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Stem Cells Vol. 24 No. 2 February 2006, pp. 230 -235
doi:10.1634/stemcells.2005-0243; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS-CHARACTERIZATION SERIES

Automated Mechanical Passaging: A Novel and Efficient Method for Human Embryonic Stem Cell Expansion

Alexis Joannidesa, Christelle Fiore-Hérichéa, Kristine Westmorea, Maeve Caldwella, Alastair Compstona, Nicholas Allenb, Siddharthan Chandrana

a Department of Clinical Neurosciences and Centre for Brain Repair, University of Cambridge, Cambridge;
b School of Biosciences, Cardiff University, Cardiff, United Kingdom

Key Words. Human embryonic stem cells • Automated mechanical passaging • Expansion

Correspondence: Siddharthan Chandran, Ph.D., M.R.C.P., Department of Clinical Neurosciences and Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CP2 2PY, United Kingdom. Telephone: 44-1223-331160; fax: 44-1223-331174; e-mail: sc222{at}cam.ac.uk


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is a need for more standardized methods of maintenance and propagation of human embryonic stem cell (hESC) cultures. Enzymatic passaging currently represents the most widely used method for expansion of hESCs. Although rapid and straightforward, this technique results in variable-sized cell clusters and significant cellular trauma, which may apply selective pressure in long-term culture. Mechanical passaging has the potential advantages of defined colony fragment sizes, reduced cellular trauma, and the possibility of selecting undifferentiated colonies for transfer. However, manual dissection of individual colonies is a prohibitively time-consuming process unsuitable for maintaining large numbers of hESCs without the use of additional chemical means. In this study we report an efficient automated method for mechanically passaging hESCs. We have used this method exclusively to maintain hESCs in long-term undifferentiated culture without the use of enzymatic digestion for longer than 100 days. This automated technique can thus be used routinely to culture hESCs in the laboratory.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human preimplantation embryos [1]. hESCs have generated considerable interest because of their potential as a source of defined cell types for studying early human development and possible application in regenerative medicine.

hESCs were initially isolated on mouse embryonic fibroblasts (MEFs) and passaged enzymatically [1]. Subsequent studies have reported a variety of methods for derivation and maintenance of hESCs [2–7]. It is apparent that passaging technique is critical for maintaining undifferentiated and karyotypically stable hESC lines. Chemical passaging represents the most widespread method for expansion of hESCs. Dissociation by this method uses enzymes such as collagenase, dispase, and trypsin or enzyme-free cell dissociation buffer [8 –11]. Although enzymatic methods readily enable large-scale expansion, emerging evidence suggests that such techniques are associated with increased rates of karyotypic abnormalities [12, 13]. In contrast, mechanical methods of passaging avoid enzymes and seem to better maintain genetic stability. In addition, mechanical expansion allows selective transfer of exclusively undifferentiated colonies. However, the comparatively laborious and time-consuming process of manual colony dissection limits the practical use of mechanical passaging in bulk hESC culture. Consequently, many studies combine both chemical and mechanical passaging methods for long-term hESC maintenance [8, 10, 14]. Indeed, for practical reasons, hESC lines initially derived and passaged by mechanical methods have been subsequently adapted for enzymatic passaging at the expense of line establishment efficiency [15].

Here we report a rapid, efficient, and automated method for bulk passaging hESCs. This technique has the advantage of being exclusively mechanical. Using this method, we have been able to maintain hESCs in undifferentiated culture for longer than 100 days. Automated mechanical passaging (AMP) thus incorporates the advantages of mechanical dissection without sacrificing the practical benefits of enzymatic passaging.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The hESC lines H9 obtained from the WiCell Research Institute (Madison, WI, http://www.wicell.org) and HuES9 (hES facility, Harvard University, Cambridge, MA, http://mcb.harvard.edu/melton/hues) were used in this study between passages 21 and 65. Cells were cultured as previously described [15–17] on 60-mm tissue-culture dishes (Corning, New York, http://www.corning.com) coated with 0.1% porcine gelatin (Sigma, St. Louis, http://www.sigmaaldrich.com) on a layer of irradiated MEFs. Culture medium (knockout serum replacement [KSR]) consisted of knockout Dulbecco’s modified Eagle’s medium supplemented with 20% serum replacement, 1% nonessential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 4 or 10 ng/ml human basic fibroblast growth factor (R&D, Minneapolis, http://www.rndsystems.com).

