Indicator Tubes: A Novel Solution for Monitoring Temperature Excursions in Biobank Storage

Abstract

Maintaining the integrity of cryogenically preserved biological materials is critical, as even brief, undetected temperature excursions in storage can compromise sample viability. Existing monitoring systems may miss transient thaw-refreeze events, posing serious quality risks. To address this, we developed and validated frozen indicator tubes that visually signal deviations from the frozen state, serving as a cost-effective backup to electronic monitors. Our first method uses an aqueous dye solution that immobilizes the dye when frozen; any thawing causes the dye to disperse, providing a clear, external visual cue of a partial or complete thaw. For ultra-low-temperature storage (-80 °C), we introduced a second method using an ethanol-based solution calibrated to indicate thaw events. This system detects temperature rises of 10 °C or more sustained for at least fifteen minutes-conditions that may jeopardize sample stability. When paired with standard monitoring systems, these indicator tubes offer an added layer of protection by providing simple, reliable, and immediate visual confirmation of critical temperature breaches. This innovation enhances confidence in cryogenic storage protocols and supports the long-term preservation of sensitive biological materials.

Keywords: biobanking; biobanking quality assurance; cellular viability; long-term cryogenic storage; specimen integrity; temperature excursion detection; temperature indicators.

Figure 1

Indicator models. Depending on the cryopreservation need, indicators were modeled to detect partial thawing following storage in a frozen environment (−20 °C) (A) and in an ultra-low-temperature environment (−80 °C) (B).

Figure 2

Indicator methods in the frozen environment (−20 °C). Frozen indicators were generated by first freezing deionized water (A,B), adding sprinkles atop the ice (C,D), and preserving at −20 °C (E,F).

Figure 3

Frozen indicator methods in the ultra-low-temperature environment (−80 °C). Frozen indicators were generated by freezing a 75% ethanol solution (A,B), generating a ‘cup’ (C–H), chilling (I), and then adding indicator dye (J,K).

Figure 4

Frozen vial validation (−20 °C). Indicators were frozen and maintained for more than 1 h at −20 °C (A), then thawed at room temperature for 5 and 8 min, respectively (B,C), and then refrozen at −20 °C (D).

Figure 5

Ultra-low-temperature vial validation (−80 °C). Ultra-low-temperature indicators were frozen and maintained for 1 h at −80 °C (A), then thawed in a controlled-rate freezer at −60 °C for 30 and 60 min, respectively (B,C), and then refrozen at −80 °C (D).

Figure 6

Partial thaw detection for the frozen indicator. Vials were thawed over time in a room-temperature environment. Within minutes, a partial thaw was detected by an increase in dye intensity and enhanced over time.

Figure 7

Partial thaw detection for the ultra-low-temperature indicator. Vials were thawed over time in a room-temperature environment. Within seconds, a partial thaw was detected by an increase in dye intensity and enhanced over time.

Figure 8

Partial thaw detection mapped. Vials were sustained for 15 min at various temperatures ranging from fully frozen (−20 or −80 °C) to temperatures that would induce a partial thaw event for frozen vials (A) and ultra-low-temperature vials (B). Using a visual bleed score (Supplementary Materials), a gradual increase in dye detection was seen as temperatures rose, with significant detection (*) at 0 °C and higher in the frozen indicators and −70 °C and higher in the ultra-low-temperature indicators.