Currently available methods for cell separation are generally based on fluorescent labeling using either endogenously expressed fluorescent markers or the binding of antibodies or antibody mimetics to surface antigenic epitopes. However, such modification of the target cells represents potential contamination by non-native proteins, which may affect further cell response and be outright undesirable in applications, such as cell expansion for diagnostic or therapeutic applications, including immunotherapy. We present a label- and antibody-free method for separating macrophages from living Drosophila based on their ability to preferentially phagocytose whole yeast glucan particles (GPs). Using a novel deswelling entrapment approach based on spray drying, we have successfully fabricated yeast glucan particles with the previously unachievable content of magnetic iron oxide nanoparticles while retaining their surface features responsible for phagocytosis. We demonstrate that magnetic yeast glucan particles enable macrophage separation at comparable yields to fluorescence-activated cell sorting without compromising their viability or affecting their normal function and gene expression. The use of magnetic yeast glucan particles is broadly applicable to situations where viable macrophages separated from living organisms are subsequently used for analyses, such as gene expression, metabolomics, proteomics, single-cell transcriptomics, or enzymatic activity analysis.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 166 28 Prague 6 Czech Republic

Department of Chemical Engineering University of Chemistry and Technology Prague Technická 5 166 28 Prague 6 Czech Republic

Department of Molecular Biology and Genetics Faculty of Sciences University of South Bohemia Branišovská 1160 31 37005 České Budějovice Czech Republic

See more in PubMed

Hochmuth R. M. Micropipette aspiration of living cells. J. Biomech. 2000, 33 (1), 15–22. 10.1016/S0021-9290(99)00175-X. PubMed DOI

Robert D.; Pamme N.; Conjeaud H.; Gazeau F.; Iles A.; Wilhelm C. Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device. Lab Chip 2011, 11 (11), 1902–1910. 10.1039/c0lc00656d. PubMed DOI

Miltenyi S.; Müller W.; Weichel W.; Radbruch A. High gradient magnetic cell separation with MACS. Cytometry 1990, 11 (2), 231–238. 10.1002/cyto.990110203. PubMed DOI

Fu A. Y.; Spence C.; Scherer A.; Arnold F. H.; Quake S. R. A microfabricated fluorescence-activated cell sorter. Nat. Biotechnol. 1999, 17 (11), 1109–1111. 10.1038/15095. PubMed DOI

Levine B. L.; Miskin J.; Wonnacott K.; Keir C. Global manufacturing of CAR T cell therapy. Molecular Therapy-Methods & Clinical Development 2017, 4, 92–101. 10.1016/j.omtm.2016.12.006. PubMed DOI PMC

Bieback K.; Fernandez-Munoz B.; Pati S.; Schäfer R. Gaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene Therapy. Cytotherapy 2019, 21 (9), 911–924. 10.1016/j.jcyt.2019.06.006. PubMed DOI

Warkiani M. E.; Khoo B. L.; Wu L.; Tay A. K. P.; Bhagat A. A. S.; Han J.; Lim C. T. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics. Nature protocols 2016, 11 (1), 134–148. 10.1038/nprot.2016.003. PubMed DOI

Law S. Antigen shedding and metastasis of tumour cells. Clinical and experimental immunology 2008, 85 (1), 1.10.1111/j.1365-2249.1991.tb05672.x. PubMed DOI PMC

Bajgar A.; Saloň I.; Krejčová G.; Doležal T.; Jindra M.; Štěpánek F. Yeast glucan particles enable intracellular protein delivery in Drosophila without compromising the immune system. Biomaterials Science 2019, 7 (11), 4708–4719. 10.1039/C9BM00539K. PubMed DOI

Soto E. R.; Caras A. C.; Kut L. C.; Castle M. K.; Ostroff G. R. Glucan particles for macrophage targeted delivery of nanoparticles. J. Drug Delivery 2012, 2012, 14352410.1155/2012/143524. PubMed DOI PMC

Rotrekl D.; Devriendt B.; Cox E.; Kavanová L.; Faldyna M.; Šalamúnová P.; Bad’o Z.; Prokopec V.; Štěpánek F.; Hanuš J. Glucan particles as suitable carriers for the natural anti-inflammatory compounds curcumin and diplacone–Evaluation in an ex vivo model. Int. J. Pharm. 2020, 582, 11931810.1016/j.ijpharm.2020.119318. PubMed DOI

Aouadi M.; Tesz G. J.; Nicoloro S. M.; Wang M.; Chouinard M.; Soto E.; Ostroff G. R.; Czech M. P. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 2009, 458 (7242), 1180–1184. 10.1038/nature07774. PubMed DOI PMC

Šalamúnová P.; Krejčí T.; Ryšánek P.; Saloň I.; Kroupová J.; Hubatová-Vacková A.; Petřík J.; Grus T.; Lukáč P.; Kozlík P. Serum and lymph pharmacokinetics of nilotinib delivered by yeast glucan particles per os. Int. J. Pharm. 2023, 634, 12262710.1016/j.ijpharm.2023.122627. PubMed DOI

