Elsevier

Cryobiology

Volume 76, June 2017, Pages 65-73
Cryobiology

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

https://doi.org/10.1016/j.cryobiol.2017.04.006Get rights and content

Abstract

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA1) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity.

First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me2SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me2SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me2SO. A mixture of Me2SO, ethylene glycol and glucose (ratio 4:2:1– termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me2SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures.

In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.

Introduction

Scientists have successfully cryopreserved a variety of cells over the last 40 years, but success has mainly been achieved by traditional slow cooling with cell suspensions in small (<10 ml) volumes [16], [45] in tube format, or in bag format in volumes of up to 200 ml [10], [17], [33]. Cooling multicellular systems down to deep sub-zero temperatures has proven far more challenging, especially when large volumes of biomass require cryo-banking. These problems began to be understood during early attempts to cryopreserve whole organs or large complex tissues. They were mainly related to the formation of extracellular ice in liquid spaces within the tissue [37], such as within small capillary blood vessels inside an organ [19], which physically destroyed the internal structure. In addition, disruptive ice propagation between inter-connected cells [1], insufficient cell dehydration and CPA penetration during slow cooling and ice-recrystallization during the warming process have all been noted. These can also make cell agglomerates more susceptible to freezing damage than individual cells. Ice formation can be avoided by vitrification, but high cooling rates must frequently be imposed to avoid toxic effects from the essentially high CPA concentrations required and to maximise the likelihood of achieving the glassy state without ice nucleation events [13]. This remains a significant challenge for large tissues or, equally, for large volumes of functionally interconnected cells such as cell spheroids, as enough time must be given for CPA to penetrate into the all the cells, including those in the core, and for the core cells to dehydrate sufficiently to avoid intracellular freezing, risking toxic effects during CPA exposure and cooling. CPA toxicity is also critical during the warming process when high concentrations of CPAs will be present when the cryogenic glasses begin to liquefy; equally the high warming rates needed to prevent devitrification and ice re-crystallisation for all cells within a sample are difficult to achieve in large volumes [22], [39].

Many of those obstacles can be overcome by Liquidus Tracking (LT), a method of achieving vitrification in an aqueous mixture by incrementally increasing concentrations of penetrating CPAs (up to 70%) at incrementally decreasing temperatures [15], [38], [47]. Cryoprotectants are known to be less toxic at lower concentrations, but also at lower temperatures [34], [48] due to decreased cell activity, reduced chemical interaction with sensitive biomolecules and reduced CPA permeation. Due to reduced CPA toxicity, and avoidance of ice nucleation in LT, large samples can be vitrified without the necessity of fast cooling rates, or when longer exposure times are required to allow for sufficient CPA penetration - for example for organ and tissue vitrification.

We have previously studied cryopreservation by slow cooling of alginate encapsulated multicellular liver cell spheroids (AELC) in developing a bioartifical liver support system [31]. Treatment volumes of between 1 and 2 L of AELC for use in a bioartificial liver device have been used in our pre-clinical studies [11] and are predicted to be needed for patient therapy. We have also shown that LT vitrification can be applied to AELC in a small volume feasibility study [40]. Our aim here was to develop a LT protocol, which would allow the cryo-banking and warming of large volumes of AELC whilst maintaining good functional recovery. To improve cell viability we have developed a low-toxicity CPA solution for LT with the requirement of low viscosity (but which – nevertheless – could suppress ice nucleation) so that it may be used within the Liquidus Tracker equipment. Additionally the development of a new stirring system substantially increased post-warming viability.

Section snippets

Materials and methods

Unless otherwise stated, all chemicals were sourced from Sigma (Poole, United Kingdom) and for cooling and warming a PlanerLiquidus Tracker Controller and a Controlled Rate Freezer (Planer, Kryo 10, Series II chamber) was used. CPA concentrations were used and reported in weight per volume (w/v) throughout this work.

CPA development

  • (i)

    Viscosity - candidate CPA combinations

It was predicted that at least 40% (w/v) penetrating CPAs and a maximum total concentration of 70% (w/v) CPA should be used to obtain sufficient vitrification for large volumes and slow cooling rates (<10°C/min) from previous feasibility studies [40]. By using the observational viscosity test at −40 °C only four solutions were identified with a similar viscosity to 70% (w/v) Me2SO when comparing (in increments of 10%) combinations of Me2SO, EG and PG with

Choice of CPA for LT

We observed here that the pumps and stirrer of the Liquidus Tracker are impaired above a viscosity of approximately 900  mPa s (Me2SO at −40 °C). Thus, viscosity was identified as the first limiting factor for developing a new LT CPA solution. Dimethyl sulfoxide (Me2SO), ethylene glycol (EG) and propylene glycol (PG) were selected as penetrating CPAs based on their relatively low viscosity and their common use in vitrification protocols. Only four combinations (in increments of 10%) of Me2SO,

Conclusion

This work has shown that it is possible to use LT to vitrify large volumes of cell therapies such as AELC in the 3-D format. Further improvements to equipment technology, especially in respect to more automatization are required. An optimized stirring and CPA inlet system, as well as the use of 3D cultures of higher cell density (>20 × 106 cells/ml beads) may further increase post-warming viability and performance in the future. In theory, the LT volume could be further up-scaled by engineering

Funding

We thank Planer Plc, the Liver Group Charity and the Welcome Trust for funding this study.

Acknowledgement

We also thank Dr. Paul Matejtschuk and Kiran Malik (NIBSC, a Centre of the Health Protection Agency) for enabling DSC analysis.

References (49)

  • F. Lahnsteiner

    The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos

    Theriogenology

    (2008)
  • F. Lahnsteiner et al.

    The cryopreservation of spermatozoa of the burbot, Lota lota (Gadidae, Teleostei)

    Cryobiology

    (2002)
  • A. Lawson et al.

    Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions

    Cryobiology

    (2011)
  • J.-H. Lee et al.

    Effect of spheroid aggregation on susceptibility of primary pig hepatocytes to cryopreservation

    Transpl. Proc.

    (2012)
  • R.L. Levin et al.

    Water transport in a cluster of closely packed erythrocytes at subzero temperatures

    Cryobiology

    (1977)
  • R. Magalhães et al.

    Influence of cell culture configuration on the post-cryopreservation viability of primary rat hepatocytes

    Biomaterials

    (2012)
  • J. Matheny et al.

    Toxicity of dimethyl sulfoxide and magnesium as a function of temperature

    Eur. J. Pharmacol.

    (1969)
  • N. Oberstein et al.

    Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods

    Theriogenology

    (2001)
  • J. Park et al.

    Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

    J. Biomech.

    (2011)
  • D.E. Pegg

    The relevance of ice crystal formation for the cryopreservation of tissues and organs

    Cryobiology

    (2010)
  • D.E. Pegg et al.

    Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method

    Cryobiology

    (2006)
  • D.E. Pegg et al.

    Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury

    Cryobiology

    (2006)
  • A.M. Saeed et al.

    Vitrification and rapid-freezing of cumulus cells from rabbits and pigs

    Theriogenology

    (2000)
  • S. Seki et al.

    Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

    Cryobiology

    (2011)
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