PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1067160)

Clipboard (0)
None

Related Articles

1.  Defining Blood Processing Parameters for Optimal Detection of Cryopreserved Antigen-Specific Responses for HIV Vaccine Trials 
Journal of immunological methods  2007;322(1-2):57-69.
Interferon-gamma (IFN-γ) ELISpot and intracellular cytokine staining (ICS) assays are routinely employed in clinical HIV vaccine trials to identify antigen-specific T cells in cryopreserved peripheral blood mononuclear cells (PBMC). Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 hours after venipuncture with that processed within eight hours, we observed on average a modest reduction in PBMC viability (∼8% decrease), a greater loss in cell recovery (∼32%), and between 36-56% loss in IFN-γ T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within eight hours of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials.
doi:10.1016/j.jim.2007.02.003
PMCID: PMC1904432  PMID: 17382342
2.  VACUTAINER® CPT™ and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples 
BMC Immunology  2006;7:11.
Background
For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNγ expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.
Results
The results indicate that cryopreserved PBMC samples tested for CMV- and HIV- specific interferon-gamma (IFNγ) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNγ response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.
Conclusion
These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV+ PBMC, as well as a viable alternative to Ficoll gradient separation.
doi:10.1186/1471-2172-7-11
PMCID: PMC1481571  PMID: 16725038
3.  Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents ▿  
The gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-γ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-γ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-γ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.
doi:10.1128/CVI.00326-08
PMCID: PMC2643552  PMID: 19091991
4.  Optimization of Storage and Shipment of Cryopreserved Peripheral Blood Mononuclear Cells from HIV-Infected and Uninfected Individuals for ELISPOT Assays 
Journal of immunological methods  2010;363(1):42-50.
Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at −70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at −70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at −70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.
doi:10.1016/j.jim.2010.09.032
PMCID: PMC3068047  PMID: 20888337
5.  Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials 
A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein– Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
doi:10.1016/j.jim.2014.04.005
PMCID: PMC4152919  PMID: 24787274
Peripheral blood mononuclear cells; Cryopreservation; Repository
6.  Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies  
Cells  2012;1(3):313-324.
Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.
doi:10.3390/cells1030313
PMCID: PMC3901099  PMID: 24710478
ELISPOT; PBMC; T cells; cryopreservation; DMSO
7.  Optimal Blood Mononuclear Cell Isolation Procedures for Gamma Interferon Enzyme-Linked Immunospot Testing of Healthy Swedish and Tanzanian Subjects▿  
Determination of antigen-specific T-cell responses is an important part of vaccine assessment. High levels of recovery, viability, and functionality of peripheral blood mononuclear cells (PBMCs) are essential for reliable assessment of cell-mediated immune responses. Here, we sought to find the cell preparation technique best suited for two clinical vaccine trial sites: Stockholm, Sweden, and Dar es Salaam, Tanzania. Standard Ficoll-Paque gradient centrifugation, BD Vacutainer cell preparation tube (CPT), and Greiner Bio-One LeucoSep tube techniques were tested. Cell yield and viability were recorded. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) testing was used to assess cell functionality. No differences in mean recovery or mean viability of fresh PBMCs were observed between Ficoll-Paque gradient centrifugation and CPT techniques as used in Stockholm. In Dar es Salaam, recovery of PBMCs isolated by use of the Ficoll-Paque gradient technique was higher than that seen with CPT (1.58 ± 0.6 versus 1.34 ± 0.4 million cells/ml of blood [P = 0.0469]), and the viability of PBMCs processed by Ficoll-Paque gradient was higher than that seen with CPT-purified cells (95.8% ± 2.3% versus 92.6% ± 4.8% [P = 0.0081]). Furthermore, LeucoSep cell separation gave higher levels of yield (1.10 ± 0.3 versus 0.92 ± 0.3 million cells/ml of blood [P = 0.0022]) and viability (95.7% ± 2.0% versus 93.4% ± 3.2% [P = 0.0012]) than Ficoll-Paque cell separation. The cells purified by the different techniques at the two sites performed equally well in IFN-γ ELISPOT assays. Both techniques generated cell preparations with excellent yield, viability, and functionality in Stockholm. In Dar es Salaam, CPT did not perform as well as Ficoll-Paque separation. In a subsequent comparison, LeucoSep performed better than Ficoll-Paque separation. Our findings emphasize the need for on-site assessment of PBMC purification techniques for optimal evaluation of cell-mediated immune responses.
