Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting

Microstructures

Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting

Microstructures

Julius OattsEmory University – Neuroscience and Behavioral Biology

August 25, 2006

Dr. Mark Bachman, Department of Electrical Engineering and Computer Science

Dr. Nancy Allbritton & Dr. Chris Sims, Department of Physiology and Biophysics

Photo Courtesy: Yuli Wang

OutlineOutline

1) Project Overview – Long term goals

2) Micro Pallet Array Fabrication

3) Cell Growth on Pallets and Laser Release

4) Virtual Wall Discussion• Effect of silanization on cell growth

5) Fluorescent Protein Coating Stability

6) Density’s effect on cell growth

7) Acknowledgements

Project OverviewProject OverviewSeparate single cells from a heterogeneous population or isolate monoclonal colonies

Expand this technology to multiple cell lines

Mock Transfection: HeLa cells on 175um base, 50um high pallets

Select

Collect

Expand

Photos Courtesy:

Grace Young

Micro Pallet Array Fabrication

Micro Pallet Array Fabrication

1.SPIN COAT

2.UV EXPOSURE

3.POST-EXPOSURE

BAKE

4.DEVELOP

Glass slide

SU-8 coating

UV Radiation

Mask

Mask (removed)

Unwanted SU-8 dissolved

Patterned, cross-linked SU-8

Cell Growth and ReleaseCell Growth and Release

O O O O

NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2

1. Protein Attachment

2. Adherent cell attachment

SU-8 (epoxy groups)

Adherent cells

Protein-SU-8 bond

OH

NH

Cell Growth and Release

Surface Protein (amine groups)

Cell Growth and ReleaseCell Growth and Release

3. Laser Pallet Release System

4. Cell/Pallet Collection

2 µJ, 10 ns pulse

Inverted Collection Well

Formation of Cavitation Bubble

Laser Release System

Released Adherent Cell on Pallet

Collection Well

Force causes pallet lift-off

Collected Adherent Cell on Pallet

Unreleased pallets are not collected

Cell Growth and Release“Virtual Wall” Discussion“Virtual Wall” Discussion

1.Deposition of Hydrophobic Silane Layer on Glass between pallets

2. Adding aqueous solution containing surface coating proteins

Protein Coating: Selective to

Pallets3. Formation of “Virtual Air Wall”

Formation of Water Droplets

“Virtual Wall” Discussion“Virtual Wall” Discussion

Top view

Side view

Cell attached to pallet

Water Droplets on Silanized Glass

Dermal Fibroblasts on 175m base pallets

Effect of Silanization on CellsEffect of Silanization on Cells

A-172 Cell Growth Day 4: (15,000 cells/mL seeding density)

0

5

10

15

20

25

30

35

40

45

50

fibr. + SU-8 + silaniz. fibr. + glass + silaniz. fibr. + glass

Ave

r. #

Cel

ls/ 4

0X F

ield

Dermal Fibroblasts Cell Growth Day 4: (20,000 cells/mL Seeding Density)

0123456789

10

fibr. + SU-8 + silaniz. fibr. + glass + silaniz. fibr. + glass

Ave

r. #

Cel

ls/ 4

0X F

ield

Silanized SU-8

Silanized Glass

Glass (control)

Protein Coating StabilityProtein Coating Stability

40 60 80 100 120 1400

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

FITC-Collagen Coated Pallet Blank Pallet Autofluorescence

y = -0.02441x + 104.51627

y = .06668x + 266.44959

Fluo

resc

ence

Inte

nsity

Time (hours)

Average Stability of FITC-Collagen Coating on a Single Pallet

Protein Coating StabilityProtein Coating Stability

Inconsistency: HeLa cells growing on collagen coated 175 µm pallets

Same Pallet Array:

Cells attached well

Poor cell adhesion

We Don’t Know.

Cause and Effect?

Cell Density’s Effect on GrowthCell Density’s Effect on Growth

Calculating Doubling Time with a Growth Curve

y(t) = y(0) * 2ntNumber of cells at time = t

Initial number of cells

Cell Cycles/Hour

Time (hours)

Solve for “n” to find Doubling Time(Doubling time = hours to complete 1 cell cycle)

Cell Density’s Effect on GrowthCell Density’s Effect on Growth

Cell Density Doubling Time

1,000 82 hours

10,000 62 hours

A-172’s

0 20 40 60 80 100-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5y

(t) = 1.57478 * 2.01226t

Ave

. #

Ce

lls/F

ield

Time (hours)

1,000 cells/mL

4 hours 24 hours 47 hours 71 hours 95 hours

0 20 40 60 80 1000

5

10

15

20

25

30

35

40

45

50y(t) = 13.8503* 2.01622t

Ave

. # C

ells

/Fie

ld

Time (hours)

10,000 cells/mL

4 hours 24 hours 47 hours 71 hours 95 hours

Cell Density’s Effect on GrowthCell Density’s Effect on Growth

0 20 40 60 80 1000

1

2

3

4

5

6

7

8

9

10

y(t)

= 2.58219 * 2.01449t

1,000 cells/mL

Ave

. # C

ells

/Fie

ld

Time (hours)

5 hours 25 hours 48 hours 72 hours 96 hours

0 20 40 60 80 1000

10

20

30

40

50

60

70

80

90

10,000 cells/mL

y(t)

= 21.35886 * 2.01999tA

ve. # C

ells

/Fie

ld

Time (hours)

5 hours 25 hours 48 hours 72 hours 96 hours

Cell Density Doubling Time

1,000 69 hours

10,000 50 hours

Dermal Fibroblasts

AcknowledgementsAcknowledgements

Professors Mark Bachman, G.P. Li (project direction)

Professors Nancy Allbritton, Chris Sims (laboratory technique, project direction)

Grace Young (laboratory technique, tissue culture, mentoring)

Yuli Wang (mentoring, pallet fabrication)

University of California, Irvine and the Undergraduate Research Opportunities Program

National Institute of Health National Science Foundation