Transcript Microplatform for Single Cell Assay

Determining Parameters of Pallet
Fabrication and
ECM Protein Coatings that Result in
Maximal Cell Viability
Nicole V. Lona
Bakersfield College
Mark Bachman, Ph.D. & G.P. Li, Ph.D.
Departments of Biomedical Engineering, Electrical Engineering, and Computer Science
Edward L. Nelson, M.D.
Department of Molecular Biology and Biochemistry
Nicholas Gunn, M.S.
Department of Biomedical Engineering
BACKGROUND INFORMATION
 Cells
 Need for Pallet Arrays
 What are Pallet Arrays?
 Fabrication of Pallet Arrays
 PEG-diacrylate Walls
CELLS
There is a need for advancement in Cancer, Stem Cell, and DNA
research
 Positive Selection: selection and isolation of a specific cell or group
of cells from a mixed population
 Purpose is to seed specific cells for Homogeneous
Population
 95% of Cells are Adherent
 Sorting of Cells is more difficult when dealing with adherent
cells due to drawbacks of available technology
• Loss of morphology
• Cell membrane damage
• Loss of viability
 3T3 GM Neu Cells
 These cells come from HER-2/neu Transgenic Mice
• Development and Histology of their tumors are similar to
those seen in Breast Cancer Patients
3T3 Neu/GM Cells
Fluorescence-Activated Cell
Sorting (FACS)
 Drawbacks
• Equipment is
expensive
• Sorts large number of
cells
• Cell surface markers
removed
Limiting Dilution
 Drawbacks
 Time consuming: 4 to 6 weeks for adequate cell count
 Excessive labor
 Limitations to neighboring cells can cause loss of viability
Laser Microdissection (LM)
 Drawbacks
 IR and UV Lights are
affected by moisture
• Cells are then fixed or
frozen (In Vitro)
 Thin thermoplastic Film
& Polyethylenenaphthalene membrane
won’t protect cells from
laser shock
• Needs to be thicker
Why Use Pallet Arrays?
 Pallets are Biocompatible
 Allows for tissue culture media to be used
• Cells will not dehydrate
 Statistics Show that cells will distribute
one cell per pallet
 More pallets than cells
 Enables positive selection
 Removal Process is 60-90% Efficient
 A laser creates a cavitation bubble at base of
individual pallets, which removes them from glass
 More than 80% efficiency when cloning cells used in
this process
What are Pallet Arrays?
 They are pedestals
that are made at the
micro scale
 Composed of 1002F
• Photoresist
• Similar to SU-8
Micro Pallet Array Fabrication
1002-F
Glass Slide
1. Spin Coat
UV Radiation
Mask
Remove Mask
2. UV
Exposure
with Mask
3. PostExposure
Bake
4. Develop
PEG-diacrylate Hydrogel Walls
 PEG stands for Polyethylene Glycol
PEG polymers are hydrophilic
Biocompatible
Resistant to protein absorption
PEG Walls
The Big Picture
 Find a faster, more affordable, and effective way
of sorting cells
 Study homogeneous populations of specific cells
 Identify Stem Cells of Breast Cancer
Acquire patient-specific
treatments

Research Objectives
 Determine:
 Which ECM protein coating is best for the
viability of cells
Whether or not the ECM protein coatings
adhere to the PEG walls
Whether or not PEG and/or PEG
Photoinitiator is toxic to cells
What Are ECM Protein Coatings?
 Extracellular Matrix Protein
Coatings
Any material part of a tissue
that isn’t a cell
Allows adhesion of cells
Which ECM Protein Coating
Worked Best?
 ECM Coatings Used
 Matrigel
Protein mixture produced by the cells of rat tumors
Marketed by BD Biosciences
• Incubated at 37 °C (body temperature)
 Fibronectin
• Binds to cell receptors
• Also allows for cell adhesion
• Incubated at 25 °C
 In Experiments both worked well, but Matrigel
was better
Cells With Fibronectin-10x
Dilute Matrigel-20x
Cells With Matrigel-5x
Were ECM Protein Coatings
Adhering to PEG Walls?
 Some Cells were adhering to the PEG
walls
Coatings may have not been washed off
 Pallets were made of PEG
Two different wells and two different
results
Cells on PEG Pallets-20x
Cells Around PEG Pallets-20x
Are PEG & PEG PhotoinitiatorToxic
to Cells When Combined?
 PEG Photoinitiator is what helps the
crosslinking in pallet fabrication
 Studies have shown that some
Photoinitiators release toxins when
exposed to UV Light
 PEG & PEG Photoinitiator are toxic
together
50x
Is PEG Alone Toxic?
 Experiment with three different
concentrations of Liquid PEG
25uL, 50uL, and 150uL
 Cells did not look healthy
 Liquid PEG is toxic
50x
Is Photoinitiator Toxic to Cells?
 Some Chambers had Photoinitiator exposed to UV light
 Other Chambers had Photoinitiator that was not exposed
to UV Light
 Different concentrations were used
 25uL
 50uL
 150uL
 The concentrations had no effect on the viability of cells
and Photoinitiator alone was not toxic
20x
Conclusion
 Both Matrigel and Fibronectin worked great for the
viability of cells
 Matrigel is preferable
 ECM coatings were adhering to PEG walls
 Were not being washed off sufficiently
 PEG and PEG Photoinitiator (liquids) are toxic when
combined
 Liquid PEG is toxic
 Photoinitiator is not toxic
 Polymerized PEG can be toxic but most likely not
Acknowledgements
 Nicholas Gunn, M.S.
 Vu Phan
 Edward Nelson, M.D.
 Richard Chang
 Mark Bachman, Ph.D.  Mark Merlo
 G.P. Li, Ph.D.
 Asheesh Divetia
 Said Shokair
 Nolan Yoshimura
 Lily Wu
 Li/Bachman Group
 UROP
 IM-SURE
QUESTIONS????