An efficient cell culture system for the studies of heterogeneous astrocytes: Time gradient digestion

Background: Astrocytes are the most abundant glial cell type in mammal brain, but there exists a lot of unknown in cell development and cell function. We aim to establish an astrocytes culture system for obtaining highly enriched primary astrocytes from the neonatal mouse brain and separating Aldh1l1⁺Gfap^- and Aldh1l1⁺Gfap⁺ cells.

New method: C57BL/J6 mouse pups at postnatal 1-4 days were used for cell preparation. Brain cortex was collected and digested with 0.25% trypsin followed by 0.5 mg/ml DNase. Cells were plated on PDL-coated flasks. After 8-10 days culture, cells were shaken at 260 rpm for 4 h at 37 ℃ to remove oligodendrocytes and microglia cells. Time gradient digestion was performed to obtain astrocyte subtypes. The digestion time was 0-2 min and 2-4 min, and 4-6 min. Flow cytometry, Immunostaining, CCK-8 assay and EdU staining was carried out to investigate the purity of the astrocytes, the ability of cell proliferation and to identify different subtypes.

Results: After shaking, percentage of oligodendrocytes significantly decreased from 22.6 ± 3.6% to 7.4 ± 1.4% (CNPase⁺ cells) and from 4.36 ± 0.6% to 0.75 ± 0.2% (Pdgfrα⁺ cells) while percentage of microglia cells reduced from 4.4 ± 0.2% to 0.6 ± 0.2%. Aldh1l1⁺Gfap^- astrocytes were the dominant cell types in 0-2 min group while Aldh1l1⁺Gfap⁺ astrocytes were the dominant cell types in 2-4 min group. Moreover, compared with Aldh1l1⁺Gfap⁺ astrocytes, Aldh1l1⁺Gfap^- astrocytes had a higher proliferative ability.

Comparison with existing methods: Aldh1l1⁺Gfap^- and Aldh1l1⁺Gfap⁺ cells were separated. The percentage of residual Tmem119 ⁺ and Gfap⁺ cells showed no significant difference. However, the percentage of Pdgfrα⁺ cells were significant decreased, and the time consuming of the new system was lower. The astrocytes acquired possess higher viability.

Conclusions: A new astrocytes culture system with time gradient digestion was established. Highly enriched primary astrocytes from the neonatal mouse brain were obtained with short shaking time. Aldh1l1⁺Gfap^- and Aldh1l1⁺Gfap⁺ cells were separated by different digestion condition. This system has advantages of high efficiency and low cost, which deserves promising application in management of astrocytes research in central nerve system.