Project Page - S Lee

PAPER TITLE:

High-throughput Screening (HTS) for Detection of Extracellular Response by Apoptosis-induced Drugs

A) BACKGROUND:

One of the areas of great interest in biological experimentation is the screening of cells to various types of microenvironmental stimuli such as combinations of matrix molecules or chemicals from a compound library. However these studies are often costly since they require the use of expensive culture reagents and samples. We hypothesize that the ideal method of performing HTS of cells should have the following properties: 1) Require minimal expertise; 2) Be cheap; 3) Be applicable to engineer various aspects of the cellular microenvironment; 4) Be easily scalable and commercializable such that it can be widely produced and distributed to the members of biology and medical communities; and 5) Minimize the time required to generate a quantifiable read-out. Here we demonstrate a platform technology that aims to achieve these properties by using a combination of microscale technologies and controlled release biomaterials.

High-throughput screening (HTS) has revolutionized the process of drug discovery and optimization of cell-microenvironmental stimuli. Typically HTS studies are performed through a combination of modern robotics, data processing and control software, liquid handling devices, and sensitive detectors. However, many high-throughput approaches currently in practice require expensive equipment, large sample volumes, long incubation times and/or extensive expertise. Thus, automation and miniaturization make it possible to screen hundreds of thousands of compounds against disease targets to find novel drugs. Microscale technologies for HTS also provide powerful tools to fabricate devices for addressing many of the challenges in drug discovery and tissue engineering. This is partially due to the emergence of techniques such as soft lithography to fabricate microscale devices without the use of expensive ‘clean rooms’ and photolithographic equipment. Soft lithography is a set of microfabrication techniques that uses elastomeric stamps, fabricated from patterned silicon wafers, to print or mold materials at resolutions as low as several hundred nanometers. Therefore, soft lithography can be used to control the topography and spatial distribution of molecules on a surface, as well as the subsequent deposition of cells.

B) HYPOTHESIS

We will develope HTS devices to control the location of adherent and non-adherent cells by selectively docking the cells within low shear stress regions created in microwells. These systems were used to perform high-throughput experimentation involving multiple cell types using multiple reagents. Especially, cell patterning can be used to precisely control the presentation of extracellular factors in the microenvironment to study cell fate and function. Extracellular factors include soluble factors, physical stimuli and insoluble factors, such as ECM. For soluble factors, such as drug candidates, the number may exceed millions while their quantity can be limited. In this case the miniaturization of the assay is necessary because of the high production price and the small scale they are prepared. To address these challenges, new methods have been developed by using pin tools, piezo tips, ultrasounds and microarray technology for nanolitre liquid handling. However, despite significant progress, major problems such as clogging, bubbling, and evaporation still exist in nanolitre liquid handling processes, especially when cells were involved. Furthermore, these approaches can be used to study the response of biomaterials and extracellular matrix (ECM) components against cells in a rapid manner.

C) SPECIFIC AIMS

We will develope a cell microarray sealed by a chemical libraries microarraied glass slide for high-throughput apoptosis screening of drug candidates. The arrayed human breast cancer cells docked in PDMS microwell were aligned and sealed with chemicals encapsulated in individual PDMS microarray spots on coverslip for addressable screening against several compounds. A single PDMS microwell coated glass slide containing 2,100 individual cell cultures provided apoptotic cell death data for human breast cancer cell. Similar responses were obtained with our assay and conventional 96-well plate assay, demonstrating that the near 100-fold miniaturization does not influence the cell death response.

Aim 1-We herein fabricate micro-platform for the high throughput technique.

Aim 2-We demonstrate apoptosis (programmed cell death) of cell in response to chemicals in their microenvironment using the HTT, which is simple, inexpensive, portable and robust that could be commercialized and used in various fields.

D) GENERAL EXPERIMENTAL APPROACH

E) DESIGN PITFALLS AND ALTERNATIVES

F) ANTICIPATED FIGURES FOR PAPER or when you have data, FIGURES FOR PAPER

Figure 1. Scheme of Two&Three-dimensional cell culture system for detecting apoptosis

Figure 2. Drug Diffusion Test of Drug (a) Drug-encapsulated hydrogel microarray (b) Aligning microarray glass slide with PDMS microwell glass slide (c) Analysis of Drug release in microwell (fluorescence to diffusion time) (d) Simulation of drug diffusion in microwell

Figure 3. PDMS microwells (a) Uniform PDMS microwell (b) MCF-7 cell (human breast cancer cell) seeding in each microwells (c) Live and dead test of MCF-7 (d) Apoptotic cell death test

G) FUTURE DIRECTIONS

PDMS microwell system will be tested for detecting apoptotic cell death because several problems were previously found out PEG microwell.