Droplet-Based Cytotoxicity Assay: Implementation of Time-Efficient Screening of Antitumor Activity of Natural Killer Cells

Overview

Journal ACS Omega

Publisher American Chemical Society

Specialty Chemistry

Date 2020 Oct 5

PMID 33015484

Citations 6

Authors

Silvia Antona

Ilia Platzman

Joachim P Spatz

Affiliations

Soon will be listed here.

Abstract

Natural killer (NK) cells are key players of the innate immune system. Due to their rapid cytotoxicity against infectious pathogens, hematologic malignancies, and solid tumors, NK cells represent solid candidates for cell-based immunotherapy. Despite the progress made in recent years, the heterogeneity in their cytotoxic behavior represents a drawback. With the goal of screening the intrinsic diversity of NK cells, droplet-based microfluidic technology is exploited to develop a single-cell time-efficient cytotoxicity assay. Toward this end, NK-92 cells are coencapsulated with hematological tumor cell lines in water-in-oil droplets of different sizes and their cytotoxic activity is evaluated. The effect of droplet-based confinement on NK cytotoxicity is investigated by controlling the droplet volume. The successful optimization of the droplet size allows for time efficiency compared to cytotoxicity assays based on flow cytometry. Additionally, the ability of individual NK-92 cells to kill multiple target cells in series is explored, expanding the knowledge about the serial killing process dynamics. The developed droplet-based microfluidic assay does not require the labeling of NK cells and represents a step toward developing of a forthcoming process for the selection of NK cells with the highest cytotoxicity against specific targets.

Citing Articles

A thermoplastic chip for 2D and 3D correlative assays combining screening and high-resolution imaging of immune cell responses.

van Ooijen H, Verron Q, Zhang H, Sandoz P, Frisk T, Carannante V Cell Rep Methods. 2025; 5(1):100965.

PMID: 39826552 PMC: 11841093. DOI: 10.1016/j.crmeth.2025.100965.

Analyzing functional heterogeneity of effector cells for enhanced adoptive cell therapy applications.

Kiel Rasmussen A, Hulen T, Petersen D, Jacobsen M, Mikkelsen M, Met O Immunooncol Technol. 2024; 24:100738.

PMID: 39629159 PMC: 11613168. DOI: 10.1016/j.iotech.2024.100738.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Tiemeijer B, Descamps L, Hulleman J, Sleeboom J, Tel J Micromachines (Basel). 2022; 13(11).

PMID: 36363930 PMC: 9692327. DOI: 10.3390/mi13111910.

Hydrogels for Single-Cell Microgel Production: Recent Advances and Applications.

Tiemeijer B, Tel J Front Bioeng Biotechnol. 2022; 10:891461.

PMID: 35782502 PMC: 9247248. DOI: 10.3389/fbioe.2022.891461.

Assessment of chimeric antigen receptor T cytotoxicity by droplet microfluidics in vitro.

Wong K, Shi J, Li P, Wang H, Jia Y, Deng C Antib Ther. 2022; 5(2):85-99.

PMID: 35441124 PMC: 9014740. DOI: 10.1093/abt/tbac008.

References

Abate A, Hung T, Mary P, Agresti J, Weitz D . High-throughput injection with microfluidics using picoinjectors. Proc Natl Acad Sci U S A. 2010; 107(45):19163-6. PMC: 2984161. DOI: 10.1073/pnas.1006888107. View

Olofsson P, Forslund E, Vanherberghen B, Chechet K, Mickelin O, Ahlin A . Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells. Front Immunol. 2014; 5:80. PMC: 3945532. DOI: 10.3389/fimmu.2014.00080. View

Kemna E, Schoeman R, Wolbers F, Vermes I, Weitz D, van den Berg A . High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel. Lab Chip. 2012; 12(16):2881-7. DOI: 10.1039/c2lc00013j. View

Boitard L, Cottinet D, Kleinschmitt C, Bremond N, Baudry J, Yvert G . Monitoring single-cell bioenergetics via the coarsening of emulsion droplets. Proc Natl Acad Sci U S A. 2012; 109(19):7181-6. PMC: 3358915. DOI: 10.1073/pnas.1200894109. View