Enzymatic Passaging
H9 cells were washed in phosphate-buffered saline (PBS) and incubated in 1 mg/ml collagenase IV (Invitrogen) for approximately 10 minutes until colonies began to curl at the periphery. The medium was replaced with KSR, and colonies were detached from the dish using a cell scraper (Falcon, San Jose, CA, http://www.bdbiosciences.com) and transferred to a 15-ml centrifuge tube. Colonies were dissociated by gentle trituration with a 5-ml pipette and replated as above. HuES9 cells were washed in PBS and incubated in 0.05% trypsin/EDTA (Invitrogen) for 3 to 5 minutes until cells detached from the plate. Trypsin was neutralized with fresh KSR, and cells were centrifuged, resuspended in fresh KSR medium, and replated onto a new feeder layer.

Automated Mechanical Passaging
The McIlwain tissue chopper (Mickle Engineering, Gomshall, Surrey, U.K.) was modified by removing a small square on the chopper arm (Fig. 1BiiiGo) to avoid contact with the side of the culture dish. A sterile razor blade was then fitted on the modified chopper arm. The culture medium was replaced with 1 ml fresh KSR before attachment to the stage of the chopper using adhesive (Fig. 1BiiGo) [18]. Colonies were then chopped at 200-µm intervals in two perpendicular directions to produce square fragments approximately 200-µm wide. The chopped colony fragments were selectively detached from the dish using a cell scraper followed by gentle pipetting and transferred onto new MEF plates (Fig. 1AGo). See also the supplementary information for a demonstration of the AMP technique.


Figure 1
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  		Figure 1. Automated mechanical passaging. (A): Schematic of the technique. (B): (i), Starting human embryonic stem cell propagated with collagenase; (ii), the McIlwain tissue chopper; (iii), modified chopper arm (arrow); (iv), phase micrograph showing a single colony after mechanical chopping. (C): Representative colony fragments after passaging using automated chopping (i, ii) and enzymatic passaging (iii–iv). Scale bar = 100 µm.

 
Reverse Transcription-Polymerase Chain Reaction
Embryonic stem cell colonies were harvested by incubating in 1 mg/ml collagenase IV for 60 minutes, selectively detached by gentle pipetting, and transferred to a 15-ml centrifuge tube. Colonies were allowed to settle by gravity for 10 minutes and washed twice with PBS. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, www.qiagen.com) according to the manufacturer’s instructions. Samples were treated with RNAse-free DNase (Qiagen) to avoid DNA contamination. cDNA was synthesized from 2.5 µg of RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo-dT primers according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out using Taq polymerase (Invitrogen). PCR products were separated on a 2% agarose gel and visualized with SYBR-Green (Invitrogen). The expression of the housekeeping gene GAPDH was used to normalize PCR reactions. Forward and reverse primer sequences, annealing temperatures and PCR cycles were as follows: POU5F1, 5'-CTTGCT-GCAGAAGTGGGTGGAGGAA-3', 5'-CTGCAGTGTGGGTT-TCGGGCA-3', 55°C, 30 cycles; SOX2, 5'-ATGCACCGCTA CGACGTGA-3', 5'-CTTTTGCACCCCTCCCATTTT-3', 60°C, 35 cycles; NANOG, 5'-TTCTTGCATCTGCTGGAGGC-3', 5'-AGTGTGGATCCAGCTTGTCCC-3', 55°C, 30 cycles; TERT, 5'-AGCTATGCCCGGACCTCCAT-3', 5'-GCCTGCAGCAGG-AGGATCTT-3', 60°C, 30 cycles; GAPDH, 5'-AGCCACA-TCGCTCAGACACC-3', 5'-GTACTCAGCGGCCAGCATCG-3', 60°C, 30 cycles.

Immunocytochemistry
hESC colonies were fixed with 4% fresh paraformaldehyde for 20 minutes at room temperature and washed three times with PBS. Fixed cells were blocked for 1 hour at room temperature with PBS/10% goat serum/0.1% Triton X-100 and then incubated overnight with Oct-4 (sc-5279, Santa Cruz, 1:100) or SSEA-4 (clone MC-813-70, DSHB, 1:50) primary antibody in PBS/2% goat serum/0.1% Triton X-100 at 4°C. After three washes in PBS, secondary antibody (goat anti-mouse, Alexa Fluor 488, 1:500) in PBS/Hoechst (1:5000) was applied for 1 hour at 37°C.

