CRYOGENIC COLD CHAIN MANAGEMENT:

PROTECTING BIO-SAMPLE TEMPERATURES USING AUTOMATION AND BEST PRACTICES

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Protecting Bio-Sample Temperatures: Using Automation and Best Practices

ABSTRACT

The implementation of an effective cryogenic cold chain ensures that all samples stay safely below their glass transition temperature (Tg H20 -135°C). Often underestimated though is how quickly samples warm when removed from a cryogenic environment. By knowing thermal excursion rates and understanding transient warming events, one can implement automation devices and best practices to ensure that all samples, including innocents, stay well below Tg at all times.

This paper presents a recommended workflow based on experimental thermal excursions using the BioStoreTM III Cryo storage system and the CryoPodTM carrier.

MATERIALS

M Brooks BioStore III Cryo (-190°C) storage system (Fig. 1)

M Brooks CryoPod Carrier (Fig. 1)

M FluidX 2mL cryo vials and standard 9 x 9 cryobox

M All vials filled 1mL with water

M Brooks CryoCep™ cryogenic vial gripper

M 36 AWG Type T Thermocouples mounted midway up the water height, touching the inner vial wall

M Measurement Computing TC-Temp data acquisition unit sampling at 0.5hz using TracerDAQ software.

RESULTS

INTRODUCTION

Biological samples are often stored in liquid nitrogen vapour phase (LN2) to ensure their temperatures stay below -135°C, the glass transition temperature of water (Tg). It is believed that enzymatic activity ceases or is most reduced when biosamples are frozen below Tg and then remain below Tg until thawed. Thus, preserving their viability until needed.

Commercial LN2 freezers available today can store samples at -190°C, but they have no built in protection or monitoring of the thousands of innocent samples that may be exposed during routine rack pulls for storage/removal operations. Additionally, keeping the samples always below Tg when outside of the LN2 freezer (i.e. around the lab) is also important and most commercially available LN2 carriers are large, cumbersome and do not have integrated temperature monitoring while homemade LN2 carriers are often unsafe.

The BioStore III Cryo storage system and CryoPod Carrier, new technologies available from Brooks Life Sciences, allow researchers to store, retrieve, transport and work with biosamples whilst keeping them safely below -135°C at all times, enabling a true cryogenic cold chain. This paper will discuss procedures and illustrate sample temperature throughout typical workflows.

PROCEDURES

M Vials were stored in the BioStore III Cryo (B3Cryo) and allowed to equilibrate

M They were requested using the system software

M The B3Cryo raised the rack and ejected the box

M The lid was removed, the vial was picked from the cryobox with the CryoCep and moved to the CryoPod, approximately 1 meter away

M The vial was removed from the CryoPod and exposed to ambient air and returned to the CryoPod

M The vial was returned back into the B3Cryo

The BioStore III Cryo (B3Cryo) has at its foundation the Chart MVE 1500P-190 LN2 freezer which has a top box temperature of -190°C, +/- 6°C. In this experiment the vial was allowed to equilibrate in the freezer to -190°C, was then retrieved from the B3Cryo system and placed into a cryobox in the CryoPod carrier. Manipulated in the CryoPod then returned to the B3Cryo. Figure 2 illustrates the vial temperature during transport from the B3Cryo, through ambient air and into the CryoPod. The total temperature rise was 34°C during the 31s exposure, approximately 1°C/min. It then took 3.6 minutes for the sample to reach -185°C in the CryoPod and 5.2 minutes to equilibrate completely at -190°C.

Figure 1

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Protecting Bio-Sample Temperatures: Using Automation and Best Practices

Despite the 34°C rise in temperature during the transfer, the sample stayed well below the critical temperature (Tg), because of the -190°C starting temperature of the LN2 freezer and the -190°C temperature of the CryoPod. Once in the CryoPod the vial immediately starts to cool back down. The cooling rate in the CryoPod was -0.14°C to -185°C and 0.1°C to -190°C.

After a vial has been moved to the CryoPod carrier, the sample may be transported to a final destination to be thawed, moved to another freezer, building or manipulated between one or two CryoPods (i.e. a tube picking operation). It is important that the sample temperature remains below Tg throughout all possible operations until it needs to be thawed. Transporting and picking/ selecting vials is a common break in the cryogenic cold chain.

Figure 2.

Figure 3

Figure 3 illustrates the vial temperature in the CryoPod and three transient exposures to ambient. This test was done to show repeatable warming and cooling of a single vial. A typical procedure would not normally expose the same vial three times in a row. One exposure represents a typical tube picking and identifying operation (moving tubes between cryoboxes or another LN2 device). In this example the vial starts at -183°C and warms to -140°C during a 35s exposure. It then takes 6 minutes to cool back down to -175°C and another 5.1 minutes to equilibrate back to -183°C. Two more vial removals for 35s were performed with very similar temperature increases and subsequent cool downs.

