Figure 4. WA09 hPSCs cultured for more than 25 passages using a hypertonic citrate dissociation solution retain their pluripotentency and karyotypic normalcy. A) Immunohistochemical and B) flow cytometric analysis for stemness antigens such as OCT4, SOX2, NANOG, SSEA4, TRA160, TRA181. C) In vitro embryoid body pluripotential differentiation assay for the three embryonic germ layers indicated by smooth muscle actin (SMA, mesoderm), alpha feto protein (AFP, endoderm) and beta III tubulin (TUJ1, ectoderm). D) In vivo teratoma pluripotency assay depicting retinal pigmented epithelium (ectoderm), cartilage (mesoderm), and gut-like epithelium (endoderm). E) G-Banding analysis of WA09 cells after 25 passages in StemPro® or 27 passages in mTeSR™1. Scale bars: 200 µM.

For research use only. HYPERStack™ is a registered trademark of Corning®; mTeSR™1 is a registered trademark of WiCell™ Research Institute; StemPro® is a registered trademark of Life Technologies CorporationAll other trademarks herein are marks of the Lonza Group Ltd. or its affiliates© Copyright 2013, Lonza Walkersville, Inc. All rights reserved. P-ISSCR2013 05/13 CD-PO032

Scalable Passaging Human Pluripotent Stem CellsYing Nie, Patrick Walsh, Diana L. Clarke, Jon Rowley, Thomas FellnerLonza Walkersville, Inc., Pluripotent Stem Cell Innovation Center, 8830 Biggs Ford Road, Walkersville, MD 21793

1. Introduction

Prevailing hPSC cultivation practices continuously enrich the undifferentiated cell population through visual selection and mechanical transfer of small undifferentiated multicellular aggregates from hPSC colonies. This labor intensive method results in substantial post-detachment cell injury and death, ultimately affecting the rate of cell expansion. Several alternative methods have been proposed and include the use of one or more enzymes to dissociate adherent hESCs and hiPSCs to single cells. However, continuous cultivation using single cell passaging methods often promotes chromosomal aneuploidy in conjunction with aberrant gene expression.

The subculture and continued maintenance of hPSCs as colony aggregates maintains lateral intercellular contacts important in cell to cell communication. This is thought to reduce cell stress, minimize spontaneous cell differentiation and promote rapid attachment and survival of cells to newly supplied substrates and matrices. Preserving this important element of conventional hPSC cultivation requires the development of a reagent that produces an equivalent colony aggregate size and the high post-dissociation viability and re-plating efficiency required for efficient pluripotent stem cell expansion.

2. Hypertonic Citrate Development and Characteristics

Our search to improve and simplify conventional hPSC dissociation methods resulted in the formulation of a scalable, hypertonic solution of sodium citrate (Figure 1). Citrate is established as a mild chelating agent. Citrate promotes cell dissociation by binding the divalent cations present in the aqueous extracellular environment and intercellular space between cells. This disrupts molecules involved in maintaining cell adhesion such as calcium-dependent cadherins and calcium- and magnesium-dependent integrins.

Citrate solutions, formulated to high osmolalities, favor the production of multicellular colony aggregates. Dissociating cultures with 1mM citrate solutions shows the preservation of colony aggregate integrity and the suppression of small colony aggregates as osmolality increases toward 800 mOsmol/kg (Figure 2). This suggests greater versatility in solution formulation and process development without reducing the colony aggregates to single cells.

Using hypertonic citrate for open, small-scale hPSC dissociation, we were able to demonstrate multiple advantages over traditional enzymatic methods (Figures 2, 3 and 4). hPSC colonies subcultured in a hypertonic citrate dissociation solution:

– Exhibited a higher post-detachment viability (97% ±1%) – Generated greater numbers of cells at each passage – Produced more uniform colony aggregate sizes – Supported the continuous generation of pluripotent cells for over 25 cell passages

3. Hypertonic Citrate Simplifies Subcultivation

References

1. Ahrlund-Richter, L. et al. Isolation and production of cells suitable for human therapy: challenges ahead. Cell stem cell 4, 20-26 (2009).2. Carpenter, M.K., Frey-Vasconcells, J. & Rao, M.S. Developing safe therapies from human pluripotent stem cells. Nature biotechnology 27, 606-613

(2009).3. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nature methods 3, 637-646 (2006).4. Draper, J.S. et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nature biotechnology 22, 53-54 (2004).5. Mitalipova, M.M. et al. Preserving the genetic integrity of human embryonic stem cells. Nature biotechnology 23, 19-20 (2005).6. Chen, G., Hou, Z., Gulbranson, D.R. & Thomson, J.A. Actin-myosin contractility is responsible for the reduced viability of dissociated human

embryonic stem cells. Cell stem cell 7, 240-248 (2010).7. Li, L., Bennett, S.A. & Wang, L. Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells.

