Why BioCoR?

Biospecimen procurers and users are separated by gaps, which are typically both phyisical (different location) and temporal (different time).  Therefore, the usefulness of a biospecimen is determined in large part by our ability to efficiently preserve the critical biological properties of the biospecimen (e.g. stabilization of the biomarkers) and its function (e.g. for therapeutic purposes).  Conventional methods of preserving biospecimens were developed in the 1970's and there has been little evolution in the techniques used to preserve biospecimens in the intervening decades.  As a result, today we are in a situation where we have very limited knowledge, technology and resources (and thus we fail) to successfully preserve the many promising diagnostic and therapeutic biospecimens we have discovered to date (see examples below).  Lack of progress in biopreservation area has resulted partially from certain misconceptions: 

Misconception #1:  We have effective methods of preservation for all the biospecimens of interest.

Reality: Many biospecimens of tremendous interest cannot be effectively preserved. The following is a partial listing of biospecimens that respond poorly to conventional preservation methods (poor recovery, brief shelf life, poor retention of function, etc.):

  • Platelets can be stored at room temperature for up to 5 days [1].
  • Granulocytes can be stored for only 24 hours [2].
  • Human embryonic stem (hESCs) cells exhibit poor post-thaw colony attachment (5-30)% and a high degree of differentiation. Current methods with the best outcome are appropriate only for small scale applications [3]. The freezing behavior of Induced pluripotent stem (IPS) cells [4] (that have similar biological properties and therapeutic potential to hESCs) has not been studied at all.
  • Cells isolated from many types of tissues (tendon, heart valve, blood vessel, etc) can be successfully cryopreserved as suspensions but the same success cannot be achieved when the cells are cryopreserved in the intact tissue [5]. Tissues for diagnostic/epidemiological applications exhibit significant changes in gene and protein expression profiles with as little as 30 minutes of ischemia [6]. These results suggest that current methods of stabilizing tissue are particularly poor and need improvement.

Reality: The growth in the number and variety of biomarkers being studied has made current methods of processing and preserving biospecimens for diagnostic/epidemiological purposes obsolete. Currently, we know that biomarkers within the same biospecimen show different degrees of susceptibility to preservation and the disproportionate loss or deactivation of one biomarker relative to another limits the usefulness of the biospecimen as a whole. For example, in plasma vitamin C and the fatty acids are extremely vulnerable to one-time, short duration exposure to room temperatures, while biomarkers for iron status (soluble transferrin receptor, ferritin, C-reactive protein and a1-acid glycoprotein) are extremely stable at room temperature[7]. Also, the specific procurement technique employed affects the stability of the biomarkers during storage. In heparinized whole blood, biomarkers of oxidative stress (malondialdehyde and esterified F2-isoprostanes) increase very significantly (~100%) over a 36 hour period while the fluorescent oxidation product levels remain unchanged [8]. Similarly, TIMP-1 values show significant variability between plasma, citrate plasma and serum collected using ethylenediaminetetraacetic acid (EDTA). BioCoR aims to develop specific preservation protocols to maximize storage stability and function while maximizing storage time.

Misconception #2: Conventional methods of preserving biospecimens that are successful are suitable for new and emerging applications.

Reality: The vast majority of cells that are cryopreserved for therapeutic applications (lymphocytes, HSCs, MSCs) use a cryopreservation solution containing 10% v/v dimethylsulfoxide (DMSO). Despite its clinical use as a cryoprotective agent, DMSO is not approved for systemic administration, including intravascular infusion. The infusion of cryopreserved cells containing DMSO into humans has been associated with various adverse events: nearly all patients receiving cryopreserved HSC transplants containing DMSO experience nausea, chills, hypotension, dyspnea and cardiac arrhythmia [9-12]. Case studies also illustrate more serious reactions in a smaller number of patients. For example, cardiac arrest [13, 14], transient heart blockage [15], neurological toxicity [16], renal failure [9, 17], and respiratory arrest [18] have been documented subsequent to infusion of DMSO-containing HSC products. Twenty to thirty years ago, the only patients receiving cryopreserved cells were leukemia patients receiving hematopoietic stem cell transplants, a one-time event to treat a life-threatening disease. Today, the number of disorders and patients being treated with cell therapies is increasing rapidly [19, 20] and many involve the infusion of multiple products. Clearly, the development of a non-toxic and effective method of preserving cells to be administered therapeutically would benefit greatly the field of cell therapy. DMSO is also used to preserve biospecimens for diagnostic and epidemiological studies. Low levels of DMSO have been associated with a series of epigenetic events such as DNA methylation and histone modification [21]. A lesser known fact is that at specific concentrations DMSO causes irreversible denaturation of macromolecules [22]. The manner by which this practice affects results of chromatin structure studies in epigenetic research is an important factor to consider. The development of effective alternatives to DMSO therefore would reduce the unknowns related to stability and quality of diagnostic/epidemiological biospecimens during storage. The development of alternatives to DMSO is one of the projects to be undertaken by BioCoR.

