Work in Progress : An Educational Kit for Introducing Microfluidics-Based Cell Adhesion Assay in Undergraduate Laboratory

The study of the adhesion behavior of cells is an active area of academic research and as such, is an increasingly important component of biomedical engineering education. However, in delivering engineering courses, it is often challenging to provide laboratory experience of cellbased adhesion assays to undergraduate students, as the lab work involved is expensive, delicate, and usually requires substantial experimental skill. This article reports the development of a novel lab experience for undergraduate students for which a self-contained microfluidic assay kit was designed to deliver controlled shear stress of fluid flow for detaching adherent cells inside microchannels. Cell adhesion strength is measured by the fraction of cells that remain adhered after the application of a defined shear stress for a fixed duration. Each prepackaged kit consists of one micro-chip containing microchannels and several cartridges containing all of the reagents including cells for each step of the assay. The kit incorporates a simple surface tension-based fluid handling mechanism that enables exchange of fluids between the micro-chip and the cartridges without any handheld pipettes or external equipment. Instructional material was also developed for the kit. In addition to a detailed procedure for the experiment and suggested observation based discussion questions, the kit includes an introduction to microfluidic technology, basic fluidic dynamics concepts, and cell adhesion biology. The instructional material is suitable for junior and senior level undergraduate students in biomedical engineering or any closely related discipline. The relative simplicity and affordability of the kit made it accessible to undergraduate students in a laboratory course, who judged the lab as a strongly positive learning experience. Cell-based assays play an important role in modern biology and biomedicine with applications in clinical diagnostics, drug development, and cell-biology research. The experience of carrying out cell-based assay in an undergraduate laboratory will help students build up the ability to make measurements on and interpret data from living systems, a critical outcome required for undergraduate programs in bioengineering or biomedical engineering in order to receive accreditation from the Accreditation Board for Engineering and Technology (ABET). However, in delivering engineering courses, it is often challenging to provide laboratory experience of cell-based assays to undergraduate students, as the lab work involved is expensive, delicate, and usually requires substantial experimental skills. We report the development of a microfluidic based assay kit to facilitate undergraduate laboratory experience of live cell measurements. It also serves as a tool to introduce microfluidic technology, a driving force in the current trend of miniaturization of analytical instrumentation. The educational kit allows students to observe and analyze the change of adhesion behavior of live cells on the channel surface under biochemical digestion. Adhesive interactions between cells and their physical environments are central in developmental biology, tissue maintenance, tissue engineering, cancer progression, and many biotechnological processes. Cell adhesion assay with a microfluidic device allows students to demonstrate their ability to address problems associated with the interaction between living and non-living materials and systems, another critical outcome required by ABET for undergraduate programs in bioengineering or biomedical engineering. The kit is a new application of the previously published microfluidic-assay platform called ‘‘Kit-On-A-Lid-Assay’’ (KOALA) in collaboration with the original inventors. As shown in Figure 1, each KOALA set consists of one KOALA micro-chip (containing 12 microchannels, called the lid) and several KOALA cartridges (called the kit). The cartridges, containing all of the reagents for each step of the assay, can be stored for extended periods of time at temperatures as low as -80° C. Each step of the assay is performed in two simple operations. First, the cartridge is prepared by quickly thawing (less than 1 min is required given the microliter volumes) and removing a protective seal. Second, the reagents from the cartridge are delivered by “clicking” it onto the micro-chip. This fluid exchange takes about 5-30 seconds, after which the cartridge can be disposed of and a new cartridge applied for each of the following steps. The use of surface tension-based methods to drive fluid exchanges in the KOALA platform permits a high level of precision without electronic actuation or external equipment. The source of cells can be from the pre-packaged cells frozen in cryopreservation fluid in a special cryopreservation KOALA cartridge, or a freshly prepared cell suspension cultured in conventional cell culture facility. Using a series of cartridges, a whole cell-based assay can be pre-loaded into the KOALA set. To apply the KOALA platform to cell adhesion assays, we designed a series of micro-channels with different channel depths to deliver different levels of shear flow stress in order to detach the adherent cells inside the micro-channel (Figure 2, right y-axis). Microscope images are used to analyze cell adhesion strength by calculating the percentage of cells that remain adhered after the application of a defined shear flow stress for a fixed duration. Practical applications of cell adhesion assays often involve assessing the effect of certain treatment (e.g., exposure to chemicals) on the ability of cells to adhere. In designing the kit, we chose to use trypsin as the biochemical treatment. Trypsin is an enzyme that weakens cell adhesion by cleaving peptides chains of the cell adhesion molecules. Students can study the change of adhesion strength of cells as a function of trypsin treatment time and temperature. This gives an opportunity for students to design experiments and build data interpretation skills from inquirybased learning. Figure 1. (A) Operation of the KOALA is as simple as pressing a cartridge onto a micro-chip. (B) Each kit consists of several cartridges prefilled with reagents, and one micro-chip containing the microchannels. Each step of the assay is completed by “clicking” one cartridge onto the micro-chip. The cells are cultured and imaged in the micro-chip. (C) An assay performed in KOALA demonstrating immunostaining capabilities (Stain 1 – Anti-tubulin (tubulin stain) / Stain 2 – DAPI (nuclear stain) / Stain 3 – Phalloidin (actin stain)) Figure 2 Experimental results (left y-axis) show that remaining cell percentage is depended on the channel depths of the fluid flow. Dashed line is data from 6min TE treatment and dashdotted line is from 3min TE treatment. Decreasing channel depth increases the shear stress of fluid flow (solid line, right y-axis) for detaching cells. As shown in Figure 2 (right y-axis), the shear stress due to fluid flow inside the microchannels increases from 160 dyn/cm to 2700/cm as channel depth decreases from 0.8mm to 0.2mm according to the Poiseuille model. We carried out experiments using mouse epithelial cell line NMμMG and 0.25% Trypsin-EDTA (TE) solution. Results in Figure 2 (left y-axis) show that the high flow shear stress in the channel with 0.2mm depth almost completely washed 0 50