The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities. In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to −25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions (‘in air’ or ‘in bulk CPA’). For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 (‘scaffold in a bulk CPA’) vs. 7.71 ± 0.43 (‘bulk CPA’, P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 (‘scaffold in a bulk CPA’) vs. 7.68 ± 0.34 (‘bulk CPA’, P < 0.0001)). At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen ‘in air’ were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 μg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively). Moreover, when frozen ‘in air’ in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend. The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.
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Understanding how 3D microarchitecture of respective TECs (e.g. inherent heterogeneities) may govern ice nucleation is an important aspect for establishing controlled cryopreservation designs. The collagen-HAP scaffolds presented also interconnected porosity (Fig. 5) that supported attachment, growth and high viability of amnion-derived mesencymal stromal cells from the common marmoset (Callithrix jacchus) (Fig. S1). The heterogeneity of the system increases not only at the expense of composition but also by increased structural complexity as for example introduced through interconnected pores. Phase transition and accompanied latent heat release may prove to have pore-to-pore character. From a cryobiology viewpoint, an approximately linear relation between the freezing point depression and the inverse mean pore size can be expected. This relationship is nevertheless difficult to quantify in scaffolds possessing relatively broad pore-size distributions. Given that scaffolds prepared by mineralization of collagen and freeze-drying contain micropores (for details see Ref. ), it could be assumed that ice formation would rather follow ‘in bulk’ scenario. However, in the absence of INA actively triggering ice formation, certain depression of freezing point contributed by CPAs confined in scaffold nanopores combined with the solute enrichment in the remaining unfrozen CPA may be anticipated. First freezing experiments with collagen-HAP scaffolds were conducted in 96-well culture plates analogous to that in which scaffolds have been freeze-casted. To reduce the edge effect, outer peripheral rows and columns (corner and edge wells) of a 96-well plate were not prefilled with experimental material. When either scaffolds alone or in combination with CPA were placed next to each other intersample latent heat dissemination was detected by the infrared camera (Fig. 7). In fact, this format allowed to provide sample records for high counting statistics and conclude that prepared scaffolds were clearly responsible for significantly lesser supercooling and far more prolonged nucleation in contrast to undercooled selected CPAs frozen alone irrespective of their composition (Fig. 8 and Table 2). Placement of collagen-HAP scaffolds into bulk CPA resulted in significantly lower ΔTs. Clearly, presence of a scaffold per se in a freezing/vitrifying solution most likely cause a tendency to the increased probability of ice nucleation and promotion of crystallization/devitrification . A further interesting feature is that under both tested conditions (bulk CPA-to-air and scaffold-to-air interface) the surface-catalyzed nucleation could be highlighted as a prevailing cause of ice nucleation.The co-author team is thankful to colleagues from the animal facility of the Lower Saxony Centre for Biomedical Engineering, Implant Research and Development for the provision of rat tails used in the fabrication of scaffolds. This work was supported by the German Research Foundation through the Cluster of Excellence REBIRTH (EXC 62/3 valid until Dec 2017, EXC 62/4 valid until Oct 2019), IP@Leibniz Program of the Leibniz University Hannover promoted by the German Academic Exchange Service (DAAD; German: Deutscher Akademischer Austauschdienst) (project code 57156199) as well as Ways to Research Program of the Leibniz University Hannover (60442522).
The co-author team is thankful to colleagues from the animal facility of the Lower Saxony Centre for Biomedical Engineering, Implant Research and Development for the provision of rat tails used in the fabrication of scaffolds. This work was supported by the German Research Foundation through the Cluster of Excellence REBIRTH (EXC 62/3 valid until Dec 2017, EXC 62/4 valid until Oct 2019), IP@Leibniz Program of the Leibniz University Hannover promoted by the German Academic Exchange Service (DAAD; German: Deutscher Akademischer Austauschdienst) (project code 57156199) as well as Ways to Research Program of the Leibniz University Hannover (60442522).
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- Hippophae rhamnoides
- Ice-nucleating agents
- Infrared thermography
- Partial freezing
- Tissue-engineered constructs