Biopreservation and Biobanking

The abstracts that follow demonstrate the broad range of timely issues addressed in the contributed oral and poster presentations within the ISBER 2020 Educational Program Series held virtually throughout 2020.

The cryobanks of agouti somatic tissues represent a promising tool for the conservation of this species and of those that are phylogenetically related and endangered. For these purposes, one strategy to guarantee the quality of samples after warming would be to choose the appropriate tissue vitrification technique. Therefore, we evaluated the effects of two different techniques, direct vitrification in cryovials (DVC) and solid-surface vitrification (SSV), on the preservation of ear somatic tissues derived from agoutis kept in a scientific center of creation. Noncryopreserved somatic tissues were used as controls. Although SSV reduced the thickness of the dermis and cartilage (p < 0.05), the epidermal thickness of these samples was observed to be similar to controls (p > 0.05). Notably, the number of fibroblasts was not altered with either technique. However, both vitrification methods led to an increase in the number of perinuclear halos, with a particularly strong increase observed in DVC-derived fragments (p < 0.05). Compared with the DVC group, SSV showed a larger number of normal chondrocytes and smaller number of degenerate chondrocytes. Furthermore, the number of empty lacunae in SSV-derived fragments remained similar to controls (p > 0.05). In summary, SSV was found to be a more efficient method for vitrifying agouti somatic tissues compared with DVC. These results are important for the proper formation of agouti somatic banks, an essential step in the study of biological resources in this species.

In the present study, four experimental groups were used: fresh embryos, cultured during in vitro maturation and in vitro culture in media supplemented with bovine serum albumin (BSA) (fresh BSA) or fetal bovine serum (FBS) (fresh FBS); and two groups of cryopreserved and thawed embryos, produced under the same conditions (frozen BSA and frozen FBS). Experiment 1 evaluated the protein source effect on embryo development and response to cryopreservation. At day 7, half of the expanded blastocysts (Bx) from each group were cryopreserved and warmed and the other half were used as controls. After warming, embryos were incubated under the same conditions for 48 hours, and the hatching rate was measured at 24 and 48 hours. The total and the apoptotic cell numbers were measured in a subset of Bx after 24 hours. Experiment 2 used the Bx of experiment 1 to compare the expression of KRT8, PLAC8, FOSL1, HSP1A1, and HSPA5 genes in hatched blastocysts at 24 and 48 hours for all groups. The FBS group showed a higher percentage (p < 0.05) of embryos (42.8% vs. 27.9%) and higher rates of Bx (75.0% vs. 63.8%) on day 7, compared with the BSA group. At 24 hours postwarming, the fresh FBS group showed the highest hatching rate (p < 0.05) in comparison with other treatments. However, at 48 hours, the hatching rate was similar (p > 0.05) among groups: fresh FBS (68.1% ± 23.3%), fresh BSA (70.0% ± 31.0%), frozen FBS (39.2 ± 27.1), and frozen BSA (38.2 ± 23.9). After 24 hours, frozen BSA showed a higher number of cells compared with frozen FBS (p < 0.05). The expression of the PLAC8 gene was higher (p < 0.05) in fresh BSA embryos compared with frozen FBS embryos at 24 hours. In the present study, BSA replacement reduced embryo development, but did not affect the response to cryopreservation. However, upregulation of the PLAC8 gene suggests that embryos cultured in BSA might have better quality to support further development.

Biobanking has become an indispensable tool for translational research and health innovations. While the field of biobanking has progressed and evolved globally, biobanking in developing Association of Southeast Asian Nations (ASEAN) countries such as the Philippines remains underrepresented because of several challenges often encountered in these low- and middle-income countries. Recently, the Philippine government has undertaken enormous efforts to advancing research and development in the country, and one of the current research pursuits is the establishment of biobanks, with the hope of attaining more discoveries and innovations in the future. Given that cancer remains a leading cause of death in the Philippines, the Philippine government supported the establishment of a cancer biobank at the Philippine General Hospital (PGH). In this study, we present a specific use case of biobanking activity at the PGH Biobank, to build a cohort of biospecimens from Filipino patients with breast, endometrial, and ovarian cancer. This initiative is part of a biomonitoring study (1) to assess environmental exposures and possible risk factors in the Philippine population and (2) to develop a system of culturing human cells from Filipino patients for subsequent in vitro studies. We discuss issues faced and the solutions developed during the implementation of the biobank. Strong research collaboration, a funding source, basic infrastructure, and appropriate technology helped initiate this pilot biobank in the Philippines. Overall, the experiences of establishing the PGH Biobank may help other institutions in low-resource countries to set up cancer biobanks.

The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at −20°C, −80°C, −196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at −20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (−80°C and −196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (Bcl6b, Erm, Dazl, and Sycp1) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor^™ media, and frozen using a slow freezing method (−1°C/min in −80°C) for 24 hours and then stored in liquid nitrogen. After 15–18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

Cryopreservation of red blood cells (RBCs) has been studied as a typical example of cryobiology methodology. To date, a mature and long-term cryopreservation process for RBCs has been developed, which has the weakness of complicated procedures due to high concentrations of glycerol (Gly). Therefore, it is still a research focus to find a new method for cryopreservation of RBCs to reduce the concentrations of cryoprotectants (CPAs). In this study, alginate hydrogels, which have been widely used in preservation research, were selected because of their advantages such as lower cost, cytocompatibility, and crosslinking occurring under mild conditions. With a variety of CPA solutions, the RBC recovery with the cryopreservation of RBCs and RBC-laden hydrogel microfibers was compared in different situations. It was found that hydrogel microfiber encapsulation enhances cryopreservation of human RBCs with low concentrations of CPAs. And Gly can be removed by washing directly with a 0.9% sodium chloride solution. This study validated the feasibility of cryopreservation of RBC-laden hydrogel microfibers. It may provide a new and evolving direction for reducing the concentrations of CPAs used in the preservation of RBCs.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

The genetic diversity of livestock is decreasing and many countries have created gene banks for ex situ–in vitro conservation of animal genetic resources (AnGR). The collection, processing, and storage of animal germplasm require substantial investment and the material collected (and associated data) is highly valuable. Therefore, quality management systems (QMSs) and practices are important. The objective of this study was to review the quality management procedures of livestock gene banks around the world to identify the general strengths and weaknesses of quality control. A survey was administered by means of an online questionnaire consisting of 54 questions, most of which were yes/no with respect to the presence of a particular aspect of quality management. The survey was distributed through networks of the Food and Agriculture Organization of the United Nations that are associated with AnGR. Ninety responses were received from 62 countries. The gene banks were predominantly public institutions, with the main goal of preventing breed extinction. Approximately 30% of the banks reported having a QMS, 15 of which involved formal certification. Many other banks have plans to implement formal quality management within the next 5 years. Regarding specific aspects of quality management, more emphasis was placed on material entering the banks than on eventual utilization. Among the banks processing and freezing material, 90% followed specific standard operating procedures, but only 24% had policies regarding provision of access to external stakeholders. Increased cooperation among livestock gene banks could improve quality management. Sharing of knowledge could standardize procedures and cooperating peers could evaluate each other's QMSs.