Alginate Sponges For 3-D Cell Culture

Smadar Cohen & Tsiona Elkayam Ben-Gurion University of the Negev

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Alginate Sponge Characteristics

Physical Highly porous (>90%); Homogenous pore size (100-200mm) and shape; Interconnecting pores Transparent; Stable Polysaccharide (algae-derived) hydrogel, easilywetted by culture medium, allowing rapid and efficient cell seeding and distribution Biocompatible with cells and the human body
Mild conditions (EDTA, Citrate buffer)

Chemical
Biological Dissolution

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

Shelf Life

Cohen_Ben-Gurion University >12 months (room temperature, low humidity)

Alginate Sponge
90% Porosity; ~200mm Pore Size

200mm

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Cell Distribution within Alginate Sponge (10mm thick)

H&E cross sections
Surface

Initial cell seeding density (1x108 cells/cm3) MTT

Center

SEM
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Bottom

3-D Culture in Alginate Sponge

Cell Type Hepatocytes- Pure Culture Hepatocytes- Co-Culture with Kupffer Cells or Non-Parenchymal Cells Mixed hepatoblasts and oval (liver stem) cells Hepatocytes - C3A Endothelial cells dermal microvascular (HDMEC) Endothelial cells umbilical vein (HUVEC) Endothelial cells dermal microvascular transfected (HMEC-1) Endothelial cells bovine aortic (BAEC) Dermal Fibroblast (NHDF) Cardiac Fibroblasts Cardiac Myocytes Cardiac Myocytes Embryonic stem cells Mesenchymal stem cells (MSC), BM MSC , CB (CD133+ enriched) Hematopoeitic stem cells (HSC) , CB, CD133+ selection Source Rat Rat Rat Human Human Human Human Cell Definition Primary, Adult Primary, Adult Primary, Neonatal Cell line, clone derivative of HepG2 Primary, Frozen Primary, Fresh Cell line (transfected with encoding SV-40 large T antigen) Primary, Frozen Primary, Frozen Primary Primary, embryonic Primary, neonatal (H-9-Wisconsin lines, Israeli lines) Primary, Bone Marrow (BM) Primary, Cord Blood (CB) Cell Source Isolation Isolation Isolation ATCC (CRL-10741) Clonetics (CC-2543) Isolation Gift5

Bovine Human Rat Rat Rat Human Human Human

Isolation Clonetics (CC-2511) Isolation Isolation Isolation Technion Isolation based on plate adherence Isolation based on plate adherence Isolation

Human Primary, CB University Cohen_Ben-Gurion

3-D Cell Applications with Alginate Sponges

Tissue engineering Bioproduction of valuable therapeutics ADMETox research Expansion of hematopoeitic and mesenchymal stem cells Stem cell differentiation
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Tissue Engineering of Cardiac Muscle

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Schwarzkopf et al, Tissue Engineering, 12 (2006)

Organization of Primary Hepatocytes as Spheroids within Alginate Sponges

Enhanced Hepatocellular Functions

FDA staining

SEM

TEM

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

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Alginate Sponges for 3-D Stem Cells

The sponge recapitulates the stem cell niche 3-D microenvironment The scaffold can be made cell specific by attaching adhesion peptides (RGD, YIGSR) The scaffold can be made a reservoir for growth factors and molecular agents important for cell growth and differentiation High cell density can be reached in the scaffold, without reaching confluence
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Growth Factor VEGF bFGF aFGF PDGF-

KA binding to M-Alginate (M-1) 6.32*106 8.62*106 2.82*107 3.53*107

Release Rate (ng/day-1) 24 20 7

10 Cohen_Ben-Gurion University

Stem Cell R&D in Alginate Scaffold

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

Alginate sponge allows colonization of undifferentiated hESC within pores Alginate 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
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Morphology of hEBs

Scaffold-borne hEBs Homogenous and Small Size No Central Necrosis

Petri-Dish Extensive Aggregation & Central Necrosis

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

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Key Features of Sponge-borne hEBs

hEBs ranging between 250-900 mm after 1 month, smaller than hEBs formed in conventional Petri dishes Spherical, homogenous in size relative to those formed in Petri dishes NO CENTRAL NECROSIS in hEBs formed in alginate sponges
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Expansion of Human Embryonic Stem Cells (hESC) in Alginate Sponges

Seeding: 0.1-1 x105/scaffold
XTT
25
Cell (10<6>/ml)

Alginate Sponge

20 15 10 5 0 0 5 10 15 Day 20 25 30 Static Scaffolds

Petri Dish

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

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Differentiation within Alginate Borne-hEBs into 3 Germ Layers

H&E

ECTODERM

ENDODERM

MESODERM

Hepatocyte-like cells (a-FP+)

Neuronal cells (nestin+)

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Case Story: Alginate vs. Collagen Scaffolds for ADMET

Collagen
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Alginate

C3A- Clone Derivative of HepG2

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C3A Cell Morphology in Alginate vs. Collagen Scaffolds

2-D Adherent Cells 3-D Spheroids

Collagen

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Alginate

Laminin Expression in C3A seeded in Alginate vs. Collagen Scaffolds

Collagen
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Alginate

C3A Spheroids Express Albumin& CK18

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CYP3A4 Metabolism in C3A Spheroids

Treatment 6b-testosterone (IU) 1.41 x 106

Cells only Cells + Inducer

302 x 106
Cells + Inhibitor Cells + Inducer + Inhibitor 5.32 x 105

6.0 x 105

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CYP1A2 Metabolism in 2D vs. 3D Cultures

Treatment

nM Resorufin produced/ 106cells/1h C3A cell C3A spheroids monolayer

0 302 2 0.7 80 20

Cells only Cells + Inducer Cells + Inhibitor Cells + Inducer + Inhibitor

0
102
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0.60.6
40 10

1A2 mRNA levels in C3A seeded in Alginate vs. Collagen Scaffolds

CYP1A2 Induction Alginate scaffold Vs. Collagen Scaffold
140 120 100

60 40 20 0 AI
Cohen_Ben-Gurion University CI A N.I

C N.I

RQ

80

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