Stem Cell Research

Figure 1. Determination of viability and cell count of mesenchymal stem cells (MSCs) for the use in cell therapies. The NucleoCounter^® instruments easily and accurately determine viability and cell count of MSCs derived from different sources e.g. lipoaspirates and bone marrow aspirates.

MSCs have a promising future in targeted regenerative cell therapies. A valuable source of MSCs comes from the adipose tissue obtained from fat biopsies and liposuction procedures, as well from bone marrow aspirates where a sufficient quantity of MSCs can be easily purified (Figure 1). Counting freshly isolated primary MSCs is challenging as the aspirate contains besides a bunch of different cells artifacts as well, including fat droplets, micelles and erythrocytes. Many counting techniques fail to discriminate between cells and artifacts or will give high variations between different users. The NucleoCounter^® instruments count only nucleated cells overcoming this challenge entirely by using a special buffer system to cope with erythrocytes and fat tissue.

Figure 2. Cell count and viability determination of peripheral blood mononuclear cells (PBMC) from whole blood using the ‘Viability and Cell Count – Blood Assay’.

• (A) Blood samples contain mesenchymal stem cells (MSC), red blood cells, platelets and leukocytes, including hematopoietic stem cells (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.

Hematopoietic stem cells (HSCs) are to date often used for allogenic or autologous stem cell transplantations for the treatment of blood or bone cancer such as leukemia and myeloma. These stem cells can give rise to all blood cells and can be easily purified from different sources, including umbilical cord blood and peripheral blood from the peripheral blood mononuclear cell (PBMC) fraction. Cell count and viability determination of freshly isolated PBMC is challenging as the patient material still contains red blood cells and automated cell counters based on bright field are limited in their ability to distinguish PBMC from red blood cells. The NucleoCounter^® instruments NC-200™, NC-250™ and NC-3000™ will only count PBMC, excluding red blood cells and platelets, as they are just weakly stained. For whole blood, the NucleoCounter^® offers the Viability and Cell Count – Blood Assay’ to cope with a very high concentration of red blood cells (Figure 2).

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 growing need for large-scale productions of human cells has spread to the recently increased demands in stem cell research and cell therapy. To solve this problem, large-scale productions turn to microcarriers. Microcarriers are supporting matrix for growing adherent cells e.g. mesenchymal stem cells (MSC). Determination of concentration and viability of cells grown on microcarriers is extremely challenging and requires time-consuming detachment of cells with trypsin. The NucleoCounter^® family instruments NC-200™, NC-250™ and NC-3000™ offer a unique protocol to count and determine viability of cells grown on microcarriers without the need of previous detachment (Figure 3). With the “Viability and Cell Count – A100 and B Assay” cells are lysed, bringing the nuclei into suspension. The total number of cells will be stained with DAPI and detected by the NucleoCounter^® instruments. Afterwards, the non-viable cells are stained with DAPI without any pretreatment based on the assumption that all dead cells without cell adhesions are free in suspension and no longer attached to the microcarriers.

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 three dimensional (3D) culture of multicellular spheroids has been widely used to mimic the physiological environment in organisms. Embryonic stem cells (ES) are forming embryoid bodies and during chondrogenic differentiation of mesenchymal stem cells (MSC) in vitro, spheroids are formed as well. Determination of the total cell count of spheroids by manual counting and with automated cell counter is impossible as there is no chance to distinguish between individual cells. The NucleoCounter^® family instruments NC-200™, NC-250™ and NC-3000™ come with an assay specifically made for spheroids (Figure 4). The use of the “Count of Aggregated Cells – A100 and B Assay” facilitates the break-up of the spheroids, ensuring a homogenous sample of single nuclei which can be stained with DAPI and detected by the NucleoCounter^® instruments.