Diabetes can change the viscoelastic properties of lymphocytes
Mechanical properties of the cells are among the most highlighted area of interests among researchers for decades. Not only many of the cells’ crucial functional characteristics such as adherence to the cellular substrate, migration abilities and morphological factors are directly influenced by their mechanical properties but also changes in these traits could have importance in diagnosis and even treatments of some serious diseases. The general mechanical properties of the cells are associated with some intercellular characteristics such as arrangement and organization of the actin fibers and cytoskeleton architecture. Any changes due to pathological conditions in the molecular and cellular processes related to these elements can alter the cells’ mechanical characteristics. In this paper, the viscoelastic properties of diabetic and normal lymphocytes were analyzed and compared by application of the iron nanoparticles attached to the cellular membrane and putting the cells in a magnetic field with certain frequency and intensity. Step force was applied to the normal and diabetic lymphocytes and their membrane displacement was tracked by special software and plotted with respect to time. Fitting the experimental data on theoretical formulation of standard linear viscoelastic model, it was demonstrated that diabetic lymphocytes have significantly different viscoelastic characteristics. The results of this paper can be of importance in assessments of diabetic lymphocytes’ abilities to fulfill their immune surveillance tasks.
KeywordsMechanical properties Viscoelastic Lymphocytes Diabetes
Cell mechanical properties play pivotal roles in vital characteristics of cells. Many of the biophysical and biological peculiarities are determined by viscoelastic properties of cells (Hayot et al. 2012; Hecht et al. 2015). For instance, it has been illustrated that interaction of a cell and the extracellular matrix is regulated by the cell’s mechanical properties (Trappmann and Chen 2013) or these mechanical traits have significant role in cell signaling (Humphrey et al. 2014). In addition, the cells’ mechanical properties can be regarded as markers of differentiation (González-Cruz et al. 2012; Mathieu and Loboa 2012), pathology (Lekka et al. 2012; Rebelo et al. 2013; Suresh et al. 2015) and transformation (Plodinec et al. 2012). Since different cell sources and different methods such as micropipette aspiration (Zhao et al. 2009), atomic force microscopy (AFM) (Cartagena and Raman 2014; Hecht et al. 2015), magnetic beads microrheometry (Bausch et al. 1998) and others have been utilized for determination of cells’ viscoelastic properties, there is a relative incongruity in results. Therefore, the mechanical properties of cells can be regarded as biomarkers that can be used in diagnosis of some diseases and analyzing the appropriate functioning of cells. In contrast to other methods of measuring viscoelastic properties, the use of magnetic field encompasses the advantage of not having direct contact with the cell body. In the methods such as AFM which include direct contact of an external tip or probe with the same dimension of a cell would lead to active cellular reaction that can easily change the mechanical properties (Guck et al. 2005). In addition, special preparations that are included in some other methods can alter the physiological and biological conditions and lead to results which are significantly different from ordinary homeostatic conditions. Using nanomagnetic adhesive beads accompanied with low-level field seems to have the lowest intervention and, therefore, gives rise to one of the most precise answers. Furthermore, magnetic-oriented approaches seem to be cheaper and simpler in comparison to other methods and so it can be widely and easily used in diagnostic and therapeutic purposes.
Lymphocytes are a small form of leukocytes that can make significant contribution in immune responses. The metabolism and natural biological processes within these cells change due to some diseases such as diabetes (Otton and Curi 2002; Otton and Curi 2002) and, therefore, it is expected to see alteration in membrane mechanical properties in normal and diabetic lymphocytes. In this research, the viscoelastic properties of normal and diabetic lymphocytes were determined and compared by low magnetic field.
Materials and methods
The lymphocytes in normal and diabetic groups were provided by Iranian Biological Research Center by ficoll method. RPMI 1640 media contained 10% FBS, 1% penicillin/streptomycin and 1 μg/mL phytohemagglutinin (PHA). The cell suspensions of two groups were incubated separately in culture medium for 18 h, followed by addition of magnetic Fe3O4 nanoparticles and glucose with certain concentration. The cells of both groups were incubated for another 24 h in CO2 incubator for absorption of the magnetic nanoparticles.
Magnetic field application
Magnetic inductance system
With the use of a function generator, the desired wave form could be produced and after amplification to 4 A the electrical current was applied to a coil. Magnetic field has the effects on magnetic nanoparticles which are connected to cell membrane. The coil has two cores. The central iron core has the radius of 2.5 cm and the length of 7 cm and the secondary Si core has the length of 2.5 cm and wrapped around the central core. The copper wire of 1.1 mm was twisted 400 times around the cores. A teslameter with the accuracy of 0.001 was located in an appropriate location for determination of the magnitude of magnetic field.
The videos are transferred into computer for processing. On the computer, four specific softwares for this purpose have been installed. The function generator software induces the desired wave form to the generator, Teslameter software that shows the magnitude of the magnetic field in micro Tesla in three Cartesian directions, video recording software that is related to the microscope and finally the software of Tracker (version 4.81) analyzing the displacement.
The parameters p1, q0 and q1 should be determined in practical experiment. Figure 2b shows the applied force. The frequency was 0.2 Hz, 4 A electrical current and the magnetic field 840 μT. The samples were exposed to this field for 15 s.
