Determining Parameters of Pallet Fabrication and

ECM Protein Coatings that Result in Maximal Cell Viability

Nicole V. LonaNicole V. LonaBakersfield CollegeBakersfield College

Mark Bachman, Ph.D. & G.P. Li, Ph.D.Mark Bachman, Ph.D. & G.P. Li, Ph.D.Departments of Biomedical Engineering, Electrical Engineering, and Computer ScienceDepartments of Biomedical Engineering, Electrical Engineering, and Computer Science

Edward L. Nelson, M.D.Edward L. Nelson, M.D.Department of Molecular Biology and BiochemistryDepartment of Molecular Biology and Biochemistry

Nicholas Gunn, M.S.Nicholas Gunn, M.S.Department of Biomedical EngineeringDepartment of Biomedical Engineering

BACKGROUND INFORMATIONBACKGROUND INFORMATION

CellsCells

Need for Pallet ArraysNeed for Pallet Arrays

What are Pallet Arrays?What are Pallet Arrays?

Fabrication of Pallet ArraysFabrication of Pallet Arrays

PEG-diacrylate WallsPEG-diacrylate Walls

CELLSCELLSThere is a need for advancement in Cancer, Stem Cell, and DNA There is a need for advancement in Cancer, Stem Cell, and DNA

researchresearch Positive SelectionPositive Selection: selection and isolation of a specific cell or : selection and isolation of a specific cell or

group of cells from a mixed populationgroup of cells from a mixed population Purpose is to seed specific cells for Homogeneous Purpose is to seed specific cells for Homogeneous

PopulationPopulation

95% of Cells are Adherent95% of Cells are Adherent Sorting of Cells is more difficult when dealing with adherent Sorting of Cells is more difficult when dealing with adherent

cells due to drawbacks of available technologycells due to drawbacks of available technology• Loss of morphologyLoss of morphology• Cell membrane damageCell membrane damage• Loss of viabilityLoss of viability

3T3 GM Neu Cells3T3 GM Neu Cells These cells come from HER-2/neu Transgenic MiceThese cells come from HER-2/neu Transgenic Mice

• Development and Histology of their tumors are similar to Development and Histology of their tumors are similar to those seen in Breast Cancer Patientsthose seen in Breast Cancer Patients

3T3 Neu/GM Cells3T3 Neu/GM Cells

Fluorescence-Activated Cell Fluorescence-Activated Cell Sorting (FACS)Sorting (FACS)

DrawbacksDrawbacks

• Equipment is Equipment is expensiveexpensive

• Sorts large number of Sorts large number of cellscells

• Cell surface markers Cell surface markers removedremoved

Limiting DilutionLimiting Dilution DrawbacksDrawbacks

Time consuming: 4 to 6 weeks for adequate cell countTime consuming: 4 to 6 weeks for adequate cell count Excessive laborExcessive labor Limitations to neighboring cells can cause loss of viabilityLimitations to neighboring cells can cause loss of viability

Laser Microdissection (LM)Laser Microdissection (LM)

DrawbacksDrawbacks IR and UV Lights are IR and UV Lights are

affected by moistureaffected by moisture• Cells are then fixed or Cells are then fixed or

frozen (In Vitro)frozen (In Vitro)

Thin thermoplastic Film Thin thermoplastic Film & Polyethylene-& Polyethylene-naphthalene membrane naphthalene membrane won’t protect cells from won’t protect cells from laser shocklaser shock

• Needs to be thickerNeeds to be thicker

Why Use Pallet Arrays?Why Use Pallet Arrays? Pallets are BiocompatiblePallets are Biocompatible

Allows for tissue culture media to be usedAllows for tissue culture media to be used• Cells will not dehydrateCells will not dehydrate

Statistics Show that cells will distributeStatistics Show that cells will distribute

one cell per palletone cell per pallet More pallets than cellsMore pallets than cells Enables positive selectionEnables positive selection

Removal Process is 60-90% EfficientRemoval Process is 60-90% Efficient A laser creates a cavitation bubble at base of A laser creates a cavitation bubble at base of

individual pallets, which removes them from glassindividual pallets, which removes them from glass More than 80% efficiency when cloning cells used in More than 80% efficiency when cloning cells used in

this processthis process

What are Pallet Arrays?What are Pallet Arrays?

They are pedestals They are pedestals that are made at the that are made at the micro scalemicro scale

Composed of 1002F Composed of 1002F • PhotoresistPhotoresist• Similar to SU-8Similar to SU-8

1.Spin Coat

2.UV Exposure with Mask

3.Post-Exposure Bake

4.Develop

UV Radiation

Glass Slide

1002-F

Mask

Remove Mask

Micro Pallet Array Fabrication

PEG-diacrylate Hydrogel WallsPEG-diacrylate Hydrogel Walls

PEG stands for Polyethylene GlycolPEG stands for Polyethylene Glycol

PEG polymers are hydrophilic PEG polymers are hydrophilic

BiocompatibleBiocompatible

Resistant to protein absorptionResistant to protein absorption

PEG WallsPEG 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-specifictreatments

Research ObjectivesResearch Objectives

Determine: Which ECM protein coating is best for the

viability of cells

Whether or not the ECM protein coatings Whether or not the ECM protein coatings adhere to the PEG wallsadhere to the PEG walls

Whether or not PEG and/or PEG Photoinitiator is toxic to cells to cells

What Are ECM Protein Coatings?What Are ECM Protein Coatings?

