MACS cell separation - the faster way to ultrapure, Dr. Katharina Winnemoeller katharinaw@miltenyibiotec.com.au

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1 MACS cell separation - the faster way to ultrapure, viable cells + Dr. Katharina Winnemoeller katharinaw@miltenyibiotec.com.au

2 About us Germany's largest independent, privately owned biotech company 1300 employees in 19 countries Cell biologists, biochemists, biophysicists, clinicians, and engineers all acting as a community 20% of staff in R&D, engineering & clinical development Manufacturing on site minimal reliance on external suppliers

3 About us Founded in Germany in 1989 Cologne (Bergisch Gladbach) corporate headquarters World market leader in magnetic cell separation (MACS) publications with products from Miltenyi Biotec

4 Worldwide sales and support

5 Technology and services Sample preparation Cell Separation Cell Culture Cell Analysis Molecular Analysis Small animal imaging Contract MFG High throughput automation

6 MACS MicroBead Technology Flexibility comes as standard

7 We are translational Miltenyi provides solution from bench to bedside

8 Cell Separation

9 MACS MicroBeads Key benefits MACS MicroBeads are 50nm superparamagnetic particles Flexible cell sorting strategies: positive selection depletion multiparameter sorting (no stripping!) Non-toxic, biodegradable: labeled cells go straightly to downstream experiments such as flow cytometry, cell culture,... For virtually any cell type: more than 350 separation reagents

10 MACS Technology Proven worldwide for more than 20 years Minimal labeling required due to column

11 Cell Separation Portfolio Stem Cells cell separation reagents Clinical 55 cytokine prod. cells B cells 46 Granul T cells 93 Immunology 287 various (MHC, ) Monos/ Mph NK cells 18 DC 31 Neuro 62 fluorochromes 11 Neural cells 21 Tumor cells 6 Whole blood 10 ES/iPS cells 17 CSC 5 HSC 18

12 Next generation MicroBeads: CD4+CD25+ T reg Isolation Kit, human CD4+CD25+CD127dim/ Regulatory T Cell Isolation Kits CD4 + T Cell Isolation Kit II, human, mouse CD8 + T Cell Isolation Kit II, human, mouse Pan T Cell Isolation Kit II, human, mouse Plasmacytoid Dendritic Cell Isolation Kit II, human New Pan Monocyte Isolation Kit, human NK Cell Isolation Kit II, mouse Naive Pan T Cell Isolation Kit, human (optional TCR γ/δ T cell depletion)

13 Whole Blood MicroBead Technology Convenience standardized Positive cell isolation directly from peripheral blood Automated with the automacs Pro Complete walkaway automation Higher recovery No RBC lysis No density centrifugation

14 A peek into the future (mouse cells): New Kits Pan-DC Enrichment Kit, mouse Pan-DC Isolation Kit, mouse PDC Isolation Kit II, mouse Classical DC Isolation Kit, mouse Monocyte Isolation Kit (BM), mouse Monocyte Isolation Kit (WB), mouse

15 Available now: fast protocols (mouse cells):

16 automacs Pro Separator Key features

17 Automated multisample cell sorting Designed and manufactured in Germany Up to 6 samples Option for automated labeling Sample cooling racks Volumes: ml (for 5, 15, 50 ml sample tubes) Samples stay at 4 degrees Choice of 12 separation programs

18 Re-usable columns For 14 days, or up to 100 separations High capacity cell sorting within a few minutes 10 5 up to 4 x 10 9 total nucleated cells up to 2 x 10 8 magnetically labeled cells up to 15 ml of whole blood

19 Numbers automacs (Pro) worldwide, 47 in Australia Up to 10 Mio cells per second

20 Programs and running time

21 automacs Pro programs Positive selection Possel Possel_s Posseld Posselds Posseld2 Posselwb positive selection in standard mode for isolation of cells with normal antigen expression and frequencies higher than 5% positive selection in sensitive mode for isolation of cells with low antigen expression and frequencies higher than 5%; positive selection in standard mode I, double-column program for isolation of rare cells in low elution volume positive selection in sensitive mode, double-column program for isolation of rare cells with low antigen expression positive selection in standard mode II, double-column program for isolation of rare cells special positive selection in double-column program for isolation of cell subsets from whole blood 4 ml/min 1 ml/min 4 ml/min 1 ml/min 4 ml/min 4 ml/min

22 automacs Pro programs Depletion Deplete Depletes Depl05 Depl025 A_depl7 A_deplS7 Depletion in standard mode for removal of cells with normal to high antigen expression and results in better target cell yield. depletion in sensitive mode I for removal of cells with low antigen expression and results in better target cell purity. depletion in sensitive mode II for removal of cells with low antigen expression and results in stringent depletion of cells depletion in sensitive mode III for removal of cells with low antigen expression and results in stringent depletion of cells special depletion in standard mode for removal of cells with normal antigen expression; used for large sample sizes where magnetically labeled cells exceed automacs Column binding capacity (2x108). special depletion in sensitive mode for removal of cells with low antigen expression; used for large sample sizes 4 ml/min 1 ml/min 0.5 ml/min) 0.25 ml/min) depends 4 ml/min

23 Results, Examples From abundant to rare cells

24 CD4 MicroBeads, human automacs program Start percentage CD4 CD4 Purity % CD4 cells in negative fraction time POSSEL 39,3% 97,9% 16,7% 2.5 min POSSEL_S 39,3% 94,7% 3,2% 5 min POSSELD 39,3% 99,6% 17,3% 7 min POSSELDS 39,3% 97,2% 2,21% 8.5 min CD4-Cy5 stained sample Results analysed by flow cytometry By Courtesy of A. Sheffold, A. Thiel - Berlin, Germany

25 Whole Blood CD14 MicroBeads, human Anticoagulated whole blood; Purity: > 98% program Posselwb /Rinse

26 7-AAD Count 7-AAD Count CD34 MicroBead Kit, human 0.37% CD34 + cells Original sample Human cord blood, Purity > 95% Program POSSELD / Rinse 95.38% CD34+ cells CD34+ cells CD34-PE CD34-PE automacs Separator, by Courtesy of Dr. Haberstock, Dr. Allam, Hautklinik Bonn, Germany

27 Maintenance and service

28 Daily Maintenance Priming: Program Rinse (automated before first separation) Shut down: Program Sleep for overnight storage (fill with Ethanol) Periodic Maintenance Exchange Columns: Every 2 weeks or after 100 separations, whatever comes first Clean pump syringe: Every 1-2 month Long term storage; (substitute columns) Service Service engineer and spare parts located in Sydney

29 automacs Pro - the best way for automated magnetic cell separation Close to 400 Products for cell separation Most flexibility positive or negative selection true Whole Blood separation, PBMC, other sources Prettiest instrument!

30 automacs Pro demo experience the difference From November 1 st you can try out the automacs Pro by yourself Until November 15 th I will be around regularly to help you with your separation Let me know what you would like to separate please let me know by tomorrow morning I will order the respective samples

31 Thank you for you attention