Cytogenetic Analysis
Chromosome number and size were determined using Giemsa-stained metaphase spreads at the Department of Haematology, Addenbrooke’s Hospital, Cambridge.

Quantification and Statistical Analysis
Colonies and passaged fragments were viewed using a Leitz microscope at high magnification (x40) for determination of cell numbers. Colony fragment sizes following passaging were obtained by determining the number of cells per cluster in a total of 50 random fields for each method over five different passages. Colony population growth was calculated by determining the number of cells per colony in 15 randomly selected fields at each time point for every condition. Colony growth curves were constructed by determining the size of 10 randomly selected colonies from each condition (on two separate occasions) 24 hours after passaging (day 1) and at two subsequent 24-hour intervals (days 2 and 3). Net expansion was calculated by dividing total cell number for a given colony at day 2 or 3 by the total cell number for the same colony at day 1. All figures and error bars are represented as mean ± standard error from the mean unless stated otherwise. For parametric analysis, a two-tailed Student’s t-test to compare population means and a two-tailed F-test to compare population variances were used. A Mann-Whitney rank-sum test to compare median values was performed for nonparametric analysis. Statistical analysis was carried out using GraphPad Prism 3.03 (GraphPad Software, San Diego, CA, http://www.graphpad.com).


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To compare enzymatic and mechanical passaging, hESC cultures were established. The H9 line had been previously passaged using collagenase (Fig. 1BiGo). Briefly, after incubation with collagenase, colonies were detached using a cell scraper, gently triturated, and replated in a 1:3 splitting ratio. To establish a mechanical passaging method, initial studies determined that single colonies could be successfully passaged by manual dissection using a scalpel blade, a finding consistent with previous observations [8, 10, 14]. However, this method is extremely time-consuming and impractical for regular use or bulk passaging. To explore whether this method could be automated, a McIlwain tissue chopper was adapted. The McIlwain tissue chopper, originally developed for sectioning fresh brain tissue, has been successfully used for passaging of human fetal neural precursor cultures [18]. The tissue chopper (Fig. 1BiiGo) was modified by removing a small square on the chopper arm (Fig. 1BiiiGo) to avoid contact with the side of the culture dish. Colony plates were fixed on the chopper stage and automatically sectioned (Fig. 1AGo and supplemental information). The McIlwain tissue chopper allows the sectioning interval to be calibrated between 50 and 950 µm. Pilot studies identified 200-µm intervals as optimal for AMP, dividing colonies into approximately 15 fragments (Fig. 1CGo), depending on the starting colony size. AMP is rapid, with a single 6-cm dish being passaged in less than 60 seconds. The chopped fragments were readily detached from the culture dish and replated at a ratio of 1:3.

A striking observation after AMP was the consistency of fragment size after chopping in contrast to the variability of size produced by conventional collagenase passaging (Fig. 1CGo). Quantitative analysis of this phenomenon was next undertaken by counting the total number of cells in randomly selected fragments (immediately after passage) at different passage numbers. A narrower size range was evident in AMP (40 to 250 cells/fragment) compared with enzymatic passaging (10 to > 500 cells/fragment). Initial analysis revealed similar mean values that were not significantly different (138 ± 8 [AMP], 133 ± 30 [collagenase]; p > .10), an observation consistent with comparable starting cell numbers. However, a large variance was apparent in collagenase-treated cultures, which was significantly different from mechanically passaged cultures (p < .0001). The scatter representation and frequency distribution confirmed large numbers of very small or large fragment sizes in collagenase-passaged cultures in contrast to the narrower distribution after AMP (Figs. 2A, 2BGo). This difference was statistically significant (median values: 148 [AMP], 31 [collagenase]; p < .0001).


Figure 2
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  		Figure 2. Quantification of mechanical and enzymatic methods of passaging. (A): Scatter representation of total cell number per fragment analyzed immediately after passaging. (B): Frequency distribution of colony fragment sizes analyzed immediately after passaging; bars in (A) represent median values; x-axis values in (B) represent the average value for each frequency interval. (C): Graph showing colony sizes of passaged human embryonic stem cell populations at 1, 2, and 3 days after passaging. (D): Growth curve showing net expansion of individual colonies in culture after passaging at days 2 and 3 (expressed relative to day 1 after passaging; see Materials and Methods).