The warming rate of the three exposures averaged 1.2°C/min. This rate is very similar to previous vial warming experiments where it was also found that placing cryogenic samples on dry ice (a common practice) actually further accelerates the warming rate1.

In all three examples the vial temperature never crossed Tg (-135°C), but did reach -140°C. Therefore, subsequent vial movements should wait for the temperature to equilibrate back to the cryogenic environment or until cold enough to protect the temperature during shorter transient exposures.

In this last experiment the samples are moved back into the B3Cryo from the CryoPod. Figure 4 illustrates the temperature of two vials as they are simultaneously picked from the CryoPod and put back into the BioStore III Cryo. The ambient exposure time of the vials were 36s and the rack was 55s. Vial 1 was placed in a cryobox surrounded by other vials that were already equilibrated in the LN2 freezer. Vial 2 was put alone into a cryobox. Notice Vial 1 warmed less, but cooled slower due to the surrounding frozen vials (insulation). Vial 1 warms 26°C and stops warming after 3.9 mins whereas Vial 2 warms 40°C and stops warming 1.3 mins after the rack is lowered back into the LN2 freezer.

Figure 4.

Neither sample warms over Tg, but both exhibit additional heating and subsequent reduced cooling rates in the LN2 freezer due to the known effect that samples still increase in temperature when put back into an LN2 freezer, largely due to the heat absorbed by the metal rack when in ambient air2.

The BioStore III Cryo includes an insulated sleeve that surrounds the rack when lifted and prevents convection which helps slow warming of the rack and samples. Figure 5 illustrates that an insulated sleeve reduces warming of exposed samples. The warming protection from the sleeve is most significant when the lab has air flow from a typical HVAC system (heating/cooling). In this case the insulated sleeve kept the samples 20°C colder over the same exposure time and procedure.

Figure 5

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Protecting Bio-Sample Temperatures: Using Automation and Best Practices

Brooks is a leading worldwide provider of automation and cryogenic solutions for multiple markets including semiconductor manufacturing and life sciences. Brooks’ technologies, engineering competencies and global service capabilities provide customers speed to market and ensure high uptime and rapid response, which equate to superior value in their mission-critical controlled environments. Since 1978, Brooks has been a leading partner to the global semiconductor manufacturing market as a provider of precision automation and cryogenic vacuum solutions. Since 2011, Brooks has applied its automation and

cryogenics expertise to meet the sample storage needs of customers in the life sciences industry, through Brooks Life Sciences. Brooks Life Sciences offerings include a broad range of products and services for on-site infrastructure for sample management at temperatures of 20°C to -190°C, as well as comprehensive outsource service solutions across the complete life cycle of biological samples including collection, transportation, processing, storage, protection, retrieval and disposal. Brooks is headquartered in Chelmsford, MA, with operations in North America, Europe and Asia.

About Brooks:

Cryopreservation & Cold Chain Solutions

Sample Storage, Lab Services & Transport

Automated Storage Systems

Informatics &Technical Solutions

Sample Consumables& Instruments

www.brookslifesciences.com ©2019 Brooks Automation, Inc. B412-19 Nov 2019

CONCLUSIONS

M A complete cryogenic cold chain that protected Tg was maintained and verified by using the BioStore III Cryo storage system, CryoPod Carrier and typical workflow procedures

M Ambient warming rates from -180°C averaged 1.2°C/sec

M CryoPod cooling rates from -140°C to -175°C averaged -0.1°C/sec

M Samples can cross Tg from -190°C in as few as 45s when exposed to an ambient environment

M Insulating the rack and samples during transient exposures slow warming

M Without automation and software, researchers should time their procedures and understand warming and cooling rates of cryogenic samples to predict sample temperatures throughout an entire workflow and then adapt their procedures to protect samples from crossing Tg.

SUMMARY OF CRYO COLD CHAIN TECHNOLOGY

M -190°C LN2 vapour phase freezer, low LN2 consumption, 20+ day safety hold time

M Automated cryobox delivery, timed, recorded and controlled

M Anytime manual access

M Insulated sleeve slows warming by blocking convection

M Sample temperature prediction software algorithims

1 Salvetti, M., Fink, J. Barlett, A. et al. (2015, May).Thermal excursions of cryogenically frozen vials (below -150°C) and the risk of rising above Tg,H2O: analyzing warm-up rates from cryogenic storage to both dry ice and ambient temperature environments. Poster presented at annual International Society of Cellular Therapy conference, Las Vegas, NV.

2 Warhurst, J., Fink, J., Holmes, T. et al. (2015 May).Protection of innocents: continued sample warm up after return to a cryogenic environment (below -150°C) following a transient ambient picking operation.Oral presentation presented at annual International Society of Cellular Therapy conference, Las Vegas, NV.