Cell proliferation in the human mammary epithelium. Differential contribution by epithelial and myoepithelial cells.

The ductal system of the human breast consists of epithelial, myoepithelial, and basal clear cells. By labeling ducts and alveoli dissected from reduction mammoplasty specimens with 3H-thymidine in vitro and labeling human breast organoids xenografted in nude mice in vivo, it was found that cellular proliferation in the human breast is virtually confined to epithelial and basal clear cells. A pulse label of 3H-thymidine in organ culture explants was followed over a period of time, and it was found that myoepithelial cells originate from a precursor cell population within the mammary epithelium after a number of cell divisions. Myoepithelial cells were not seen to divide when fully mature.     

Human breast epithelium transplanted into nude mice

Summary Epithelial components of the normal human breast and their response to hormonal manipulation have been studied in the nude mouse. Six to eight week-old female athymic nude mice were used as the recipients of enzymatically prepared breast organoids, composed of ductal and lobuloalveolar structures. After 12 weeksin situ in the mouse mammary fat pad the human breast tissue retains its normal morphology as demonstrated by the presence of myosin positive myoepithelial cells and keratin positive luminal cells. Monoclonal antibodies M8 and M18 raised to components of the human milk fat globule membrane give a similar staining pattern in the xenografted organoids to that seen in the donor tissue. On mating the recipient female nude mice, the human breast tissue responds with both an increased³H-thymidine labeling index and -lactalbumin production. This model in conjunction with in vitro studies is, therefore, suitable for the study of extrinsic and intrinsic factors controlling differentiation and morphogenesis in the human breast.     

改良型混合酶消化法培养小鼠乳腺上皮细胞

背景：目前采用的A型胶原酶和胰蛋白酶混合酶分离乳腺细胞团的方法操作复杂,实验条件要求高。 目的：观察改良型混合酶消化法能否成功进行乳腺上皮细胞的体外培养。 方法：采用Ⅰ型、Ⅱ型胶原酶和胰蛋白酶(1∶1∶1)混合消化直接用眼科剪剪碎的小鼠乳腺组织,37 ℃振荡消化获取乳腺细胞团,并采用差速贴壁法去除成纤维细胞,将细胞团接种于细胞培养瓶进行培养。 结果与结论：倒置显微镜下观察显示,改良型混合酶消化法能获得较多的细胞团,且培养12 h后细胞团绝大多数贴在细胞瓶壁,培养72 h后细胞团完全铺开融合成片,细胞呈典型的铺路石样。免疫荧光细胞化学染色结果显示,培养细胞角蛋白18呈阳性反应。说明应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞。     

Concept for organ engineering: a reconstruction method of rat liver for in vitro culture

In the past decade, there have been remarkable advances in tissue engineering technology toward the goal of creating organoids in vitro from cells and cellular scaffolding. Indeed, tissue-engineered organoids such as skin and cartilage, each with comparatively simple architectures, are presently at the clinical stage. However, conventional tissue engineering techniques have not allowed for the reconstruction of an organoid that mimics an organ of complex architecture of abundant vascular networks. We established a method for organ engineering that can remodel a rat liver into a reconstructed organoid without separating the majority of liver cells by a continuous three-step perfusion. The liver was perfused through its vascular system with a buffered balanced salt solution to cleanse blood from the organ, with a collagenase/dispase medium to deconstruct cellular scaffolds, and with a culture medium containing collagen type I to reorganize the multicellular architecture. The reconstructed organoid was then prepared by excising the perfused liver from the rat and culturing it at 37 degrees C for 2 h. Histologically healthy parenchymal hepatocytes expressing albumin were observed in the excised organoid even after culture for 3 weeks. Furthermore, a fibroblast-implanted organoid was prepared by using a culture medium containing suspended fibroblasts in the third step of the perfusion procedure, demonstrating the efficacy of heterogeneous cells for the reconstruction of an organoid. This method may be applicable to the formation of organoids from other organs, such as kidney and spleen, each of which have abundant capillaries, and therefore the method provides a novel concept for the development of lab-grown organs, i. e., organ engineering.     

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.     

