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author:("Shu, huiquan")
1.  Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum 
PLoS ONE  2014;9(1):e86018.
An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 106-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.
PMCID: PMC3899086  PMID: 24465845
2.  Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study 
PLoS ONE  2014;9(1):e85675.
Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.
Methods and Findings
We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.
CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.
PMCID: PMC3893217  PMID: 24454917
3.  Cryopreservation of Mycobacterium tuberculosis Complex Cells 
Journal of Clinical Microbiology  2012;50(11):3575-3580.
Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of “live” cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.
PMCID: PMC3486236  PMID: 22933596
4.  A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures 
Biopreservation and Biobanking  2011;9(4):355-362.
Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem–Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (Vb=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, −5°C, −10°C, and −20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, −10, −20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10−3 cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C–12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.
PMCID: PMC3247705  PMID: 22232706
5.  Development of a Reliable, Low-cost, Controlled Cooling Rate Instrument for the Cryopreservation of Hematopoietic Stem Cells 
Cytotherapy  2010;12(2):161-169.
An optimal cooling rate is one of the critical factors influencing the survival of cells during cryopreservation. In this paper we describe a novel device, named the box-in-box, which was developed for optimal cryopreservation of human hematopoietic stem cells (HSC). This work presents the design of the device, a mathematical formulation describing the expected temperature histories of samples during the freezing process, along with actual experimental results of thermal profile tests. In experiments, when the box-in-box device was transferred from room temperature to a −80 °C freezer, a cooling rate of −1~−3.5 °C/min, which has been widely used for the cryopreservation of HSC, was achieved. In order to further evaluate this device, HSC cryopreservation was compared between the box-in-box device and a commercially available controlled rate freezer (CryoMed). The experimental data, including total cell population and CD34+ hematopoietic progenitor cell recovery rates, viability, and cell culture colony assays, showed that box-in-box worked as well as CryoMed instrument. There was no significant difference in either survival rate or the culture/colony outcome between the two devices. In conclusion, the box-in-box device can work as a cheap, durable, reliable and maintenance-free instrument for the cryopreservation of HSC. This concept of a box-in-box may also be adapted to other cooling rates to support cryopreservation in a wide variety of tissues and cells.
PMCID: PMC3022343  PMID: 19929459
cryopreservation of hematopoietic stem cells; box-in-box; controlled cooling rate freezer; colony assay

Results 1-5 (5)