Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
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DOI：
10.3791/3814
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发表时间：
2012-08-01
影响因子：
1.2
通讯作者：
Byrnes, Kimberly R.
中科院分区：
文献类型：
--
作者：
Tamashiro, Tami T.;Dalgard, Clifton Lee;Byrnes, Kimberly R.
小胶质细胞约占哺乳动物脑细胞总数的12%。虽然神经元和星形胶质细胞被认为是神经系统的主要细胞类型，但小胶质细胞通过监测组织中的碎片和病原体并通过吞噬活性维持实质中的稳态，在正常脑生理学中发挥重要作用1，2。小胶质细胞在许多损伤和疾病条件下被激活，包括神经退行性疾病，创伤性脑损伤和神经系统感染（3）。在这些活化条件下，小胶质细胞增加其吞噬活性，经历形态学和增殖变化，并主动分泌活性氧和氮物质、促炎趋化因子和细胞因子，通常活化旁分泌或自分泌环（4-6）。由于这些小胶质细胞反应有助于神经系统疾病的发病机制，因此有必要对小胶质细胞进行研究。由于脑细胞的异质性，在体内实验中很难获得足够的高纯度小胶质细胞样品材料。目前对小胶质细胞的神经保护和神经毒性功能的研究需要常规的技术方法来持续地产生具有足够产量的纯的和健康的小胶质细胞用于研究。我们提出，在文本和视频中，一个协议，以分离纯初级小胶质细胞的混合神经胶质细胞文化的各种下游应用。简言之，该技术利用新生大鼠幼仔的分离脑组织产生混合神经胶质细胞培养物。在混合的神经胶质培养物达到汇合后，通过短暂的振荡从培养物中机械分离原代小神经胶质细胞。然后将小胶质细胞以高纯度铺板用于实验研究。该方法的原理和方案已在文献中描述（7，8）。此外，分离原代小胶质细胞的替代方法也有很好的描述（9-12）。可以通过密度梯度离心分离均质化的脑组织以产生原代小胶质细胞（12）。然而，离心是中等长度的（45分钟），并且可能导致细胞损伤和活化，以及导致富集的小胶质细胞和其他细胞群体。另一种方案已被用于通过在培养时长时间（16小时）振荡来分离各种生物体中的原代小胶质细胞（9-11）。振摇后，离心培养基上清液以分离小胶质细胞。这种较长的两步分离方法也可能干扰小胶质细胞的功能和激活。我们在实验室中主要使用以下小胶质细胞分离方案，原因如下：（1）原代小胶质细胞比永生化啮齿动物小胶质细胞系更忠实地模拟体内生物学，（2）标称机械破坏使潜在的细胞功能障碍或活化最小化，以及（3）不需要混合的神经胶质细胞培养物传代就可以获得足够的产量。重要的是要注意，该方案使用的是来自新生大鼠的脑组织，使用幼鼠分离小胶质细胞和使用老年大鼠分离小胶质细胞可以显著影响分离的小胶质细胞的产量、激活状态和功能特性。有证据表明，衰老与小胶质细胞功能障碍，神经炎症增加和神经退行性病变有关，因此以前的研究使用离体成人小胶质细胞来更好地了解小胶质细胞在神经退行性疾病中的作用，其中衰老是重要参数。然而，离体小胶质细胞不能在培养物中长时间保持。因此，虽然该方案延长了培养中的初级小胶质细胞的寿命，但应该注意的是，小胶质细胞的行为与成人小胶质细胞不同，并且当转化为体内环境时，应仔细考虑体外研究。
Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection (3). Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop (4-6). As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.The principle and protocol of this methodology have been described in the literature (7,8). Additionally, alternate methodologies to isolate primary microglia are well described (9-12). Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia (12). However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture (9-11). After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.