Single cell culture wells (SiCCWells).

摘要

Single cell analysis is an increasingly important part of biomedical research and diagnostic medicine. Cells are known to be dynamic organisms and when they are influenced by the cellular micro-environment produce a spectrum of responses. Bulk cellular techniques can lead to the loss of information by averaging signals together from populations that are inhomogeneous. Bulk cellular techniques also typically require the disruption or destruction of cellular process. Because of this there are few, if any, options to monitor a cell in real time in vitro. The dichotomy of single cells analysis is that the bulk signal cannot be neglected for the individual cellular response. Rather the bulk signal must be built out of the individual responses. Due to this, single cell resolution must be coupled with high through put automated system that can discretely isolate cells, deliver chemistry, sample the microenvironment, and maintain cells in long term cell culture.;We present an integrated platform for high volume single cell analysis built on the femtopump technology. The femtopumps use a combination of micropatterned materials to restrict electro-osmotic flow to a small cross section. The result is a robust platform technology on which a variety of microchemical total analysis systems can be built. Because of the patterned electro-osmotic technology these devices offer a high degree of precision and repeatability for in vitro experimentation.;We present a femtopump-based micro total analysis system, the Single Cell Culture Well or SiCCWell. The SiCCWell design provides for vacuum assisted cell seeding, long term culture, manipulation of the microenvironment through fluid delivery, and continuous monitoring of cellular processes through fluid sampling. The SiCCWells have demonstrated the ability to capture and deliver fluid to cells in vitro at a rate of approximately 10 femtoliters per second. The SiCCWells have also demonstrated the capacity for electro-osmotic generation of microdroplets. The SiCCWell design provides for high throughput single cell monitoring. Current designs are thought capable of generating data at single cell resolution from up to 12500 cells per square centimeter.;In addition, the theory, design, and practical implementation of an electro-osmotic gradient generator is presented. Chemotaxic gradients are a valuable way to model the in vivo cellular microenvironment to understand how chemical and environment cues affect the behavior of cells. The gradient generator uses a highly modified SiCCWell fabrication to give discrete activation sites to which differing volumes of fluid can be delivered. The gradient generator is capable of varying the flow rate per channel to create geometric and arbitrary gradient patterns with a high level of precision and repeatability.;The femtopump-based technologies are an exciting new platform technology that is highly customizable for wide variety of applications. The SiCCWell microchemical total analysis platform is an example of the opportunities in single cell analysis presented by this technology.