PneumaCult™ 肺泡类器官培养基

人肺泡类器官扩增和分化的细胞培养基

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产品号 #100-0847_C

人肺泡类器官扩增和分化的细胞培养基

产品优势

生理相关性。使用体外培养系统模拟人体肺泡生理学,该系统重现了体内ATII和ATI细胞的关键特征。
生物库。利用培养基的生物样本库功能冻存类器官以及重启类器官培养。高产量。使用支持ATII类器官传代和长期扩增的培养基最大限度地提高初始样本的产量。
简单。使用方便的设计和易于使用的方案生成成熟的ATII和完全分化的ATI类器官。
可靠。使用通过质量控制测试的标准化培养基来确保实验可重复性。

产品组分包括

PneumaCult™肺泡类器官培养基（产品号：#100-0847）；

PneumaCult™肺泡类器官扩增基础培养基，450 mL；
PneumaCult™肺泡类器官扩增10X添加物，50 mL；
PneumaCult™肺泡类器官扩增100X传代添加物，1.5 mL；

PneumaCult™肺泡类器官分化培养基（产品号：#100-0861）；

PneumaCult™肺泡类器官分化基础培养基，90ml；
PneumaCult™肺泡类器官分化10X添加物，10ml

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总览

从人肺泡上皮细胞中生长、扩增和分化类器官。与传统使用的动物模型不同，肺泡类器官模拟体内肺泡的区域特异性，使其成为肺泡生物学研究、传染病研究和药物筛选的理想模型。使用PneumaCult™肺泡类器官培养基，可长期高效地传代和扩增人肺泡上皮II型（ATII）细胞形成类器官，并进一步将这些ATII类器官分化为肺泡上皮I型（ATI）细胞，用于下游应用。

使用PneumaCult™肺泡类器官扩增（AvOE）培养基在10代内可将ATII细胞扩增10000倍；显著减少对供体组织的依赖。可以通过将在 PneumaCult™ AvOE 培养基中培养的类器官冷冻保存为单细胞，灵活地在任何传代暂停培养。该培养基含有三组分、无血清配方可提高实验的可重复性，扩增获得的类器官保持了典型的ATII细胞表型特性，包括自我更新的能力、预期的标记物表达（例如HT2-280和SP-C）以及向ATI细胞分化的谱系潜力。PneumaCult™AvOE培养基也可用于生成肺泡单层，用于后续气-液界面（ALI）培养，具体操作可参见我们的标准流程。

使用PneumaCult™肺泡类器官分化（AvOD）培养基，可将在AvOE 培养获得的 ATII 类器官诱导分化为 ATI 细胞。
该两组分PneumaCult™AvOD培养基可在短短的10天内实现分化，纯度高达 85% 以上。分化后的细胞表现为 ATII 标志物显著降低，而 ATI 标志物（如 RAGE/AGER、HT1-56 和 GPRC5a）显著上调。

PneumaCult™AvOE和AvOD培养基均与原代分离的新鲜或冷冻保存的ATII细胞以及高质量的商业化肺泡上皮细胞源兼容。

欢迎访问我们的肺类器官学习中心，进一步了解肺类器官培养体系与应用。

Data Figures

PneumaCult™ Alveolar Organoid Media workflow diagarm illustrating the expansion and differentiation stages of the protocol.

Figure 1. Generation of Alveolar Organoids Using PneumaCult™ Alveolar Organoid Media

The PneumaCult™ Alveolar Organoid Media workflow is a two-stage protocol. During the expansion stage, primary isolated or cryopreserved human ATII single cells are seeded in PneumaCult™ Alveolar Organoid Expansion (AvOE) Seeding Medium. On day 2 - 3, after a full-media change, cultures are expanded using PneumaCult™ AvOE Medium to obtain mature ATII organoids. In the differentiation stage, ATII organoids are cultured for 10 additional days using PneumaCult™ Alveolar Organoid Differentiation (AvOD) Medium to generate ATI organoids.

Brightfield image of alveolar organoids and a bar graph of organoid forming efficiencies of ATII organoid cultures.

Figure 2. PneumaCult™ Alveolar Organoid Expansion Medium Supports High Organoid Forming Efficiency

Organoid forming efficiency (OFE) serves as a functional measure of the health of an organoid culture and the performance of the media. ATII organoids were expanded in PneumaCult™ AvOE Medium (160 cells seeded per dome). (A) Brightfield image taken at day 9 using a 4X objective. The entire dome was captured in the image, each organoid formed was counted, and indicated (total 44; OFE=27.5%). (B) Bar graph showing OFE of ATII organoid cultures derived from three different donors (means ± SD). OFE = (Organoids Formed/Cells Seeded) x 100%.

Comparison of the cumulative doublings for alveolar organoids cultured in PneumaCult™ and published formulation.

Figure 3. PneumaCult™ AvOE Medium Supports Long-Term Expansion and Cryopreservation For Biobanking

(A) Box and whiskers plot of the cumulative doublings at each passage (n=4 independent donors) for organoids expanded in PneumaCult™ AvOE Medium (orange) and published formulation (gray). The means are connected by a line. Long-term passaging of alveolar organoids and over 10000x fold expansion was achieved by both PneumaCult™ AvOE Medium and published formulation. (B) Bar graph showing population doublings (mean ± SD) of organoids generated from cells previously cryopreserved at early and late passages, and expanded in PneumaCult™ AvOE Medium. The cultured organoids retained their expansion potential after thawing.

