**3.** *In vitro* **models**

However, it is not difficult to find primary CNS cell cultures from rodents such as mice and rats, embryonic cells, mainly neurons, and oligodendrocytes are limited.

It can be more difficult for isolating cells from adult animals and also from the human brain [36]. Because of these limitations, cell lines are used in most of the studies [37]. Among many cell lines, glial cell lines are used frequently because of a key role in the explanation of the mechanisms involved in health and disease; however, it is important to ensure the cell lines have properties like *in vivo* conditions [38].

Microglial cell activation may be seen in active demyelinating lesions. Also, it can be seen in pre-active lesions, remyelination areas, and the normal-looking white matter [39]. For finding the functions of these cells in MS, isolation of microglia from embryonic or early post-natal animals was done before [40]. However, it must be mentioned that in the field of MS and other neurodegenerative disorders, using young animal cells may not be relevant to study chronic diseases that happened in older animals and humans [41, 42]. Thus, few studies are using aged microglial cells isolated from adult animals such as rhesus monkeys and humans. Notably, because of many limitations such as ethical aspects, using cells isolated from post-mortem are often in humans and primates [42, 43].

An important issue with respect to the use of microglia cell cultures is what it tells us about the pathogenesis of MS. Based on previous studies, primary human microglia cultures derived from MS brain tissue versus healthy brain have the major advantage of revealing pathways involved in the disease process [44]. Another major problem is microglial cells like other cells of the innate immune system, which reacts quickly to any danger or foreign signals. Therefore, the use of fast isolation methods for revealing the initial trigger of microglia activation is critical [45]. Studies using a detailed transcriptomic analysis in the cuprizone animal model of MS found that in the early stages of demyelination, a microglia phenotype supportive of regeneration is observed [46]. It stays to be determined if this is also observed in tissues affected by multiple sclerosis, which are normally available only after a very long continuance of the disease. Nevertheless, multiple sclerosis lesions disappear throughout the disease and early stages of lesion development (microglia clusters considered pre-active lesions) are honored even after a long continuance of the disease. Detailed studies using animal models may provide important facts about the role of microglia in lesion development in MS [47].

Oligodendrocytes are diligent in the production of myelin in the CNS [48]. In CNS tissues, various development stages of oligodendrocytes can be observed, including the pre-progenitor, progenitor, pro-oligodendrocyte, and juvenile oligodendrocytes. Each of these steps can be identified *in vitro* and *in vivo* through the expression of several different molecules such as proteins involved in myelin structure and production. It can be possible to maintain primary rodent oligodendrocytes *in vitro* for up to several weeks by several methods [49]. According to previous findings, when these cells proliferate and differentiate depending on the culture medium *in vitro*, the features of these cells may change during subcultures. Thus, re-checking the characteristics of passaged cells is very important [50].

In general, isolation of primary oligodendrocytes is dependent upon their ability to not adhere to culture plates. This feature has benefits because by gently shaking isolated cells from the CNS, microglia and adherent atrocities can be separated from floating oligodendrocytes [51]. A disadvantage of the primary cell culture of oligodendrocytes is that they are usually only available in small numbers. However, mature oligodendrocytes can be obtained when glia progenitors are cultured in serum-free media or by differentiating stem cells [52].

Other important cells that change with MS include astrocytes. In the injured brain, the shape of astrocytes turns into hypertrophic form and the scar tissue as typical of chronic MS lesions generate [53, 54]. When astrocytes are damaged, they are at the risk of losing the ability to maintain the blood–brain barrier (BBB), thus contributing to additional damage [55]. Astrocytes also contribute to the repair process by secreting growth factors, so they have the ability to promote regeneration [56, 57]. By now, several human and animal cell lines are available, as well as primary astrocyte cultures. Primary astrocyte cultures often grow slowly. Therefore, it is the advantage of astrocytes not only being suitable for repeated subcultures, but also suitable for cryopreservation [58]. Many studies using the cell lines of astrocytes are available. Usually, they have been derived from rodents or isolated from human brain tissue with astroglioma. A weakness of astrocytic cell lines is that they respond in a different way in comparison with primary cultures [59]. Several protocols allow obtaining primary astrocytes from adult tissues or post-mortem fetal as well as from biopsies or resected brain tissue in neurosurgery cases. While primary human astrocytes are attractive to culture, care must be taken to ensure the adequate removal of microglia that frequently contaminate primary astrocyte cultures and may influence responses in culture [60].

In MS research, the increasing awareness that axonal damage and neurodegeneration contribute to the progression of the disease has prompted researchers to use neuronal cells. One ordinary cell line is the neuroblastoma cell line is SH-SY-5Y that can be differentiated with retinoic acid (RA), while the HCN and the NT2 cell lines are differentiated with brain-derived growth factor (BDNF). But it has to be considered that these cells cannot express many markers of mature neuronal cells. These cell lines are also slow to proliferate *in vitro and* require expensive growth factors [61, 62]. Absolutely, it looks like finding more neuronal cell lines is necessary for a better MS search.
