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.

中文翻译：

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用于研究异质星形胶质细胞的高效细胞培养系统：时间梯度消化

背景

星形胶质细胞是哺乳动物大脑中最丰富的神经胶质细胞类型，但在细胞发育和细胞功能方面存在许多未知。我们的目标是建立一个星形胶质细胞培养系统，用于从新生小鼠脑中获得高度富集的原代星形胶质细胞，并分离 Aldh1l1 ⁺ Gfap ^-和 Aldh1l1 ⁺ Gfap ⁺细胞。

新方法

出生后 1-4 天的 C57BL/J6 小鼠幼崽用于细胞制备。收集大脑皮层并用 0.25% 胰蛋白酶消化，然后用 0.5 mg/ml DNase 消化。将细胞接种在 PDL 包被的烧瓶上。培养8-10天后，37℃、260rpm振荡4h，去除少突胶质细胞和小胶质细胞。进行时间梯度消化以获得星形胶质细胞亚型。消化时间分别为 0-2 分钟和 2-4 分钟，以及 4-6 分钟。进行流式细胞术、免疫染色、CCK-8 测定和 EdU 染色以研究星形胶质细胞的纯度、细胞增殖能力并鉴定不同的亚型。

结果

摇动后，少突胶质细胞的百分比从 22.6 ± 3.6% 显着下降到 7.4 ± 1.4%（CNPase ⁺细胞）和从 4.36 ± 0.6% 到 0.75 ± 0.2%（Pdgfrα ⁺细胞），而小胶质细胞的百分比从 4.4 ± 0.2% 下降。到 0.6 ± 0.2%。Aldh1l1 ⁺ Gfap ^-星形胶质细胞是 0-2 分钟组的主要细胞类型，而 Aldh1l1 ⁺ Gfap ⁺星形胶质细胞是 2-4 分钟组的主要细胞类型。而且，与Aldh1l1 ⁺ Gfap ⁺星形胶质细胞相比，Aldh1l1 ⁺ Gfap ^-星形胶质细胞具有更高的增殖能力。

与现有方法的比较

分离 Aldh1l1 ⁺ Gfap ^-和 Aldh1l1 ⁺ Gfap ⁺细胞。残留的 Tmem119 ⁺和 Gfap ⁺细胞的百分比没有显着差异。然而，Pdgfrα ⁺细胞的百分比显着降低，并且新系统的耗时更低。获得的星形胶质细胞具有更高的生存能力。

结论

建立了一种新的星形胶质细胞时间梯度消化系统。从新生小鼠脑中获得高度富集的原代星形胶质细胞，只需很短的摇晃时间。Aldh1l1 ⁺ Gfap ^-和 Aldh1l1 ⁺ Gfap ⁺细胞通过不同的消化条件分离。该系统具有高效、低成本的优点，在中枢神经系统星形胶质细胞的管理研究中值得应用。