Cohen_Ben-Gurion University

Alginate SpongesFor 3-D Cell Culture

Smadar Cohen & Tsiona Elkayam

Ben-Gurion University of the Negev

Cohen_Ben-Gurion University

Alginate Sponge Characteristics

PhysicalHighly porous (>90%); Homogenous pore size (100-200m) and shape; Interconnecting poresTransparent; Stable

ChemicalPolysaccharide (algae-derived) hydrogel, easily-wetted by culture medium, allowing rapid and efficient cell seeding and distribution

BiologicalBiocompatible with cells and the human body

DissolutionMild conditions (EDTA, Citrate buffer)

Cell RecoveryEasy, no cell damage, maintaining surface cell markers (FACS analysis)

Shelf Life>12 months (room temperature, low humidity)

Cohen_Ben-Gurion University

Alginate Sponge90% Porosity; ~200m Pore Size

200m

Cohen_Ben-Gurion University

Cell Distribution within Alginate Sponge (10mm thick)

Surface

Bottom

Center

Initial cell seeding density (1x108 cells/cm3)

H&E cross sections

MTT

SEM

Cohen_Ben-Gurion University

3-D Culture in Alginate Sponge

Cohen_Ben-Gurion University

3-D Cell Applications with Alginate Sponges

Tissue engineering Bioproduction of valuable therapeutics ADMETox researchExpansion of hematopoeitic and mesenchymal stem cellsStem cell differentiation

Cohen_Ben-Gurion University

Tissue Engineering of Cardiac Muscle

Schwarzkopf et al, Tissue Engineering, 12 (2006)

Cohen_Ben-Gurion University

Organization of Primary Organization of Primary Hepatocytes as Spheroids within Hepatocytes as Spheroids within

Alginate SpongesAlginate Sponges

FDA staining SEM TEM

Enhanced Hepatocellular Functions

Glicklis et al (2000, 2004); Dvir-Ginzberg et al (2003, 2004);

Cohen_Ben-Gurion University

The sponge recapitulates the stem cell nicheniche 3-D microenvironmentmicroenvironment

The scaffold can be madeThe scaffold can be made cell specific cell specific by attaching adhesion peptides (RGD, YIGSR)

The scaffold can be madeThe scaffold can be made a reservoir a reservoir for growth factors and molecular agents important for cell growth and differentiation

High cell density High cell density can be reachedcan be reached in the scaffold, without reaching confluence

Alginate Sponges for 3-D Stem Alginate Sponges for 3-D Stem CellsCells

Cohen_Ben-Gurion University

Growth Factor

KA binding to

M-Alginate (M-1)

Release Rate

(ng/day-1)

VEGF6.32*106 24

bFGF8.62*106 20

aFGF2.82*107 7

PDGF-ββ3.53*10710

Cohen_Ben-Gurion University

Expansion and a better control over differentiation of Human Human Embryonic Stem (hES) CellsEmbryonic Stem (hES) CellsExpansion of Hematopoietic stem Hematopoietic stem cells (HSC)cells (HSC)Expansion and induced differentiation of Mesenchymal Mesenchymal stem cells (MSC)stem cells (MSC)

Stem Cell R&D Stem Cell R&D in Alginate Scaffoldin Alginate Scaffold

Cohen_Ben-Gurion University

Human Embryonic Stem Cells (hESC)

Alginate sponge allows colonization of undifferentiated hESC within poresAlginate sponge pore size controls the aggregation of human embryoid bodies (hEB)The alginate sponge maximizes hES cell expansion and has a better control over cell differentiation

Cohen_Ben-Gurion University

Morphology of hEBs

Scaffold-borne hEBsHomogenous and Small Size

No Central Necrosis

Petri-DishExtensive Aggregation &

Central Necrosis

Gerecht-Nir et al, Biotechnol. Bioeng. (2004)

Cohen_Ben-Gurion University

Key Features of Sponge-borne hEBs

hEBs ranging between 250-900 m after 1 month, smaller than hEBs formed in conventional Petri dishesSpherical, homogenous in size relative to those formed in Petri dishesNO CENTRAL NECROSIS in hEBs formed in alginate sponges

Cohen_Ben-Gurion University

Expansion of Human Embryonic Stem Cells (hESC) in Alginate Sponges

4-Fold higher expansion in scaffold-borne EBs relative to Petri-dish

XTT

0

5

10

15

20

25

0 5 10 15 20 25 30

Day

Cell

(10<

6>/m

l) Static

Scaffolds

Petri Dish

Alginate Sponge

Seeding: 0.1-1 x105/scaffold

Cohen_Ben-Gurion University

Differentiation within Alginate Borne-hEBs

into 3 Germ Layers

ECTODERM

MESODERM

ENDODERM

H&E

Neuronal cells (nestin+)Hepatocyte-like cells (FP+)

Cohen_Ben-Gurion University

Case Story: Alginate vs. Collagen Scaffolds for

ADMET

Collagen Alginate

Cohen_Ben-Gurion University

C3A- Clone Derivative of HepG2

Cohen_Ben-Gurion UniversityCollagen Alginate

C3A Cell Morphology in Alginate vs. Collagen

Scaffolds

3-D Spheroids2-D Adherent Cells

Cohen_Ben-Gurion University

Laminin Expression in C3A seeded in Alginate vs. Collagen Scaffolds

Collagen Alginate

Cohen_Ben-Gurion University

C3A Spheroids Express Albumin& CK18

Cohen_Ben-Gurion University

Treatment6-testosterone (IU)

Cells only1.41 x 106

Cells + Inducer30±2 x 106

Cells + Inhibitor5.32 x 105

Cells + Inducer + Inhibitor6.0 x 105

CYP3A4 Metabolism in C3A Spheroids

Cohen_Ben-Gurion University

TreatmentnM Resorufin produced/ 106cells/1h

C3A cell monolayer

C3A spheroids

Cells only02 ±0.7

Cells + Inducer30±280 ±20

Cells + Inhibitor00.6±0.6

Cells + Inducer + Inhibitor10±240 ±10

CYP1A2 Metabolism in 2D vs. 3D Cultures

Cohen_Ben-Gurion University

1A2 mRNA levels in C3A seeded in Alginate vs.

Collagen Scaffolds

CYP1A2 Induction Alginate scaffold Vs. Collagen Scaffold

0

20

40

60

80

100

120

140

C N.IA N.IC IA I

RQ

Cohen_Ben-Gurion University

Summary- ADMET

3-D culture of C3A in alginate scaffolds induces spheroid formationSpheroids reveal high levels of hepatocellular functions (protein secretion, drug metabolism and detoxification)This construct is suitable for HTS initial drug screening (ADMET)

Cohen_Ben-Gurion University

Summary- Why Alginate Sponges?

•Ready to use for 3-D cell Culture•Algae-derived hydrogel•Optimized for specific cell types •Enhances cell activity •Degrades without changing local pH •Prolonged shelf life •Dimensionally stable in culture