Caen O, Schutz S, Jammalamadaka M, Vrignon J, Nizard P, Schneider T . High-throughput multiplexed fluorescence-activated droplet sorting. Microsyst Nanoeng. 2019; 4:33. PMC: 6220162. DOI: 10.1038/s41378-018-0033-2. View

Siegler E, Zhu Y, Wang P, Yang L . Off-the-Shelf CAR-NK Cells for Cancer Immunotherapy. Cell Stem Cell. 2018; 23(2):160-161. DOI: 10.1016/j.stem.2018.07.007. View

Papadopoulos N, Dedoussis G, Spanakos G, Gritzapis A, Baxevanis C, Papamichail M . An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. J Immunol Methods. 1994; 177(1-2):101-11. DOI: 10.1016/0022-1759(94)90147-3. View

Bhat R, Watzl C . Serial killing of tumor cells by human natural killer cells--enhancement by therapeutic antibodies. PLoS One. 2007; 2(3):e326. PMC: 1828617. DOI: 10.1371/journal.pone.0000326. View

Konry T, Dominguez-Villar M, Baecher-Allan C, Hafler D, Yarmush M . Droplet-based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine. Biosens Bioelectron. 2010; 26(5):2707-10. PMC: 3141325. DOI: 10.1016/j.bios.2010.09.006. View

10.

El Debs B, Utharala R, Balyasnikova I, Griffiths A, Merten C . Functional single-cell hybridoma screening using droplet-based microfluidics. Proc Natl Acad Sci U S A. 2012; 109(29):11570-5. PMC: 3406880. DOI: 10.1073/pnas.1204514109. View

11.

Mandal A, Viswanathan C . Natural killer cells: In health and disease. Hematol Oncol Stem Cell Ther. 2015; 8(2):47-55. DOI: 10.1016/j.hemonc.2014.11.006. View

12.

Mehta R, Rezvani K . Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer. Front Immunol. 2018; 9:283. PMC: 5818392. DOI: 10.3389/fimmu.2018.00283. View

13.

Vanherberghen B, Olofsson P, Forslund E, Sternberg-Simon M, Khorshidi M, Pacouret S . Classification of human natural killer cells based on migration behavior and cytotoxic response. Blood. 2013; 121(8):1326-34. DOI: 10.1182/blood-2012-06-439851. View

14.

Clausell-Tormos J, Lieber D, Baret J, El-Harrak A, Miller O, Frenz L . Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms. Chem Biol. 2008; 15(5):427-37. DOI: 10.1016/j.chembiol.2008.04.004. View

15.

Eyer K, Doineau R, Castrillon C, Briseno-Roa L, Menrath V, Mottet G . Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring. Nat Biotechnol. 2017; 35(10):977-982. DOI: 10.1038/nbt.3964. View

16.

Segaliny A, Li G, Kong L, Ren C, Chen X, Wang J . Functional TCR T cell screening using single-cell droplet microfluidics. Lab Chip. 2018; 18(24):3733-3749. PMC: 6279597. DOI: 10.1039/c8lc00818c. View

17.

Platzman I, Janiesch J, Spatz J . Synthesis of nanostructured and biofunctionalized water-in-oil droplets as tools for homing T cells. J Am Chem Soc. 2013; 135(9):3339-42. PMC: 3806295. DOI: 10.1021/ja311588c. View

18.

Zamir E, Frey C, Weiss M, Antona S, Frohnmayer J, Janiesch J . Reconceptualizing Fluorescence Correlation Spectroscopy for Monitoring and Analyzing Periodically Passing Objects. Anal Chem. 2017; 89(21):11672-11678. PMC: 5677728. DOI: 10.1021/acs.analchem.7b03108. View

19.

Khorshidi M, Vanherberghen B, Kowalewski J, Garrod K, Lindstrom S, Andersson-Svahn H . Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro. Integr Biol (Camb). 2011; 3(7):770-8. DOI: 10.1039/c1ib00007a. View

20.

Somanchi S, McCulley K, Somanchi A, Chan L, Lee D . A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry. PLoS One. 2015; 10(10):e0141074. PMC: 4619620. DOI: 10.1371/journal.pone.0141074. View