is used to stain all the cells and these are detected using the NucleoCounter instruments.

Later, DAPI is used again to stain the non-viable cells without any need for pretreatment, supposing that the dead cells without cell adhesions are not linked to the microcarriers and are freely suspended.

Figure 4. Cell count determination in spheroids. (A) Cells growing in spheroids will be heavily aggregated. By the use of Solution A100 and B, spheroids will be disaggregated and the membranes permeabilized allowing for staining of nuclei with DAPI. (B) Image cytometry of cells stained with DAPI shows the total cell count. C) The accompanying NucleoView software allows the user to verify that all cells have been counted correctly.

The physiological environment in organisms is usually replicated using the 3D culture of multicellular spheroids. Embryonic stem cells (ES) are actually forming embryoid bodies, and spheroids are formed during chondrogenic differentiation of mesenchymal stem cells (MSC) in vitro.

The manual counting process or automated cell counter cannot be used to establish the total cell count of spheroids, because separate cells cannot be distingushed. The NucleoCounter instruments such as NC-250, NC-200, and NC-3000 are offered with an assay that is primarily made for spheroids (refer Figure 4).

The application of the Count of Aggregated Cells A100 and B Assay makes it easy to break-up the spheroids and so guarantees an even sample of single nuclei. DAPI can be used to stain these nuclei, and the NucleoCounter system can be employed to detect them.

Source

Stem Cells Counting

This information has been sourced, reviewed and adapted from materials provided by ChemoMetec A/S.

For more information on this source, please visit ChemoMetec A/S.

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Guide to Determining Viability and Concentration of Stem Cells Grown on Microcarriers - AZoM