isolation of cells from murine dermis

从小鼠真皮淋巴毛细血管中分离内皮细胞

The lymphatic system plays a pivotal role in human physiology. It is considered a vital homeostatic factor, that facilitates important functions, such as the maintenance of tissue-plasma fluid balance, immune surveillance, and lipid absorption1, as well as recently-identified functions, such as the repair ability of the heart2 and regenerative capacity of bone and hematopoiesis3. Despite the significant role of the lymphatic system, several aspects of the role of the lymphatics, as well as the molecular mechanisms governing certain physiological parameters and responses remain elusive. Moreover, lymphatic vascular defects are triggering or deteriorating factors in certain pathophysiological conditions. Well-known examples are the primary and secondary lymphedemas, depending on the genetic or non-genetic origin of the disease, respectively, while the role of the lymphatic system on primary tumor growth and metastatic dissemination is pivotal, as it can act as a conduit for metastasis and modulator of immune functions1.
The lag in the study and knowledge of the lymphatic compared to the blood vasculature is mainly due to the later discovery of the lymphatic system in comparison with the blood vascular system and the delay in the identification of lymphatic-specific molecular markers. This has greatly improved in recent decades, leading to the alteration of the conventional picture of the lymphatics at steady state and in diseased conditions1. Similar to angiogenesis, lymphangiogenesis is the formation of new lymphatic vessels from pre-existing ones and plays a pivotal role in the development of a wide spectrum of diseases4,5,6. However, unlike angiogenesis, the investigation of lymphangiogenesis has been limited to mostly in vivo models focusing on developmental vessel formation and structural defects in pathological models. The isolation of the lymphatic endothelial cells is important for in vitro studies, and this can be provided by the presented protocol.
Several protocols for lymphatic endothelial isolation from mice have been developed, which require the use of fluorescence-activated cell sorting7. The advantage of the cell sorter, where available, is the higher degree of purity, contrary to bead-based isolation, with the latter providing a higher yield. The protocol utilizes the expression of lymphatic endothelial hyaluronan receptor 1 (LYVE-1), a lymphatic endothelial marker, important for cell trafficking within the lymphatic vessels4,8. Since LYVE-1 expression is limited to the lymphatic capillaries and not the collecting lymphatic vessels, the technique is suitable for the isolation of lymphatic endothelial cells specifically from the lymphatic capillaries, contrary to other lymphatic markers, such as podoplanin, which are expressed uniformly in all lymphatic endothelial cells9. For the protocol, other important lymphatic markers that were not transmembrane, such as Prox1, were excluded.
As the physiological outcome was lymphangiogenesis, which is usually initiated at the lymphatic capillaries, LYVE-1 was selected as a target due to its selectivity in these cells and because the selected antibody provided a high yield. The enzymes used were collagenase type II, which is generally known to be more effective in collagen dissociation than type I10, and dispase, a broader protease, regularly used for dermis-epidermis separation11; however, other enzymes for tissue separation have been reported12,13 and can be used. The goal of this protocol is to describe a method for dermal lymphatic endothelial cell isolation from lymphatic capillaries of adult mice with high yield using magnetic bead purification, especially in places where fluorescence-activated cell sorting is not readily available. The method is suitable for applications where the purity of lymphatic endothelial cells can be compromised for downstream applications.

作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

小鼠真皮淋巴内皮细胞分离

从小鼠真皮中分离细胞

在安乐死的小鼠的生物安全柜内执行该方案，适当剃须并用70%乙醇消毒。用剪刀在动物的腹部做一个 0.5 英寸的浅切口。沿中线纵向切至颈部和下腹部。此外，进行横向切口以剥开皮肤。使用钝镊子轻轻地将皮肤与周围组织分开，同时绕过动物的躯干，在颈部和下腹部横向切割皮肤。小心地将皮肤置于真皮面朝下，置于 100 毫米组织培养板中。并确保皮肤尽可能拉伸。向板中加入 6 毫升分散酶溶液，确保皮肤完全覆盖。然后，盖上盘子。用石蜡膜包裹板。将板在室温下孵育 10 分钟，使外植体变平。第二天，使用移液管从含有分离皮肤的板中取出液体。然后，在镊子的帮助下，将真皮与表皮分离，同时从中去除任何可见的脂肪组织。并将分离的真皮放入干净的组织培养板中。在培养板中的真皮中加入一毫升含有 1X 抗生素 - 抗真菌溶液的 DMEM。倾斜板以将液体积聚在板的一侧。然后，将真皮切成一到两平方毫米的碎片。并将它们转移到一个新的 50 毫升锥形管中。向试管中加入 10 毫升 II 型胶原酶溶液。在 37 摄氏度下孵育两个小时，以 30 至 50 rpm 的速度连续搅拌以消化真皮。通过 70 微米细胞过滤器过滤细胞悬浮液。并以 250 克离心 5 分钟。弃去上清液后，将沉淀重悬于含有抗生素 - 抗真菌剂的10毫米DMEM溶液中。如前所述，在离心之前通过 40 微米细胞过滤器过滤细胞悬浮液。将所得细胞沉淀重悬于一毫升完全 LEC 培养基中。然后，将细胞铺在胶原包被的 60 毫米组织培养板中的完全 LEC 培养基中。每两天更换一次培养基，用PBS洗涤细胞两次，并加入新鲜的LEC培养基。从小鼠真皮中分离的细胞的低倍率图像显示混合细胞群。

Research

JoVE Journal

Biology

Isolation of Cells from Murine Dermis

162 次观看. 在安乐死的小鼠的生物安全柜内执行该方案，适当剃须并用70%乙醇消毒。用剪刀在动物的腹部做一个 0.5 英寸的浅切口。沿中线纵向切至颈部和下腹部。此外，进行横向切口以剥开皮肤。使用钝镊子轻轻地将皮肤与周围组织分开，同时绕过动物的躯干，在颈部和下腹部横向切割皮肤。小心地将皮肤置于真皮面朝下，置于 100 毫米组织培养板中。并确保皮肤尽可能拉?...

视频: 从小鼠真皮中分离细胞

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...