Cell adhesion & migration 6, 59-70 (2012).

Figure 1. The simplified process of subculturing hPSCs in an open culture platform. A comparison of the subculturing process using traditional hPSC methods or a hypertonic citrate dissociation solution.

Figure 2. Osmolality affects the size of dissociated WA09 hPSC colonies cultured on Matrigel in mTeSR™1. After 5 days of cultivation, colonies were dissociated following 20 minutes of treatment time with various 1mM sodium citrate solutions formulated at increasing osmolalities. Cultures were also dissociated into a single-cell suspension and mechanically for comparison. Images were produced from dissociated culture samples. Overlaid histograms represent a quantitative approximation of detached cell aggregate size performed using image analysis.

Figure 3. Direct comparison of dissociation reagents on WA09 hPSCs cultured on Matrigel in StemPro® or mTeSR™1. A) Post-dissociation viabilities of hESCs using the hypertonic citrate solution, non-enzymatic manual scraping, dispase or collagenase. B) actual multi-passage cell counts, and C) the expected culture scale required to accommodate extrapolated multi-passage expansion. All conditions, n=3.

4. Hypertonic Citrate Promotes Dissociation As Multicellular Aggregates

5. Hypertonic Citrate Allows for Rapid Expansion of hPSCs

6. Hypertonic Citrate Is Safe for Long-term Passaging

AcknowledgementsWe thank Melody Crouse, David Jones and Sonya Boulware for their help in formulating the hypertonic citrate solutions, Barry Simon for his advice on formula optimization, Huan Tran for technical advice on cell imaging, Randy Cramer, Dan Ragland, Chi Ritchie and Jim Miller for performing teratoma formation assays and Xinghui Tian for technical advice on cell characterization and teratoma assay development.

A

C

Passage 4Passage 3Passage 2Passage 1 Passage 5Citrate

Dispase

Collagenase

No Enzyme

Million Billion Trillion

010

304050

80

100

Viab

le C

ells

Citrate Dispase Collagenase

mTeSR™1 StemPro®

No Enzyme

20

6070

90

B

B

StemPro® mTeSR™1

OCT4

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

0

100

200

300

400

100 101 102 103 104

99.2%

Coun

t

SSEA4

TRA-1-60

TRA-1-81

A

StemPro® mTeSR™1

OCT4

Sox2

Nanog

SSEA4

TRA-1-60

TRA-1-81

C

SMA

AFP

TUJ1

D

Mesoderm

Endoderm

Ectoderm

1. Remove culture medium and rinse cells with 1X calcium- and magnesium-free bu�ered saline solution (Cells are on the bottom of the plate)

remove rinse solution

remove dissociation solution

remove enzymatic solution

3. Rinse cell aggregates from surface with fresh culture medium

2. Add citrate solution to cells for 5-15 minutes

Return cells to incubator

Hypertonic solution technique(Lonza product)

5. Extract suspended cells

Distribute cells on new culture plates

1. Remove enzyme from controlled temperature storage

2. Measure quantity desired

3. Dilute as directed and sterilize by �ltration

4. Remove culture medium and apply solution to cells

6. Rinse and scrape cells from plates in fresh culture medium

5. Incubate solution on cells at 37°C

8. Centrifuge cell suspension

9. Remove solution and add fresh culture medium to desired seeding density

4. Collect cell aggregates and dilute to desired seeding density with fresh culture medium

7. Collect Cells

Enzymatic solution technique(standard process)

E StemPro® (Passage 25) mTeSR™1 (Passage 27)

Passage 4Passage 3Passage 2Passage 1 Passage 5Citrate

Dispase

Collagenase

No Enzyme

0 1x107 2x107

Passage 5

0 10 20 30

>1 x4 x30

T-FlaskPlate

x10 x77

Cultu

re S

cale

HYPERStack™

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

0

10

20

30

%

<1

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Citrate Optimization Size Referencem0smol/kg

270

420

570

720

Size

Incr

ease

with

Osm

olal

ity

Mechanical Clump Passage

Single-CellPassage

Cell Number Per Colony

BioResearch

91.1% ±4.4% 91.1% ±1.8%

99.8% ±0.1% 98.0% ±1.6%

94.0% ±1.8% 81.1% ±1.0%

91.8% ±2.7% 85.9% ±0.7%