Extracellular Matrix Protein Extracellular Matrix Protein CoatingsCoatings

Any material part of a tissue Any material part of a tissue that isn’t a cellthat isn’t a cell

Allows adhesion of cellsAllows adhesion of cells

Which ECM Protein Coating Which ECM Protein Coating Worked Best?Worked Best?

ECM Coatings UsedECM Coatings Used MatrigelMatrigel

Protein mixture produced by the cells of rat tumors Protein mixture produced by the cells of rat tumors

Marketed by BD BiosciencesMarketed by BD Biosciences• Incubated at 37 Incubated at 37 °C (body temperature)°C (body temperature)

FibronectinFibronectin• Binds to cell receptorsBinds to cell receptors• Also allows for cell adhesionAlso allows for cell adhesion• Incubated at 25 Incubated at 25 °C°C

In Experiments both worked well, but Matrigel In Experiments both worked well, but Matrigel was betterwas better

Cells With Fibronectin-10x

Dilute Matrigel-20x

Cells With Matrigel-5x

Were ECM Protein Coatings Were ECM Protein Coatings Adhering to PEG Walls?Adhering to PEG Walls?

Some Cells were adhering to the PEG Some Cells were adhering to the PEG wallswallsCoatings may have not been washed offCoatings may have not been washed off

Pallets were made of PEGPallets were made of PEGTwo different wells and two different Two different wells and two different

resultsresults

Cells on PEG Pallets-20x

Cells Around PEG Pallets-20x

Are PEG & PEG PhotoinitiatorToxic Are PEG & PEG PhotoinitiatorToxic to Cells When Combined?to Cells When Combined?

PEG Photoinitiator is what helps the PEG Photoinitiator is what helps the crosslinking in pallet fabricationcrosslinking in pallet fabrication

Studies have shown that some Studies have shown that some Photoinitiators release toxins when Photoinitiators release toxins when exposed to UV Lightexposed to UV Light

PEG & PEG Photoinitiator are toxic together

50x

Is PEG Alone Toxic?Is PEG Alone Toxic?

Experiment with three different Experiment with three different concentrations of Liquid PEGconcentrations of Liquid PEG25uL, 50uL, and 150uL25uL, 50uL, and 150uL

Cells did not look healthyCells did not look healthy

Liquid PEG is toxicLiquid PEG is toxic

50x50x

Is Photoinitiator Toxic to Cells?Is Photoinitiator Toxic to Cells?

Some Chambers had Photoinitiator exposed to UV lightSome Chambers had Photoinitiator exposed to UV light

Other Chambers had Photoinitiator that was not exposed Other Chambers had Photoinitiator that was not exposed to UV Lightto UV Light

Different concentrations were usedDifferent concentrations were used 25uL25uL 50uL50uL 150uL150uL

The concentrations had no effect on the viability of cells The concentrations had no effect on the viability of cells

and Photoinitiator alone was not toxicand Photoinitiator alone was not toxic

20x20x

ConclusionConclusion Both Matrigel and Fibronectin worked great for the Both Matrigel and Fibronectin worked great for the

viability of cellsviability of cells Matrigel is preferableMatrigel is preferable

ECM coatings were adhering to PEG wallsECM coatings were adhering to PEG walls Were not being washed off sufficiently Were not being washed off sufficiently

PEG and PEG Photoinitiator (liquids) are toxic when PEG and PEG Photoinitiator (liquids) are toxic when combinedcombined

Liquid PEG is toxicLiquid PEG is toxic

Photoinitiator Photoinitiator is not toxic is not toxic

Polymerized PEG can be toxic but most likely notPolymerized PEG can be toxic but most likely not

AcknowledgementsAcknowledgements

Nicholas Gunn, M.S.Nicholas Gunn, M.S. Edward Nelson, M.D.Edward Nelson, M.D. Mark Bachman, Ph.D.Mark Bachman, Ph.D. G.P. Li, Ph.D.G.P. Li, Ph.D. Said Shokair Said Shokair Lily WuLily Wu UROPUROP IM-SUREIM-SURE

Vu PhanVu Phan Richard ChangRichard Chang Mark MerloMark Merlo Asheesh DivetiaAsheesh Divetia Nolan YoshimuraNolan Yoshimura Li/Bachman GroupLi/Bachman Group

QUESTIONS????QUESTIONS????