Research Article: Dual Dependence of Cryobiogical Properties of Sf21 Cell Membrane on the Temperature and the Concentration of the Cryoprotectant

Date Published: September 4, 2013

Publisher: Public Library of Science

Author(s): Jianye Wang, Kaixuan Zhu, Gang Zhao, Jian Ren, Cui Yue, Dayong Gao, Xiaoming He.

http://doi.org/10.1371/journal.pone.0072836

Abstract

The Sf21 cell line is extensively used for virus research and producing heterologous recombinant proteins. To develop optimal strategies for minimizing cell injury due to intracellular ice formation and excessive volume shrinkage during cryopreservation, the fundamental transport properties including the osmotic inactive volume (Vb), the hydraulic conductivity (Lp), and the glycerol permeability (Ps) of Sf21 cell membrane at 25, 15, 5 and −2°C were characterized using a micro-perfusion chamber. The effects of temperature on the hydraulic conductivity and the glycerol permeability of Sf21 cell membrane, reflected by the activation energies, were quantitatively investigated. It was found that the hydraulic conductivity decreases along with the increase of the final CPA concentration at a given temperature, and quantitative analysis indicates that the hydraulic conductivity has a significant linear attenuation along with the increase of the concentration of glycerol. Therefore, we incorporate the concentration dependence of the hydraulic conductivity into the classic Arrhenius relationship by replacing the constant reference value of the hydraulic conductivity at the reference temperature with a function that is linearly dependent on the CPA concentration. Consequently, the prediction of the Arrhenius relationship is improved, and the novel Arrhenius relationship could be very important to the development of optimal strategies for cell cryopreservation.

Partial Text

The Sf21 cell line, derived from the ovary of the fall armyworm (Spodoptera Frugiperda) by Vaughnet at 1977 [1], is a host of many viruses, and when combined with baculoviruses, is a powerful platform technology that is widely used for the manufacture of viral particles and heterologous recombinant proteins [2], [3], [4]. This robust expression system is able to produce large amounts of different proteins, with applications from basic research (protein-protein interaction, multiple-affinity protein purification, ultrasensitive mass spectrometry, and protein structures) [5] to clinical medicine (routine diagnostic tests, therapeutic protein drugs for various diseases, etc.) [6], [7], [8].

Figure 2A shows the Sf21 cells adhered to the micro-channel in the initial state at −2°C, and the transient response of a representative cell (indicated by a red box in the left figure) during the osmotic shift from PBS (291 mOsm) to 3×PBS (784 mOsm) at −2°C was shown in Figure 2B at a 10 second intervals. The measured cell volume changes at four different temperatures and the corresponding fitting processes are comparatively shown in Figure S1. Table 1 summarizes the values of the inactive cell volume (Vb) and the hydraulic conductivity at four different temperatures. The mean value of the inactive cell volume is 0.518 and the standard error is 0.089, n = 25. The Vb of the Sf21 is similar to mammalian ovarian tissues (0.5 V0) [26], [27], [28].

The osmotically inactive volume, the hydraulic conductivity and the glycerol permeability coefficient of Sf21 cells were determined experimentally at 25, 15, 5 and −2°C. The parameters of both the K-K model and the 2-p model of Sf21 cells were fitted in the presence of 1.0, 1.5, 2.0 M glycerol at 25, 15, 5 and −2°C. We found that Lp sharply decreases with the increase of the CPA concentration at high temperature, while the reduction is relatively less at lower temperatures. The Lpg of Sf21 cells decreases with the increase of glycerol concentration in the solution, while such trend was not observed for the ELp of Sf21. The EPs and Psg increase slightly with the increase of the glycerol concentration. We proposed that the hydraulic conductivity has a significant linear correlation with the concentration of glycerol for the Sf21 cells and the Lg values correlate well with temperature and the molar concentration of glycerol. The volume changes were predicted using the cryobiological parameters in the experiment. The concentration dependence of the hydraulic conductivity was incorporated into the classic Arrhenius relationship by replacing the constant reference value of the hydraulic conductivity at the reference temperature with a function that is linearly dependent of the CPA concentration. Further comparison between the predictions of the extended formula with the experimental data indicates that this formula was adequate at least for the Sf21 cells.

 

Source:

http://doi.org/10.1371/journal.pone.0072836