Establishing a culture system that supports in vitro expansion of adult microglia

Introduction: The vast majority of in vitro research on microglia are based on cells isolated from neonatal animals (3-5 days of age). Studying microglia of adults has been limited by the lack of a suitable culture system that supports their growth. In this study, we describe a protocol for growing microglia of adults based on modifications of the technique for culturing microglia isolated from neonatal rats. Methods: Mixed glia isolated from adult rats (age range of 1 month to 3 years old) were seeded in culture flasks coated with poly-L-lysine. Cells were maintained in DMEM media supplemented with insulin-transferrin-selenium (ITS) and recombinant human macrophage colony-stimulating factor (M-CSF). Mild trypsinisation was carried out to isolate microglia from mixed glia culture. Results: Microglia cells of adult rats were successfully grown in vitro. For the expansion of adult microglia, it was observed that coating the cell culture flasks with poly-L-lysine was crucial to encourage cell adherence. The substitution of insulin in culture media with ITS was found to improve cell yield and reduced the number of days required for culture from 28 days to 14 days. Addition of M-CSF to cell culture medium, along with the improvisations described above provided the best adult microglia cell yield (2.91 ± 0.56 × 10 6 cells) compared to the technique of replating cells (0.91 ± 0.65 × 10 6 cells; p<O.O5). Conclusion: Optimisation of primary cell culture technique by coating culture flasks with poly-L-lysine, supplementation of culture medium with ITS and M-CSF allowed microglia of adult rats to be successfully cultured in vitro

Effects of Age on Microglial Responses to Cellular Stress Induced by Lipopolysacharides and Beta Amyliod

Microglia are the resident macrophages of the central nervous system (CNS). In the normal CNS, they are in a resting condition, characterised by low expression of MHC class II (MHC II) and co stimulatory molecules such as CD40. Following activation by various stressors, microglia acquire an inflammatory phenotype and the continuous activation of microglia is thought to exacerbate neuronal damage. It is also believed that with increasing age, the inflammatory response of microglia becomes uncontrolled. In this study, the effects of age on microglia responses were determined by culturing microglia from Sprague Dawley rats of 6 days (neonates), 2-4 months (adult) and 3 years (old). Microglia were then activated with lipopolysaccharide (LPS; 1μg/ml) or beta amyloid (Aβ; 25 or 50μg/ml) and assessed for expression of CD40 and MHC II activation markers, nitric oxide (NO) production and proliferative capacity at pre-determined time points. Adult microglia were difficult to culture, therefore ptimisations were carried out to support and enhance cell growth by coating culture flasks with poly-L-lysine and supplementing culture with insulin-transferrin-selenium (ITS) and macrophage colony stimulating factor (M-CSF). Expression of CD40 and MHC II was determined by immunophenotyping. Basal expression of CD40 and MHC II was higher in older microglia compared to neonates. Similarly, after activation with LPS and Aβ, CD40 and MHC II expression was higher in older cells. Upon LPS stimulation, the CD40 and MHC II expression was as follows:neonates, 11.3 ± 1.8% and 4.2 ± 2.3%; 2-4 months, 32.4 ± 25.2% and 21.2 ± 8.8%; 3 years, 26.4 ± 15.3% and 21.1 ± 12.6% respectively. Upon stimulation with Aβ, the CD40 and MHC II expression was: neonates, 50.3 ± 19.8% and 3.6 ± 1.4%; 2-4 months, 63.9 ± 24.5% and 36.9 ± 7.4%; 3 years, 75.2 ± 24.1% and 15.1 ± 11.5% respectively. The higher expression of CD40 and MHC II in older microglia is indicative of its potential for subsequent activation of T cells. Using the Griess assay and [3H]-thymidine roliferation assay, it was observed that the NO production and proliferation rate was induced at 48hrs and 18hrs time point respectively. Under resting condition, older microglia released marginally higher NO compared to neonates (p<.05); however, upon stimulation, older microglia released significantly lower (p<.05) NO production compared to neonates. Under resting condition, the NO production was as follows: neonates, 2.0μM; 2-4 months, 6.9μM and 3 years, 5.3μM. Upon stimulation with LPS and 50μg/ml Aβ, the NO production was: neonates, 29.3 and 60.2μM; 2-4 months, 11.9 and 9.9μM; 3 years, 9.9 and 5.7μM respectively. As for the proliferation capacity,older cells displayed significantly lower proliferation (p<.05) both when resting and upon stimulation, compared to neonates. In the resting condition, 2-4 months and 3 years old microglia recorded readings of 39929 cpm and 37328 cpm lower compared to neonates. Upon stimulation with LPS and 50μg/ml Aβ, proliferation rate of older microglia were consistently lower compared to neonates. Thus, as a result of our work, we demonstrated differences between primary neonatal and adult microglia responses to LPS and Aβ stimulation by comparing activation profiles, NO and proliferation rate. We demonstrated that although older cells exhibit a higher activation state upon stimulation (based on CD40 and MHC II expression), their functional aspects such as NO production and proliferation remain significantly low compared to neonat

Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid

A challenge for studies involving microglia cultures is obtaining sufficient cells for downstream experiments. Macrophage colony-stimulating factor (M-CSF) has been used to improve yield of microglia in culture. However, the effects of M-CSF on activation profiles of microglia cultures are still unclear. Microglia activation is characterised by upregulation of co-stimulatory molecules and an inflammatory phenotype. The aim of this study is to demonstrate whether M-CSF supplementation alters microglial responses in resting and activated conditions. Microglia derived from mixed glia cultures and the BV-2 microglia cell line were cultivated with/without M-CSF and activated with lipopolysaccharide (LPS) and beta amyloid (Ab). We show M-CSF expands primary microglia without affecting microglial responses to LPS and Ab, as shown by the comparable expression of MHC class II and CD40 to microglia grown without this growth factor. M-CSF supplementation in BV-2 cells had no effect on nitric oxide (NO) production. Therefore, MCSF can be considered for improving microglia yield in culture without introducing activation artefacts

Evaluating anticancer and immunomodulatory effects of spirulina (Arthrospira) platensis and gamma-tocotrienol supplementation in a syngeneic mouse model of breast cancer

Nutrition can modulate host immune responses as well as promote anticancer effects. In this study, two nutritional supplements, namely gamma-tocotrienol (γT3) and Spirulina, were evaluated for their immune-enhancing and anticancer effects in a syngeneic mouse model of breast cancer (BC). Five-week-old female BALB/c mice were fed Spirulina, γT3, or a combination of Spirulina and γT3 (Spirulina + γT3) for 56 days. The mice were inoculated with 4T1 cells into their mammary fat pad on day 28 to induce BC. The animals were culled on day 56 for various analyses. A significant reduction (p 0.05) differences in the expression of MIG-6, Cadherin 13, BIRC5, and Serpine1 upon combined feeding. This showed that combined γT3 + Spirulina treatment did not show any synergistic anticancer effects in this study model

Bone marrow-derived mesenchymal stem cells modulate BV2 microglia responses to lipopolysaccharide.

The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (p < .05). Co-culturing MSC with BV2 cells at different ratios revealed interesting dynamics in NO production. A high number of MSC significantly increases NO in co-cultures whilst a lower number reduces NO. The increased NO levels in co-cultures may be MSC-derived, as we also show that activated BV2 cells stimulate MSC to produce NO. Cell-cell interaction is not a requirement for this effect as soluble factors released by activated BV2 cells alone do stimulate MSC to produce high levels of NO. Although NO is implicated as a mediator for T cell proliferation, it does not appear to play a major role in the suppression of microglia proliferation. Additionally, MSC reduced the expression of the microglial co-stimulator molecule, CD40. Collectively, these regulatory effects of MSC on microglia offer insight into the potential moderating properties of MSC on inflammatory responses within the CNS