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Multichannel DeviceThe device was initially prepared by inserting tubing (25-gauge) into the devices’ ports: one for the source, one for the drain. HEK-293 cells were cultured for several weeks. Themultichannel device inlet was connected to a 5 cc syringe containing fresh media

(DMEM, 10% FBS, 1% Penicillin-Streptomycin antibiotic, 1% glutamine) which was introduced into the device at a flow rate of 50uL/min using a syringe pump(Harvard PHD2000). After media exchange and trypsinization, cells were introduced into device inlet with fresh media at a flow rate of 5uL/hr. Flow was maintained until cells were seen trapped in the majority of trapping channels, at which point the device was placed in an incubator and connected to a syringe pump to perfuse with media at a rate of 5uL/hr. Images for initial cell trapping as well as those obtained 24 and 48 hours after trapping were taken using an Olympus BX41 microscope under 40x and 100x magnification.

Gradient DeviceBuffer solution was introduced into the culture chamber at a flow rate of 100uL/min. After filling, the inlet and outlet ports of the culture chamber were capped with plugs. Fluorescein at a concentration of 72uM was flowed into the source channel while a blank buffer solution (DPBS) was flowed into the sink channel at rates of .1uL/min, 1uL/min, 5uL/min, 10uL/min, and 20uL/min. The device was permitted to reach equilibrium in between changes in flow rate. This was done for both gradient designs.

Genetically identical cells within a given population can exhibit heritable genetic variation across several generations. When observing and studying these cells as an ensemble, as might be done in a cell culture flask, much of the generation-dependent variations are lost from view. Cell lineage is difficult to track when the cells are allowed to divide in a manner that gives rise to a chaotic geometric shape. To track variation in cells from generation-to-generation, we have designed a microfluidic device that makes it possible to track multiple cell lineages by trapping single cells and forcing them through spatial confinement to grow in linear chains for as many divisions as possible (Figure 1). This is of particular importance in regard to cancer stem cells, which exhibit asymmetric cell division, giving rise to stem cell renewal or differentiation into particular subtypes based on cell generation.

Cell differentiation can be initiated by exposure to signaling molecules and proteins such as Wnt3. In developing tissue, these initiators are introduced to cells in the form of concentration gradients. To study in vitro the effect of gradients of Wnt3 protein on cell fate, we have designed a microfluidicgradient generating device that minimizes shear stresses which could be detrimental to adherent cells. Consequently, a design that minimizes convective flow into the culture chamber was chosen. Cells were successfully loaded into the trapping array, and it appears that

several divisions resulted. This was the first time mammalian cells were cultured inside of this unique geometric confinement system. Future: To discriminate between genuine cell division and possible multiple loading events: time lapse imaging, implementation of dual inlet version of device, possible incorporation of valve system, use of colon cancer stem cells.Gradient device design with increased spacing between transverse channels yielded more reliable gradients.Future: Culture cells in culture chamber, replace fluorescein with Wnt3.

Motivation

Materials and Fabrication

Data

Discussion

Gradient Device

D. Weitz et. al., “Tracking lineages of single cells in lines using a microfluidic device,” PNAS, vol. 106, no. 43, pp. 18149–18154, Oct. 2009.G. Vunjak-Novakovic et. al., “Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of b-catenin signaling,” Lab Chip, vol. 10, no. 23, pp. 3277–3283, 2010.S. Heilshorn, et. al., “Endothelial cell polarization and chemotaxis in a microfluidic device,” Lab Chip, vol. 8, no. 8, pp. 1292–1299, 2008.M. Yang, et. al., “Generation of linear and non-linear concentration gradients along microfluidic channel by microtunnel controlled stepwise addition of sample solution,” Lab Chip, vol. 7, no. 10, pp. 1371–1373, 2007.

References

AcknowledgementsDr. Xiling Shen, ECE Department Cornell University, Ithaca NY 14853 Dr. Shivaun Archer, BME Department Cornell University, Ithaca N 14853

Multichannel Device

Procedure

Multichannel Device:CAD Design :

L-Edit softwareMaster Preparation:

Resist Application: P-20 3k rpm, 1k rpm/s, 30s; S1813 3k rpm, 1k rpm/s, 30sBake 60s @ 115C

Exposure: EV620 contact aligner, hard contact mode, 2.2s exposureDevelop: MIF720 120secDescum: Branson Asher 10minEtch: Unaxis 770 Bosch EtcherMetrology: Depth confirmed as 16um with stylus profilometerPost-processing: FOTS monolayer deposited using MVD100

Device:Dow Corning Sylgard-184 PDMS (10:1), bake 120min @ 80CBonded to glass slide using Harrick oxygen plasma chamber

Gradient Device:CAD Design :

L-Edit softwareMaster Preparation:

Resist Application: P-20 3k rpm, 1k rpm/s, 30s; S1827 3k rpm, 1k rpm/s, 30sBake 60s @ 115C

Exposure: EV620 contact aligner, hard contact mode, 5s exposureDevelop: MIF720 120sDescum: Branson Barrel Asher 10minEtch: Unaxis 770 Bosch EtcherMetrology: Depth confirmed as 50um with stylus profilometerPost-processing: FOTS monolayer deposited using MVD100

Device:Dow Corning Sylgard-184 PDMS (10:1), bake 120min @ 80C Bonded to glass slide using oxygen plasma chamber

Microfluidic Devices for Tracking Cell Lineage and DifferentiationEric Sharpsteen Onondaga Central School District, Nedrow NY 13120

Erik A. Zavrel Department of Biomedical Engineering, Cornell University, Ithaca NY 14853

Figure 1: Microfluidic device versus ensemble cell culture.

.1uL/min 1uL/min 5uL/min 10uL/min 20uL/min

Gradient Device

.1uL/min 1uL/min 5uL/min 10uL/min 20uL/min

Figure 5a

Figure 5b

Figure 5c

Figure 5d

Multichannel Device

Figure 2a

Figure 2b

Figure 2c

Figure 3a Figure 3b

Figure 3c

Figure 4c Figure 4b Figure 4a

Figure 2a: single inlet multichannel CAD layout.Figure 2b: dual inlet multichannel CAD layout. Figure 2c: dual inlet multichannel device cast in

PDMS captured by Nikon SMZ1000 Stereomicroscope.

Figure 3a: 50um transverse channel spacing gradient device CAD layout.

Figure 3b: 125um transverse channel spacing gradient device CAD layout.

Figure 3c: 50um transverse channel spacing gradient device cast in PDMS capturedby Nikon SMZ1000 Stereomicroscope.

Figure 4: 16um wide trapping channels

Figure 4a: 0 hour loading image captured by Olympus BX41 at 100x magnification. L/R 1, 1, 1 cell.

Figure 4b: 24 hour loading image captured by Olympus BX41 at 100x magnification. L/R 4, 1, 3 cells.

Figure 4c: 48 hour loading image captured by Olympus BX41 at 100x magnification. L/R 4, 1, 3 cells.

Figure 5a: series of 5 second exposure fluorescent gradient images of device with 50um spacing between transverse channels.

Figure 5b: series of 5 second exposure fluorescent gradient images of device with 125um spacing between transverse channels.

Figure 5c: Plot of normalized fluorescent intensity verses channel distance, data analysis performed with ImageJ software. Data obtained from device with 50um spacing between transverse channels.

Figure 5d: Plot of normalized fluorescent intensity verses channel distance, data analysis performed with ImageJ software. Data obtained from device with 125um spacing between transverse channels.