3-D printing provides a novel approach for standardization and reproducibility of freezing devices

Abstract

Cryopreservation has become an important and accepted tool for long-term germplasm conservation of animals and plants. To protect genetic resources, repositories have been developed with national and international cooperation. For a repository to be effective, the genetic material submitted must be of good quality and comparable to other submissions. However, due to a variety of reasons, including constraints in knowledge and available resources, cryopreservation methods for aquatic species vary widely across user groups which reduces reproducibility and weakens quality control. Herein we describe a standardizable freezing device produced using 3-dimensional (3-D) printing and introduce the concept of network sharing to achieve aggregate high-throughput cryopreservation for aquatic species. The objectives were to: 1) adapt widely available polystyrene foam products that would be inexpensive, portable, and provide adequate work space; 2) develop a design suitable for 3-D printing that could provide multiple configurations, be inexpensive, and easy to use, and 3) evaluate various configurations to attain freezing rates suitable for various common cryopreservation containers. Through this approach, identical components can be accessed globally, and we demonstrated that 3-D printers can be used to fabricate parts for standardizable freezing devices yielding relevant and reproducible cooling rates across users. With standardized devices for freezing, methods and samples can harmonize into an aggregated high-throughput pathway not currently available for aquatic species repository development.

Keywords: 3-D printing; Aquatic; Cryopreservation; Freezing rates; High-throughput; Standardized freezing devices.

Fig. 1

Diagram of the components of a prototype 3-D printed freezing device. A: Upper and lower freezing platforms (140 × 140 mm, L × W) with 50-columns (40-mm columns not shown). B: Raft lifter with polystyrene raft (152 × 152 mm × 13, L × W × D). The raft had a center-cut square that could be inserted or removed depending on desired freezing rates. C: Cut-away view of the nestable insulated shipper with insulated bio foam container.

Fig. 2

The positioning of data logger thermocouples used during freezing curve tests. All thermocouples were inserted into containers filled with Hanks’ balanced salt solution (300 mOsmol/kg). A: 0.5-ml French straws; B: 0.25-ml French straws; C: 2-ml Cryovials; D: 0.5-ml Nunc Bank-it tubes. A thermocouple (X) was used to record the ambient positional temperature profiles for A and B.

Fig. 3

Maximum temperatures recorded after 30 min for specific heights above liquid nitrogen within the double polystyrene boxes. These heights were below the highest position of the tallest device configuration. Therefore, all samples were exposed to positional temperatures at or below −80 °C.

Fig. 4

Representation of cooling curves for different container configurations. The freezing device was designed to provide a range of reproducible cooling rates for four containers: 0.5-ml French straws, 0.25-ml French straws, 2-ml Cryovial, and 0.5-ml Nunc Bank-it tubes. A range of cooling rates was produced by various combinations of rafts area (155 cm² and 542 cm²) and column height (40 mm, 50 mm, or none). Such cooling curves could be used for reference among different users. See Table 1 for average cooling rates.