Development of a radioimmunoassay for human milk-fat-globule membrane glycoprotein. Its quantitation in spent media from both primary and established mammary and non-mammary epithelial cell lines

A radioimmunoassay (RIA) was developed for the quantitative measurement of a glycoprotein which was purified from human milk-fat-globule membrane (MFGM) and termed MFGM-pg 70. The assay with a sensitivity of detecting 30 ng of MFGM-pg 70/ml was employed to quantitate the levels of MFGM-gp 70 shed in supernatants from primary cultures of normal and malignant human breast cells and from various established cell lines of human mammary including myoepithelial and fibroblast and non-mammary malignant epithelial cells. The RIA for MFGM-gp 70 showed that the amount of antigen shed was much higher in supernatants from normal mammary epithelial cells compared with their malignant counterparts grown in primary culture and with those from established cell lines of malignant mammary epithelial cells. No detectable antigen was found in supernatants from cultures of normal myoepithelial-like cell lines or primary cultures of fibroblast cells from breast, or cell lines of squamous carcinomas of head and neck and tongue, renal cell carcinoma and teratoma. Trypsinization of mammary epithelial cells from both primary and established lines resulted in the release of most of the antigen from the surface of the cells, suggesting the presence of this molecule on the cell's surface. Following trypsinization, approximately 99% of the cells was viable, indicating that the release of the antigen in supernatants was due to shedding and not cell death. The levels of MFGM-pg 70 in spent media were unaffected by the lactogenic hormones such as prolactin or insulin. The RIA for MFGM-gp 70 provides a sensitive and quantitative means to in vitro study the synthesis of a membrane glycoprotein from human mammary epithelium.     

Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue

Reconstruction of the female breast after cancer surgery is a demanding task where the methods used today suffer from several disadvantages. In the present study we have investigated the possibility to use tissue engineering methods to regenerate human autologous breast tissue. Human mammary epithelial cells and preadipocytes were derived from breast tissue biopsies from healthy women undergoing reduction mammoplasty, and the two celltypes were co-cultured with conventional cell culture methods as well as in 3-D matrices. The study shows that it is possible to harvest both human mammary epithelial cells and preadipocytes in a single session, propagate several subcultures, and that the cells maintain a normal intercellular distribution and growth-pattern when co-cultured in a 3-D collagen gel. We propose that growth and formation of a tissue closely resembling normal human breast tissue be readily obtained in the described in vitro cell culture set-up using basic tissue engineering principles. This concept may be of great importance in the development of new methods for reconstruction of the human breast.     

Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum.

Microvascular endothelial cells from rat lungs were cultured in serum-free medium supplemented with an endothelial growth substance, insulin, hydrocortisone and so on. Five to seven days after plating, cultured cells formed a monolayer. They were identified as endothelial cells by morphology and by positive immunohistochemistry for factor VIII-related antigen, a marker for endothelia cells. Differences between gelatin coated culture plates and plastic culture plates in endothelial cell proliferation were evaluated. Cells plated on uncoated plastic plates had a spindle-shaped morphology and did not express factor VIII-related antigen. Two types of medium, serum-free medium containing endothelial growth substance and basal medium supplemented with 20% fetal calf serum, were also compared in primary culture. In contrast with the serum-free medium, cells cultured in the serum-containing medium showed fibroblast-like morphology and did not express factor VIII-related antigen. These results suggest that a gelatin substratum and serum-free medium containing endothelial growth supplement are necessary for in vitro proliferation of microvascular endothelial cells isolated from rat lungs. The culture method and conditions outlined here allow the proliferation of pure microvascular endothelial cells from rat lungs. It may be useful in studying hematogenous metastasis to the lung and the role of microvascular endothelium in other pulmonary disease.     

Selection of medium for serum-free primary culture of adult rat hepatocytes

To select a suitable medium for serum-free primary culture of adult rat hepatocytes, ten commercially-available synthetic media were compared for their ability to maintain the cells under serum-free and serum-supplemented conditions with special reference to attachment, survival and albumin secretion. It was found that Williams' medium E and DM-160 medium were the best among the ten media for maintaining hepatocytes under serum-free conditions in primary culture.     