Plots showing ATII and ATI cell marker expression in alveolar organoids cultured in PneumaCult™ Media.

Figure 4. PneumaCult™ Alveolar Organoid Media Maintains High Levels of ATII and ATI Cell Marker Expression

(A) Flow cytometric gating of fluorescence minus one (FMO) controls for HT2-280 and HT1-56 markers (top), stained cells from organoids expanded in PneumaCult™ AvOE Medium (middle), and subsequently differentiated in PneumaCult™ AvOD Medium (bottom). PneumaCult™ AvOE Medium maintains both the ATII marker expression (HT2-280) and lineage potential to differentiate into ATI cells (HT1-56) in PneumaCult™ AvOD Medium. (B) Organoids derived from 4 independent donors, expressing high levels of HT2-280 were cultured in PneumaCult™ AvOE Medium or published formulation, and compared at each passage. Dashed line depicts 50% of starting HT2-280++%. (C) Scatter plot illustrating the percent change in HT2-280 expression between P0 and P5 from the same donors. Organoids cultured in PneumaCult™ AvOE Medium maintained higher levels of HT2-280 marker expression, compared to those cultured using published formulation. Mean is displayed as a solid line. * = p<0.05 in a one-tailed paired T-test (n=4).

Immunostained alveolar organoids showing expression of ATI and ATII markers.

Figure 5. Immunohistochemistry Confirms Expression of ATII and ATI Cell Markers in Alveolar Organoids

(A) Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) express the ATII markers, HT2-280 (green) and pro-surfactant protein C (pro-SPC, yellow). When further differentiated in PneumaCult™ AvOD Medium (bottom), the levels of these ATII markers are down-regulated, while the ATI marker RAGE (red) is up-regulated. (B) Differentiated organoids also express high levels of ATI marker GPRC5a (yellow).

Immunostained alveolar organoids showing expression of SARS-CoV-2 entry markers.

Figure 6. Organoids Cultured in PneumaCult™ Alveolar Organoid Media Express TMPRSS2 and ACE2, Key Markers for SARS-CoV-2 Entry

Organoids from three donors expanded in PneumaCult™ AvOE Medium (top) and differentiated in PneumaCult™ AvOD Medium (bottom) express proteins associated with SARS-CoV-2 entry, TMPRSS2 and ACE2. While ACE2 was expressed in all donors and conditions, TMRPSS2 expression was donor-dependent.

Heatmap comparing gene expression in alveolar organoids cultured in PneumaCult™ and published formulation.

Figure 7. Gene Expression Analysis of Organoids Cultured in PneumaCult™ Alveolar Organoid Media Shows Presence of Alveolar Markers

Normalized gene expression heatmap of organoids expanded in PneumaCult™ AvOE Medium and differentiated in PneumaCult™ AvOD Medium compared to those grown using a published formulation. The presence of canonical ATII and ATI genes as well as SARS-CoV-2 key entry markers, ACE2 and TMPRSS2, are shown. Published RNA-Seq gene expression datasets of primary alveolar epithelial cells (including ATI and ATII cells, from Mikami et al. 2021, GSE186359) and primary ATII cells (Jacob and Kotton, 2017, GSE96642) were used as controls.

Figure 8. ATII Cells Show Optimal Marker Expression When Cultured for 7 Days As Submerged or ALI Cultures

The generation of alveolar type 2 (ATII) cultures at the air-liquid interface (ALI) on cell culture inserts is a two-stage protocol. In the first stage, ATII cells from two separate donors were seeded in PneumaCult™ Alveolar Organoid Expansion (AvOE) Medium as organoid cultures for 10 days. Single cells were then harvested and seeded into ECM-coated inserts and cultured to Day 7 or Day 14. Cultures were placed at ALI on day 4 and cultured for an additional (C, D) 3 or (G, H) 10 days. Cells were fixed on the membrane in 4% PFA and stained for the ATII-specific marker HT2-280 (green), as well as the ATI-specific markers RAGE (red) and GPRC5a (magenta) and counterstained with DAPI (blue). In both donors, cultures in (A, B) submerged and (C, D) ALI for 7 days express HT2-280 with some RAGE, and little GPRC5a. Additional growth for 7 days in (E, F) submerged cultures resulted in a decrease in HT2-280 and an increase in RAGE, indicating differentiation into ATI cells. (G, H) Growth for an additional 7 days at the ALI resulted in some decrease in HT2-280, but expression was more stable than in submerged culture conditions. This data suggests that the ideal culture conditions for ATII cells on cell culture inserts are for (C, D) 7 days, with 3 days at ALI; however these cultures can still be maintained for up to 14 days (10 days of ALI) with minimal loss of ATII markers. Note scale is the same for XY and ZX figures.

Figure 9. Alveolar Cells Grown on Cell Culture Inserts Maintain Expression of HT2-280 and Increase Expression of HT1-56

ATII cells from three separate donors were seeded from organoid domes onto ECM-coated cell culture inserts and cultured to Day 7. Expression of the ATII marker HT2-280 and the ATI marker HT1-56 was determined as the percentage of viable singlets by flow cytometry. The data demonstrates HT2-280 high positive expression decreases slightly but not significantly, while HT1-56 high positive expression increases significantly (ANOVA with Dunnett’s post hoc test, where ** = p < 0.01; n = 3) when alveolar cells are cultured on cell culture inserts compared to organoid cultures. There were no differences between ALI (3 days) and always submerged cultures in either HT2-280 or HT1-56 expression. Symbols represent different donors across the conditions.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Applications

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