doi:10.1128/CVI.00161-07
PMCID: PMC2292660  PMID: 18287577
8.  Improved Post-Thaw Recovery of Peripheral Blood Stem/Progenitor Cells Using a Novel Intracellular-like Cryopreservation Solution 
Cytotherapy  2009;11(4):472-479.
Background
Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate freezing and liquid nitrogen storage have become “routine” practice in many cell processing facilities, there is clearly room for improvement, as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of DMSO that can contribute to the morbidity and mortality of the transplant therapy.
Methods
This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor™ (BioLife Solutions, Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 × 107 cells/1 ml sample vial were prepared for cryopreservation in the following solutions: 1) FHCRC standard – Normosol-R, 5% HSA, 10% DMSO, and 2) CryoStor™ CS10 (final diluted conc. of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of one week. Vials were then thawed and evaluated for TNC, Viability, CD34, and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose.
Results
The PBSC samples frozen in CryoStor™ CS10 yielded significantly improved post-thaw recoveries for total viable CD34+, CFU, and viable granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8 fold increased recovery of viable CD34+ cells (P = 0.005), a 1.5 fold increase in CFU-GM numbers (P = 0.030), and a 2.3 fold increase in granulocyte recovery (P = 0.045).
Discussion
This study indicates that use of CryoStor™ for cryopreservation can yield significantly improved recovery and in vitro functionality of the stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while also not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary, however these findings imply use of CryoStor™ for cryopreservation might ultimately result in improved engraftment for those patients with lower content of CD34+ cells in their PBSC collections, along with reducing the requirement for additional apheresis collections, and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.
doi:10.1080/14653240902887242
PMCID: PMC3021966  PMID: 19499402
Cryopreservation; CryoStor; CD34+; CFU-GM; PBSC
9.  Enzyme-Linked Immunospot Assay for Detection of Human Respiratory Syncytial Virus F Protein-Specific Gamma Interferon-Producing T Cells 
Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 106 PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/106 PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.
doi:10.1128/CVI.00736-13
PMCID: PMC4018879  PMID: 24574540
10.  Effects of Blood Transportation on Human Peripheral Mononuclear Cell Yield, Phenotype and Function: Implications for Immune Cell Biobanking 
PLoS ONE  2014;9(12):e115920.
Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum), biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC) and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9): at a distal location (shipped overnight) and in the central laboratory (processed immediately). PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might be that despite changes due to overnight shipment, highly standardized central processing (and analysis) could be superior to multicentric de-central processing with more difficult standardization.
doi:10.1371/journal.pone.0115920
PMCID: PMC4277394  PMID: 25541968
11.  Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials 
Background
Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC.
Patients/Methods
EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45− CD34+CD133+/−VEGFR2+/− and EC were gated for CD45−CD146+CD34+/−VEGFR2+/−.
Results
Cryopreservation for 7 days at −80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at −65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45− CD34+CD133+VEGFR2+) were enumerated as 0.2 ± 0.06%, late EPC(CD45−CD146+CD34+VEGFR2+) as 0.6 ± 0.1% and mature EC (CD45−CD146+CD34−VEGFR2+) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at −80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant.
Conclusions
Our data indicate that cryopreservation at −80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.
doi:10.4172/2155-9864.1000158
PMCID: PMC4193669  PMID: 25309814
Endothelial progenitor cells; Endothelial cells; Flow cytometry; Cryopreservation; Angiogenesis
12.  Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy 
Cancer Immunology, Immunotherapy  2012;61(11):2021-2031.
Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.
doi:10.1007/s00262-012-1262-0
PMCID: PMC3493671  PMID: 22527251
DC; Dendritic cells; Cryopreservation; Cellular immunotherapy; Cell yields
13.  Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data 
Introduction
Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).
Methods
The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed.
Results
The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties.
