Optimised isolation and characterisation of adult human astrocytes from neurotrauma patients
Introduction
In the central nervous system, astrocytes are the major class of glial cells and represent the main cellular constituent, distributed throughout the brain and spinal cord. In some areas of the brain, they are estimated to comprise from 25% up to 50% of the total volume, thus outnumbering the neurons in humans (Sofroniew and Vinters, 2010; Montgomery, 1994). As the name suggests, these cells have a distinctive shape with star-shaped and finely branching processes. According to their distribution and differences in their morphologic appearance, they have been divided into two main subtypes, protoplasmic and fibrous (Montgomery, 1994; de Majo et al., 2020; Bedner et al., 2019; Kettenmann and Verkhratsky, 2011). From the end of 19th century, this classification is still valid today. Besides their territorial organisation, according to various brain regions and variations in morphology, astrocytes differ also in the physiological characteristics, including the glutamate transporter and expression of proteins, such as glial fibrillary acidic protein (GFAP), membrane potential and potassium conductance (de Majo et al., 2020; Nimmerjahn, 2009). The GFAP is a prototypical marker for immunocytochemical identification of astrocytes as it is a sensitive and reliable marker (Sofroniew and Vinters, 2010; Kimelberg, 2004a; Lee et al., 2008; Sharif and Prevot, 2012). It is the major component of glial fibrils specific for astrocytes in the central nervous system (Condic et al., 2014). GFAP is one of the intermediate filament proteins, including vimentin, actin, nestin, and others that are important for cyto-architectural functions. It is essential in reactive astrogliosis and in glial scar formation. There are different isoforms of GFAP that may be expressed in a heterogeneous manner in both healthy and pathological conditions. However, it is important to note also some limitations of GFAP as an astrocyte marker. Besides local and regional variability that is regulated by a number of signalling molecules, GFAP labels only reactive astrocyte, responding to central nervous system injuries and may not be immunohistochemically detectable in astrocytes in healthy central nervous system tissue. Other molecular markers that have been used for immunocytochemical identification of astrocytes include GLAST, a glutamate aspartate transporter, which shows the most widespread expression in astrocytes among astrocyte markers. Other most popular markers for astrocytes include S100B, which belongs to the family of calcium binding proteins, glutathione peroxidase, GLT-1 (EAAT2 in humans) glutamate transporter, glutamine synthetase and astrocyte specific water channel, aquaporin 4 (AQP4) (Sofroniew and Vinters, 2010; Montgomery, 1994; Nimmerjahn, 2009; Kimelberg, 2004a; Lee et al., 2008; Sharif and Prevot, 2012; Condic et al., 2014; Hansson, 1988; Temple and Alvarez-Buylla, 1999; Wang and Bordey, 2008).
Astrocytes have long been considered as supporting and structural cells for the neurons, playing primarily passive roles in the nervous system (Khakh and Sofroniew, 2015; Araque et al., 1999). Nevertheless, this viewpoint has been gradually changing. The recent evidence has stressed their importance in playing complex and diverse roles in the central nervous system, such as synaptic transmission, information processing in neural circuits and functions (Sofroniew and Vinters, 2010; Shandra and Robel, 2019; Sofroniew, 2005). Additionally, they make widespread contacts with blood vessels, participate in the maintenance of the neural microenvironment, the guidance and support of neuronal migration during development and serve as antigen-presenting cells in the modulation of immune reactions (Montgomery, 1994).
