The use of an inexpensive processing aid device (the Mouse PAD) to facilitate rodent tissue banking

One of the few places in the world where the space radiation environment can be simulated and its impact on tissues and living organisms studied is at the National Space Radiation Laboratory (NSRL) located in Brookhaven National Laboratories, New York [1]. NSRL staff reports that an hour of this experimental radiation costs $5800 [1], and this amount does not account for the additional costs and challenges associated with transporting and housing the animals between research institutions. These experimental animals undergo whole-body irradiation and thus all tissues from these animals are valuable for various types of investigation.

The novelty of the experimental conditions (space radiation) and the limited availability of these organs elsewhere prompted our establishment of a repository of over 5000 tissues from mice exposed to space radiation. The majority of the tissues and organs from these experimental animals were collected and preserved. To establish this repository, we developed a device, the mouse Processing Aid Device (Mouse PAD) that streamlines the preparation of materials, tissue collection and tissue processing. The Mouse PAD is affordable, easy to use and produces high-quality, consistent results. It can also be easily adapted by others to establish their own unique rodent biorepositories.

Materials & methods

Animals

All procedures were approved by both the home institution and Brookhaven National Laboratory Institutional Animal Care and Use Committees (IACUCs).

Male C57BL/6J mice (5 months of age) were purchased from Jackson laboratories. All animals were randomly assigned an experimental group, then transported to NSRL, where they were subjected to full-body irradiation or were ‘sham irradiated’. All animals were then transported back to the home institution and periodically evaluated for physiological changes over the next 11–18 months. At the end of the observational period, all mice were euthanized and their organs and tissue collected and stored.

Processing aid device: design & printing

The Mouse PAD was designed in Autodesk Inventor (Autodesk, CA, USA), exported as a .STL file, and then 3D-printed using 3dprinterOS, an online printing software (3dprinteros.com). The device was printed using polylactic acid in an Ultimaker 2 printer (Ultimaker, Geldermalsen, Netherlands). The design.STL files are available for download and modification as labs require [2].

Euthanasia & surgical tissue harvest

Mice were euthanized singly under isoflurane by exsanguination via direct cardiac puncture as approved by our institutional IACUC. All materials for tissue and organ harvesting – including 1-ml Luer-Lok syringes and 25G, 1½″ needles (Becton Dickinson, NJ, USA), surgical instruments (Metzenbaum scissors and tissue forceps), a Styrofoam board to serve as the dissecting area, liquid nitrogen for flash freezing tissue, and appropriate vials (such as cryovials or microcentrifuge tubes) primed with EDTA for blood collection – were prepared in advance. One assembled Mouse PAD was used for each surgical procedure.

Appropriate vials were inserted into the mouse PAD in preparation for collection. The vials were either empty or formalin-filled, depending on the preservation requirements of the planned downstream tissue analysis. Exsanguination was performed using a percutaneous subxiphoid approach and direct cardiac puncture, and the blood collected in tubes with 10 μl EDTA (0.5M, pH 8.0) on ice. Forceps were then used to grip the xiphoid process, using the Metzenbaum scissors to cut laterally on both sides (clamshell approach) and caudally (sternotomy) to open the thoracic cavity. The heart was excised first and flash frozen. The right and left lung, the thymus and the aorta were then excised. Once all organs were removed from the thoracic cavity, a cut was made from the diaphragm to the right ankle to open the abdominal cavity and expose the skeletal muscle of the right leg. The liver and bladder were removed. The surgeon then excised, in order, the spleen, left kidney, intestines, right kidney and skeletal muscle. The animal was then rotated to the prone position, and the eyes were removed by simple enucleation. A superior cut was then started at the base of the skull, followed by a shallow cut up the sagittal suture, and the whole brain then carefully removed. As the organs were excised, each was placed into its corresponding vial in the mouse PAD.

Results & discussion

Overview of experimental design

Each assembled mouse PAD (Figure 1A) consists of a labeled display lid (Figure 1B) depicting the outline of a dissected mouse. This design was chosen in order to provide the surgical technician with a reference for tissue placement. The lid of the device has labeled cavities designed to hold cryovials of appropriate size for each sample (larger for liver and intestines) in the general anatomical location of the specific organs to be harvested. These cavities in the lid align with appropriately sized supports in the base (Figure 1C) to hold all cryovials securely without assistance. The base can also be packed with ice if desired, to keep samples cold and decrease ischemic time. The display lid required approximately 9 h to print and the base for holding the tubes required approximately 20 h.

