Modern biomedical research requires access to high quality specimens of human tissue with or without extensive clinical annotation. Multiple types of organizations have developed to supply human tissues to support biomedical research. These organizations follow different models including the specific models of 1) prospective collection, 2) tissue banking, and 3) tissue collection associated with clinical trials as well as the model of 4) a tissue resource that incorporates features of the other models. These types of organizations devoted to supplying tissues for research have chosen different goals to meet the different tissue and informational needs of the investigators to whom they supply tissue. In order to provide high quality tissues to support research, all models should rely on a strong quality assurance program with extensive quality control of the tissues being provided to support research. In addition to facilities which collect, process, store and provide tissues, the need for a rigorous QA program applies to all resources and infrastructures used to support biomedical research. The UAB Tissue Collection and Banking Facility which provides human tissue to support biomedical research has been functioning and developing since 1979. To our knowledge, similar programs in providing tissues from animals are less developed, but could easily follow the models which UAB and other institutions providing human tissues have established, including the approaches of UAB and others to QA and QC. This manuscript reviews the current concepts of QA and QC in use in organizations supplying tissue to support biomedical research as well as new approaches in QA and QC that have been proposed.
A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.
Light microscopy method offers unique abilities for the determination of membrane transport properties of either single or multiple cells. A stream imaging system composed of a microfluidic device, a charge-coupled device camera, and a microscope has been developed to study the osmotic behavior of multiple cells in response toward their extracellular environment. Cells of interest were first mixed with the desired extracellular medium and streamed into a microchannel. The microchannel confines the movement of the cells in a monolayer and allows cells to move along the flow direction only. The cells then pass through a sensing zone where the images of cells were capable of being captured under a microscope. Using mouse dendritic cells (mDCs) as a model system, the membrane transport properties were investigated. The kinetics volume changes of mDCs under various extracellular conditions at room temperature (22°C) were analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane. This prototype system directly allows us to observe, trace, capture, and store the sample information in terms of number, concentration, dynamic size, or shape for further analyses and documentations. We believe that the system has the potential of being used as a stand-alone equipment, or integrated into a lab-on-a-chip system, or embedded into commercialized instruments.
Breast cancer research frequently requires frozen cancer tissue samples for potential biomarker detection. Ninety-one breast cancer frozen tumor tissue samples were examined for factors contributing to the proportion of the tumor cells per sample. The strongest predictor of high proportion tumor cells was tumor grade as determined by the Scarff-Bloom-Richardson scoring system, followed by tumor size. Because the low-grade tumors had fewer tumor cells per specimen, the type of surgery and experience level of the sample collector was not significant. This variable should be considered by investigators when evaluating potential biomarkers.
Genetic resource banks and assisted reproductive technologies support the conservation of endangered or threatened species. In this study we assessed two procedures to cryopreserve skin biopsies from live brown bears. Skin biopsies were taken from six live, anesthetized brown bears. Single biopsies (n = 3) of each animal were cut into small pieces and assigned to one of the three experimental groups: freezing, vitrification, or untreated fresh. There were no differences on cell attachment. However, both freezing and fresh culture allowed for higher cell proliferation (p < 0.05) and less days to reach 70% to 80% confluence (p < 0.03) than vitrification. Skin biopsies from brown bears can be preserved long term, allowing fibroblasts to proliferate in culture. Slow freezing was effective to cryopreserve skin biopsies from brown bears.
The purpose of this study was to systematically examine blastocoelic fluid reduction prior to vitrification and its potential benefits. In addition, we compared artificial collapse (AC) by laser pulse to a mechanical method. Mouse and dicarded human blastocysts were used in this study. Blastocysts were collapsed using either a 10 MS Pulse with a laser (LAC) or else mechanical Puncture with a microneedle (MAC). Blastocysts were vitrified on cryoloops using a two-step ethylene glycol/dimethyl sulfoxide protocol. We examined the effects of AC on specific Outcome parameters such as overall survival, reexpansion, cell proliferation, and DNA damage. Unlike others, we report overall high survival rates with expanded blastocysts even without fluid reduction. We did detect a significant increase in blastomeres showing signs of DNA damage in the control group (13%) in comparison to blastocysts AC prior to vitrification (LAC 3%, MAC 5%; p < 0.001). Control blastocysts exhibited a lower rate of reexpansion. Within 3 h of warming, 73%, and 81%, respectively of mechanically or laser collapsed blastocysts were fully reexpanded as compared to only 53%, of control blastocysts (p < 0.001). Overall blastomere count was also significantly lower in control blastocysts (CT 103+32, MAC 121 + 37, LAC 134 + 35; p < 0.0001). Early blastocysts with smaller blastocoelic volumes did not benefit from any further reduction Of fluid volume. AC can reduce DNA damage and enhance postwarming reexpansion and cell proliferation in expanding blastocysts. The laser method also appeared to be effective and may offer some advantages over mechanical collapse.
