Department of Population Sciences, Beckman Research Institute at City of Hope, 1500 E. Duarte Rd, Duarte, CA, 91010, USA.
Irell and Manella Graduate School of Biological Sciences, City of Hope, 1500 E. Duarte Rd, Duarte, CA, 91010, USA.
Breast Cancer Res. 2023 Jan 18;25(1):6. doi: 10.1186/s13058-022-01595-z.
A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes.
We demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2-5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3-5 days to maintain subconfluent density.
When compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components.
Postconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.
在人乳腺上皮细胞（HMEC）培养中，一个挑战是随着时间的推移维持竞争的腔、肌上皮和祖细胞谱系的代表性。随着细胞在培养中复制，肌上皮细胞在连续传代中逐渐主导培养物的组成。这种组成上的漂移给研究腔和祖细胞带来了挑战，因为腔和祖细胞是大多数乳腺癌亚型的潜在起源细胞。
我们展示了在 HMEC 上进行汇合后培养的应用。汇合后培养需要在没有传代的情况下培养 HMEC 2-5 周，但要在低应激 M87A 培养基中频繁喂食。相比之下，标准 HMEC 培养需要每隔 3-5 天进行酶消化亚培养，以维持亚汇合密度。
与标准 HMEC 培养相比，汇合后培养产生了更多比例的腔细胞和 c-Kit+祖细胞。汇合后培养物发展出独特的多层形态，与标准培养物中的细胞相比，单个细胞显示出降低的物理变形性。汇合后细胞的基因表达分析显示出谱系特异性标记物和细胞外基质成分的表达增加。
汇合后培养是改变 HMEC 谱系组成的一种新颖、有用的策略，通过增加腔和祖细胞的比例代表性。我们推测，汇合后培养创造了一个更接近生理状态的细胞组成的微环境，并且更容易分离稀有细胞亚型。因此，对于使用 HMEC 进行乳腺癌研究的研究人员来说，汇合后培养是一种有价值的工具。
