Supercritical drying of vascular endothelial growth factor in mesenchymal stem cells culture fluids

https://doi.org/10.1016/j.supflu.2019.104710Get rights and content

Highlights

  • VEGF in mesenchymal stem cells culture fluids was processed by supercritical drying.

  • VEGF tertiary structures were preserved after supercritical drying.

  • VEGF by supercritical drying showed good structural stability in an aqueous solution.

  • Growth factors by supercritical drying showed good efficacy on cell regeneration.

Abstract

Mesenchymal stem cells (MSCs) culture fluids are frozen and thawed for long-term storage when needed for medical purposes. However, stresses accompanied with freezing and thawing can disrupt the tertiary structures of proteins, which need to be in a certain state to show their therapeutic effect, by affecting their internal interactions. Supercritical drying of MSCs culture fluids using carbon dioxide was conducted to resolve these problems. Various process parameters were controlled to improve process efficiency in the water displacement step which takes place prior to supercritical drying. Among the proteins contained in the MSCs culture fluids, vascular endothelial growth factor (VEGF) was selected as a representative material to optimize the process parameters. Analyzed results show that the tertiary structures of VEGF via supercritical drying are well preserved. In addition, the structural stability and bioactivity of VEGF when re-dissolved in an aqueous environment were better than those obtained from freeze drying.

Introduction

Mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into various cells such as osteocyte, chondrocyte, adipocyte, myocyte, and neuron-like cells [[1], [2], [3]]. MSCs modulates allogeneic immune cell response and induces successful tissue regeneration [[1], [2], [3], [4], [5], [6]]. Moreover, it has been reported that chemical secretions such as growth factors, gelatins, cytokines, microvesicles, and exosomes from MSCs also show therapeutic effects [[7], [8], [9], [10]]. This means MSCs culture fluids can also be utilized for medical purposes. MSCs culture fluids are generally frozen and thawed when needed. In aqueous solutions, tertiary structures of proteins, which means three dimensional structures of molecules and have an important role in function of molecules [11], can collapse due to unfolding and aggregation induced by various causes such as temperature, pressure, pH, co-solvents, co-solutes, and salts, which decrease protein efficacy [[12], [13], [14], [15], [16], [17], [18]]. However, maintaining a frozen state consumes a lot energy and also requires appropriate infrastructure. In order to resolve these problems, drying of MSCs culture fluids is beneficial in terms of energy required for storage and long-term storage stability [[19], [20], [21], [22]].

One representative drying method currently considered in the medical science is freeze drying (lyophilization) [[20], [21], [22], [23]]. Freeze drying is a method in which the fluid to be dried is frozen by lowering the temperature, and then sublimated to gas by lowering the pressure below the triple point. Unlike spray drying, which is also a commonly used method, freeze drying avoids utilization of high temperatures and therefore can prevent the unfolding of protein [21,24]. However, in freeze drying, the risk of denaturation of protein due to exposure to low temperature also should be taken into account [20,25,26]. Also, protein denaturation can occur due to disruption of interactions maintaining protein tertiary structures by dehydration [12,14,20,22,[27], [28], [29], [30]]. Therefore, appropriate excipient may be required to preserve the protein structures [12,14,20,22,25,[27], [28], [29], [30]]. In addition, a lot of energy is needed to maintain low temperatures during the long process time.

Supercritical drying has been considered as an alternative technology to overcome the drawback of the freeze drying [21,24,31,32]. In supercritical drying, drying occurs through a phase transition from a liquid phase to a gas phase via a supercritical phase which is achieved above the critical temperature and pressure. When drying progresses in presence of interface between two phases, capillary force is inevitably occurred by surface tension. This force affects structure of proteins directly or causes aggregation of proteins so that denaturation of proteins occurs [12,[33], [34], [35], [36]]. This surface tension-induced protein denaturation can be avoided through supercritical drying as a surface tension free process, because surface tension between liquid phase and supercritical phase and between gas phase and supercritical phase does not exist. Water is a commonly used as solvent for protein drugs, but has a severe supercritical conditions of critical temperature (647 K) and critical pressure (22.1 MPa). Thus, supercritical drying of water alone cannot avoid temperature induced denaturation of proteins. Therefore, it is necessary to displace water with a fluid with a critical temperature lower than the denaturation temperature of the proteins. Carbon dioxide (CO2) is a widely used compressed fluid due to its moderate supercritical conditions of critical temperature (304 K) and critical pressure (7.39 MPa), stability, non-toxicity to the human body, and low cost. Many studies have used supercritical CO2 to dry various drugs [21,24,31,32,[37], [38], [39], [40]].

However, the solubility of water in CO2 is low for CO2 to displace water in a short time to avoid long-term exposure to an aqueous environment. Therefore, a co-solvent that can solubilize both water and CO2 can be used to assist water displacement [[41], [42], [43], [44], [45]]. Moreover, to reduce process time of water displacement, utilization of a capillary nozzles can help enhance mass transfer by increasing the contact area of two immiscible phases [46,47].

The aim of this study is to dry proteins included in MSCs culture fluids using supercritical drying to preserve protein tertiary structure. For an overall process efficiency, ethyl alcohol anhydrous (EtOH) was used as a co-solvent to assist water displacement. The tertiary structure of proteins dependence on the process parameters were investigated through the characterization of one of the protein in MSCs culture fluids, vascular endothelial growth factor (VEGF). Analyses of the products were compared to that of a freeze dried product to demonstrate the validity of supercritical drying.

Section snippets

Preparation of mesenchymal stem cells culture fluid

The mesenchymal stem cells (MSCs) were cultured in 1000 cells/cm2 density using T175 culture flask with phenol red-free, choline chloride-free medium with 4% human platelet lysate. During the culture, medium was changed every 2 days. At 80% to 90% confluency, the culture flask was washed with Dulbecco’s phosphate buffered saline (DPBS) 3 times removing all the medium on the flask wall. The culture flask was conditioned for 72 h after adding 20 cm3 of phenol red-free, choline chloride-free, and

Results & discussion

The experimental conditions and analysis results are shown in Table 1, Table 2, Table 3, Table 4. Experiments were conducted by changing only one process parameter at a time.

Conclusion

In this study, MSCs culture fluid was processed by water displacement to CO2 and supercritical drying. Various process parameters of water displacement such as total flow rate, temperature, pressure, and feed ratio of EtOH to CO2 were optimized to minimize yield loss with tertiary structures preservation of VEGF. When compared to freeze dried samples, samples via supercritical drying showed higher structural stability of in aqueous environments. The cell regeneration efficacy of the growth

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was financed by SCM lifescience Co. Ltd. (project No. 0458-20170056).

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      To aid in water removal, ethanol was utilized as a co-solvent. Similar concentrations of VEGF were observed in reconstituted supercritically processed and freeze-dried samples at 0 days in aqueous solution, but a rapid reduction in stable VEGF was observed in the freeze-dried samples after 14 days due to a loss in tertiary structure, and not in the supercritically dried samples [39]. Park et al. produced similar results using the precipitation from compressed antisolvent (PCA) method, and a distinct difference in morphology and stability between VEGF processed with PCA and freeze-drying can be seen from the SEM images and graphs in Fig. 7 [40].

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