Transformation of B cells by Epstein-Barr Virus (EBV) is used by the CCR at Coriell Institute for Medical Research to produce renewable cell lines and DNA to further genomic and proteomic studies of inherited and complex diseases alike. Optimal and efficient transformation requires an accurate assessment of the titer of the virus in each virus preparation. However, current methods to determine EBV titer assess transformation efficiency using large-scale biological assays ending in the establishment of lymphoblastoid cell lines (LCLs). This method determines the virus dilution capable of producing LCL outgrowth but does not determine virion number and optimal dilution will vary with the preparation. Therefore, we developed a real-time PCR method to detect and quantify EBV DNA. In addition to quantifying the titer of the viral supernatants, we evaluated both the biological activity of the viral dilutions as measured by LCL outgrowth and the use of diluted EBV viral stocks to transform previously frozen and untransformed lymphocytes using days to cryopreservation as a comparative end-point. To this end, we determined that there was no difference in time to cryopreservation/transformation of lymphocytes exposed to undiluted or diluted viral stocks. The Real-Time PCR assay provides a method to assess the quality and virion number of new lots of virus without the need for large-scale and time-consuming biological assays. With this information, we are able to determine quickly and efficiently the titer of new virus preparations and to optimize and extend the usage of existing virus stocks.
A major obstacle in using intracellular Sugars for the cryopreservation of mammalian cells is the inability of cells to synthesize or actively accumulate these Sugars. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. This Study examines the interactions of trehalose-containing liposomes with human red blood cells (RBCs). Unilamellar liposomes were synthesized using an extrusion method to contain trehalose in the aqueous core. Liposomal preparations were labeled with a lipophilic fluorophore rhodamine B chloride (R18) at quenching concentrations (4 mol%), or the 5(6)-carboxy fluorescein [5(6)-CF] marker to label the intraliposomal aqueous phase. Flow cytometry and fluorescent microscopy were used to assess the interactions between fluorescently labeled liposomes and RBCs. The delivery of liposomal contents into RBCs was assessed by spectrophotometric measurement of intracellular trehalose. The results of this study show that the transfer Of fluorescence from liposome to RBC population is clue, to both liposomal adsorption and fusion. The merger of membranes and lateral diffusion Of liposomal lipids into the RBC bilayer resulted in the spatial separation of membrane R18 fluorophores, and therefore, a dequenching effect, marked by a 344 +/- 11% increase of the RBC mean fluorescence intensity. The ability of 5(6)-CF and R18 liposomes to deliver their aqueous contents into RBCs, was confirmed by intracellular trehalose measurement. After treatment with 5(6)-CF liposomes, RBC trehalose concentration was 0.1.8 +/- 0.03 mM, whereas R18 liposomes delivered 0.96 +/- 0.07 mM trehalose. The results from this study strongly support the hypothesis that liposomes can be used as trehalose delivery vesicles. Delineating the mechanism of liposomal interaction with RBCs is an important step toward establishing the use of liposomes as tools for the intracellular delivery of stabilizing disaccharides for biopreservation purposes.
Based on Boutron's semi-empirical crystallization theory, a quantitative microscopic imaging method was developed to determine the quantity of ice in thin films of vitrification solutions. A universal equation was obtained for various vitrification solutions. The new method was applied to determine the ice quantity and critical cooling rates (Vcc) for two new vitrification solutions. Results reveal that the new quantitative imaging method is reliable. This new method has several advantages including the fact that it is cheaper than differential scanning calorimetry (DSC) and that it can be used to study vitrification solutions with Vcc values extending beyond the maximum cooling rate of a typical DSC system. Also, this method should permit quantitative imaging of the volume fraction of ice in space and time in microscope samples.