Conclusions
We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.
doi:10.1186/scrt398
PMCID: PMC4055140  PMID: 24438697
14.  Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells 
Stem Cells and Development  2010;19(4):513-522.
Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco’s modified Eagle’s medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (106 cells/mL) of P1 ASCs were frozen overnight in a −80°C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37°C water bath (1–2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37°C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, ∼84% ± 5% and ∼84% ± 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.
doi:10.1089/scd.2009.0173
PMCID: PMC3139530  PMID: 19788372
15.  Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide 
BMC Biotechnology  2012;12:49.
Background
Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed.
Results
The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods.
Conclusion
A 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC.
doi:10.1186/1472-6750-12-49
PMCID: PMC3465236  PMID: 22889198
Mesenchymal stem cells; Cryopreservation; Controlled rate freezing; Hydroxyethyl starch
16.  IFN-γ production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1*0401 and HLA-DQ8 
Arthritis Research  1999;2(1):75-84.
IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.
Introduction:
Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5].
It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses.
Aims:
To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum.
Methods:
Forty-five patients who met the 1987 American College of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera.
Heparinized blood was density gradient separated and PBMCs were cultured at 1 × 106/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 μ g/ml), killed influenza virus (Vaxigrip, Pasteur Mérieux, Lyon, France; diluted 1 : 1000) or purified protein derivative (PPD; 10 μ g/ml). CII was heat-denatured in 56°C for 30 min.
Cell supernatants were collected after 7days and IFN-γ contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study.
Results:
PBMCs from both RA patients and healthy control individuals responded with inteferon-γ production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for healthy control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-γ response to both native and denatured CII than did healthy control individuals (P = 0.02 and 0.04, respectively).
Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-γ responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II.
The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6,7,8]. That no difference between the RA group and the control group was recorded in CII-induced IFN-γ production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-γ production and the PPD-induced and killed influenza virus-induced IFN-γ production, and compared these fractions. A highly significant difference between the RA and healthy control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-γ values as the denominator (Fig. 2b).
When comparing the compensated IFN-γ response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8).
Discussion:
No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach.
We identified an association between the IFN-γ response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404).
Conclusion:
CII, a major autoantigen candidate in RA, can elicit an IFN-γ response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.
PMCID: PMC17806  PMID: 11219392
collagen type II; human leucocyte antigen-DR; IFN-γ; rheumatoid arthritis; T cell
17.  Analysis of the Viability of Umbilical Cord blood Stem Cells 
The investigations on sources and viability of stem cells are important as stem cell transplantation has shown promising results in diseases like leukemias and lymphomas. Umbilical cord blood samples were collected in a closed aseptic system. The samples were diluted with phosphate buffered saline, treated with ficol and centrifuged at 15,000 rpm for the recovery of progenitor stem cells.The stem cells were cryopreserved with different media Containing DMSO, patient’s serum and human albumin.The viability of the cells was studied by dye exclusion method. Suitable media are necessary for optimal cryoprotection and prevention of cryoinjury DMSO is essential for improved cryopreservation and post-thaw quality. The addition of a protein additive also provides a protective effect. The medium containing DMSO, DMEM and patient’s serum proved to be the most effective for cryopreservation and viability as high as 82.4% was achieved after one year. The unique findings of the present study are that the addition of patient’s serum enhances the cryoprotection and cord blood stem cells can be stored at -20°C for the duration up to two months instead of the requirement of storage at ultralow temperature at -186°C.
PMCID: PMC3908141  PMID: 24693042
UCB; Viability; Stem cells; Media
18.  Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization▿  
The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
doi:10.1128/CVI.00342-08
PMCID: PMC2725535  PMID: 19515870
19.  Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution 
World Journal of Hepatology  2012;4(5):176-183.
AIM: To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.
METHODS: The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7.
RESULTS: The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r2 = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.
CONCLUSION: The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.
doi:10.4254/wjh.v4.i5.176
PMCID: PMC3365437  PMID: 22662286
Human hepatocytes; Viability; Cytochrome P540; Dimethylsulphoxide; Cryoprotectant; Cryopreservation
20.  OPTIMIZED CRYOPRESERVATION METHOD FOR HUMAN DENTAL PULP-DERIVED STEM CELLS AND THEIR TISSUES OF ORIGIN FOR BANKING AND CLINICAL USE 
Cryobiology  2009;59(2):150-157.
Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120 hours after tooth extraction, and that cryopreservation of early-passage cultured DPSC leads to high-efficiency recovery post thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me2SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2 × 106 cells/mL. It was further established that DPSC can be stored at −85°C or −196°C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.
doi:10.1016/j.cryobiol.2009.06.005
PMCID: PMC2757034  PMID: 19538953
Mesenchymal stem cells; dental pulp stem cells; adult stem cells; cryopreservation; tissue engineering; stem cell banking
21.  A practical method for preparation of pneumococcal and nontypeable Haemophilus influenzae inocula that preserves viability and immunostimulatory activity 
BMC Research Notes  2013;6:522.
Background
Convenience is a major reason for using killed preparations of bacteria to investigate host-pathogen interactions, however, host responses to such preparations can result in different outcomes when compared to live bacterial stimulation. We investigated whether cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) permitted investigation of host responses to infection without the complications of working with freshly prepared live bacteria on the day of experimental challenge.
Findings
S. pneumoniae and NTHi retained >90% viability following cryopreservation in fetal calf serum for at least 8 weeks. Host responses to live, cryopreserved (1 week and 4 weeks), heat-killed or ethanol-killed S. pneumoniae and NTHi were assessed by measuring cytokine release from stimulated peripheral blood mononuclear cells (PBMCs). We found that cryopreserved bacteria, in contrast to heat-killed and ethanol-killed preparations, resulted in comparable levels of inflammatory cytokine release from PBMCs when compared with fresh live bacterial cultures.
Conclusion
Cryopreservation of S. pneumoniae and NTHi does not alter the immunostimulatory properties of these species thereby enabling reproducible and biologically relevant analysis of host responses to infection. This method also facilitates the analysis of multiple strains on the same day and allows predetermination of culture purity and challenge dose.
doi:10.1186/1756-0500-6-522
PMCID: PMC3867214  PMID: 24321049
Ethanol-killed; Heat-killed; Live bacteria; Cryopreservation; S. pneumoniae; NTHi; Immunostimulation; PBMCs
22.  Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae 
PLoS ONE  2013;8(11):e78668.
The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.
doi:10.1371/journal.pone.0078668
PMCID: PMC3823882  PMID: 24244336
23.  Cryopreservation Decreases Receptor PD-1 and Ligand PD-L1 Coinhibitory Expression on Peripheral Blood Mononuclear Cell-Derived T Cells and Monocytes▿  
Clinical and Vaccine Immunology : CVI  2009;16(11):1648-1653.
The B7-CD28 immunoglobulin superfamily of costimulatory and coinhibitory ligands and their cell receptors play a critical role in modulating immune responses. Imbalances in these immune regulatory signals occur in pathological conditions characterized by chronic antigenic stimulation. Clinical studies often rely on the use of cryopreserved peripheral blood mononuclear cells (PBMC) to evaluate cellular immune responses. The impact of cryopreservation on these coinhibitory ligands and their cell receptors is unknown. In our studies, cryopreservation significantly reduced the expression of both PD-1 and PD-L1 on PBMC-derived CD3+/CD8+ T cells and CD45+/CD14+ monocytes obtained from adult control subjects. Blockade of PD-1, PD-L1, and PD-L2 using both freshly isolated and cryopreserved PBMC led to higher levels of phytohemagglutinin (PHA) and Candida-induced gamma interferon (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) with no effect on IL-10 production. Coinhibitory signaling blockade of freshly isolated, PHA-stimulated PBMC from normal adult controls and human immunodeficiency virus (HIV)-infected subjects led to increased production of IL-4 and IL-5. Candida-stimulated PBMC preferentially induced IFN-γ and TNF-α production, with reduced production of IL-2 and IL-10. This is in contrast to high levels of IFN-γ, IL-2, and TNF-α production with PHA-stimulated cells. The effects of coinhibitory blockade on PHA and Candida-induced lymphoproliferation were varied, with freshly isolated PBMC from adult control subjects and HIV-infected patients yielding higher levels of lymphoproliferation in response to PD-1/PD-L1 blockade. Immune function studies employing cryopreserved cells may lead to increased T-cell effector cytolytic and regulatory immune responses.
