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 morphologyLoss of morphology Cell membrane damageCell membrane damage Loss of viabilityLoss 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 PatientsDevelopment 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 expensiveEquipment is expensive Sorts large number of cellsSorts large number of cells Cell surface markersCell surface markers removed 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)Cells are then fixed or frozen (In Vitro)  Thin thermoplastic Film & Polyethylene- naphthalene membrane won’t protect cells from laser shock Needs to be thickerNeeds to be thicker

Why Use Pallet Arrays?  Pallets are Biocompatible  Allows for tissue culture media to be used Cells will not dehydrate Cells will not dehydrate  Statistics Show that cells will distribute one cell per pallet 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 Photoresist Similar to SU-8 Similar 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 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  to cells  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) Incubated at 37 °C (body temperature)  Fibronectin Binds to cell receptors Binds to cell receptors Also allows for cell adhesion Also allows for cell adhesion Incubated at 25 °C 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.  Edward Nelson, M.D.  Mark Bachman, Ph.D.  G.P. Li, Ph.D.  Said Shokair  Lily Wu  UROP  IM-SURE  Vu Phan  Richard Chang  Mark Merlo  Asheesh Divetia  Nolan Yoshimura  Li/Bachman Group

QUESTIONS????