THREE-DIMENSIONAL GROWTH AND FUNCTION OF NEURAL TISSUE IN DEGRADABLE POLYETHYLENE GLYCOL HYDROGELS - PowerPoint PPT Presentation

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

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... Testing platform for drug delivery * pre/post encapsulation ... Study: Culture both monolayer and 3-D hydrogel cultures Measure DNA, ATP content ... – PowerPoint PPT presentation

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Title: THREE-DIMENSIONAL GROWTH AND FUNCTION OF NEURAL TISSUE IN DEGRADABLE POLYETHYLENE GLYCOL HYDROGELS


1
THREE-DIMENSIONAL GROWTH AND FUNCTION OF NEURAL
TISSUE IN DEGRADABLE POLYETHYLENE GLYCOL 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 HWANG
2
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
3
POLYMER SCAFFOLDS
Grafting as disease treatment limited by graft
survival integration
? (viability) ? (retain functionality)
Cell line chosen for this study undifferentiated
(embryonic) murine neural precursor cells (NPC)
4
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
5
RATIONALE
NPCs merits - in vitro expansion before
transplantation ? unlimited NPC source - from
previous studies successful transplantation
into adult rats adequate chemical
microenvironment in adult CNS ECM
3D 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

6
MATERIALS AND METHODS BIG PICTURE
  • Overall Goals
  • 1) Construct gel ? determine degradation with
    mechanical tests
  • Incubate cells in gel
  • 2) Cell imaging
  • Stain for in gel viability
  • Stain for in gel bioactivity (monitor calcium
    level)
  • 3) Biochemical analysis after gel/cell lysed
  • DNA levels
  • ATP levels

Does hydrogel restrict cell growth? Does hydrogel
affect viability?
7
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

8
MATERIALS AND METHODS STAINING
Confocal Imaging Gels vibrotome sectioned
a) Stained for live/dead Calcein-AM Calcein-AM
membrane permeable Cleaved calcein
fluoresces, membrane impermeable Ethidium
bromide Fluoresces red after binding DNA
b) Stained for calcium Fluo-3 calcium
indicator GABA applied to cells Laser excited
Fluo-3 measures calcium (GABA response)
9
MATERIALS AND METHODS DNA, ATP
Obtaining cytosolic material Hydrogel
homogenized with lysis buffer
disrupts polymer gel
Biochemical Analysis
a) DNA content quantified with PicoGreen Assay
b) Protein content analyzed with Western Blot -
glial fibrillary acidic protein (GFAP) - beta
tubulin III
c) Immunocytochemical identification with
antibodies (directly on gel) - GFAP - beta
tubulin III - nestin - fibronectin -
synaptophysin
10
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 week Study 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
11
DOES HYDROGEL AFFECT VIABILITY?
12
DOES HYDROGEL AFFECT VIABILITY?
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
Questions How old is monolayer reference?
Day 16 monolayer ATP/DNA ratio?
13
CELL AGGREGATION IN HYDROGEL (DAYS lt12)
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
14
CELL AGGREGATION IN HYDROGEL
Mesh size increases 3x days 10-12 Gel completely
hydrolyzed day 16
15
TISSUE FORMATION (DAYS gt 12)
Plexus formation Days 10, 12, 14, 16
Temporal control achieved with different polymers
16
CELL / TISSUE MORPHOLOGY IN HYDROGEL
  • Initial growth is proliferation, not
  • migration, based hence clusters
  • First 12 days (Fig. 3b)
  • Processes start to form
  • Processes wrap around cluster core
  • (not penetrate hydrogel)
  • Core 17/-4 um thick
  • Days 13-14 (Fig. 3d)
  • Processes grow radially
  • Rapid hydrolysis of hydrogel
  • Mesh size increases 3x
  • Process length 52um into hydrogel

17
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
18
ECM IN HYDROGEL
During development Neurite receptors bind
ECM ECM provides traction force for neurite
extension
19
CELL DIFFERENTIATION IN HYDROGEL
- Neural precursors forms neurons or glia - Beta
tubulin III ? neurons - GFAP ? glia
Immunocytochemistry staining of hydrogel sections
revealed - Day 0 2.6106 cells 66 cells
beta tubulin III positive No GFAP - Day 16
7.3106 cells (3.5x increase) 35 cells beta
tubulin III positive (Fig. 4b) 38 cells GFAP
positive (Fig. 4a)
20
CELL DIFFERENTIATION IN HYDROGEL
- Neural precursors forms neurons or glia - Beta
tubulin III ? neurons - GFAP ? glia
Immunocytochemistry staining
Western Blot
21
CELL FUNCTION IN HYDROGEL
Method observe cellular response to GABA to
assay functionality 1) Fluo-3 tags calcium
within cells 2) GABA transmitter applied to
cells 3) Response ? cellular calcium influx ?
visible with Fluo-3
Observations Cells functional Fast and slow
response types
Did not mention proportion functional
Typical calcium response
22
SUMMARY
- Achieved temporal control over gel
degradation tissue formation
- NPC generates limited (but sufficient) ECM
- NPC proliferates AND differentiates
- 3-D scaffold not physical obstacle
- Proliferation/viability better than 2-D culture
23
CELL / TISSUE MORPHOLOGY IN HYDROGEL
Choice of graph? Does this mean at day 7 60
cells at 30um clusters and 80 cells at 45um
clusters? Or at day 16 20 cells at 20um
clusters and 80 cells at 70um clusters ?
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