ASCB-Alginate Sponge_SC 1206

8/10/2019 ASCB-Alginate Sponge_SC 1206

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Cohen_Ben-Gurion University

Alginate Sponges

For 3-D Cell Culture

Smadar Cohen & Tsiona ElkayamBen-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

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

efficient cell seeding and distribution

Biological Biocompatible with cells and the human body

Dissolution Mild conditions (EDTA, Citrate buffer)

Cell Recovery Easy, no cell damage,

maintaining surface cell markers (FACS analysis)

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


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Alginate Sponge90% Porosity; ~200mm Pore Size

200mm


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Cell Distribution within

Alginate Sponge (10mm thick)

Surface

Bottom

Center

Initial cell seeding density

(1x108cells/cm3)

H&E cross

sections

MTT

SEM


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3-D Culture in Alginate Sponge

Cell SourceCell DefinitionSourceCell Type

IsolationPrimary, AdultRatHepatocytes- Pure Culture

IsolationPrimary, AdultRatHepatocytes- Co-Culture with KupfferCells or Non-Parenchymal Cells

IsolationPrimary, NeonatalRatMixed hepatoblasts and oval (liverstem) cells

ATCC (CRL-10741)Cell line, clone derivative of HepG2HumanHepatocytes - C3A

Clonetics(CC-2543)

Primary, FrozenHumanEndothelial cellsdermal microvascular (HDMEC)

IsolationPrimary, FreshHumanEndothelial cellsumbilical vein (HUVEC)

Gift5Cell line (transfected with encodingSV-40 large T antigen)

HumanEndothelial cellsdermal microvascular transfected(HMEC-1)

IsolationPrimary, FrozenBovineEndothelial cellsbovine aortic (BAEC)

Clonetics (CC-2511)Primary, FrozenHumanDermal Fibroblast (NHDF)

Isolation

Primary

Rat

Cardiac Fibroblasts

IsolationPrimary, embryonicRatCardiac Myocytes

IsolationPrimary, neonatalRatCardiac Myocytes

Technion(H-9-Wisconsin lines, Israeli lines)HumanEmbryonic stem cells

Isolation based on plateadherence

Primary, Bone Marrow (BM)HumanMesenchymal stem cells(MSC), BM

Isolation based on plateadherence

Primary, Cord Blood (CB)HumanMSC , CB (CD133+ enriched)

Isolation

Primary, CB

Human

Hematopoeitic stem cellsHSC CB CD133+ selection


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3-D Cell Applications

with Alginate Sponges

Tissue engineering

Bioproductionof valuable therapeutics

ADMEToxresearch

Expansionof hematopoeitic andmesenchymal stem cells

Stem cell differentiation


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Tissue Engineering of Cardiac Muscle

Schwarzkopf et al, Tissue Engineering, 12 (2006)


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Organization of Primary Hepatocytes

as Spheroids within Alginate Sponges

FDA staining SEM TEM

Enhanced Hepatocellular Functions

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


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The sponge recapitulates the stem cellniche

3-D microenvironment

The scaffold can be madecell specific byattaching adhesion peptides(RGD, YIGSR)

The scaffold can be madea reservoirfor

growth factors and molecular agentsimportant for cell growth and differentiation

High cell density can be reachedin the

scaffold, without reaching confluence

Alginate Sponges for 3-D Stem Cells


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Release Rate

(ng/day-1)

KAbinding to

M-Alginate (M-1)

Growth

Factor

246.32*106VEGF

208.62*106bFGF

72.82*107aFGF

103.53*10

7

PDGF-


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Expansion and a better control over

differentiationof Human Embryonic

Stem (hES) Cells

Expansion of Hematopoietic stem

cells (HSC)

Expansion and induced differentiation

of Mesenchymal stem cells (MSC)

Stem Cell R&D

in Alginate Scaffold


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Human Embryonic Stem Cells (hESC)

Alginate sponge allows colonizationof

undifferentiated hESC within pores

Alginate sponge pore sizecontrols theaggregationof human embryoid

bodies (hEB)

The alginate sponge maximizes hEScell expansionand 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, smallerthan hEBs

formed in conventional Petri dishes

Spherical, homogenous in sizerelative

to those formed in Petri dishes

NO CENTRAL NECROSISin hEBs

formed in alginate sponges


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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/ml)

Static

Scaffolds

Petri Dish

Alginate Sponge

Seeding: 0.1-1 x105/scaffold


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Differentiation within Alginate Borne-hEBs

into 3 Germ Layers

ECTODERM

MESODERM

ENDODERM

H&E

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


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Case Story:

Alginate vs. Collagen Scaffolds for ADMET

Collagen Alginate


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C3A- Clone Derivative of HepG2


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Cohen_Ben-Gurion UniversityCollagen Alginate

C3A Cell Morphology in

Alginate vs. Collagen Scaffolds

3-D Spheroids2-D Adherent Cells


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Laminin Expression in C3A seeded in

Alginate vs. Collagen Scaffolds

Collagen Alginate


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C3A Spheroids Express Albumin& CK18


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6b-testosterone (IU)Treatment

1.41x 106

Cells only

302 x 106Cells + Inducer

5.32

x 105

Cells + Inhibitor

6.0 x 105

Cells + Inducer +

Inhibitor

CYP3A4 Metabolism in C3A Spheroids


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nM Resorufin produced/

106cells/1h

Treatment

C3A spheroidsC3A cellmonolayer

2 0.70Cells only

80 20302Cells + Inducer

0.60.60Cells + Inhibitor

40 10102

Cells + Inducer +

Inhibitor

CYP1A2 Metabolism in 2D vs. 3D Cultures


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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

R

Q


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Summary- ADMET

3-D culture of C3A in alginatescaffolds induces spheroidformation

Spheroids reveal high levels ofhepatocellular functions(proteinsecretion, drug metabolism anddetoxification)

This construct is suitable for HTSinitial drug screening (ADMET)


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Summary- Why Alginate Sponges?

Ready to use for 3-D cell Culture

Algae-derived hydrogelOptimized for specific cell types

Enhances cell activity

Degrades without changing local pHProlonged shelf life

Dimensionally stable in culture

Documents

ASCB-Alginate Sponge_SC 1206