Tissue Analogues and Disease Modeling Laboratory:
Capabilities for tissue modeling, simulated environments and gene expression profiling
Susana B. Zanello, Ph.D.
Universities Space Research Association
3D Tissue culture technologies
a) Spheroids: multicellular aggregates in liquid culture
hepatocytes
b) Cell aggregates in Matrigel-like subtrate
Chondrocytes derived from IV
discs, expressing abundant CS,
KS, Col I and Col II
(Gruber & Hanley, Musculoskelet Disord
2000; 1:1)
c) Stratified cell culture
Porcine corneal section
Reconstructed step-by
step human corneal
model
(Reichl et al, Br J Ophthalmol
2004)
d) Rotating Wall Vessel (RWV)
• Developed by NASA
•Based on clinorotation (ESA uses the Random Positioning machine), nullifies
gravity
•Minimal shear force
•Coaxial oxygenator rotates at same angular velocity
• Vessel and liquid medium rotate as
a solid body, resulting in
counterbalancing forces
•Rotational speed is adjusted to
aggregate size
•
•
•
•
•
Advantageous features of the
NASA bioreactor
Cells establish cell‐cell contacts (and cell‐
substrate contacts) critical for cell signaling
Spatial freedom to acquire organotypic 3D‐
structures, resembling native tissue
Mass transfer of nutrients and oxygen is limited by the static nature of some cultures, solved by the RWV.
Microgravity simulation
Long term cultures
Tissue culture assembly steps
ASSEMBLY of 3 – DIMENSIONAL TISSUE ASSEMBLY into
HIGH ASPECT ROTATING WALL VESSEL (STLV)
CELL/TISSUE
ALIQUOT
2 – D TISSUE CULTURE FLASKS
CELL CLONAL
EXPANSION
TRYPSINIZE
MICROCARRIER BEADS
CELL COUNT
106/ ml.
LOAD CELLS
and BEADS
RWV
MICROSCOPIC AND
GENETIC/PROTEOMIC
ANALYSIS
DETERMINE OPTIMAL
CELL DENSITY for RWV
CONICAL TUBE
with CELLS
3
Goodwin
Simulation of space environment
Microgravity
Other conditions
Examples:
Radiation
Δ pO2
Thermal stress
Nutritional stress
Analysis:
•Histology
•Proteomics (tissue or secreted
proteins)
•Gene expression
epithelium
Keratocytes
ECM layers
stroma
endothelium
Example: 3D- Corneal model
B
A
2
1
Microcarrier
bead
Vimentin (+) keratocytes
C
D
E
3
1
5
4
Chondrotin-4-sulfate
Chondrotin-6-sulfate (+)
cells
fibronectin
Goodwin, T.
Prototypic
Experimental
Design
Human corneal analog
Hypoxia/hyperoxia regimen
pO2
(mmHg)
Pumped O2
Pumped
O2
175
150
110
Reduced mix
2
8
10
Expression profiling
Gene expression
Proteomic analysis
Days
PROTEOMICS
Diagram of the current core proteomic technologies as well as the four key
applications they drive in advancing biology and medicine
Drake, T. A. et al. J. Lipid Res. 2007;48:1-8
Copyright ©2007 American Society for Biochemistry and Molecular Biology
2D GEL ELECTROPHORESIS
(+)
(-)
(-)
high
pI
M.W.
Individual
proteins
(+)
low
IEF strip
Protein extract
Second dimension separation
pH 3
pH 10
Normal cultured keratocytes
GRP-78
k1,
k2, k3
Cofilin-1
Cofilin-1
GRP-78
kc3
kc2
kc1
Vimentin cluster
keratoconus
Zanello et al, CER 2006
Comparison
Cut spot
In-gel
digestion
Peptides
Mass
spectrometry
The ProteinChip System
(Ciphergen, now commercialized by BioRad)
Comprised of:
• ProteinChip Arrays: capture proteins from mix due to different chromatographic chemistries
• ProteinChip Reader: laser desorption/ionization TOF mass spectrometer, calculates MW and mass.
• ProteinChip Software: controls the reader and collects and analyzes data
• Range of detection: small molecules to big proteins (1000 Da‐
500 kDa)
SELDI
ProteinChip
® Array
Technology
Reddy & Dalmasso, J Biomed Biotechnol. 2003
Reference intervals for 70 protein analytes in plasma
Anderson, N. L. (2002)
Mol. Cell. Proteomics 1: 845-867
Copyright ©2002 American Society for Biochemistry and Molecular Biology
LUMINEX TECHNOLOGY
•Wide range of multiplexed bioassays: able to detect
up to 100 analytes simultaneously
•Immunoassays, receptor-ligand interactions, enzyme
assays, nucleic acids
•Currently we do a “multiplexed ELISA in solution”
concentration (pg/ml)
MMP-13 levels in human chondrocytes
2000
1500
Control
1000
Treatment
500
0
0
10
20
29
time (days)
GENE EXPRESSION PROFILING
High‐throughput (HT) approaches
• “Next‐generation” sequencing includes multiple fast and efficient sequencing platforms with various applications • Study of the transcriptome is now possible with sensitivity down to a single molecule of mRNA
• Microarrays: Human Genome U133 Plus 2.0 Genechip, Affymetrix
qPCR validation(real time semi‐quantitative PCR)
PCR basics
Real time PCR
Real time PCR fluorescent chemistries
HP
RT
1
B2
M
β2
TG
F
TG
Fβ
1
IG
F1
IL8
IL6
bF
GF
bp
VE
GF
Cytokine RT-PCR products from human
chondrocyte 3D cultures
500
125
75
•* Notes:Each product shown in duplicate, from “control” and “treated” samples, respectively
• In green, housekeeping genes
VE-cad
L19
“Sharp” melting curves are critical for adequate quantitation using SYRBR green
Semi-quantitative (normalized) qPCR relative to control condition
Treatment
TaqMan primers/probes targeted Genes
+
Toll-like receptor pathways related
genes screened by PCR arrays
(SABioasciences)
Subtotal = approx 110 genes studied
+
Microarray analysis
Total = more than 1000 genes analyzed !!!
Is this too much information?
How can we use this information to build an organized
gene network and cellular pathway from expression data?
LITERATURE BASED
DATA MINING
EXPERIMENTAL
•Expression profiles
Bioinformatic tools
Elucidation of biological
pathways and control
mechanisms underlying a
biological response
•Known pathways
•Known gene networks
•Prot-prot interactions
•Prot-DNA interactions
Comparison of network analysis platforms
Cline et al, Nature Protocols 2007
Cellular assays
3D-HCH cultures
and conditions
DNAmicroarrays
Differentiallyexpressed genes
TaqMan and
PCRArrays
Luminex
MMP levels
Integrated Bioinformatics
Expression profiling+pathway/network mapping
GenMAPP
Pathways
Hypotheses
Candidates for validation
Acknowledgements
Tissue Analogues Laboratory
JSC Core Lab Facility
•Dr. Thomas Goodwin
•Dr. Dianne Hammond
•Maureen McCarthy
Bioinformatic Websites used:
• www.cytoscape.org
•www.genmapp.org
•www.wikipathways.org