FTIR and confocal Raman microspectroscopy were used to measure interactions between albumin and ice in situ during quasi-equilibrium freezing in dimethyl sulfoxide (DMSO) solutions. At temperatures of −4 and −6 °C, albumin was found to be preferentially excluded from the ice phase during near-equilibrium freezing. This behavior reversed at lower temperatures. Instead, DMSO was preferentially excluded from the ice phase, resulting in an albumin concentration in the freeze-concentrated liquid phase that was lower than predicted. It is hypothesized that this was caused by the albumin in the freeze-concentrated liquid getting adsorbed onto the ice surface or becoming entrapped in the ice phase. It was observed that, under certain freezing protocols, as much as 20% of the albumin in solutions with starting concentrations of 32–53 mg/mL may be adsorbed onto the ice interface or entrapped in the ice phase.
Biorepositories worldwide collect human serum samples and store them for future research. Currently, hundreds of biorepositories across the world store human serum samples in refrigerators, freezers, or liquid nitrogen without following any specific cryopreservation protocol. This method of storage is both expensive and potentially detrimental to the biospecimens. To decrease both cost of storage and the freeze/thaw stresses, we explored the feasibility of storing archival human serum samples at non-cryogenic temperatures using isothermal vitrification. When biospecimens are vitrified, biochemical reactions can be stopped, the specimen ceases to degrade, and macromolecules can be stabilized without requiring cryogenic storage. In this study, 0.2, 0.4, or 0.8 M trehalose; 0, 0.005 or 0.01 M dextran; and 0 or 10% (v/v) glycerol was added to human serum samples. The samples were either dried diffusively as sessile droplets or desiccated under vacuum after they are adsorbed onto glass microfiber filters. The glass transition temperatures (Tg) of the desiccated samples were measured by temperature-ramp Fourier Transform Infrared (FTIR) spectroscopy. Sera samples vitrified at 4 ± 2 °C when 0.8 M trehalose and 0.01 M dextran were added and the samples were vacuum dried for two hours. Western immunoblotting showed that vitrified serum proteins were minimally degraded when stored for up to one month at 4 °C. About 80% of all proteins were recovered after storage at 4 °C on glass microfiber filters, and recovery did not decrease with storage time. These results demonstrated the feasibility of long-term storage of vitrified serum at hypothermic (and non-cryogenic) temperatures.
Despite abundant research conducted on cancer biomarker discovery and validation, to date, less than two-dozen biomarkers have been approved by the FDA for clinical use. One main reason is attributed to inadvertent use of low quality biospecimens in biomarker research. Most proteinaceous biomarkers are extremely susceptible to pre-analytical factors such as collection, processing, and storage. For example, cryogenic storage imposes very harsh chemical, physical, and mechanical stresses on biospecimens, significantly compromising sample quality. In this communication, we report the development of an electrospun lyoprotectant matrix and isothermal vitrification methodology for non-cryogenic stabilization and storage of liquid biospecimens. The lyoprotectant matrix was mainly composed of trehalose and dextran (and various low concentration excipients targeting different mechanisms of damage), and it was engineered to minimize heterogeneity during vitrification. The technology was validated using five biomarkers; LDH, CRP, PSA, MMP-7, and C3a. Complete recovery of LDH, CRP, and PSA levels was achieved post-rehydration while more than 90% recovery was accomplished for MMP-7 and C3a, showing promise for isothermal vitrification as a safe, efficient, and low-cost alternative to cryogenic storage.Cancer is one of the leading causes of mortality, accounting for approximately 23% of all deaths in the U.S. each year 1 . Early detection and continuous monitoring for recurrence are essential for a positive prognosis, as it is at its initial stages that the disease is most responsive to therapeutic intervention. Early detection focuses on diagnosing the disease before clinical symptoms arise; for example, by detecting the presence of certain cancer biomarkers found in bodily fluids such as the blood 2,3 . Therefore, studies focusing on discovery of highly sensitive and specific cancer biomarkers have become increasingly prevalent [3][4][5] . In spite of the advances in fast and sensitive analytical detection methodology and the vast amount of research conducted evaluating thousands of molecular signatures as potential biomarkers for cancer (detailed in more than 150,000 reports published to date), less than two dozen biomolecules have currently been approved for clinical use by the Food and Drug Administration (FDA) 6,7 . An even smaller number is found in the blood, which is home to more than 10,000 potential biomarkers 8,9 . One of the main reasons for the inefficient and slow progress is the poor informational quality of the collected human biospecimens (tissue samples, bodily fluids, etc.) used in biomarker detection and validation studies. A significant fraction of the collected biospecimens is known to be compromised due to sub-optimal handling and storage conditions 10,11 . Biomarker development is composed of a series of phases including discovery, verification, and clinical validation, which require large numbers of high quality biospecimens 12 . For this purpose, millions of "archival" biospecimens are continuously ...
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