Hormone-responsive 3D multicellular culture model of human breast tissue

A hormone-responsive 3D human tissue-like culture system was developed in which human primary mammary epithelial cells (MECs) were co-cultured with two types of predominant mammary stromal cells on silk protein scaffolds. Silk porous scaffolds with incorporated extracellular matrix provided a compatible environment for epithelial structure morphogenesis and differentiation. The presence of stromal cells promoted MEC proliferation, induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast, only alveolar structures were observed in monocultures. The alveolar structures generated from the heterotypic cultures in vitro exhibited proper polarity similar to human breast tissue in vivo. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the heterotypic cultures, where casein-α and -β mRNA expression were increased significantly. Additionally, this 3D multicellular culture model displayed an estrogen-responsive physiologically relevant response, evidenced by enhanced cell proliferation, aberrant morphology, changes in gene expression profile and few polarized lumen structures after estrogen treatment. This culture system offers an excellent opportunity to explore the role of cell-cell and cell-substrate interactions during mammary gland development, the consequences of hormone receptor activation on MEC behavior and morphogenesis, as well as their alteration during neoplastic transformation in human breast tissue.     

大脑皮质类器官培养及其在中枢神经系统疾病中的应用

文题释义： 大脑皮质类器官：利用三维细胞培养的方法,在合适的条件下将多能干细胞从单细胞生成一种能发育成神经上皮并具有类似活体大脑组织的复杂结构。 自我重组：像胚胎干细胞或诱导性多能干细胞这一类的多能干细胞,具有自发发育成某些原始结构的趋势。 背景：大脑组织是人体发育最复杂的结构,许多人类大脑疾病难以在动物体内重现,因此建立大脑发育的体外模型具有非常重要的研究价值。最近几年随着干细胞领域的飞速发展,出现了一种人工组织培养技术,即通过体外三维培养的方式,将干细胞从单个细胞生成一种复杂的、类似活体组织的结构,这种结构称为类器官。 目的：文章从大脑类器官的培养现状、发生机制、组织学特征及在神经系统疾病中的应用等多个方面综述了近年来大脑类器官的研究进展,并分析了目前大脑类器官的研究缺陷,旨在为相关领域的研究提供参考。 方法：由第一作者检索1998年1月至2019年6月中国知网、万方、PubMed数据库相关文献,检索词为“类器官、大脑类器官、胚胎干细胞、诱导性多能干细胞、神经发生、大脑皮质、发育、神经退行性疾病”“organoids,cerebral organoids,embryonic stem cells,induced pluripotent stem cells, neurogenesis,cerebral cortex,development,neurological diseases,self-organization”,纳入45篇文献进行综述。 结果与结论：利用多能干细胞自我重组的特性及添加神经诱导因子的方法,在体外可以有效产生大脑皮质类器官。作为一种全新的生物培养技术,大脑类器官在研究活体组织的发育、疾病形成的机制、组织替代疗法以及药物实验等方面都有很大的研究和应用价值。 ORCID: 0000-0002-8700-3030(范文娟) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Cell culture of the normal human mammary gland cultivated in monolayer – A mini systematic review

The mammary glands are constituted of different cell types. For example, the epithelial cells appear as the target in many studies since they produce and secrete milk during lactation and are the origin of many human breast cancers. Mammary gland biology is characterized by dynamic tissue growth, function and regression phases, which are understood mainly due to tissue culture studies. Cell culture is probably one of the most used in vitro scientific models, and the most common research model is still the two-dimensional (2D) culture system. Different approaches and conditions have been tested and used to improve the isolation, growth, yield and maintenance of viability of mammary gland cells. Therefore, our study aimed to explore and summarize the cell culture techniques with normal human mammary gland cells cultured in a monolayer. A search strategy was conducted using the electronic databases ‘PubMed’, ‘Scopus’ and ‘Virtual Health Library’. The search was carried out using the keywords ‘cell culture’ and ‘mammary gland’ and ‘human’. The main search was carried out by two authors between July and August 2021. In addition, we performed a review matrix elaborated in a spreadsheet to organize and systematize information about each article for inclusion. A total of 11 studies were included in the review and have conducted qualitative analyses on them. Although studies of these cells have been reported since the 1970 s, most found are from the last decade and are largely carried out in the USA. In addition, it was possible to verify the Human Mammary Epithelial Cells (HMEC) primary culture obtained from breast surgery as the main cell type studied. These cells are cultivated in Dulbecco’s Modified Eagle Medium (DMEM) and M87A medium with diverse supplements. Finally, there was a diversity in the use of dissociation reagents and a lack of information about cryopreservation. We have observed detailed methodological information about these study models, which would propose further investigations.     

