Application Note 17GrowDex®-alginate hydrogel used for3D co-culture of HepG2 and SK-HEP-1
cells and as a cell-carrier system forcoating surgical sutures
Patrick Laurén
University of Helsinki, Finland

INTRODUCTION

MATERIALS

Non-animal originated hydrogel nanomaterials have great potential in biomedical
applications due to their versatility and soft-tissue like properties. With the ability tosimulate native tissue function, hydrogels are potentially well suited for e.g. cellular therapy
applications. Earlier studies with GrowDex® have shown it to be an optimal culture matrix
for several different cell types. In this study we investigated the rheological properties ofthe GrowDex-alginate hydrogels and performed cell cultures with HepG2 and SK-HEP-1
cell lines as a co-culture model within GrowDex-alginate hydrogel and on the surface,respectively. In addition, suture coating with GrowDex-alginate with HepG2 cells andperformance of the coated sutures on pig liver tissue was demonstrated.

• GrowDex 1.5% (Cat No. 100 100 005, UPM)
• Sodium alginate (Cat No. W201502, Sigma), calcium chloride anhydrous (Cat No.
C4901, Riedel-de-Haen), barium chloride * 2 H20 (Cat No. 529591, Sigma)• HepG2 (HB-8065™, ATCC) and SK-HEP-1 cells (HTB-52™, ATCC)• DMEM culture medium (Cat No. 11965092, Gibco)• Rat collagen I (Cat No. 3443-003-01, Trevigen)• CellTracker™ Green CMFDA (Cat No. C2925, Molecular Probes) and Red CMPTX (CatNo. C34552, Molecular Probes)• Surgical sutures Velosorb™ Fast 3-0 (Cat No. CV916, Covidien)

METHODSRESULTS

Figure 1. A) Storage modulus (G')
and B) loss modulus (G’’) values
showed increased strength in the
GrowDex-alginate network whencompared to native GrowDex.

1.

Preparation of GrowDex-alginate and co-culturing of
HepG2 and SK-HEP-1 cells
a. GrowDex-alginate hydrogel was prepared by adding and mixing sodium alginate
with GrowDex stock hydrogel. The mixture was left to stabilize for 24 h. The final
GrowDex-alginate mixture contained 8% (w/v) sodium alginate and 1.35% (w/v)
GrowDex.
b. HepG2 cells were encapsulated in GrowDex-alginate threads by suspending thecells in GrowDex-alginate (cell density 1043 cells/µl) and dispensing the mixture
with a syringe and a 22G needle into 68 mM calcium chloride crosslinking solution
for 3 min and 20 mM barium chloride solution for additional 5 min.
c. For SK-HEP-1 cell seeding, the GrowDex-alginate-HepG2 threads were treated with
1 mg/ml type I collagen by pipetting collagen on the threads to cover them fully andincubating at 37°C for 30 min. The threads were transferred into low attachment
culture plates, SK-HEP-1 cell suspension (1.2 million cells/ml) was introduced on
top of GrowDex-alginate-HepG2 threads, and incubated at 37°C for 2 h withsubsequent shaking of the plate every 30 mins.
d. The well plates were incubated at 37°C from 48 h up to 2 weeks and the culture
medium was replaced every 48 h.

2.

Coating of surgical sutures with GrowDex-alginate-HepG2 hydrogela. Surgical sutures (Velosorb) were coated with GrowDex-alginate-HepG2 by inserting
the suture through the syringe barrel and needle orifice, filling the syringe with
GrowDex-alginate-HepG2 hydrogel and feeding the suture through the needle atslow rate ensuring the formation of an even hydrogel layer with 15000 cells per cm/suture.
b. Coated sutures were treated with 68 mM calcium chloride crosslinking solution for 3min and 20 mM barium chloride solution for additional 5 min.

3.

Imaging of the cells
a. Cells within and on top of the threads were imaged with confocal microscopy (LeicaTCS SP511 HCS A, HC PL APO10x/0.4 objective, HeNe 633 nm abd DPSS
561nm lasers) with cellular dyes CellTracker™ Green CMFDA and Red CMPTX.

The addition of sodium alginate 8% (w/v) increased the GrowDex storage- and loss
modulus contributing significantly to coating strength which was clearly seen in frequency
sweeps of the hydrogels (Fig. 1). Confocal microscopy showed nearly 100% cell viability
throughout the 2 weeks incubation period within and on the surface of the coating. Typicalmorphologies in the cell co-culture of spheroid forming HepG2 (inside coating) andmonolayer type SK-HEP-1 (on top of coating) were observed (Fig. 2). Surgical sutures weresuccessfully coated with GrowDex-alginate-HepG2 hydrogel, the coating remained intact
during suturing operation with pig liver, and the cells remained viable (Fig. 3).

Figure 2. SK-HEP-1 cells seeded on the surface of the GrowDex-alginate threads after 48h
incubation (green). HepG2 cells (red) within the threads grew individually or in very smallclusters; however, surface growth showed typical cluster and epithelial morphology of bothcell lines.

Figure 3. Surgical sutures coated with GrowDex-alginate-HepG2 and sewn three times
through a pig liver segment indicating intact suture coating and live HepG2 cells within thecoating matrix.
CONCLUSIONSREFERENCES

GrowDex-alginate hydrogel is suitable matrix for 3D co-culture of HepG2 and SK-HEP-1
cells. Cells can be cultured within and on top of GrowDex-alginate threads. In addition,
surgical suture coatings from GrowDex-alginate were fabricated as biomedical devices to be
used as cell-carrier systems in cellular therapy. GrowDex-alginate suture coatings could help
to overcome the limitations related to cellular therapy, such as low cell survival rates and cell
distribution out of target tissue. Cell-containing hydrogel coated sutures can be used as cellcarrier systems for cellular based therapy and post-surgical treatment, including oral mucosa
repair and ulcer treatment, and e.g. Crohn’s disease.

1.

Laurén P et al. 2017 Nanofibrillar cellulose-alginate hydrogel coated surgical sutures
as cell-carrier systems. PLoS ONE 12(8)You can order products online at: www.upmbiomedicals.com/store.
Or contact us at biomedicals.sales@upm.com for a quotation or to place an order.

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