PBMC viability & live/dead cell concentrations

Fast, Accurate PBMC Count & Viability

Cellometer provides cell sample analysis results that include: Cell count Cell concentration Cell viability Cell diameter and size distribution Cell images, and more

Benefits of Cellometer Dual Fluorescence Viability Assays: 20 ul per measurement is fit for primary cells having very low sample volume No lysing of red blood cells saves time and eliminates extra sample preparation step Improve accuracy and eliminate errors Ensure user-to-user consistency Store data automatically

Importance of Accurate PMBC Counts

Peripheral blood mononuclear cells are used to measure immunological functions. Assays include proliferation, cytotoxicity and cytokine production. The use of cryopreserved PBMCs is critical both in clinical trials and biomedical research.

In cancer immunotherapy, leukapheresis is often used to isolate immune system cells from a patient's blood. Cells are then expanded or exposed to different agents depending on the vaccine design. Active cells are then infused back to the patient. Cell concentration and viability are essential parameters to monitor throughout the manufacturing process.

Ficoll is routinely used to isolate mononuclear cells from bone marrow, peripheral blood, and umbilical cord blood. Monocytes and lymphocytes form a buffy coat under a layer of plasma. Cells are collected and washed. Typically, some residual platelets or red blood cells are mixed in the mononuclear cells.

The functional assays on cryopreserved PBMC are associated with viability of the cells. Viability thresholds should be used in clinical trials in order to obtain reliable results of functional assays.

Characteristics of PBMC experiments Live PBMC concentration and viability are routinely measured for cell speration, processing and cryopreservation REFERENCES
"Preliminary Report: Evaluation of Storage Conditions and Cryococktails during Peripheral Blood Mononuclear Cell Cryopreservation", L.M. Cosentino, W. Corwin, J.M Baust, N.Diaz-Mayoral, H.Cooley, W. Shao, R. Van Buskirk, and J.G Baust, Cell Preservation Technology, Volume 5 Number 4, 2007

"Viability and Functional Activity of Cryopreserved Mononuclear Cells", A. Weinberg, L. Zhang, D. Brown, A. Brice, B.Polsky, M. S. Hirsch, S. Owens, and K. Lamb Clincal and Diagnostic Laboratory Immunology, July, 2000, P714-716

"Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples", V. Laroche, D. H. McKenna, G. Moroff, T. Schierman, D. Kadidlo, and J. McCullough, Transfusion, Vol., 45, Dec. 2005.

"Viability and Revovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study", C. A. Kleeberger, R. H. Lyles, J. B. Margolick, C. R. Rinaldo, J. P. Phair, and J. V. Giorgi, Clinccal and Diagnostic laboratory Immunology, Vol. 6, No. 1, Jan. 1999.

Limitations of Cell Count & Viability by Trypan Blue

Challenges

Image-based cell counters rely on software to process cell images captured by a digital camera. Trypan blue stained cells appear fully dark in color while viable cells have a clear bright center. Software algorithms are able to identify the two populations to calculate cell concentration and % viability. Mononuclear cells mixed with platelets or red blood cells can interfere with the accurate identification of the live cells or trypan blue stained dead cells. Additional steps including lysing of the red blood cells are required to get accurate results with bright field counting of trypan blue stained cells. Optimization of cell size gating are also needed to avoid counting platelets.

Solution

Dual Fluorescence PBMC Concentration and Viability using Cellometer Vision

The Acridine Orange/Propidium Iodide (AO/PI) viability assay is a rapid, highly linear, functuionally correlated assay that is superior to conventional viability measurement by trypan blue exlusion. (HPC viability measurement: trypan blue versus acridine orange and propidium iodide, K. Mascotti, J. McCullough, and S. R. Burger, Blood Components, Transfusion, Volume 40, June 2000)

Cell Count & Viability by Dual Fluorescence

Dual Fluorescence Viability Assay Principle

The AO/PI or AO/EB (Ethidium Bromide) viability assay is a rapid, highly linear, functionally correlated assay that is superior to conventional viability measurement by trypan blue exclusion.

How Cellometer Vision Does Dual Fluorescence Measurement

Assay Performance - Counting Range, Repeatability and Consistency

Measure cell concentration linearity and consistency using fresh PBMC

• Produce PBMC sample with serial dilution using PBS
• Stain PBMC sample at each dilution with acridine orange and ethidium bromide mixture
• Load 10 Cellometer counting chambers for each dilution
• Measure live / dead cell concentration and viability using Cellometer Vision

PBMCs dilution series showed linearity of R² = 0.9991

PBMC dilution series results

Dilution factor	Live PBMC (cells/ml)	Std (n=10)	%CV
1	2.2E+07	7.6E+05	3.5
0.5	1.1E+07	3.4E+05	3.0
0.25	5.0E+06	2.4E+05	4.8
0.125	2.6E+06	1.6E+05	6.2
0.0625	9.7E+05	5.3E+04	5.4
0.03125	4.1E+05	5.6E+04	13.9
0.015625	2.1E+05	2.9E+04	14.1

• PBMC was measured from live cell concentration of 2x10⁷ to 2x10⁵.
• Coefficient of variation ranged from 3.5% to 14.1%, which are in acceptable range.

Measure cell viability using fresh PBMC

Fresh PBMC sample was stored at 4°C. At day 1, day 4 and day 7, cell sample was removed for viability measurement at each time point, using the following procedure:

• Stain PBMC with acridine orange and ethidium bromide mixture
• Load 10 Cellometer counting chambers for each dilution
• Measure viability using Cellometer Vision

When fresh PBMCs sample was stored in 4°C, viability was reduced with storage time.

Lysing RBC For Cell Count or Not

After Ficoll separation, there are some residual red blood cells mixed with PBMC, making manual counting under a microscope difficult. Before counting cells, red blood cells are lysed using ACK lysis buffer.

Following process typically takes about 15 minutes. (PACTG PBMC Processing, Cryopreservation, and Thawing Method)

• Resuspend the cell pellet in 5-7 ml ACK lysis buffer and let stand for 3 minutes
• Fill tube with serile PBS
• Centrifuge at 250 xg for 10 minutes
• Aspirate supernatant
• Resuspend PBMC pellet in PBS
• Proceed to count viable PBMC's using trypan blue viability manual counting method

To eliminate the lysing process, nuclear staining dyes are used. Mature mammalian red blood cells don't contain nuclei, only live and dead mononucleared cells produce fluorescence signal.



Cellometer Vision employs this method to automate viable live / dead PBMC concentration and viability measurement without the need to lyse RBC.

How to Select Cell Counters

Depending on the cell sample conditions, either Cellometer Vision or Cellometer Auto T4 may be the best solution for you. Cellometer Vision is useful for measuring concentration and viability of messy samples because of its fluorescence capability, while Cellometer Auto T4 is best used for clean samples, where trypan blue viability is sufficient.

• Live/dead cell concentration and viability from samples with varied degree of contamination by platelets, red blood cells
• Live cell concentration for highly viable samples
• Clean sample after isolation
• Willing to do sample preparation, such as lysing RBC for each measurement

Sample Conditions	Stain	Measurement	Cellometer	Imaging mode
Messy sample	AO/EB (AO/PI)	Live/dead cell count & viability	Vision	Dual Fluorescence
Clean sample	Trypan blue	Live/dead cell count & viability	AutoT4	Bright-field only