HEK 293 cell suspension culture using fibronectin-adsorbed polymer nanospheres in serum-free medium

Previously, we reported on suspension culture of anchorage-dependent animal cells using plain polymer nanospheres in serum-containing medium. For commercial cell culture, it is more advantageous to use serum-free medium than serum-containing medium. To culture anchorage-dependent animal cells using polymer nanospheres in serum-free medium, the nanospheres need to be coated with cell adhesion proteins. In this study, we utilized fibronectin-adsorbed polymer nanospheres for suspension culture of anchorage-dependent animal cells in serum-free medium. Fibronectin was adsorbed onto poly(lactic-co-glycolic acid) nanospheres (433 nm in average diameter) by immersing the nanospheres in fetal bovine serum. The nanospheres were used to culture human embryonic kidney (HEK) 293 cells in serum-free medium in stirred suspension bioreactors. Nanospheres attached between HEK 293 cells and promoted cell aggregate formation compared with culture without nanospheres. Most cells in the aggregates were viable over a 10-day culture period. Importantly, the use of poly(lactic-co-glycolic acid) nanospheres promoted the cell growth significantly, compared with culture without nanospheres (3.8- vs 1.8-fold growth). The nanosphere culture method developed in this study removes the time-consuming and costly process of adaptation of anchorage-dependent animal cells to suspension culture in serum-free medium. This culture method may be useful for the large-scale suspension culture of various types of anchorage-dependent animal cells in serum-free medium.     

无血清培养促进脂肪干细胞向血管内皮细胞分化

背景：无血清培养对大鼠脂肪干细胞向血管内皮细胞诱导分化影响的报道甚少。 目的：观察无血清培养大鼠脂肪干细胞后向血管内皮细胞诱导分化的情况。 方法：采用酶消贴壁培养法获得雄性SD大鼠脂肪干细胞,传代培养至第3代。实验组细胞无血清培养24 h,对照组用含体积分数10%胎牛血清的L-DMEM完全培养基培养。然后应用血管内皮细胞诱导培养基培养3周。 结果与结论：大鼠脂肪干细胞经体外培养可呈多角形或梭形贴壁生长,并能稳定传代。传代后大鼠脂肪干细胞极低表达细胞表面标志CD31。大鼠脂肪干细胞定向血管内皮细胞诱导分化后细胞呈鹅卵石样,CD31阳性表达明显上升且实验组明显高于对照组。实验组诱导后大鼠脂肪干细胞能够吞噬Dil标记的乙酰化低密度脂蛋白及在基质胶上形成2D小管样结构,其能力明显强于对照组。结果证实无血清培养可促进体外大鼠脂肪干细胞向血管内皮细胞诱导分化。     

适合人肌母细胞的无血清培养基

背景：肌母细胞体外培养大多使用添加胎牛血清培养基,存在很多不足,开发无血清培养基,更加稳定、安全、经济,有广泛的应用前景。 目的：初步探索适合人肌母细胞培养的无血清培养基。 方法：配制含胎牛血清培养基 (DMEM/F12 1∶1添加胰岛素样生长因子1、碱性成纤维细胞生长因子和体积分数20%胎牛血清),间充质干细胞无血清培养基组(无血清培养基、2%血清替代物、2 mmol/L L-谷氨酰胺,人脐带间充质干细胞,细胞纯度大于99%,UltraCULTURETM添加血清替代物,谷氨酰胺)。自制无血清培养基组(GibcoTM添加胰岛素样生长因子1,碱性成纤维细胞生长因子,谷氨酰胺)。3种培养基分别对人肌母细胞分别进行培养,观察肌母细胞的形态、免疫组织化学的鉴定,计算克隆的形成率,绘制细胞生长的曲线,MTT检测肌母细胞的活性,比较3种培养基培养肌母细胞增殖、分化的差异。 结果与结论：各组细胞形态上无明显差异;各组免疫组织化学鉴定肌母细胞纯度均达99%;含胎牛血清培养基组和自制无血清培养基组克隆形成率显著高于间充质干细胞无血清培养基组(P < 0.01),含胎牛血清培养基组克隆形成率显著高于自制无血清培养基组(P < 0.01);含胎牛血清培养基组与自制无血清培养基组细胞数远高于间充质干细胞无血清培养基组;自制无血清培养基组细胞活性显著高于含胎牛血清培养基组和间充质干细胞无血清培养基组(P < 0.01)。自制无血清培养基组可有效用于肌母细胞的培养,在促进肌母细胞早期贴壁、增殖上还需进一步优化。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

