Cellular Therapeutics - Flow Cytometry in the Vanguard

A new review article published in the September issue of Clinical Cytometry by scientists from Advanced Therapeutics at the Scottish National Blood Transfusion Service highlights the central importance of flow cytometry in the development and qualification of advanced cell therapeutics.

The authors emphasise focusing on good practice and then the identity, stability and function of the cell therapeutic.  They stress the avoidance of over-complication. For example, they encourage minimal sample manipulation (a core tenet of BioStatus's own product development) and cite the progressive introduction of no-lyse, no-wash protocols, viability dyes, and counting beads as having contributed greatly to the improved accuracy of flow cytometric assays for what they stratify as minimally-manipulated (e.g. T cell depletion in allogeneic BMT), simple (e.g. monocyte-derived dendritic cells) and complex (e.g. CAR-T) cell therapeutics.

Translation from research to a GMP environment benefits from high dimension, multi-laser flow cytometers for simple-as-possible assays thereby avoiding unnecessary and unwanted compensation. Suitable instrument selection is, of course, key along with suitable procedures for their on-going validation and performance monitoring.

The authors then move to the challenging topic of release criteria for the cell "product", which is exemplified by the authors in the specific case of phagocytic macrophages (detailed in Fraser et al., 2017).

The authors encourage special attention be paid to minimal sample manipulation, highly accurate sequential gating, dead cell exclusion and the addition of counting beads.

Together, the different considerations raised in the review are foundational to the analysis for cell therapies.

A number of assay examples are described, by increasing level of complexity:

1. ISHAGE CD34 stem cell enumeration
Following the principles outlined they chose chromophores (Vio-Blue-CD34, FITC-CD45 and DRAQ7 as viability dye) to avoid unwanted compensation issues having the three chromophores excitation separated on the MACS Quant 10
(This example is also used on the cover of the journal issue.)

2. Determination of very rare antigen-specific T-cells 
Again, the panel was designed to eliminate as much need for compensation as possible (PE-Cy5.5-CD45, VioGreen-CD8, Phycoerythrin-EBNA-3 Tetramer HLA-A3 and DRAQ7) along with scatter properties and applying a sound gating logic, on the MACS Quant 10

3. Purity and quality assessment of clinical CD14 cell selection
An antibody panel of CD14, CD15, CD16 and CD45 with DRAQ7 for % live cells and to ascertain removal of neutrophils (a CD45-intermediate / CD15-strong phenotype) in the preparative isolation of CD14 monocytes for differentiation to macrophages, on the FACS Canto II

4. Memory T cell definition, combination surface phenotype & cytokine expression profile
Again including a viability measure, using a fixable viability dye since cells require permeabilisation for the cytokine measurement, on the FACS Canto II

The authors' selection of DRAQ7 as the viability dye of choice in the first three of these example assays highlights its preferential characteristics: far-red fluorescence and therefore separation from commonly used fluorophores, its further potential for dead cell exclusion based on a diagonal gate (or "virtual" channel) due to its dual-excitation in cases where compensation would otherwise be required, binary separation of live and dead cells, and convenience - water-soluble, ready-to-use.

WHERE TO BUY

References:
Campbell, John DM, and Alasdair R. Fraser. "Flow cytometric assays for identity, safety and potency of cellular therapies." Cytometry Part B: Clinical Cytometry 94.5 (2018): 569-579.

Fraser, Alasdair R., et al. "Development, functional characterization and validation of methodology for GMP-compliant manufacture of phagocytic macrophages: A novel cellular therapeutic for liver cirrhosis." Cytotherapy 19.9 (2017): 1113-1124.

Drink your milk! Aiding gut immune homeostasis

Scientists from Wageningen University and Friesland Campina have investigated the immuno-modulatory properties of bovine lactoferrin (LF), an abundant glycoprotein of bovine milk.

Overall, they conclude that LF can play a role in immune homeostasis in the human GI tract, based on the findings that LF modifies dendritic cell (DC) differentiation and reduces the response by DC to TLR ligands.

In one experiment to study whether LF modulates the dendritic cell (DC) differentiation monocytes were differentiated into monocyte-derived DC by culturing them with IL-4 and GM-CSF and with high, low or no LF.

Following the three cell culture regimes, cells were analysed with the following fully-compensated antibody panel and associated chromophores: CD86, V450; anti-CD14, FITC; CD1a, PerCP-Cy5.5; PD-L1, PE-Cy7; HLA-DR, APC-efluor780.  After antibody staining, DRAQ7 was added and incubated for 10 minutes to label the leaky i.e. non-viable cells, for exclusion in the subsequent flow cytometric analysis by BD Biosciences FACSCanto II.

Given the presence of three red/far-red chromophores in the panel DRAQ7-positive events would have been excluded using DRAQ7's unique capacity to be detected in a distinct population in a bivariate plot of the available pairs of red/far-red channels including the unused FL5, without resort to compensation.  Otherwise, the panel would not readily permit the use of conventional viability dyes such as PI, 7-AAD and DAPI.

This is, then, a representative example of the "virtual" channel exclusion of dead cells.  This is described in detail in a recent white paper:
Channel Free Dead Cell Exclusion with DRAQ7™ - White Paper (PDF)
and a poster presentation at CYTO 2018:
https://tinyurl.com/CYTO2018poster

WHERE TO BUY

Reference:
Perdijk, O., van Neerven, R., van den Brink, E., Savelkoul, H., & Brugman, S. (2018). Bovine Lactoferrin Modulates Dendritic Cell Differentiation and Function. Nutrients, 10(7), 848.