Development and validation of a sperm-freezing device created using 3D printer technology

Abstract

Objective: To develop and evaluate the effectiveness of a 3D-printed prototype to hold semen straws during the freezing process under safe and reproducible conditions.

Methods: A prototype capable of holding ten straws in liquid nitrogen vapor (LN2) was 3D printed. A second support that is commonly used was assembled from pieces of expanded polyethylene (EPS), respecting the identical distance between the straws and the LN2 surface. Temperatures were registered with a thermocouple placed inside a straw. Semen samples were frozen in the presence of cryoprotectant using the prototype (n=20) and the EPS support (n=20) in two independent series of measurements. Sperm parameters (motility, vitality, and DNA fragmentation) were measured for fresh and frozen-thawed samples.

Results: The temperature cooling curves measured on the prototype were remarkably reproducible. The prototype material withstood over 300 freezing cycles without damage. The mean motility and vitality of fresh (64.2%, 72.0%) and frozen-thawed (25.7%, 38.8%) samples were significantly different (p<0.001) using either support. Recovery rates of motility, vitality, and sperm DNA fragmentation in frozen-thawed sperm samples were equal regardless of straw position on the prototype or support type used.

Conclusions: The developed device allows a homogeneous, quantifiable, reproducible cooling of the straws in liquid nitrogen vapor. The recovery rates are comparable to those reported in the literature for both tested supports. The designed 3-D printed prototype favors the safe handling of the straws, an explicit way of describing freezing conditions, and a better intra-operator and inter-laboratory reproducibility of the cryopreservation process.

Keywords: 3D printer; cryopreservation; liquid nitrogen vapor; reproducibility; sperm.

Figure 1

Views of the straw holder, consisting of a hollow parallelepiped resting on a floating base. A/B: Three-dimensional projection of the prototype, and C: 3D-printed straw holder. The indentations allow ten straws to be placed securely in the horizontal position.

Figure 2

Hand-made device made of expanded polystyrene (EPS) used to support straws with seminal samples for floating in N2L vapor during cryopreservation. Side view on A and top view on B.

Figure 3

Temperatures recordings by two thermocouples (Akzo Instrumentos, Brazil), one inserted in a control straw in position 5 of the device and the second in ambient air. Three cooling periods are monitored: 1) for 20 min in a refrigerator, 2) for ten minutes in an expanded polystyrene container containing liquid nitrogen, the support is then in fluctuation mode, 3) for a variable period in liquid nitrogen (LN2) the straws being then released from the support.

Figure 4

Boxplot of the motility of fresh, thawed, and after 24h of culture at 37°C, using EPS support and ABS prototype, and recuperation rates (R)

Figure 5

Boxplot of the vitality of fresh, thawed, and after 24h of culture at 37°C, using EPS support and ABS prototype, and recuperation rates (R)