THREE-DIMENSIONAL GROWTH AND FUNCTION OF THREE-DIMENSIONAL GROWTH AND FUNCTION OF NEURAL TISSUE IN DEGRADABLE POLYETHYLENE NEURAL TISSUE IN DEGRADABLE POLYETHYLENE

GLYCOL HYDROGELSGLYCOL HYDROGELS

M.J. Mahoney, K.S. AnsethDepartment of Chemical and Biological Engineering and the Howard Hughes Medical Institute

University of Colorado,Campus Box 424, Boulder, CO 80309, USA

MARK HWANGMARK HWANG

POLYMER SCAFFOLDS - BACKGROUND

Uses:- Model tissue ECM environment in vitro- Observation platform for:

* cell-cell interaction* cell-ECM interaction

- Testing platform for drug delivery* pre/post –encapsulation

- Tissue replacement/grafting therapy

Key Factors:- Mesh size- Scaffold chemistry

POLYMER SCAFFOLDS

Grafting as disease treatment limited by:graft survivalintegration

(viability) (retain functionality)

Cell line chosen for this study:undifferentiated (embryonic) murine neural precursor cells

(NPC)

Graft research focus for CNS diseaseScaffolds support neural cell - growth- differentiation- function

- CENTRAL NERVOUS SYSTEM

STUDY GOALS

Assess effect of 1) degradable hydrogel

on

2) neural precursor cell (NPC) viability +

3) NPC differentiation +

4) 3D tissue morphology +

given

5) mesh size that changes with time

RATIONALE

NPCs merits- in vitro expansion before transplantation unlimited NPC

source- from previous studies

successful transplantation into adult ratsadequate chemical microenvironment in adult

CNS ECM3D v. 2D scaffold- directly implant 3D scaffold- cells must be dislodged from 2D substrate

shear forces

PEG hydrogel- non-immunogenic- tolerated in CNS- degradable- protein scaffolds (e.g. collagen) hard to control

MATERIALS AND METHODS: BIG PICTURE

Overall Goals1) Construct gel determine degradation with mechanical

tests

Incubate cells in gel

2) Cell imagingStain for in gel viabilityStain for in gel bioactivity (monitor calcium level)

3) Biochemical analysis after gel/cell lysedDNA levelsATP levels

Does hydrogel restrict cell growth?Does hydrogel affect viability?

MATERIALS AND METHODS: CELLS and GEL

Cell Culture-Embryonic forebrain removed + digested (rat)-Single cells cultured on:

1) poly ornithine coated cover slips + media (control)2) 3-D hydrogel construct

Hydrogel Preparation-Components

PEG-Macromer (10wt%)cells + mediaphotoinitiator (0.05wt%)

-Exposed to UV light 10 min

-Gel massed when dry/wet, calculate compressive modulus over time(degradation rate)

MATERIALS AND METHODS: STAINING

Confocal Imaging:

Gels vibrotome sectioned

a) Stained for live/deadCalcein-AM

Calcein-AM = membrane permeableCleaved calcein fluoresces, membrane impermeable

Ethidium bromideFluoresces red after binding DNA

b) Stained for calciumFluo-3 = calcium indicator

GABA applied to cellsLaser excited Fluo-3 measures calcium (GABA response)

FUNCTION

VIABILITY

MATERIALS AND METHODS: DNA, ATP

Biochemical Analysis

a) DNA content quantified with PicoGreen Assay

c) Immunocytochemical identification with antibodies (directly on gel)- GFAP- beta tubulin III- nestin- fibronectin- synaptophysin

Obtaining cytosolic materialHydrogel homogenized with lysis buffer

disrupts polymer gel

b) Protein content analyzed with Western Blot- glial fibrillary acidic protein (GFAP)- beta tubulin III

DOES HYDROGEL RESTRICT CELL GROWTH?

Differences between monolayer (plate) and hydrogel cultures

- 2-D v 3-D access to nutrients- physical obstruction in 3-D gel

Is the hydrogel a physical barrier to growth?Are nutrients directed toward replication or creating space?

1 weekStudy: Culture both monolayer and 3-D hydrogel cultures

Measure DNA content (reflects population size)

Observations: No statistical difference in DNA content between both culture types (with p-value 0.05)

Conclusion: Cells proliferate in early hydrogel equally well

DOES HYDROGEL AFFECT VIABILITY?

