Development of Optimal Biopreservation Methods and Technology for Cellular Therapy and Clinical Diagnosis
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Successful biopreservation of bio-samples, including DNA/RNA, proteins, bio-fluids, cells, tissues and organs, is vital for researches and clinical trials, enabling the diagnosis of disease, drug development and cellular therapy. Challenges in biopreservation (particularly cryopreservation) include 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. In this dissertation, a few novel techniques are developed to determine the fundamental cryobiological properties of cells, optimize cryopreservation protocols and the applications of biopreservation in cellular therapy and disease diagnosis. The optimal cryopreservation protocol for a cell type is determined by the inherent cryobiological characteristics of the cells, including the cell membrane permeabilities to water and the CPA at different temperatures, the osmotically inactive cell volume fraction, the activation energy of water transport across cell membranes, the osmotically inactive cell volume fraction, osmotic tolerance limit, sensitivity to the CPA toxicity, intracellular ice formation (IIF) temperature, and others. To determine these cryobiological properties, two methods are proposed and applied: microfluidic perfusion channel and differential scanning calorimetry (DSC) measurements. For the microfluidic perfusion channel method, human vaginal mucosal immune cells (T cells and macrophages) were chosen because of their importance in HIV vaccine research and poor long-term preservation with current protocols. Using the micro-fabricated channel, cells can be trapped in the channel and observed under microscopy. Different solutions (2xPhosphate-buffered saline [PBS], 3xPBS, and CPA solutions) were fed into the channel. In response to the perfusion solutions, the cell volume changed (shrank or shrank then expanded). The volume excursion history of individual cells was recorded. After image analysis, the data were simulated to evaluate the cell membrane properties. The results showed that T cells and macrophages had relatively low membrane permeabilities, implying a low optimal cooling rate for these cells. Comparing four different CPAs (Dimethyl sulfoxide (DMSO), glycerol, propylene glycol and ethylene glycol), showed that glycerol crossed the cell membranes very slowly; therefore, it cannot be used for the cryopreservation of these cells. DMSO and propylene glycol could be good CPA options. Tests of CPA cytotoxicity demonstrated similar results. The results also showed that T cells were more susceptible to stresses than macrophages. While it is technically challenging to use the micro-perfusion channel method at temperatures below the freezing point, DSC can be used to evaluate the cell membrane properties at any sub-zero temperatures. Based on the fundamental theory developed by Devireddy et al., a "slow-fast-fast-slow" cooling program is used for cell suspensions. In the first slow cooling process, the measured heat release of ice crystallization includes the crystallization of extracellular water and the water that is transported from inside of the cells due to extracellular ice formation and consequently the osmolality gradient occurrence across the cell membranes. During the repeated fast cooling processes, cells are assumed to be killed and lysed. Some water will be bound to the proteins and cell debris of the lysed cells. Therefore, in the last slow cooling step, the heat release of ice crystallization will be lower than that of the first slow cooling step. The thermogram difference between the first and the last slow cooling steps can be analyzed to obtain the water transport history across cell membranes in the suspension during freezing; therefore, cell membrane properties at any temperature during cooling can be predicted. This approach was optimized and applied to measure the membrane properties of PBMC lymphocytes (including T cells and monocytes). The results were then applied in theoretical simulation to predict the optimal cooling rate for the cells. The prediction was consistent with the standard operating procedure (SOP) for PBMC cryopreservation. Another challenge in cryopreservation and cellular therapy is the addition and removal of CPA with minimal injury to the cells. In order to overcome this problem, a multi-functional cell processor based on semi-permeable hollow fibers and "dilution-filtration" working principle was developed. A novel approach of electrical conductivity measurement was also proposed for real-time, online monitoring of the residual CPA concentration in the cell suspension. The results showed that this system can be applied for successful cell concentration (control of the cell suspension volume) and removal of CPA with much lower cell loss. Furthermore, this automatic device with closed fluid loop can save labor significantly, reduce risk of contamination and decrease cell damage. Cryopreservation of bacteria and freeze-drying of proteins were also studied in this dissertation. Mycobacterium tuberculosis (MTB) was studied due to the challenges in tuberculosis diagnosis. Tuberculosis is the second leading infectious disease (only after HIV) causing death in the world. Fast and accurate diagnosis of MTB is still challenging. Cryopreservation of MTB cells is very important for pathological research, diagnosis and drug development. Due to the small size of MTB cells and the complex cell wall/membrane configuration, it is challenging to study their fundamental cryobiological properties. So, MTB cells were cryopreserved under systematically varied conditions and the effects on recovery were measured. The results showed that among all the parameters, slow cooling rate is the most important for successful MTB cryopreservation. Inconsistencies were found between the results of microbiological culturing and BacLight Live/Dead staining, implying that suboptimal cryopreservation might not cause severe damage to cell wall and/or membrane, but instead cause intracellular injury, which leads to the loss of cell viability. For long-term preservation of the MTB IgY antibodies, freeze-drying was applied. The results showed that the lyophilized IgY antibodies can be well preserved for up to 13 months at either room temperature or 4°C. Plans for further experiments are also presented in this dissertation, including cryopreservation and vitrification of mucosal immune cells and tissues based on fundamental researches and predicted protocol, multi-center evaluation of the developed techniques and protocols, and further optimization of the multi-functional cell processor.
- Mechanical engineering