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A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

ABSTRACT: Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

SUBMITTER: Somanchi SS

PROVIDER: S-EPMC4619620 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

Somanchi Srinivas S SS McCulley Kelsey J KJ Somanchi Anitha A Chan Leo L LL Lee Dean A DA

PloS one 20151022 10

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a signif ...[more]

PMID: 26492577

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Project description:BackgroundNatural Killer (NK) cell-based therapies represent a ground-breaking opportunity for the treatment of solid tumors and hematological malignancies. NK cell manufacturing under good manufacturing practice (GMP) is complex and requires attentive assessment the product's safety and efficacy through quality control (QC). Release testing includes monitoring of in vitro cell expansion, differentiation, purity, phenotype, and cytotoxicity. As NK cells are biologically active products, the establishment of potency methods is particularly relevant; surrogate or improper assays can lead to rejection of qualifiable batches or to release of products that falsely meet potency specifications, potentially causing low efficacy during clinical trials. As cell-based therapeutics are highly heterogeneous, no universal guidelines for product characterization are available, and developers must invest significant effort in establishing and validating robust and fit-to-purpose assays. In this study, we describe the qualification procedure of a flow cytometry-based analytical method to assess in vitro potency of GTA002 NK cells, to be applied to oNKord®/inaleucel allogeneic off-the-shelf NK cell product from Glycostem Therapeutics, undergoing a Phase I/IIa clinical trial in acute myeloid leukemia (AML) patients (NCT04632316).MethodsFirst, we established multi-color flow cytometry panels to quantitatively determine the count of effector (E) GTA002 cells and leukemia target (T) K562 cells alone and in co-culture at different E:T ratios (10:1, 3:1, 1:1). Effector potency was then qualitatively expressed as percentage of cytotoxicity. Next, we defined protocols for method qualification to assess the pivotal features of the assays, including accuracy, precision, linearity, range, specificity, robustness, and carryover; quantitative acceptance criteria were determined for all parameters. Results of the qualification procedure are reported and discussed against pre-defined acceptance criteria.ResultsOverall, our methods show robust performance across all parameters, ensuring QC-compliant assessment of NK cell potency as part of the release test panel for clinical batches. Notably, we identified relevant aspects to address when progressing towards method validation to support pivotal clinical studies.ConclusionsThis article provides a "case-study" of how analytical method development for cell therapeutics is planned and executed from early clinical stages, anticipating the need to establish robust procedures to overcome scientific and regulatory challenges during method validation.

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A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

Publications

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

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