Application Note 2

Recovery of pluripotent stem cellspheroids from GrowDex®
by enzymatic digestion
Yan-Ru Lou (Ph.D.)
University of Helsinki, Finland
INTRODUCTION

Recovery of cells or spheroids for downstream processing once a live cell assay has beenconcluded can be advantageous for many cell based studies. Additional data relating togene or protein expression can be generated or cells used for further experimental work
such as in multi-dose studies. Here we have made use of GrowDase™ a cellulase enzyme
preparation, to recover spheroids cultured in GrowDex. GrowDase is an enzyme thatspecifically degrades cellulose without affecting animal or human cells, as they do notcontain cellulose. The degraded products of GrowDex are nontoxic sugars. However,before using GrowDase in the 3D culture we optimized the GrowDex concentration with H9GFP, WA07 and iPS(IMR90)-4 cell lines. AlamarBlue assay was used to assess cell viability
and any possible toxicity of the GrowDase enzyme. Removal of GrowDex was visualized bycalcofluor white stain, which is a cellulose-binding fluorochrome. The use of H9-GFP cells
enabled us to monitor the spheroid structure after the enzymatic removal of the hydrogel
using confocal microscopy.

MATERIALS•••••METHODSGrowDex staining and live cell imaging using a confocal microscope.
To evaluate the enzymatic removal of GrowDex, calcofluor white stain was added tothe culture to stain the nanocellulose fibers following the manufacturer’s instructions. GFP
fluorescence of the live H9-GFP cells and calcofluor stained GrowDex were visualized usinga Leica TCS SP5II HCS A confocal microscope with Argon 488nm and UV 405nm lasers,respectively.

H9-GFP modified from H9 (WA09, WiCell) WA07 and iPS(IMR90)-4 (WiCell)mTeSR1 medium (STEMCELL Technologies)
GrowDex 1.5% (UPM)
Calcofluor white stain (Sigma FLUKA)
GrowDase 5 mg/ml (UPM)

Degradation of GrowDex hydrogel with GrowDase.
GrowDase enzyme was used to recover the 3D hPSCs and spheroids from the hydrogel
which were then subsequently sub-cultured using a 2D platform following the recovery
process. The optimal working temperature of the enzyme is 45–50°C, which is not suitablefor human cells, therefore incubations were performed at 37°C. Before GrowDase treatment
the old mTeSR1 medium was removed, and GrowDase diluted in mTeSR1 medium wasadded to the samples. Various concentrations of GrowDase were evaluated during this study
by incubating the samples over 24 h period. Following enzymatic removal of GrowDex thehPSC spheroids were collected for further cultivation.

RESULTS

The 3D spheroids stayed intact after treatment with all concentrations of the GrowDase
enzyme tested. It was noted that GrowDase concentrations below 200 µg/mg (µgenzyme/mg cellulose in GrowDex) were insufficient to remove the hydrogel completely.

Fig. 1. Enzymatic removal of the NFC hydrogel. (A, B) Mitochondrial metabolic activity ofWA07 and iPS(IMR90)-4 cells (A) and H9-GFP cells (B) as a function of the concentrationof GrowDase. Cells cultured on a standard platform were treated with GrowDase at variousconcentrations. The mitochondrial metabolic activity was determined by an AlamarBlue
assay, and the increase in fluorescence intensity after a 24 h treatment with GrowDase ispresented. Four (A) or six (B) biological samples for each condition were prepared. Theresults are expressed as mean ± standard deviation (SD). (C) Removal of GrowDex by 0,50, 200, and 500 µg/mg is visualized by calcofluor white staining of GrowDex, whichis represented in blue. H9-GFP cells are seen in green. Adapted with permission from (1).
Copyright 2014, Mary Ann Liebert, Inc.

CONCLUSIONSREFERENCE

In this study we have demonstrated that GrowDex can be removed from cell samples
by enzymatic digestion using the GrowDase enzyme. Here spheroids were successfully
recovered from the 3D samples for further downstream processing or re-culture. In addition,
no significant impact on cell viability was observed as a result of performing the recovery
process, making the use of GrowDex in combination with GrowDase as an ideal system for
3D culture and cell recovery.
1.

Lou Y-R et al. 2014 “The Use of Nanofibrillar Cellulose Hydrogel As a Flexible ThreeDimensional Model to Culture Human Pluripotent Stem Cells. STEM CELLS AND
DEVELOPMENT 23:4, pp. 380-392.

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