Liver ischemia-reperfusion (I/R) injury is a major complication of liver resection and transplantation. Cytokine release by activated Kupffer cells (KCs) play a central role in the inflammatory cascade of I/R. injury There is evidence that glycine may protect against liver I/R injury by inhibition of KC activity. However, its effect on bile flow an established marker of hepatic function, and bile composition is not known. A rabbit model of hepatic lobar warm I/R was used. Under general anesthesia, the sham group (n = 6) underwent laparotomy alone for 7 h. The I/R group (n = 6) underwent 60 min of left and median lobe inflow occlusion and 6 h of reperfusion. The glycine + I/R group (n = 6) underwent a same procedure to controls after receiving glycine 5 mg/kg intravenous infusion for over 15 min. Bile flow was collected, measured, and analyzed by proton magnetic resonance spectroscopy. Glycine prevented the significant reduction in bile flow seen in I/R at 6 h reperfusion (145.0 +/- 11.4 vs. 108.3 +/- 28.2 mu L/min/g, p = 0.011). It reduced phosphatidylcholine shedding (1.2 +/- 0.8 vs. 3.0 +/- 0.5 mu mol/L, p = 0.001) and lactate surge (8.1 +/- 4.3 vs. 26.3 +/- 78 mu mol/L, p < 0.001) compared to I/R. Glycine significantly stimulated bile acid (17.9 +/- 2.8 vs. 8.9 +/- 2.1 mu mol/L, p = 0.001), pyruvate (1.3 +/- 0.3 vs. 0.7 +/- 0.1 mu mol/L, p = 0.005), glucose (3.9 +/- 0.9 vs. 1.6 +/- 0.6 mu mol/L, p = 0.007), and acetoacetate (0.7 +/- 0.1. vs. 0.4 +/- 0.1 mu mol/L, p = 0.009) release in bile on reperfusion compared to I/R. Glycine induced a reduction of liver I/R results in increased bile flow and altered bile composition.
Cell Preservation TechnologyVol. 6, No. 1 From the Editor's DeskISBER: Best Practices for Repositories and Trends at the Institute for Problems of Cryobiology and MedicineJ.G. BaustJ.G. BaustSearch for more papers by this authorPublished Online:1 Apr 2008https://doi.org/10.1089/cpt.2008.9996AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "ISBER: Best Practices for Repositories and Trends at the Institute for Problems of Cryobiology and Medicine." , 6(1), p. 1FiguresReferencesRelatedDetailsCited ByLong-term archival of environmental samples empowers biodiversity monitoring and ecological research10 May 2022 | Environmental Sciences Europe, Vol. 34, No. 1Long-term stabilization of DNA at room temperature using a one-step microwave assisted process23 March 2021 | Emergent Materials, Vol. 5, No. 2Analysis of the methods for projection of spaces for good manufacturing practice29 December 2019 | South East European Journal of Architecture and Design, Vol. 2019Tagging of Test Tubes with Electronic p-Chips for Use in Biorepositories Wlodek Mandecki, Wesley M. Kopacka, Ziye Qian, Von Ertwine, Katie Gedzberg, Maryann Gruda, David Reinhardt, and Efrain Rodriguez1 August 2017 | Biopreservation and Biobanking, Vol. 15, No. 4Analysis of Heterogeneous Concepts during Projection of Facilities of Good Manufacturing Practice and Human Biobank11 November 2015 | South East European Journal of Architecture and DesignMarking Shifts in Human Research Ethics in the Development of Biobanking5 September 2014 | Public Health Ethics, Vol. 8, No. 1Establishing and managing a periodontal biobank for research: the sharing of experience7 August 2014 | Oral Diseases, Vol. 21, No. 1Informed Consent for Biobanking Research: Cancer Patient Recruitment from Rural Communities in Maine17 April 2013 | Biopreservation and Biobanking, Vol. 11, No. 2The evolution of biobanking best practicesClinica Chimica Acta, Vol. 413, No. 19-20Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples29 December 2011 | Biopreservation and Biobanking, Vol. 9, No. 4Is There a Duty to Share? Ethics of Sharing Research Data in the Context of Public Health Emergencies17 February 2011 | Public Health Ethics, Vol. 4, No. 1Setting up a Prospective Thyroid Biobank for Translational Research: Practical Approach of a Single Institution (2004–2009, Pasteur Hospital, Nice, France)7 April 2011 | Biopreservation and Biobanking, Vol. 9, No. 1Principles of Research Tissue Banking and Specimen Evaluation from the Pathologist's Perspective18 December 2010 | Biopreservation and Biobanking, Vol. 8, No. 4Designing and managing a flexible and dynamic biorepository system: a 15-year perspective from the CPCRA, ESPRIT, and INSIGHT clinical trial networksCurrent Opinion in HIV and AIDS, Vol. 5, No. 6A hands-on overview of tissue preservation methods for molecular genetic analyses9 March 2010 | Organisms Diversity & Evolution, Vol. 10, No. 1Tumor Grade Predicts Tumor Proportion in Fresh Frozen Tissue Samples for Breast Cancer Research12 November 2008 | Cell Preservation Technology, Vol. 6, No. 3 Volume 6Issue 1Mar 2008 Information© 2008 Mary Ann Liebert, Inc.To cite this article:J.G. Baust.ISBER: Best Practices for Repositories and Trends at the Institute for Problems of Cryobiology and Medicine.Cell Preservation Technology.Mar 2008.1-1.http://doi.org/10.1089/cpt.2008.9996Published in Volume: 6 Issue 1: April 1, 2008PDF download