Classically, astrocyte cultures are acquired from the rodent brain (Nimmerjahn, 2009; Foo et al., 2011). In spite of their usefulness, the animal models cannot be directly translated to study similar processes in humans. Recent evidence has suggested important differences between rodent and human astrocytes (Foo et al., 2011; Thomsen et al., 2015). As a result, a culture of human astrocytes is desirable. The human sources for astrocyte isolation include the adult and neonatal brain (Sharif and Prevot, 2012; Condic et al., 2014). The differentiation of neonatal astrocytes may be incomplete because of a lack of normal differentiation signals and distinct gene expression properties. Consequently, neonatal astrocytes are considered more activated than adult cells (Sharif and Prevot, 2012; John, 2012; Nakagawa and Schwartz, 2004). This is especially important when using the cell culture in the study of neurodegenerative diseases. The experimental results from neonatal cells cannot be directly conveyed to the adults, highlighting the superiority of adult astrocyte culture in these cases. Additionally, factors that may influence the astrocyte isolation and increase case-to-case variability include age differences in neonatal donors and different conditions of donor tissue (Sofroniew and Vinters, 2010; Sharif and Prevot, 2012; Oberheim et al., 2009; Kimelberg, 2004b; Chaboub and Deneen, 2012). Moreover, the tissue source for the isolation of adult astrocytes is much more readily available in comparison to neonatal brain tissue, which is taken at 9–22 weeks of age during elective abortions. Not all foetuses are suitable for the isolation. Where abortion is performed after a medical procedure, the tissue is not suitable, since the pharmaceutical agents used for foetal death may alter cell viability and thus hamper the development of primary cell culture (Sharif et al., 2006; Minchev et al., 2019). On the other hand, adult tissue is easily accessible, since many more surgical procedures are performed that may provide the tissue for experimentation. The transport to the laboratory may vary and is usually longer in brain samples taken during abortion. In adults, the tissue is usually more stable as it is taken during the biopsies and reaches the laboratory much quicker (Sharif and Prevot, 2012; Jakovcevski et al., 2009; Rustenhoven et al., 2016; Giffard and Ouyang, 2009).
The aim of this study was to establish a new, simple, efficient and reproducible protocol for isolation and culturing of a highly enriched primary astrocyte culture, obtained from adult cerebral tissue and to evaluate astrocyte functions in vitro. Our established astrocytes are among the few such cell cultures in the world and, to the best of our knowledge, the first originating from this region.
Section snippets
Tissue source
The tissue for human astrocyte isolation was obtained following cranial operations during brain necrectomy in adult neurotrauma patients. Permission for human brain tissue utilisation has been obtained from the ethical committee and a written informed consent from the patient’s relatives was acquired before the experiment (ethical approval number KME/98/14). The cortical tissue has been taken in sterile conditions from the cortical and subcortical regions of the right frontoparietal lobes.
Results
The astrocyte culture described in the experiment consisted of rapidly growing cells that were isolated from cerebral tissue of an adult donor. The primary culture of astrocytes was 100% confluent after one month. The cells were then split in a ratio of 1:3 and after seven days, a 95% confluent culture of the first passage was obtained. We have subcultured the cells to the fifth passage. A part of these cells was stored in liquid nitrogen. A viability of 95% was observed when the cells were
Discussion
Astrocytes are key cells in the central nervous system (Bedner et al., 2019; Kettenmann and Verkhratsky, 2011; Nimmerjahn, 2009). They are involved in many important functions under physiological and pathological circumstances. Primary cultures of astrocytes represent an important target for basic and translational neuroscience research, especially for the in vitro cell models (Nimmerjahn and Bergles, 2015).
The primary cell cultures of astrocytes have been isolated from various sources, usually
Conclusions
The demonstrated isolation process is simple, quick and economical, allowing viable long-term primary astrocyte cell culture. The cultured cells showed the expression of major astrocytic markers and may represent an important new tool for in vitro studies. The availability of such a system will permit the study of cell properties, biochemical aspects and the potential of therapeutic candidates for traumatic and neurodegenerative disorders in a well-controlled environment.
CRediT authorship contribution statement
Lidija Gradisnik: Conceptualization, Methodology, Writing - original draft. Uros Maver: Data curation, Investigation. Roman Bosnjak: Supervision. Tomaz Velnar: Writing - review & editing.
Declaration of Competing Interest
None.
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