A Microfluidic Platform for Human Epidermal Keratinocyte Cytotoxicity Assays
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2009-04-23
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Abstract
Linear dilution is a method to create linearly varying concentrations of a solution. Linear dilutions are commonly used in biological studies where the threshold concentration at which a physiological reaction occurs is unknown, whether it be a minimum effective or a maximum tolerable dosage. In this dissertation we present a summary of the approaches used for creating dilutions with microfluidics followed by a detailed methodology for constructing a proportional mixing linear dilution microfluidic device. The microfluidic device presented here is made with a rapid and inexpensive microfabrication method, soft lithography. The device is capable of generating nine linearly varying dilution values with an R-squared value exceeding 0.999 and the linearity of the dilutions is independent of the input flowrate, making it a very robust approach to creating linear dilutions. With the model presented the device can be expanded to an arbitrary number of dilutions with commensurate savings in reagent usage and time.
Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body’s defensive barrier and are used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0% - 99.6% viability at 72 h under medium perfusion ranging from 0.025 – 0.4 microliters per minute. HEK maintained this viability while ~100% confluent a level not possible in 96 well plates. Microscale HEK cultures offer the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new discoveries in toxicological screening methods and wound healing techniques.
A microfabricated cell curtain is presented that facilitates cellular assays. The cell curtain is defined as a poly(dimethylsiloxane) (PDMS) wall that extends from the ceiling of a cell culture microchamber to within microns of the chamber floor. Curtain use is demonstrated by observing monolayer human epidermal keratinocyte (HEK) colonies for 48 hrs longer than possible with non-curtained microfluidic chambers. The curtains were further characterized by integrating them into a 96-chamber high-throughput microfluidic cell culture device. As proof of concept, this device was used to assay a range of ethanol dilutions spanning 0 – 22% in cell culture medium. Cells exposed to 12% ethanol or less for 30 min would recover to 85% viability at 24 hr, while cells exposed to higher concentrations had viabilities below 10%. The data also showed that cells exposed to 6% ethanol or less grew in population size, 8% ethanol exposure stunted growth, and higher concentrations led to population loss. Curtain use permitted high initial cell seeding densities and increased the amount of time cells can be cultured compared to multi-well plates.
It has been established that dermal exposure to jet fuel and its aromatic / aliphatic hydrocarbon components cause cytotoxicity. Two markers of this are cell viability and cytokine release. Measuring cell viability detects failure of some mechanism in the cell indicating eventual death. Cytokine release from HEK is an indicator of inflammation. Specifically, the cytokine interleukin-8 (IL-8) is released by the cell and attracts neutrophils, lymphocytes, T-cells and basophils, all part of an immune response. Significant work has been done to establish the HEK in vitro cell culture model and protocols to predict cytotoxicity. However, these can be expensive and time consuming. In this research we design a microfluidic device capable of simplifying the procedures for detecting those two forms of cytotoxicity, viability and IL-8 release. This device has been used to determine a highest non-toxic dose (HNTD) value and 50% lethality dose (LD-50) of the aromatic hydrocarbon cyclohexyl benzene. Results indicate that the HNTD value is 0.01% CHB and the LD-50 value is greater than 10% CHB in cell culture medium. The device has also been used to show that microfluidic HEK cultures can generate enough cytokines to be detected using a Bio-Plex commercial assay. These two capabilities, combined with a linear or serial dilution microfluidic device, constitute a high throughput device capable of reducing the time, labor, reagent and tissue use over conventional methods.
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keratinocyte, cytotoxicity, dilution, microfluidics, PDMS
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Degree
PhD
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Biomedical Engineering