Cumulative osmotic stress models have been previously used to successfully describe the dehydration kinetics of recalcitrant seeds. For example, Liang and Sun demonstrated that a cumulative stress model effectively described the dehydration rate-dependence of recalcitrant seed viability under various drying conditions. In contrast, most studies describing the functionality of mammalian cells following drying conditions have been end-point oriented, describing cell viability as a function of the final moisture content reached or the duration of drying. This study applies a thermodynamics-based water metric similar to the Liang and Sun model to describe the viability of J774 mouse macrophage cells as a function of both moisture content and time of drying. Cells were incubated in full-complement DMEM media containing 50 mM trehalose, to enable trehalose loading by endocytosis. Treated cells and untreated controls that were not previously incubated in trehalose were dried in hypertonic (508 mOsm) and isotonic (308 mOsm) solutions of trehalose (200 mM) in phosphate-buffered saline (PBS). Various levels of dehydration were achieved by placing droplets of cell suspension in a desiccator for time periods up to 2 h. Cells were then immediately rehydrated and viability was assessed 45-min after rehydration. Cell viability was evaluated using a combination of Trypan Blue staining for membrane integrity of detached cells and Calcein AM-ethidium bromide fluorescence as a live-dead assay for attached cells. The cellular response was then evaluated as a function of cumulative osmotic stress, defined as the integral of the deviation in osmolality from isotonic conditions as a function of time. The results of this modeling suggested that significant cell injury was occurring in a moderate osmolality range, and that modulation of osmotic stresses in this range could lead to improved processing outcomes.
Cell therapy requires proper techniques to store cells in good viability conditions. Liquid nitrogen containers may store cells indefinitely, but samples may suffer cross-contamination or bacterial contamination when they are manipulated. Cultured human chondrocytes are commonly employed in cell therapy. In this study, we show a simple system to store them at room temperature. We investigated 12 strains of cultured chondrocytes. First, we studied four strains, which were stored in different conditions. Later, we Studied the other eight strains, which were maintained in the optimal viability condition obtained from the first experiment (room temperature with DMEM and HAM-F12 as maintenance medium). Using HAM-F12 as the maintenance medium, the viability after 15 days was similar to that of the control cells. After 30 days, the average viability was 87%. In both cases, cells were able to reach exponential growth when restored to 37 degrees C. They could also synthesize specific cartilage extracellular matrices after being cultured in agarose gel. As a result, they could be used for therapeutic purposes.
Tumor banks are the only means for researchers to study in situ human lesions in a reproducible manner. Large amounts of money are spent at national and international levels for proper collection, in respect of both quality control and ethics, most importantly for shared use of the human samples. Preservation as whole tissue, and not simply as extracted DNA or RN, allows additional scientific use of the samples. However, economic issues influence long-term development of biobanks since low-temperature cryopreservation with long-term storage of numerous samples and transfers avoiding RNA degradation are particularly costly. We optimized freeze-drying protocols for tumor samples dedicated to molecular analyses in our biobank using directly snap-frozen samples as references. We then compared different long-term storage conditions for preservation of nucleic acids in tumor samples to corresponding normal tissues and human cell lines. When stored for 1 year at room temperature with dessicant and no light, cell and tissue samples allowed for the extraction of nucleic acids, retaining their initial quality and Suitable for quantitative molecular analyses. An extended use of the freeze-drying process in tumor banks could favor a larger scientific use of the samples and the standardization of preanalytical methods in molecular biology while reducing the cost of safe long-term preservation and transfers.