Saloň I.; Hanuš J.; Ulbrich P.; Štepánek F. Suspension stability and diffusion properties of yeast glucan microparticles. Food and Bioproducts Processing 2016, 99, 128–135. 10.1016/j.fbp.2016.04.010. DOI

Ruphuy G.; Saloň I.; Tomas J.; Šalamúnová P.; Hanuš J.; Štěpánek F. Encapsulation of poorly soluble drugs in yeast glucan particles by spray drying improves dispersion and dissolution properties. Int. J. Pharm. 2020, 576, 11899010.1016/j.ijpharm.2019.118990. PubMed DOI

Šalamúnová P.; Saloň I.; Ruphuy G.; Kroupová J.; Balouch M.; Hanuš J.; Štěpánek F. Evaluation of β-glucan particles as dual-function carriers for poorly soluble drugs. Eur. J. Pharm. Biopharm. 2021, 168, 15–25. 10.1016/j.ejpb.2021.08.001. PubMed DOI

Mirza Z.; Soto E. R.; Hu Y.; Nguyen T.-T.; Koch D.; Aroian R. V.; Ostroff G. R. Anthelmintic activity of yeast particle-encapsulated terpenes. Molecules 2020, 25 (13), 2958.10.3390/molecules25132958. PubMed DOI PMC

Baert K.; De Geest B. G.; De Rycke R.; Da Fonseca Antunes A. B.; De Greve H.; Cox E.; Devriendt B. β-glucan microparticles targeted to epithelial APN as oral antigen delivery system. J. Controlled Release 2015, 220, 149–159. 10.1016/j.jconrel.2015.10.025. PubMed DOI

Patel A.; Asik D.; Snyder E. M.; Dilillo A. E.; Cullen P. J.; Morrow J. R. Binding and release of FeIII complexes from glucan particles for the delivery of T1MRI contrast agents. ChemMedChem. 2020, 15 (12), 1050–1057. 10.1002/cmdc.202000003. PubMed DOI

Figueiredo S.; Moreira J. N.; Geraldes C. F. G. C.; Rizzitelli S.; Aime S.; Terreno E. Yeast cell wall particles: a promising class of nature-inspired microcarriers for multimodal imaging. Chem. Commun. 2011, 47 (38), 10635–10637. 10.1039/c1cc14019a. PubMed DOI

Hauser A. K.; Mathias R.; Anderson K. W.; Hilt J. Z. The effects of synthesis method on the physical and chemical properties of dextran coated iron oxide nanoparticles. Mater. Chem. Phys. 2015, 160, 177–186. 10.1016/j.matchemphys.2015.04.022. PubMed DOI PMC

Aysan A. B.; Knejzlík Z.; Ulbrich P.; Šoltys M.; Zadražil A.; Štěpánek F. Effect of surface functionalisation on the interaction of iron oxide nanoparticles with polymerase chain reaction. Colloids Surf., B 2017, 153, 69–76. 10.1016/j.colsurfb.2017.02.005. PubMed DOI

Vollmers A. C.; Mekonen H. E.; Campos S.; Carpenter S.; Vollmers C. Generation of an isoform-level transcriptome atlas of macrophage activation. J. Biol. Chem. 2021, 296, 10078410.1016/j.jbc.2021.100784. PubMed DOI PMC

Rattigan K. M.; Pountain A. W.; Regnault C.; Achcar F.; Vincent I. M.; Goodyear C. S.; Barrett M. P. Metabolomic profiling of macrophages determines the discrete metabolomic signature and metabolomic interactome triggered by polarising immune stimuli. PLoS One 2018, 13 (3), e019412610.1371/journal.pone.0194126. PubMed DOI PMC

Specht H.; Emmott E.; Petelski A. A.; Huffman R. G.; Perlman D. H.; Serra M.; Kharchenko P.; Koller A.; Slavov N. Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2. Genome Biol. 2021, 22 (1), 50.10.1186/s13059-021-02267-5. PubMed DOI PMC

Grützkau A.; Radbruch A. Small but mighty: How the MACS®-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years. Cytometry, Part A 2010, 77 (7), 643–647. 10.1002/cyto.a.20918. PubMed DOI

Radbruch A.; Mechtold B.; Thiel A.; Miltenyi S.; Pflüger E. High-gradient magnetic cell sorting. Methods in cell biology 1994, 42, 387–403. 10.1016/S0091-679X(08)61086-9. PubMed DOI

Zborowski M.Physics of magnetic cell sorting. In Scientific and clinical applications of magnetic carriers; Springer, 1997; pp 205–231.

Navrátil O.; Lizoňová D.; Slonková K.; Mašková L.; Zadražil A.; Sedmidubský D.; Štěpánek F. Antibiotic depot system with radiofrequency controlled drug release. Colloids Surf., B 2022, 217, 11261810.1016/j.colsurfb.2022.112618. PubMed DOI

Neyen C.; Bretscher A. J.; Binggeli O.; Lemaitre B. Methods to study Drosophila immunity. Methods 2014, 68, 116–128. 10.1016/j.ymeth.2014.02.023. PubMed DOI

DRSC FlyPrimerBank (flyrnai.org).