Background

Mesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival.

Methods

PBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 10⁷/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50–60,000 cells/well and cultured in 96 well plates for 4–48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0–5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader.

Results

PBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased <20% at the highest PHA concentrations. Co-culture with MSCs suppressed PBMC proliferation dependent upon MSC passage number, source, and prior growth conditions. Total time to complete the ATP assay was under an hour including incubations. With minimal manipulations in the assay, intra- and inter- assay variations averaged 11.1 and 15.7% respectively.

Conclusions

Direct co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.

中文翻译：

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使用混合淋巴细胞的简化增殖测定，用于评估人间充质干细胞的免疫调节活性

背景

间充质基质细胞（MSCs）已被提议用于治疗急性呼吸窘迫综合征（ARDS），移植物抗宿主病（GVHD），伤口愈合和创伤。建立共识是，MSC的免疫调节对于治疗潜力很重要。MSC在体外抑制外周血单个核细胞（PBMC）增殖，可能反映出在体内抑制PBMC炎症反应的能力。当前通常用于评估MSC效能的混合淋巴细胞反应（MLR）分析通常依赖于直接共培养或间接培养，使用transwell系统在有丝分裂非活性MSC的存在下监测分离的PBMC的增殖。PBMC的增殖可通过几种方法进行监测，包括掺入放射性标记核苷酸，羧基荧光素标记的PBMC的BRDU标记和ELISA分析或流式细胞术。在这里，我们提出了一种在直接共培养系统中使用MSC与未修饰的MSC进行简化的分析，并使用发光ATP分析来评估PBMC和MSC增殖/存活的方法。

方法

通过在LeucoSep管中通过Ficoll-Paque离心从新鲜的抗凝全血中分离出PBMC。收集8至10个供体的分离的PBMC，并以1×10 ⁷冷冻保存。/ ml在50％RPMI培养基，10％DMSO，40％人AB血清中。将源自骨髓，脂肪组织或脐带的MSC（分别为BM-MSC，Ad-MSC，UC-MSC）从50-60,000个细胞/孔开始连续稀释，并在96孔板中培养4–48 h。各自的媒介。在第0天，将MSC洗涤，重悬于PBMC培养基（含10％FBS，2 mM谷氨酰胺，10 mM HEPES，pH 7.4的RPMI）中，并在有或没有植物血凝素存在或不存在的情况下与每孔150,000新鲜融化的PBMC一起孵育。 A（PHA，0–5μg/ ml）。使用生物发光试剂Cell Titer-Glo（Promega）对ATP含量进行定量，从而评估了MSC（粘附的）和PBMC（非粘附的）的增殖情况。培养上清液含有PBMC，而洗涤的贴壁细胞主要是MSC。

结果

响应PHA刺激的PBMC增殖导致ATP显着增加72 h，比未刺激的PBMC增长> 6倍，后者未显示增加。在最高PHA浓度下，MSC增殖降低了<20％。与MSC共培养可抑制PBMC增殖，具体取决于MSC的传代次数，来源和先前的生长条件。包括孵育在内，完成ATP测定的总时间不到一个小时。在测定中进行最少的操作后，测定内和测定间变化的平均值分别为11.1和15.7％。

结论

将未修饰的活MSC与新鲜融化的PBMC直接共培养，可以轻松轻松地可靠地确定MSC的免疫抑制作用。可以确定分级的反应，从而可以比较MSC制剂之间的效价，就像比较新鲜融化和培养的MSC以及干扰素-γ许可的MSC一样。使用96孔板测定法，与典型的流式细胞仪测定相比，通常所需的PBMC少得多。这种简化的测定可以在72小时内进行，而无需照射细胞，也不需要专门的设备。