Applications in Bioprocessing

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 increased demands in bioprocessing. To solve this problem, large-scale productions turn to microcarriers. Microcarriers are support matrix for growing adherent cells. 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 1). 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.

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Figure 2. Monitoring the whole process from leukapheresis to the formulated product. With the NucleoCounter^® instruments it is easy to monitor all different steps of the purification, expansion and formulation of CAR-T cells using the same instrument to ensure precise and reliable results.

Promising approaches in cellular therapies have been developed using T cells expressing chimeric antigen receptors (CAR). Adoptive CAR-T cell therapy involves the collection of and enrichment of the desired T cell subset and genetic modification by viral transfection to create engineered CAR-T cells. The engineered CAR-T cells are then expanded in large-scale and infused back into the patient. Easy and user-adaptable protocols allow performing cell counting directly from leukapheresis as well as from purified and expanded CAR-T cells using the same instrument during bioprocessing giving a highly un-biased count (Figure 2).

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Mammalian cell lines are often used for recombinant protein production of industrially relevant proteins. One method to follow the expression of recombinant proteins is to co-express a fluorescent protein such as green fluorescent protein (GFP) under the same promoter and determine the amount of GFP. ChemoMetec A/S offers a fast and easy assay for testing GFP transfection with the NucleoCounter^® NC-3000™ (Figure 3). Cells are stained with Hoechst 33342 and propidium iodide (PI) to define the total cell population and the dead cell population together with the population expressing GFP. Easy coupling between the image obtained and the scatter plots or histograms allows for very precise determination of the gate settings used for counting of populations (Figure 4). The NucleoCounter^® NC-3000™ offers an all-in-one platform for evaluating GFP expression efficiency.

Figure 5.

Cell count determination in spheroids.

• (A) Cells growing in spheroids will be heavily aggregated. By the use of reagents 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.

Certain cell lines grow in floating spheres. As cells growing in spheroids are not homogenous in solution, measurements of the optical density will not give a direct measure for the concentration of cells. 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-202™, NC-250™ and NC-3000™ come with an assay specifically made for spheroids (Figure 5). 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.

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Figure 6. NucleoCounter^® instruments count the same despite location or production date. After assembly each instrument is during the QC process calibrated against a master instrument. We provide reagents that allow our customers to perform 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 (Figure 6). This is because each instrument is calibrated against a master instrument during the QC process.

Figure 7. The NucleoCounter^® instruments are designed to be easily implemented in GMP and 21 CFR Part 11 compliant laboratories. The in-built protocols for IQ/OQ/PQ ensure control of consistent operation.

Standardized PDF reports (Figure 7), user control and audit trails allow for documentation of all events. Together with the high reproducibility and consistency in measurements, the NucleoCounter^® instruments are the ideal choice for automated cell counting in GMP and 21 CFR Part 11 compliant laboratories.

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