doi:10.1128/CVI.00259-09
PMCID: PMC2772387  PMID: 19726615
24.  Substantial Improvements in Performance Indicators Achieved in a Peripheral Blood Mononuclear Cell Cryopreservation Quality Assurance Program Using Single Donor Samples▿  
Storage of high-quality cryopreserved peripheral blood mononuclear cells (PBMC) is often a requirement for multicenter clinical trials and requires a reproducibly high standard of practice. A quality assurance program (QAP) was established to assess an Australia-wide network of laboratories in the provision of high-quality PBMC (determined by yield, viability, and function), using blood taken from single donors (human immunodeficiency virus [HIV] positive and HIV negative) and shipped to each site for preparation and cryopreservation of PBMC. The aim of the QAP was to provide laboratory accreditation for participation in clinical trials and cohort studies which require preparation and cryopreservation of PBMC and to assist all laboratories to prepare PBMC with a viability of >80% and yield of >50% following thawing. Many laboratories failed to reach this standard on the initial QAP round. Interventions to improve performance included telephone interviews with the staff at each laboratory, two annual wet workshops, and direct access to a senior scientist to discuss performance following each QAP round. Performance improved substantially in the majority of sites that initially failed the QAP (P = 0.002 and P = 0.001 for viability and yield, respectively). In a minority of laboratories, there was no improvement (n = 2), while a high standard was retained at the laboratories that commenced with adequate performance (n = 3). These findings demonstrate that simple interventions and monitoring of PBMC preparation and cryopreservation from multiple laboratories can significantly improve performance and contribute to maintenance of a network of laboratories accredited for quality PBMC fractionation and cryopreservation.
doi:10.1128/CVI.00214-06
PMCID: PMC1797705  PMID: 17050740
25.  Identification of Endogenous HLA-A2–Restricted Reactivity Against Shared Melanoma Antigens in Patients Using the Quantitative Real-Time Polymerase Chain Reaction 
Summary
This study was conducted to determine whether reactivity to melanoma cells of pretreatment peripheral blood mononuclear cells (PBMCs) from patients with metastatic melanoma correlated with subsequent response to treatment with interleukin-2 (IL-2). The sensitivity of the quantitative real-time polymerase chain reaction (PCR) assay was optimized, including the total number of cells used (3 × 106 in 1 mL), the responder-to-stimulator cell ratio (5:1), the optimal time to incubate PBMCs with tumor (2 h), the appropriate tumor stimulators (melanoma cell lines differing only in the expression of histocompatibility leukocyte antigen [HLA-A2]), the duration of recovery in the culture of PBMCs after cryopreservation (18–24 h), and the medium used (Iscove, 10% human AB serum). Using this optimized assay to detect HLA-A2–restricted antitumor reactivity in the pretreatment PBMCs from patients with melanoma, positive reactive responses were detected in 7 of 28 patients with an objective clinical response to IL-2 therapy compared with 6 of 21 positive reactive responses in nonresponding patients. None of 12 healthy donors were positive in this study. Thus, there was no significant difference in the reactivity of pretreatment PBMCs when responders were compared with nonresponders, although the melanoma patients had an increased incidence of response compared with healthy donors (p = 0.05). The PBMCs from 11 of the 13 melanoma patients with pretreatment HLA-A2–restricted antimelanoma reactivity were tested against a panel of transfectants expressing known shared melanoma antigens. Anti–MART-1 reactivity was detected in the pretreatment PBMCs of three patients. It thus appears that some melanoma patients are immunologically primed to antigens expressed on the tumor surface, although the HLA-A2–restricted antimelanoma activity detected in this real-time PCR assay was not predictive of patients’ responses to IL-2 therapy.
PMCID: PMC2424231  PMID: 11924911
Melanoma antigens; Histocompatibility leukocyte antigen; Immunotherapy; Quantitative real-time polymerase chain reaction

Results 1-25 (1067160)