Cell therapeutic applications
Determination of cell count and viability
Abstract
Enumeration of cells in cell therapy research and manufacturing is challenging. In contrast to traditional bioprocesses that rely on the expansion of cell lines in a bioreactor, autologous cell therapies are made from individual donor tissues undergoing multistep manufacturing processes. Thus, substantial sample diversity is observed during manufacturing of cell therapies. Under similar conditions, traditional cell counting based on bright field microscopy and trypan blue exclusion fails to deliver consistent cell count and viability measurements. The NucleoCounter® counts cells by identifying nuclei stained with DNA-binding fluorescent dyes, resulting in a signal that is both strong and specific to cells. Thereby, the NucleoCounter® can quantify cell numbers and viability under difficult conditions often encountered in cell therapy processing, such as the presence of red blood cells, sorting beads or cell clumps. With its GMP-ready software, the NucleoCounter® is ideal for cell therapy manufacturing.
Table of contents:
Diagram of a traditional manual cell counting workflow compared to the NucleoCounter® NC-202™. Manual cell counting includes at least two pipetting steps, manual microscopy, counting and calculation. With NucleoCounting the user only needs to load the Via2-Cassette™ by pressing the piston; the rest of the procedure is performed by the instrument.
Traditional cell counting methods can give high variation, especially between operators. This variation is particularly pronounced for manual cell counting, while automated cell counters that rely on manual focus are also subjected to operator bias. The automated cell counter NucleoCounter® NC-202™ uses a fixed focus and employs a disposable Via2-Cassette™ that standardizes sampling, cell staining and counting chamber loading. The NucleoCounter® NC-202™ automatically acquires fluorescent images, performs image analysis and provides the user with a total cell count and viability. This data is electronically stored and can be implemented as part of a 21 CFR Part 11 setup.
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Cell Count and viability determination of peripheral blood mononuclear cells (PBMC) from whole blood using the ‘Viability and Cell Count – Blood Assay’.
Image 1: Blood samples contain mesenchymal stem cells (MSC), red blood cells (erythrocytes), platelets and leukocytes. 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.
Image 2: Image cytometry of a stained whole blood sample shows the total count (green) and the dead count (blue).
Image 3: Example from the NucleoView™ software, used with the NucleoCounter® NC-200™, which allows the user to verify that all cells have been counted correctly.
Monitoring the whole process from leukapheresis to the formulated product using NucleoCounter® instruments.One of the most promising approaches in cellular therapies is immunotherapies using engineered T cells expressing chimeric antigen receptors (CAR). During all steps of clinical CAR-T cell manufacturing, the viability and cell count has to be determined precisely. The NucleoCounter® instruments offer easy and robust determination of the viability and cell count even between different users.
Cell count determination in spheroids in NucleoCounter® systems.
Cells growing in spheroids will form large aggregates. Using Solution A100 and B, cell membranes dismantle and spheroids disaggregate leaving nuclei in suspension. Nuclei can then be stained with DAPI and automatically counted.
For the use in cell therapies, Mesenchymal stem cells (MSCs) can be isolated from different sources, e.g. lipoaspirates and bone marrow aspirates. The NucleoCounter® instruments allow for accurate determination of cell count and viability.
Mesenchymal stem cells (MSCs) are currently used for the treatment of different diseases, mostly focused on bone diseases, cardiac diseases, musculoskeletal disorders and disorders affecting the blood system. These cells have a promising future in targeted regenerative autologous and allogeneic cell therapies because of their high proliferative capacity and differentiation potential. But determining the viability and cell count of freshly isolated MSCs is challenging as the samples often contain artifacts, e.g. erythrocytes and fat droplets. The NucleoCounter® instruments overcome these challenges entirely by using a special buffer system to cope with erythrocytes and fat tissue.
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Microcarriers serve as a scaffold that adherent cells can attach to, allowing them to proliferate while a bioreactor keeps the cell-microcarrier complex freely suspended in the media.
Mesenchymal stem cells (MSC) have a promising future in targeted regenerative cell therapies because of their high proliferative capacity and differentiation potential. Currently, the expansion of MSC is performed using two-dimensional (2D) cultures in cell culture flasks. One promising approach in cell therapy is the large-scale cultivation and expansion of MSC on microcarriers. The NucleoCounter® instruments offer an accurate and reliable method for determining cell count and viability of cells grown in microcarrier cultures.
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Figure 6. NucleoCounter® instruments count consistently despite location or production date. After assembly, each instrument is calibrated against a master instrument during the QC process. We provide reagents that allow our customers to perform a regularly scheduled operation and performance qualifications.
A common problem with conventional cell counting is data discrepancies between users, departments and sites. All NucleoCounter® instruments count the same regardless of location or production date. This is because each instrument is calibrated against a master instrument during the QC process.
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