骨形态发生蛋白7通过PI3K/Akt通路可抑制人髓核细胞的凋亡

背景：骨形态发生蛋白7可促进人髓核细胞的细胞外基质合成,减缓椎间盘退变。近年来,有学者提出其可能通过对抗髓核细胞凋亡从而发挥上述作用,但其进一步的分子机制一直未被详细阐明。 目的：观察骨形态发生蛋白7对无血清诱导下发生凋亡的人髓核细胞产生的作用及其对PI3K/Akt通路的影响,分析并探讨骨形态发生蛋白7抑制人髓核细胞凋亡的分子机制。 方法：通过改良Pfirrmann分级及相关条件选取12例患者获取椎间盘组织,采用酶消化法获取人髓核细胞后分组实验,以含体积分数15%胎牛血清的培养基正常培养的髓核细胞设为空白组;使用无血清培养基培养   48 h诱导凋亡作为阳性对照组;在无血清条件下,通过加入不同剂量的骨形态发生蛋白7以及同时添加PI3K/Akt通路拮抗剂LY294002形成处理组和拮抗组。使用流式细胞术检测各组细胞凋亡率;免疫荧光观察p-Akt表达;蛋白印迹法检测Akt,p-Akt,BAD和Caspase 9等通路蛋白的表达变化。 结果与结论：无血清凋亡诱导下,流式细胞术结果显示,随着骨形态发生蛋白7处理浓度上升,髓核细胞凋亡率明显下降,加入LY294002共同作用后细胞凋亡率再次升高(P < 0.05)。p-Akt免疫荧光和蛋白印迹法检测结果进一步表明,与凋亡阳性对照组相比,加入骨形态发生蛋白7的实验组中,p-Akt表达明显增加,其下游凋亡相关蛋白BAD、Caspase 9蛋白表达减少(P < 0.05),而在同时加入Akt通路拮抗剂LY294002后,p-Akt蛋白表达下降而凋亡相关蛋白的表达又恢复到相对较高的水平(P < 0.05)。结果证明,骨形态发生蛋白7在无血清诱导的人类髓核细胞凋亡中通过激活PI3K/Akt通路,拮抗了BAD-Caspase 9相关的细胞凋亡过程,抑制了髓核细胞的退变。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Isolation and growth of human mammary epithelial cells

Summary Techniques for the isolation and culture of human mammary epithelial cells are described. The isolation procedure consists of dissection followed by partial enzymatic digestion with collagenase and hyal-uronidase and subsequent filtration to separate the epithelial clusters from the digested stromal elements. Culture procedures utilizing two different growth media are presented. A serum-free medium, MCDB170, permits long-term growth (45 to 60 population doublings) of a pure epithelial population; a less defined medium, MM, yields fewer population doublings but increased expression of some mammary-specific properties.     

Techniques for establishing human epithelial cell cultures: sensitivity of cell lines for propagation of herpesviruses

The development of tissue culture systems for propagation of human epithelial cells has aided the investigation of events that lead normal epithelial cells to become neoplastic. The establishment of human epidermal keratinocyte and human bronchial epithelial cell lines and their usefulness for oncogenic virus assay systems are described. In addition, cultivation of primary epithelial culture from oral hairy leukoplakia and normal salivary gland biopsies in a serum-free medium is discussed.     

Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue

Human mammary glands arise from multipotent progenitor cells, which likely respond both to cell-autonomous and to extrinsic cues. However, the identity of these cues and how they might act remain unclear. We analyzed HER1 ligand effects on mammary morphogenesis using a three-dimensional organoid model generated from human breast tissue that recapitulates both qualitatively and quantitatively the normal ductal network in situ. Strikingly, different HER1 ligands generate distinct patterns of cell fate. Epidermal growth factor (EGF) causes a massive expansion of the myoepithelial lineage. Amphiregulin, in contrast, enables normal ductal development. These differences cannot be ascribed to preferential apoptosis or proliferation of differentiated cell populations, but are dependent on HER1 signal intensity. Inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) effector RSK prevents the EGF-induced myoepithelial expansion. Notably, mouse mammary organoids are much less responsive to HER1 ligands. Little is known about the myoepithelial lineage or about growth factor effects on mammary progenitor differentiation, and our studies provide an important window into human mammary development that reveals unexpected differences from the mouse model.