Mesenchymal Stem Cells (MSCs)

Consistent cell count & viability for development & manufacturing

Mesenchymal stem cells (MSCs) are used in regenerative medicine due to their high differentiation potential combined with a low graft-versus-host-disease response. The MSCs can be generated from the patient being treated (autologous) or from genetically distinct patients (allogeneic).

Interest in studying MSCs originated after discovering that these cells could be differentiated by bone, fat, cartilage, muscle and neurons and would therefore be suitable candidates for regenerative therapies.

Potential clinical applications for MSC transplantation range from tissue regeneration to therapy for immune disease such as graft-versus-host disease. Furthermore, MSCs secrete anti-apoptotic and angiogenic cytokines and growth factors which can support the healing process after myocardial infarction or in wound healing. Valuable sources of MSCs are aspirates from bone marrow, adipose tissue, umbilical cord blood, placenta and Wharton’s Jelly.

MSCs have a promising future in allogeneic and autologous cell therapies but counting these cells is challenging due to the vast amount of tissue material. This material may erroneously be counted as proliferating cells by other cell counting methods (see below for further explanation).

Overcoming challenges for reliable MSC count & viability

For MSC therapy manufacturing, cells are isolated after liposuction or from the bone marrow and are then purified, expanded, formulated, and administered to the patient.

The NucleoCounter® instruments provide a robust and precise method to effectively determine viability and total cell count of MSCs derived from different sources. Adipose-derived MSCs can be isolated from the stromal vascular fraction (SVF) after liposuction, whereas another valuable source of MSCs comes from bone marrow aspirates.

MSCs may be directly used or further expanded and used after long-term storage for autologous or allogeneic therapies. At each stage of production, cell count and viability can be reliably determined using NucleoCounter® instruments.

The Via2-Cassette™ combines cell sampling, staining and counting chamber loading in one step, ensuring consistency and robustness between different users. Cell processing GMP facilities can benefit from the NucleoCounter® system’s consistent results and user-friendly technology.

Counting adipose-derived mesenchymal stem cells in lipoaspirates

Adipose-derived MSCs can proliferate in culture and can be isolated from the stromal vascular fraction (SVF) after fat biopsies and liposuctions. This fraction contains a variety of cells such as MSCs, stromal cells, endothelial cells, adipocytes, erythrocytes or red blood cells (RBCs), as well as lipid droplets and micelles. For cell therapeutic applications, the SVF can be directly used for patient treatment.

It is crucial to precisely quantify the total cell count and viability for further applications. Many other counting techniques fail to discriminate between cells and artefacts. Furthermore, an excessive amount of cell debris causes stickiness resulting in a high degree of cell aggregation. Also, the tissue-derived aspirates are non-homogenous and will give highly varying results between users. To overcome these problems, the NucleoCounter® instruments offer an effective protocol to determine cell count and viability of adipose-derived MSCs.

For determining the total cell population of the SVF with a NucleoCounter® instrument, the cell suspension is treated with a lysing solution, lysing cells and other membrane-enclosed particles for cell nuclei staining with DAPI using the Via2-Cassette™. In the second step, the non-viable cells are stained with DAPI without needing any pretreatment.

To count SVF cells, you lyse the cells and count them for the total cell count. A sample is stained with DAPI and counted for the dead cell count.

Due to the specificity of DAPI binding to DNA, NucleoCounter® instruments only count the cells containing nuclear DNA. Therefore, they disregard artefacts often observed in the SVF that interfere with the accurate measurement, such as cell fragments, micelles, micro vesicles, fat droplets. This also includes cells without DNA content like erythrocytes and platelets.

Counting bone marrow-derived MSCs

To isolate bone marrow derived MSCs, bone marrow biopsies or aspirates are taken. Determining cell count and viability of MSCs directly from bone marrow samples is especially challenging, as these contain great levels of erythrocytes, which interfere with conventional automated and manual counting methods.

In some cases where the ratio of erythrocytes to nucleated blood cells, or white blood cells (WBCs), exceeds a certain threshold, it may be advised to lyse the erythrocytes.

To count MSCs directly easily and reliably in bone marrow aspirates, the NucleoCounter® instruments offer a fast protocol to handle erythrocytes in the sample. Erythrocytes are lysed by adding blood lysis buffer to the bone marrow aspirate and incubating for a short period.

Easy-to-use & ready for GMP environments

The NC-View™ software is GMP-ready as it supports electronic signatures, adapted protocols, and automated PDF reports

The user-friendly software accompanying a NucleoCounter® automatically reports the cell viability, total count, live count, and dead count. NC-View™ enables you to inspect the fluorescence image and to verify counting.

The automated cell counters are ready to use within GMP facilities applying 21 CFR Part 11-related regulations.

With its all-in-one workflow and built-in pipette, the unique Via2-Cassette™ ensures consistent cell sampling and eliminates production variation between users. Additionally, the cassettes are individually volume-calibrated, for the most precise cell count calculation. As a result, due to their design, the cassettes avoid human error otherwise introduced through mixing reagents and sample loading. This translates into a precise and reproducible cell count which far exceeds the trypan blue exclusion method.