Sorting of MSCs with CD271 and MSCA-1 antibodies in a closed-cartridge system using the MACSQuant® Tyto® Sorter

Kathrin Godthardt¹, Nadine Chelius¹, Jens Gaiser¹, Christian Schmidt², Ronny Schulz², Andreas Bosio¹, and Sebastian Knöbel¹ ¹Miltenyi Biotec GmbH, , , ²Universität , Medizinische Fakultät, Klinik und Poliklinik für Orthopädie, Unfallchirurgie und Plastische Chirurgie, Leipzig, Germany

Expansion of MSCs isolated by flow sorting Introduction 3 or plastic adherence Mesenchymal stem cells (MSCs) have raised great contaminations and sample-to-sample carryover. Sorted MSCs and PA-MSCs were cultivated in MSC- well as PA-MSCs met ISCT criteria showing high expectations in a number of clinical trials studying Here, we demonstrate the possibility to sort Brew GMP Medium in order to assess their growth expression levels of CD105, CD90, and CD73, while the regeneration of bone, cartilage, and heart or CD271+MSCA-1 (TNAP)+ MSCs from human BM curves over 33 days of expansion (fig. 3A). Cell quality CD14, CD20, CD34, and CD45 (Non-MSCs) was low autoimmune diseases including GvHD, Crohn’s samples using the MACSQuant Tyto. The clonogenic after expansion was investigated by flow cytometry (fig. 3B, example for sorted MSCs). Morphology during disease, and diabetes. MSCs are an extremely rare potential was compared between the sorted MSC MSC marker expression analysis using the MSC MSC expansion was as expected (not shown). cell type in cord blood, adipose tissue, and bone fraction, CD271– cells, and MSCs obtained by plastic Phenotyping Kit (Miltenyi Biotec). Sorted MSCs as marrow (BM, 0.01%). Several groups worked on the adherence (PA-MSCs). Furthermore, we analyzed identification of MSCs, using markers such as CD271, the expansion potential with and without sorting. A Anti-MSCA-1 (TNAP), CD73, and STRO-1. Sorted and PA-MSCs were analyzed regarding their Recent technological advances like the MACSQuant® potential to suppress T cell proliferation. The gentle PA-MSCs Sorted MSCs Tyto®, a benchtop microfluidic flow sorter, enable the cell sorting process on the MACSQuant Tyto resulted 1.2×1010 purification of cells in a fully closed, sterile cartridge, in a highly pure population of viable and functional which completely eliminates the risk of external CD271+MSCA-1+ MSCs. 1.2×109

1.2×108

1.2×107

6 Results Number of MSCs 1.2×10

1.2×105 Sorting of CD271+MSCA-1+ MSCs 1.2×104 1 10 18 22 26 33 Days of cultivation Density gradient centrifugation (DGC) was performed with 50–100 mL human BM in a closed system using Isotype Control MSC/Non-MSC marker the automated CliniMACS Prodigy®. Subsequently, B bone marrow mononuclear cells (BM-MNCs) were stained with CD271-PE and Anti-MSCA-1-APC and

sorted for MSCs on the MACSQuant® Tyto® (fig. 1A) e cell number e cell number e cell number e cell number using a single-use cartridge in a two-step sorting number 100.00%-# 99.98%-# 99.98%-# 0.09%-# 8 cell Relative Relativ approach. In total, a maximum of 5×10 BM-MNCs Relativ Relativ Relativ including 1–8×104 MSCs were isolated. Depending -1 01 10¹10² 10³ -1 01 10¹10² 10³ -1 01 10¹10² 10³ -1 01 10¹10² 10³ on the volume, the sort took 3–3.5 h. Samples before XXXCD105-PEYYYxxxyyy CD90-FITCXXXYYYxxxyyy XXXCD73-APCYYYxxxyyy Non-MSC-PerCPXXXYYYxxxyyy and after sorting were analyzed via flow cytometry using the MACSQuant® Analyzer 10 (fig. 1B). The Figure 3 purity of target cells increased from 0.03% (±0.02%) Figure 1A before sorting to 91% (±3%) after sorting (fig. 1C). T cell suppression assay B C Before sorting 4 10³

100 T cell-suppressive potential of sorted MSCs and loaded particles (MSC Suppression Inspector, 10² 0.04%-# PA-MSCs after expansion (P3) was analyzed by Miltenyi Biotec). T cells were co-cultured with MSCs 10¹ flow cytometry. CD4+CD25– T cells were labeled in different ratios. T cell-suppressive potential was 80 CD271-PE

CD271-PE with CellTrace™ Dye to monitor T cell division after observed for all conditions (fig. 4). 1 0 stimulation with CD2, CD3, and CD28 anti­body– -1 -1 0 1 10¹10² 10³ 60

AfterAnti-MSC sortingA-1-APC 10³ 40

10² 98.06%-# T cells only PA-MSCs Sorted MSCs 20 10¹ yield,Purity, viability [%] 80 CD271-PE

CD271-PE 1 0 0 -1 Purity* Purity* Yield Viability 60 -1 0 1 10¹10² 10³ before sorting after sorting Anti-MSCA-1-APC Anti-MSCA-1-APC *among leukocytes

40 Figure 1 (continued)

of T cells (Median) T cells of 20 Colony-forming unit fibroblast (CFU-F) assay Violet fluorescence CellTrace 2 0 T cells T cells 1:1 1:5 1:10 1:100 1:1000 unstimulated stimulated The clonogenic potential of the sorted CD271+ A MSCA-1+ cell fraction (Sorted MSCs) was compared T cell: MSC ratio 6 to CD271– cells and MSCs obtained by plastic 1×10 adherence (PA; PA-MSCs). Cells were Giemsa-stained 1×105 after 10 days of culture in MSC-Brew GMP Medium Figure 4 cells

(fig. 2B) and colony-forming unit fibroblast (CFU-F) 6 1×104 numbers were determined (n=3). The CFU-F numbers increased by more than three orders of magnitude 1×103 when CD271+MSCA-1+ MSCs were sorted as com­ pared to PA (fig. 2A). A significant depletion of Conclusion 1×102 clonogenic potential was detected in the CD271– cell fraction (fig. 2A). 1×101 We have shown the potential of the MACSQuant® of the isolated cells. Sorted and expanded Tyto® Sorter for the isolation of the rare CD271+ CD271+MSCA-1+ MSCs are viable and functional, as + Number of CFU-F per 1×10 per CFU-F of Number 1×10 0 MSCA-1 MSC population from human BM-MNCs. shown by their high clonogenic and T cell–sup­pres­ PA-MSCs CD271– cells Sorted MSCs The gentle sorting conditions and the sterile, fully sive potential. Furthermore, multi-parameter cell closed system of the MACSQuant Tyto Cartridge sorting with the MACSQuant Tyto might also enable provides an optimal basis for subsequent expansion the sorting of MSC subpopulations. B

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