 
We next examined growth of colonies derived by enzymatic and mechanical passaging. Population analysis at days 1 to 3 after passaging revealed comparable mean cell number per colony between the two methods (p > .10 for all time points). However, a significantly larger variability of colony cell number persisted in collagenase-passaged cultures compared with AMP after seeding (Fig. 2CGo; p < .02 for all time points).

Serial examination of individual colonies at days 1 through 3 after passaging showed no significant difference in net expansion between the two methods (D2: 2.21 ± 0.14 [AMP], 1.96 ± 0.15 [collagenase]; D3: 4.61 ± 0.27 [AMP], 4.01 ± 0.37 [collagenase]; values relative to day 1; p > .10 for both time points; Fig. 2DGo).

Cultures were passaged approximately every 3 to 4 days and, to date, have been expanded exclusively by AMP for over 100 days. AMP-passaged cells could also be successfully freeze-thawed. Cytogenetic analysis showed maintenance of a stable karyotype by AMP (data not shown). In addition to the H9 line, HuES9 was also successfully propagated with AMP for multiple passages.

Spontaneous differentiation was occasionally observed in hESC cultures. However, substantially less differentiation was found in cultures maintained by AMP. This may reflect the uniformity of size achieved by the technique, given that differentiation was predominantly evident within the centre of large colonies. Furthermore, expression analysis revealed maintenance of pluripotency of hESC cultures with AMP. Semiquantitative reverse transcription (RT)-PCR did not reveal any significant differences in the expression of POU5F1, NANOG, SOX2, or TERT between the two methods (Fig. 3AGo). Immunocytochemistry confirmed that mechanically passaged colonies remained positive for Oct-4 and SSEA-4 (Figs. 3B, 3CGo).


Figure 3
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  		Figure 3. Maintenance of pluripotency with mechanical passaging. (A): Reverse transcription–polymerase chain reaction analysis showing maintenance of pluripotency markers in the two methods, with no reverse transcriptase addition as a control. (B, C): Phase and immunomicrographs showing phase-contrast (i), Hoechst (ii), and Oct-4 (Biii) or SSEA-4 (Ciii) staining of human embryonic stem cell expanded using automated mechanical passaging. Scale bar = 100 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report a novel method for bulk expansion of hESCs by automated mechanical chopping that is both rapid and maintains greater uniformity of colony size.

In contrast to mouse embryonic stem cell cultures, hESC cultures are comparatively more challenging to maintain and expand. hESC cultures can be expanded by two methods: chemical and mechanical. To date, bulk passaging has generally used chemical methods. However, chemical methods have two potential drawbacks. Chemical dissociation precludes ready removal of differentiated colonies. Recent evidence also suggests an increased incidence of karyotypic instability after repeated chemical passaging [12, 13, 19]. Mechanical passaging, in contrast, permits selective colony dissection and thus maintenance of undifferentiated colonies [14]. Two recent reports also provide evidence of comparative cytogenetic resilience upon mechanical passaging, which is lost upon switching to chemical dissociation and expansion [12, 13]. Large-scale mechanical passaging is limited, however, by the time-consuming nature of individual colony dissection. There is thus a need for standardized and rapid mechanical methods for expanding hESC cultures.

We have previously reported successful mechanical sectioning of human-derived neural precursors using an automated tissue chopper [18]. A modification of this method allowed bulk long-term expansion of undifferentiated hESC colonies. Critically, this method permits the generation of near-uniform fragment sizes, an important consideration for maintaining homogeneous cultures. This may also be pertinent to limiting differentiation of colonies, increased density being potentially associated with a greater propensity for spontaneous differentiation [12]. The reported automated method also maintains uniform colony size expansion with comparable doubling times to those observed in parallel enzymatically passaged cultures. In addition, over a prolonged period in culture, mechanically chopped hESCs maintained a normal karyotype and expression of pluripotent markers.

In summary, we report an automated mechanical passaging method that provides a useful and reproducible method for maintaining large numbers of hESCs.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Craig Secker provided valuable technical assistance in adapting the tissue chopper. Isabelle Bouhon and Jane Anderson provided valuable technical assistance. The SSEA-4 antibody, developed by Davor Solter and Barbara Knowles, was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa. This work was supported by an MRC Stem Cell Strategic Grant. A.J. is supported by a Merck Sharpe and Dohme Neuroscience Studentship, the Cunning Fund, and the University of Cambridge MB/PhD program.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 4, 2005; accepted for publication September 6, 2005.



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