巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

摘要

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

摘要

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

引言

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages.

Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages ¹. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function ². These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS ^2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages ⁶.

During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

研究方案

白细胞分离是根据人类主体的卫生机构审查委员会的国家机构认可的研究方案进行。

1.分离的单核细胞冷冻保存

1. 得到单核细胞由人类供体的白细胞分离，并通过如在参考文献^7,8中所述的单核细胞的连续逆流离心淘析丰富的单核细胞。得到约100×10 ⁶淘洗细胞（约80 - 90％的单核细胞）在由制造商指定的淘析缓冲。算使用血球细胞。
2. 在15ml聚丙烯管中离心的单核细胞在300 xg离心 在室温下5分钟。
3. 除去上清液并轻轻悬浮随后二甲基亚砜FBS中的细胞达到90％的FBS / 10％二甲亚砜的终浓度和50×10 ^6个细胞/ ml。
4. 将每个毫升细胞悬液ŤØ个别冷冻管。
5. 放置冷冻管在细胞冷冻容器和转移至液氮冷冻管贮槽用于长期保存前转移到-80℃冷冻24小时。

2.单核细胞分化成巨噬细胞

1. 通过快速转移到37℃水浴中，并在细胞悬浮液解冻约70％，然后立即除去解​​冻细胞的离心管。
2. 后立即在室温下完全融化，转移1毫升离心管内容到50ml的37℃温热罗斯韦尔园区纪念研究所（RPMI）有2mM L-谷氨酰胺，50纳克/毫升的M-CSF，25纳克/毫升1640培养基白细胞介素-10（IL-10），和10％FBS（完全培养基）。
3. 转印25毫升单核细胞悬浮液到每两个75cm ²的塑料细胞培养瓶中的。
4. 孵育培养物中，用5％ _二氧化碳 / 95％空气的37℃的细胞培养培养箱中48小时。
5. 冲洗立方米ltures 3次用10ml的RPMI 1640培养基（预热至37℃），轻轻除去培养基，以避免撞出任何松散附着的细胞。
6. 后续漂洗，加入新鲜完全培养基，培养基改变每2天，直到单核细胞分化，增殖，足以成为融合。这需要大约一个星期的文化。

3.收获巨噬细胞实验启动

1. 用10毫升漂洗分化的巨噬细胞在烧瓶3次预热37℃的Dulbecco磷酸盐缓冲盐水不含Ca ²⁺和Mg ²⁺加入10ml预热的37℃的0.25％胰蛋白酶-乙二胺四乙酸（EDTA）溶液之前。
2. 此时的巨噬细胞的约90％应具有圆形和拆卸15分钟 - 在10细胞培养孵化地方烧瓶中。由烧瓶镜检验证这一点。
3. 加入10 mL RMPI 1640培养基含有10％FBS送顶部胰蛋白酶。
4. 从烧瓶中转移巨噬细胞悬浮到50ml的聚丙烯管中，离心300×g离心5分钟，并弃去上清液。
5. 轻轻重悬巨噬细胞在1ml完全培养基。
6. 混合10微升巨噬细胞悬浮液与10微升锥虫蓝溶液，并确定使用血球巨噬细胞数量和生存力（典型地> 95％）。
7. 根据细胞计数（通常为约15 - 18×10 ^5个巨噬细胞），加入以达到所需接种细胞密度额外的完全培养基。注意:在每一个12孔板的孔1.5ml介质1×10 ^5个巨噬细胞将产生一个近汇合培养。
8. 以下播种，过夜孵育的巨噬细胞中，用5％ _二氧化碳 / 95％空气的37℃的细胞培养孵化器，以允许细胞附着。然后，开始所需的实验。

结果

与台盼蓝染色⁹确定的新鲜或冷冻保存的单核细胞的存活力大于95％。 图 1和图2在较低和较高的倍率进行比较，分别新鲜和冷冻（ 即冷冻）的单核细胞分化的进展成巨噬细胞。注意，新鲜比较了冷冻保存的单核细胞显示分化的单核细胞不传播，但仍然圆形的亚群。相朗讯液泡发生在这两个条件下，单核细胞衍生的巨噬细...

讨论

定义生成巨噬细胞类型可以澄清一些研究巨噬细胞生物学当调查员获得的结果相互矛盾的。使用各种培养条件和分化因子，以产生初级人类巨噬细胞可以导致非常不同的巨噬细胞的类型，可能无法由研究人员可以理解一个事实。例如，巨噬细胞有时从人单核细胞用无血清，单独的人血清，人血清补充有M-CSF，FBS单独，或含M-CSF或GM-CSF的FBS的生成。如先前报道和如我们还观察到，单核细胞为一个星期...

材料

Name	Company	Catalog Number	Comments
Cellbind 12-well culture plate	Corning	3336
CELLSTAR, T-75 flask, tissue culture treated	Greiner Bio-One North America	658157
RPMI 1640 culture medium	Cellgro Mediatech	15-040-CM	warmed to 37 °C
L-Glutamine	Cellgro Mediatech	25-005-CI
Fetal bovine serum	Gibco	16000-036
M-CSF	PeproTech	300-25
GM-CSF	PeproTech	300-03
IL-10	PeproTech	200-10
DMSO	Sigma	D2650
Cryovial	Thermo Scientific	375418
DPBS without Ca2+ and Mg2+	Corning cellgro	21-031-CV
0.25% Trypsin-EDTA	Gibco	25200-056
50 ml polypropylene conical tube	Falcon	352070
Trypan Blue	Lonza	17-942E
Neubauer-improved bright light hemocytometer	Paul Marienfeld GmbH & Co. KG	610031	http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html
CoolCell LX cell freezing container	BioCision	BCS-405	other freezing containers also should  be adequate for this step
Liquid Nitrogen Storage System, CryoPlus 1	Thermo Scientific	7400	any liquid nitrogen tank should be adequate