hPSCcultivation and

expansion

hPSCcryopreservation

hPSC quality control• Immunophenotyping• Pluripotency assay• Genomic stability

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Standardized QC assays and (automated) expansion of pluripotent stem cells

Annett Kurtz, Andrea Bretz, Hannah Johannsen, Tristan Kuchler, Frank Jüngerkes, Annika Last, Andreas Bosio, and Sebastian KnöbelMiltenyi Biotec GmbH, Bergisch Gladbach, Germany

IntroductionHuman pluripotent stem cells (hPSCs) hold great promise for dis-ease modeling, drug discovery, and clinical applications. In this regard, working with highly pluripotent, quality-controlled cell stocks during development is crucial to ensure reproducible ex-perimental conditions. We established a workflow encompassing i) stable expansion of hPSCs using a xeno-free cultivation medium, ii) assessment of pluripotency by quantitative flow cytometry using a defined marker combination, iii) flow cytometric assessment of differen-tiation potential, based on lineage-specific, complete media, as well as iv) cryopreservation of hPSCs using a chemically defined medium. Following this workflow, hPSCs could be stably expanded over 20 passages with persistent, high expression of pluripotency markers and almost no expression of differentiation markers.

Cultured hPSCs showed the typical morphology and retained a stable karyotype. Quantitative flow cytometry analysis repro-ducibly confirmed the cells’ potential to differentiate into all three germ layers. hPSCs were recovered effectively after cryopreser-vation. After thawing, a 20-fold hPSC expansion could be achieved, and cells displayed high pluripotency marker expres-sion in p1.Thus, the workflow assures standardized, robust hPSC expansion and includes characterization and quality control (QC) of the ex-panded cells as well as efficient cryopreservation. The flow cy-tometry–based QC strategy was also successfully applied for characterization of hPSCs cultivated automatically in the closed system of the CliniMACS Prodigy®, which is of major relevance for generation of master cell banks (MCB) and working cell banks (WCB) for future clinical cell manufacturing.

hPSCs frozen in the animal component–free, chemically defined StemMACS Cryo-Brew showed a reproducible recovery of 88% and high viability of 93% after thawing (fig. 3A, clone 1, n = 3). Cells rapidly recovered in p1 after thawing (n = 3), almost imme-

diately reached a standard doubling time of 27–28 h (fig. 3B, clone 1, n = 3), and displayed a normal morphology (fig. 3C, clone 1) and high expression level of pluripotency markers (fig. 3D, clone 1).

Clone 1 was expanded automatically in the closed system of the CliniMACS Prodigy, on Laminin-521 in iPS-Brew GMP Medium un-til p3 (fig. 4A, n = 3). Afterwards, the flow cytometry–based QC was applied. Immunophenotyping revealed that these expanded cells expressed pluripotency markers comparably to the manually expanded control cells (96–99%, control: 98–99%), with almost no

expression of the differentiation marker (<0.1% SSEA-1+TRA-1-60–, control: <0.1% SSEA-1+TRA-1-60–) (fig. 4B). The differentiation potential of the automatically expanded cells was also compara-ble to the manually expanded control (fig. 4C, n = 3). Additionally, karyotype analyses of cells expanded in the CliniMACS Prodigy were without pathological findings (data not shown).

Figure 1A illustrates the workflow combining hPSC expansion, QC of the expanded cells using immunophenotyping, pluripotency as-say (differentiation into all three germ layers) and karyotype analy-sis, as well as cryopreservation. Two hPSC lines were expanded for 20 passages on Matrigel® in StemMACS™ iPS-Brew XF. Cells had a

stable doubling time of 24–27 h as assessed for single-cell passag-ing during long-term cultivation (fig.1B). Using StemMACS iPS-Brew XF hPSCs could be cultivated as single cells or cell clusters and displayed the typical morphology (fig. 1C, clone 1).