Misconception #3:  Current technology for cell preservation is adequate

Reality: Existing technology does not permit us to monitor biospecimen quality during transportation and long storage. Ideally, we would want to continuously monitor biospecimen quality at all times during processing and storage and use these measurements to qualify (or disqualify) a specific biospecimen. Unfortunately, there is no direct method of monitoring biospecimen quality, in particular at low temperatures. Currently, the best measure of biospecimen ‘quality’ is the temperature history of the sample. During cryopreservation, cells must traverse temperatures ranging from physiological (~37oC) to liquid nitrogen (~ -196oC). The post-thaw survival of the cells is influenced by the manner in which this is accomplished. The cooling rate experienced during freezing directly influences the post-thaw viability of cells [23, 24]. The temperature at which ice forms in the extracellular environment also influences the post-thaw viability of a cell [25]; 3) During storage, the shelf-life of the biospecimen is determined by the storage temperature (the higher the storage temperature, the shorter the shelf-life) [26, 27]. Temperature fluctuations resulting from introduction and removal of samples from storage have been shown to influence the post-thaw recovery of a biospecimen [28]. All of these factors demonstrate the importance of recording and monitoring temperature (and its history) as a measure of quality during freezing, storage and transportation.

Current technology for temperature measurement during processing for storage (freezing) is also limited. For samples frozen using a controlled rate freezer, the temperatures of the chamber and one dummy sample are recorded. The temperature history for all of the samples in the freezer is implied from the freezing behavior of the ‘dummy’ sample. However, studies demonstrate that during controlled rate freezing, cooling rates and temperatures at which ice forms in the extracellular solution can vary from sample to sample and between cooling runs for the same cooling program [25, 29]. During transportation, a single probe is placed in the shipping container and it is assumed that the temperature is uniform throughout the container. Similarly, a single temperature probe in the storage unit is used to represent the temperature of all samples in the Dewar. Entire liquid nitrogen storage units have been disqualified for use because the temperature at one point in the device reached the maximum acceptable temperature, resulting in all the specimens in the device to be considered unusable. BioCoR will develop wireless/batteryless temperature sensors to permit continuous monitoring of biospecimens during freezing, shipment and storage.

Reality: Current methods of processing biospecimens for preservation can result in significant losses. Children receiving cryopreserved umbilical cord blood (UCB) cannot tolerate the infusion of cells cryopreserved using DMSO. Current strategies for DMSO removal involve centrifugation of cells to form a pellet at the bottom of a bag; the supernatant is expressed and replaced with fresh wash solution and this process is repeated. The entire washing process takes 1.5-2 hours in the clinical lab (a significant amount of time) and losses of 27-30% of nucleated cells are reported [9, 30]. Cell losses can occur due to mechanical stresses on the cells during both centrifugation and expression of the supernatant.  Washing to remove DMSO requires significant intervention of a skilled operator in order to minimize losses to the levels quoted in literature. Cord blood harvests are single-time events with considerable effort made to maximize the number of cells harvested. Thus, cells lost during processing cannot be recovered, and the transplantation of low cell count has been shown to have a significant negative influence on transplant outcome [31, 32]. BioCoR will develop microfluidic devices capable of removing cryoprotective solutions with minimal cell losses.

The number of UCB units transplanted per year in children is relatively small (~thousands/year) but this example illustrates the time, labor and the loss of sample that can occur during a single step of processing for preservation. A RAND report on biorepositories conservatively estimated that annual accumulation of biospecimens exceeds 20 million specimens per year in the US alone [33]. Therefore, improvements in processing and biopreservation technologies are required to translate into improved biospecimen quality, reduced losses and reduced costs for preserving millions of biospecimens.

 

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