Study: Culture both monolayer and 3-D hydrogel culturesMeasure DNA, ATP content (reflects population size)

Observations:

Conclusion: Hydrogel viability significantly greater as measured by DNA content

MONOLAYER (24h)

1486 ng ATP7386 pg DNA

143 pg ATP / pg DNA

DEAD CELLS

1 pg ATP8230 pg DNA

6 pg ATP / pg DNA

HYDROGEL (24h)

190 pg ATP / pg DNA

DOES HYDROGEL AFFECT VIABILITY?

Questions: How old is monolayer reference? Day 16 monolayer ATP/DNA ratio?

Column 1: healthy monolayer reference

201 pg ATP / pg DNA

Column 2: dead cells

Column 3: monolayer culture (24h) 143 pg ATP/ pg DNA

Column 4: hydrogel culture (24h) 190 pg ATP/ pg DNA

Column 5: hydrogel culture (16d) 215 pg ATP / pg DNA

CELL AGGREGATION IN HYDROGEL (DAYS <12)

Day 0: single cells distributed uniformly throughout gel

Day 3: single cells and cell clusters (~ 20 um)

Day 7: cell clusters (~ 30 um) Actively dividing

CELL AGGREGATION IN HYDROGEL

Mesh size increases 3x days 10-12Gel completely hydrolyzed day 16

TISSUE FORMATION (DAYS > 12)

Plexus formation:Days 10, 12, 14, 16

Temporal control achieved with different polymers

CELL / TISSUE MORPHOLOGY IN HYDROGEL

- Initial growth is proliferation, not migration, based ~ hence clusters

- First 12 days (Fig. 3b)Processes start to formProcesses wrap around cluster

core(not penetrate hydrogel)

Core ~ 17+/-4 um thick

- Days 13-14 (Fig. 3d)Processes grow radiallyRapid hydrolysis of hydrogelMesh size increases 3xProcess length ~52um into

hydrogel

CELL / TISSUE MORPHOLOGY IN HYDROGEL

Fig. 1b- Mesh size inversely proportional to modulus

- Processes penetrate hydrogel at 2 wks- PEG 2.5 glycolide decreases time to 1 wk- PEG 2 lactide increases time to 3 wks

Possible to achieve temporal control of tissue growth in 3-D

ECM IN HYDROGEL

During development:Neurite receptors bind ECMECM provides traction force for neurite extension

Normal Neurite ECM

YYY

Hydrogel ECMNYN

ECM molecules- Laminin- Fibronectin- Collagen IV

CELL DIFFERENTIATION IN HYDROGEL

- Neural precursors forms neurons or glia- Beta tubulin III neurons- GFAP glia

Immunocytochemistry staining of hydrogel sections revealed:

- Day 0 ~ 2.6*10^6 cells66% cells beta tubulin III positiveNo GFAP

- Day 16 ~ 7.3*10^6 cells (3.5x increase)35% cells beta tubulin III positive (Fig. 4b)38% cells GFAP positive (Fig. 4a)

CELL DIFFERENTIATION IN HYDROGEL

- Neural precursors forms neurons or glia- Beta tubulin III neurons- GFAP glia

Immunocytochemistry staining Western Blot

CELL FUNCTION IN HYDROGEL

Method: observe cellular response to GABA to assay functionality

1) Fluo-3 tags calcium within cells2) GABA transmitter applied to cells3) Response cellular calcium influx visible with

Fluo-3Observations:Cells functionalFast and slow response

types

Typical calcium response

Did not mention proportion functional

SUMMARY

- Achieved temporal control over gel degradationtissue formation

- NPC generates limited (but sufficient) ECM

- NPC proliferates AND differentiates

- 3-D scaffold not physical obstacle

- Proliferation/viability better than 2-D culture

CELL / TISSUE MORPHOLOGY IN HYDROGEL

Choice of graph?

Does this mean at day 7:~ 60% cells at 30um clustersand~ 80% cells at 45um clusters?

Or at day 16:~ 20% cells at 20um clustersand~ 80% cells at 70um clusters

?