QC of the expanded cells is exemplified for clone 1 in figure 2. Im-munophenotyping was performed using a multicolor flow cytom-etry protocol1 based on well-known pluripotency markers and a differentiation marker. Analysis in passages 5, 10, 15, and 20 showed persistently high expression of pluripotency markers TRA-1-60, SSEA-4, SSEA-5, Sox2, and Oct-4 (92–99%) and almost no expres-sion of differentiation marker SSEA-1 (0.1% SSEA-1+TRA-1-60–) (fig. 2A, C). During development of the flow cytometry protocol, clone 1 was differentiated into neural lineage and analyzed using this marker panel. All pluripotency markers were down-regulated except for Sox2, which is also a marker for neural precursors, where-as SSEA-1 was up-regulated (fig. 2B). This confirmed the reliability of the chosen markers for hPSC immunophenotyping. Additionally, PSA-NCAM and Pax6 were stained to prove the neural identity. Differentiation potential was assessed in passages 5, 10, 15, and 20 using the StemMACS Trilineage Differentiation Kit, which supports

directed differentiation into all three germ layers based on lin-eage-specific, complete media. Quantitative flow cytometry analy-sis2 confirmed a stable capacity of the cells to differentiate into CD140b+ vascular smooth muscle or CD144+ endothelial cells (me-soderm), CXCR4+Sox17+ definitive endoderm cells (endoderm) and Sox2+PAX-6+ neuroectoderm cells (ectoderm) during long-term cultivation (fig. 2D, E). During development of the differentiation kit, clone 1 was cultivated in either StemMACS iPS-Brew XF, mTeSR™ 1, or Essential 8™ Medium and subsequently differentiated. Regard-less of the PSC expansion medium used, the overall differentiation efficiency was the same for each germ layer (fig. 2F). Thus, the po-tency assay can be used in combination with various PSC cultiva-tion media for QC. At last, genomic stability after long-term cultiva-tion was confirmed by karyotype analysis. No abnormalities were observed (fig. 2G).

We established a workflow which allows:• efficient and stable expansion of hPSCs,• standardized characterization and QC of the expanded cells

by immunophenotyping as well as assessment of differentiation potential and genomic stability,

• efficient hPSC cryopreservation including rapid recovery of highly pluripotent cells.

QC of hPSCs can also be used to monitor cell cultures maintained under standardized, automated, closed-system conditions, for example, during generation of master cell banks (MCB) and work-ing cell banks (WCB).

hPSCs show high recovery, normal doubling time and high pluripotency marker expression after cryopreservation in StemMACS™ Cryo‑Brew3

Results

Conclusion and outlook

hPSCs can be stably expanded as single cells or cell clusters using StemMACS™ iPS‑Brew XF1

QC of hPSCs can be standardized using immunophenotyping, pluripotency testing by StemMACS™ Trilineage Differentiaton Kit, and karyotype analysis2

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS® GMP Products are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. For regulatory status in the USA, please contact your local representative. MACS GMP Products are manufactured and tested under a quality system certified to ISO 13485 and are in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. The CliniMACS® System components, including Reagents, Tubing Sets, Instruments, and PBS/EDTA Buffer, are designed, manufactured and tested under a quality system certified to ISO 13485.In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in humans the national legislation and regulations – e.g. for the EU the Directive 2004/23/EC (“human tissues and cells”), or the Directive 2002/98/EC (“human blood and blood components”) – must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System.In the US, the CliniMACS CD34 Reagent System, including the CliniMACS Plus Instrument, CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved; all other products of the CliniMACS Product Line are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE).CliniMACS MicroBeads are for research use only and not for human therapeutic or diagnostic use. CliniMACS, CliniMACS Prodigy, MACS, the MACS logo, and StemMACS are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.

References1. Miltenyi Biotec (2016) Multicolor flow cytometry analysis of human pluripotent stem cell cultures. 2. Miltenyi Biotec (2017) StemMACS™ Trilineage Differentiation Kit – Protocol for flow analysis.

Both articles are available as PDF at www.miltenyibiotec.com

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Average expression profile over 20 passages

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Quality control of hPSCs cultivated under standardized, automated, closed‑system conditions using the CliniMACS Prodigy® 4