All the tests were performed three times and in each test at least ten cells from each group were selected. T test-paired statistical analysis was done for the three constants of the Eq. 3 and P value below 0.05 was set as the criterion of significant difference.
The constants of the standard linear model of viscoelastic material for normal and diabetic lymphocytes
Constants of Eq. 3
0.0074 ± 0.0008
0.2522 ± 0.0036
0.1852 ± 0.03145
0.8528 ± 0.0752
0.0003 ± 4.1 × 10−5
0.0175 ± 0.00277
The mechanical properties of cells and tissues have attracted many scientists’ and researchers’ attention. It has been demonstrated that changes in mechanical characteristics of cell can be one of the best criteria in early diagnosis of many diseases. In addition, cellular well-functioning is attributed directly to mechanical properties of those cells. Due to these facts, recently some papers have been published on measuring viscoelastic properties of lymphocytes. To the best of our knowledge, this is the first time that the viscoelastic properties of normal and diabetic lymphocytes were measured and analyzed by this approach. While many researchers use micropipette aspiration or AFM method, in this research the viscoelastic properties of normal and diabetic lymphocytes have been investigated by application of magnetic field on iron nanoparticles-loaded cells. In this research, the viscoelastic properties of normal and diabetic lymphocytes were assessed and obtained. Magnetic iron nanoparticles have been added to the culture medium of the lymphocytes with certain concentration and by application of magnetic field in the graph of membrane displacement with respect to the time was plotted. By utilization of standard linear viscoelastic model, the mechanical properties of normal and diabetic lymphocytes were investigated and compared.
The results demonstrated that diabetes can change the mechanical properties of lymphocytes. Any physical (Rebelo et al. 2013; Rianna and Radmacher 2016) and chemical (Peetla et al. 2013) alterations at the cells’ surface can readily lead to intervention in cells’ vital functions, malfunction of entering and exit process of the necessary chemicals and even disorder in secretion of enzymes, proteins and other substances. As the previous researches have illustrated that diabetes can change some pivotal traits of lymphocytes such as metabolism rate (Otton and Curi 2002) or apoptosis (Otton et al. 2004), and also because of the interconnected physical and functional characteristics of the cells it was expected to see different mechanical properties for normal and diabetic lymphocytes. Our results have proven this hypothesis.
There are evidences that demonstrate the close relationship between the mechanical properties of cells’ membrane and the organization and arrangement of their actin cytoskeleton (Lekka et al. 2011, 2012). It means that any change in actin fibers’ arrangement and organization will cause change in mechanical properties of the whole cell. In addition, there are sufficient clues which illustrate the direct connection between the actin cytoskeleton remodeling properties and the cell motility. Past research works have shown that the remodeling of the dynamic filament meshwork is one of the primary influential factors in the cells’ migration abilities (Gardel et al. 2010; Ridley et al. 2003). Therefore, the mechanical characteristics of cells including viscoelastic properties can be regarded as one of the essential parameters which depict the migration capability. This issue even becomes more highlighted for lymphocytes as their immune surveillance tasks are closely interconnected with their motile abilities (Dupré et al. 2015).
The results of this paper show a significant difference between the viscoelastic properties of normal and diabetic lymphocytes. Based on the previous discussion, this means a discrepancy exists between some vitally important characteristics of normal and diabetic lymphocytes such as their migration ability and, therefore, it can jeopardize diabetic lymphocytes’ efficiency in immune responses.
By the hypothesis of regarding the architecture of the cytoskeleton as one the primary influential factors in total mechanical properties of a cell, we can associate the changes in viscoelastic properties of diabetic lymphocytes to the molecular and chemical mechanism which control the structure of cytoskeleton. Many proteins such as the family of ERM (ezrin, radixin, moesin) which contribute as actors in remodeling of cytoskeleton (Chen et al. 2013) can change in diabetic situation (Nishida et al. 2014).
In this research, the viscoelastic properties of normal and diabetic lymphocytes have been investigated and significant differences have been identified. It has been understood that the mechanical properties of healthy and diseased lymphocytes are different in terms of their energy storing and dissipating rates. While cellular migration includes continuous membrane bending and deformation, it can be deduced that the diabetic cells have different migration pattern. Finally, while the most important function of the lymphocytes necessitates their facilitated movement, diabetic cells seem to have problems in performing their tasks.
The authors would like to thank warmly all the individuals who contributed in the quarantine laboratory of the national genetic bank center.
Compliance with ethical standards
Conflict of interest
This study was not funded by any company or Grant. Each individual author declares that she/he has no conflict of interest. Author Neda Parvanehpour declares that he has no conflict of interest. Author Shahrokh Shojaei declares that he has no conflict of interest. Author Vahid Faghihi Rezaei declares that she has no conflict of interest. Author Siamak Khorramymehr declares that she has no conflict of interest. Author Vahabodin Goodarzi declares that he has no conflict of interest. Author Fatemeh Hejazi Jahromi declares that she has no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The reseach methodology has been checked to be compiled with ethical standards.
Informed consent was obtained from all individual participants included in the study.
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