Mouse PAD use

In our laboratory, most tissue collections are conducted by two technicians, one being the surgeon and the second the tissue processor. During the surgical time, the tissue processor prepares materials for exsanguination, places the next animal into the isoflurane chamber, loads labeled vials into an empty Mouse PAD, and stores the completed vials in racks for later organization into cryo-boxes. Using the Mouse PAD, we procured approximately 5000 tissues and organs from over 350 irradiated mice. The Mouse PAD keeps all tubes organized and stationary, freeing the tissue processor's hands to prepare the materials, Mouse PAD and animal for the following procedure. Thus the surgical technician is allowed to focus solely on dissection and organ collection. The surgical technician simply places the excised tissue into the easily visualized vial, without the need for assistance or direction from the processor. Upon completion of the first surgical procedure, the processor may quickly remove the full Mouse PAD and replace it with a newly prepared Mouse PAD. While the surgical technician starts the next surgery, the processor can quickly cap the vials containing the procured tissue, move them to cold storage, and start preparing for the next procedure. The primary benefit of the Mouse PAD is that all the tissues are kept in order, and the risk of committing errors (such as placing tissue into the wrong tubes) is reduced.

Efficiency testing: surgical procedure duration

To determine the effect of the Mouse PAD on processing efficiency, we measured the duration of surgical procedures in different scenarios with and without the Mouse PAD. First, we examined whether the Mouse PAD provided a benefit with two experienced technicians (Figure 2A). In this scenario the use of the Mouse PAD slightly slowed the procedure. This was possibly due to the few seconds required to preload the device with the tubes.

While no benefit was observed with two experienced processors, use of the Mouse PAD significantly improved the processing time when two inexperienced technicians (Figure 2B) or a single experienced processor (Figure 2C) were harvesting tissue. With two inexperienced technicians, the use of the Mouse PAD reduced the harvest time from an average of 9 min 13 s (n = 8) to an average of 7 min 13 s (n = 6) (Figure 2B). Furthermore, the Mouse PAD helped in the scenario where only one experienced technician was available and acted in the roles of both surgeon and processor (Figure 2C). During these solo procedures, the Mouse PAD reduced the average harvest time from 10 min 9 s (n = 4) to 8 min 29 s (n = 4).

The Mouse PAD reduced dissection time by up to 21.7% of the original required time. Use of the PAD made harvesting much more efficient and was key to the development of our rodent biobank. The Mouse PAD enabled 2 technicians to process as many as 18 animals in a single workday.

A lack of established procedures in biobanking has previously been reported to contribute to variability between sample collection procedures that hinders the reproduction of study findings [3]. The Mouse PAD is useful in minimizing ischemic time and reducing variability in the time it takes for dissection and preservation, thereby making these procedures more efficient and consistent.

The placement of excised organs into the correct vials during a hectic surgical dissection session has been the rate-limiting step and the most frequent source of frustration and variability during organ procurement. The visual reminders of the Mouse PAD helped to reduce the risk of these unintended processing errors – for example, putting a tissue sample into the incorrect vial – as demonstrated in the decreased time required for the process. Furthermore, the Mouse PAD makes the collection process efficient even in the situation where only one technician is available.

A change in biobanking team personnel can also affect sample quality and consistency, just as human behavior can affect the collection and processing steps. The use of the Mouse PAD allows an inexperienced team member to quickly learn and adapt to the harvesting procedure. This is especially helpful in cases where language barriers exist between team members, given the visual nature of the device. The device also serves as a checklist of sorts in these situations, allowing an inexperienced surgeon an easy view of which organs have already been excised, and which they must still dissect. Utilizing the PAD during personnel transitions will help to maintain the quality and consistency of the biobank's samples (Table 1).

Future perspective

The use of 3D-printed devices is becoming more commonplace in many research labs, because of its affordability in prototyping; many institutions are making 3D printers widely available to their associated researchers. The example of the Mouse PAD for tissue banking demonstrates the utility of this technology and its wide range of applications, while also improving the organization and efficiency of past tissue banking techniques.