Development of cell confinement and manipulation microfluidics and methods for optimal biopreservation

Abstract

Biopreservation, especially cryopreservation, of bio-samples, includeing DNA/RNA, proteins, bio-fluids, cells, tissues and organs, has attracted more and more attentions, because of its great support in clinical research and trials. The successful biopreservation of bio-samples enables the cellular therapy, drug development and diagnosis of disease. To achieve the best biopresration result, various of factors and steps need to be optimized, including selection of the optimal cryoprotective agent (CPA), successful addition of CPA, optimization of the cooling protocol, thawing of the frozen samples, removal of CPA after thawing, and others. Cryobiologists rely on the theoretical interpretation of bio-heat and mass transfer and novel measurement techniques to design optimal cryopreservation protocol. In this dissertation, cell confinement and manipulation devices were developed to determine the fundamental cryobiological properties of cells, and a novel across cell membrane temperature dependent mass transfer model were investigated with measurement methods proposed. Determianation of the intrinsic cryobiological characteristics of the cells is the very first steps of cryopreservation protocol optimization, which includes the cell membrane permeabilities to water and the CPA at different temperatures, the osmotically inactive cell volume, the activation energy of water/CPA transport across cell membranes, osmotic tolerance limit, sensitivity to the CPA toxicity and others. To determine these cyobiological properties, two kinds of microfluidic device are proposed and applied: a microfluidic perfusion channel and non-contact cell confinement and manipulation platform. Studying the phase contrast microscopy of cell volume excursion history when perfused with solutions is the common method to evaluate the cell membrane properties, which requires the measurement devices equiped with features as followed: (1) steadily trap cells for long time; (2) change the extracellular media cell exposed to; (3) control and monitor the extracellular media temperature and the whole cell volume excursion history can be recorded and easily analyzed. The microfluidic perfusion channel is used to determine the cell membrane transport properties of human vaginal mucosal immune cells (T cells and macrophages), because of their importance in HIV vaccine research. The cell membrane permeabilities to four different CPAs (Dimethyl sulfoxide (DMSO), glycerol, propylene glycol and ethylene glycol) at room temperature are measured, indicating that DMSO and propyleneglycol could be a potencial CPA options, while glycerol is not a good choice for these cells as to the slow across membrane transport of which. Though a low cost and easy to operate tool the microfuidic perfusion channel is, the challenges, like the intense images processing effort, due to the shadow from the blocker, lack of on-chip temeprature control, remain to be solved for better measurement precision and efficiency. To overcome the challenges unsolved in microfludic perfusion channel, a cell confinement and manipulation platform with instananous flow and local temperature control was developed. Numerical simulation of in-channel laminar flow coupled heat transfer was coducted to exam various heater desgins, to achieve the best temperature uniformity and heating efficiency. The fabrication protocol was developed and tested to provide the optimal device performance of the integrated microfludics. With the developed cell confinement and manipulation platform, two practices of cell membrane properties measurement were executed, with both the traditional mass transfer model under static temperatures and the originally proposed temperature dependent mass transfer model. With the assist of the numerical simulation of temperature dependent mass transfe model, the temperature profile during the measurmenet was identified and used for the estimation of activation energy. The further development vista beyond the projects covered in this dissertation was also discussed, including the integration of on-chip active cooling design, options to scale up current system, and some other applications, for instance exploring new potencial CPAs.