Although the disaccharide trehalose has been shown to protect cells during freezing and desiccation when present intracellularly, a major obstacle to using intracellular sugars for biopreservation applications is the impermeability of plasma membranes. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. Previous work has shown that the mechanism of red blood cell (RBC)–liposome interaction includes both liposome fusion with the RBC membranes, as well as tight adsorption of the vesicles onto the RBC surface. However, the fusion efficiency of liposomes was low, with only micromolar concentrations of trehalose delivered to the RBC cytosol. The purpose of this study is to enhance the efficacy of liposomes’ delivery of trehalose into RBCs with minimal detrimental effects on RBC membrane quality, by manipulating liposome physical properties and liposome–RBC incubation conditions. Charged and uncharged un...
BACKGROUND: In many cohort studies, biological specimens are being stored without specific plans for analyses. In the Norwegian Mother and Child Cohort Study biological specimens (DNA, plasma, and whole blood) are stored on 96-well plates and as a result may undergo multiple freeze-thaw cycles. METHODS: To explore the impact of multiple freeze-thaw cycles on chemical constituents, we conducted a quality control study using pooled EDTA-plasma. Over a two-year period, samples stored at -80 degrees C were subjected up to 100 freeze-thaw cycles. Specimens were analyzed in triplicate for sodium, cholesterol, triglycerides, vitamin E, aspartate aminotransferase (AST), and free fatty acids. We assessed the percent change of analyte concentration from the values for the first freeze-thaw cycle, because this is the baseline for all stored specimens. RESULTS: With the exception of free fatty acids, there was little change over the first 10 freeze-thaw cycles. A majority of analytes showed no significant changes until 30 freeze-thaw cycles. After 30 freeze-thaw cycles, the largest percent change was observed for free fatty acids (+32%), AST (+21%), and triglycerides (-19%). CONCLUSIONS: Human plasma can go through several freeze-thaw cycles before analysis without influencing sample integrity for the selected analytes.
The National Institute of Standards and Technology (NIST) environmental specimen banking system consists of two environmental specimen banks (ESBs): the National Biomonitoring Specimen Bank established in 1979, and the Marine Environmental Specimen Bank established in 2002. Both facilities were specifically designed to store environmental specimens over long periods of time (50-100 years) and in such a way that future researchers could use these specimens to answer questions regarding trends in newly recognized environmental contaminants and verification of past analytical results. The NIST environmental banking system maintains collections of human liver specimens, human blood serum and blood spots, human diet samples, marine sediments, fish tissues, mussels, oysters, marine mammal tissues, and bird eggs and feathers collected as part of several monitoring and research programs supported by the U.S. Government. The NIST environmental banking system emphasizes: (1) carefully designed (and published) collection and banking procedures, (2) cryogenic storage to ensure sample stability, (3) high efficiency particulate air (HEPA)-filtered clean air conditions in the sample preparation and freezer rooms, (4) cryogenic homogenization systems for sample preparation, (5) computerized sample inventory and tracking system, (6) computerized security and monitoring systems, and (7) redundancy to minimize sample loss due to equipment or system failure. The NIST ESBs provide a resource of research specimens that are used to address questions regarding temporal and geographic trends in environmental contamination (including documentation of newly recognized contaminants), changes in ecosystem structure and function, genetic separation of populations of animals, and the health status of marine animals.
Clinical trials using dendritic cells (DCs) to treat cancer patients have generated promising results in recent years. However, even simple aspects of this therapy are still not well understood, including the storage and distribution of manufactured vaccines. These processes are essential and must be elucidated in order to reduce costs. We evaluated the effects of different storage conditions on vaccine functionality using mixed lymphocyte reaction (MLR). Vaccine storage at 4°C for up to 72 h had no significant effect on vaccine activity. Shipping to distant places is possible, if vaccines are kept at 4°C and used up to 3 days after manufacture date.
