Flow Cytometry: Applications - BioProcess

Bioprocess Applications

A wide range of easyCyte systems is now able to address the demands of bioprocess. The easyCyte 5HT HPL is uniquely suited for simple viability determinations and precise cell counts, while the easyCyte 8HT provides additional capabilities for more complex analyses. To learn more about instruments and applications suited for bioprocess, please read the selections below.

Count & Viability Testing

The Guava easyCyte 5HT HPL instrument and ViaCount kit are an ideal combination for the Bioprocess framework to perform Cell Counting and Viability analysis.
The Guava easyCyte 5HT HPL System provides absolute cell counts and offers other high-demand features, such as five parameters (3 fluorescent colors, 2 light scatter) and a blue laser for access to commonly used fluorescent dyes. The built-in 96-well Autosampler allows walk-away automation to obtain more data for complex biological studies (cell phenotyping, signaling, cytokine production, compound screening).
The Guava easyCyte 5HT HPL System runs all Guava Assays and uses GuavaSoft software for data acquisition and analysis. The Viacount software module includes 21 CFR part 11 enabling features for this procedural requirement.
Evaluation in a bioprocess environment assayed 3 common bioprocess cell lines, at both low and high concentrations with Viacount on the Guava versus Trypan Blue. Results demonstrated fast, reproducible cell counting & viability assessment across all cell lines and cell concentrations tested.

A recent internal study validates this method for bioprocess development or process monitoring (see Technical Brief TB2134EN00).

Accuracy of Viacount method on easyCyte 5HT HPL versus Trypan Blue

Recovery versus Trypan Blue	Expected cell concentration	Total cell concentration recovery (%)	Viability Recovery (%)
CHO	10e5	99.5	96.1
CHO	10e7	101.9	101.8
ANJ2	10e5	111.9	94.4
ANJ2	10e7	103.4	90.9
VERO	10e5	101.6	96.4
VERO	10e7	98.3	90.5
Average		102.8	95.0
Standard Deviation		4.8	4.2

Viability and cell concentrations were tested with Viacount for three separate bioprocess cell lines on the guava easyCyte HT HPL. As shown above, results were well within the range of acceptance criteria and showed concordance with expected cell concentrations of all samples tested.

Viacount Assay Precision Testing on easyCyte 5HT HPL versus Trypan Blue

		EasyCyte 5HT hpl
CV (%)	Expected cell concentration	Total cell concentration CV (%)	CV: difference between high cell abd low cell concentration	Viability CV (%)	CV: difference between high cell and low cell concentration
CHO	10e5	4.4	.09	1.4	-1.2
	10e7	3.5		2.6
ANJ2	10e5	6.6	3.5	1.6	-4.8
	10e7	3.1		6.4
VERO	10e5	6.2	2.2	1.2	-5.2
	10e7	4		6.4
		Trypan blue
CHO	10e5	17.3	5.1	1.2	-4.4
	10e7	12.2		5.6
ANJ2	10e5	29.1	14.8	3.1	-13.6
	10e7	14.3		16.7
VERO	10e5	19.8	9.2	1.1	-11.7
	10e7	10.6		12.8

CVs were evaluated for viability and cell concentration results across three different bioprocess cell lines at low and high concentrations. Guava easyCyte results show the best agreement with the acceptance criteria of the study as compared to Trypan Blue. Differences of CV between high and low concentration for Guava are lower than differences of CV of Trypan Blue for total cell concentration and viability.

Description of the easyCyte 5HT HPL system used in this bioprocess application are listed here:

Guava easyCyte 5HT Base System (PN 0500-4005) with HPL option (0500-4109) for bioprocess analysis

The ViaCount Assay analyzes cells based on differential permeabilities of two DNA-binding dyes. The nuclear dye stains only nucleated cells, while the viability dye brightly stains dying cells. Debris is excluded from results based on negative staining with the nuclear dye.

The blue population of cells show a significant amount of annexin V staining (bottom plot), indicating that intermediate levels of staining with the viability dye correlates with apoptosis.

Increased reproducibility in ELISPOT assays when using Guava ViaCount assay for cell counting.

Guava ViaCount assay provides accurate cell counts. Comparisons with an impedance counter and manual trypan blue hemocytometer counting indicated greater linearity and accuracy over a rang of cell concentrations. Expected cell counts were determined using an FDA-cleared reference counting bead method.