Megalin,Proton Pump Inhibitors and the Renin–Angiotensin System in Healthy and Pre-Eclamptic Placentas

Soluble Fms-like tyrosine kinase-1 (sFlt-1) is increased in pre-eclampsia. The proton pump inhibitor (PPI) lowers sFlt-1, while angiotensin increases it. To investigate whether PPIs lower sFlt-1 by suppressing placental renin–angiotensin system (RAS) activity, we studied gene expression and protein abundance of RAS components, including megalin, a novel endocytic receptor for prorenin and renin, in placental tissue obtained from healthy pregnant women and women with early-onset pre-eclampsia. Renin, ACE, ACE2, and the angiotensin receptors were expressed at identical levels in healthy and pre-eclamptic placentas, while both the (pro)renin receptor and megalin were increased in the latter. Placental prorenin levels were upregulated in pre-eclamptic pregnancies. Angiotensinogen protein, but not mRNA, was detectable in placental tissue, implying that it originates from maternal blood. Ex vivo placental perfusion revealed a complete washout of angiotensinogen, while prorenin release remained constant. The PPI esomeprazole dose-dependently reduced megalin/(pro)renin receptor-mediated renin uptake in Brown Norway yolk sac epithelial cells and decreased sFlt-1 secretion from placental villous explants. Megalin inhibition blocked angiotensinogen uptake in epithelial cells. In conclusion, our data suggest that placental RAS activity depends on angiotensinogen taken up from the maternal systemic circulation. PPIs might interfere with placental (pro)renin-AGT uptake/transport, thereby reducing angiotensin formation as well as angiotensin-induced sFlt-1 synthesis.     

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。     

体外培养前后脐血CD34~+细胞差异表达基因的筛选

目的筛选扩增培养前后脐血CD34+细胞差异表达基因。方法将新鲜分离的CD34+细胞设为对照组,经静态和动态培养系统扩增的CD34+细胞设为试验组,应用基因差异显示技术(DDRT-PCR)比较两组之间的基因表达差异,对差异表达基因片段进行克隆、测序及生物信息学分析,并且通过半定量RT-PCR方法验证基因差异表达的真实性。结果应用差异显示技术筛选出5个差异表达基因,其中4个为已知功能基因,1个为未知功能基因,对其中2个基因RAN和DC24进行半定量RT-PCR检测表明,在静态和动态体系扩增培养后的CD34+细胞内,RANmRNA的表达水平分别是新鲜分离细胞的1.521和3.978倍,DC24mRNA的表达水平分别是新鲜分离CD34+细胞的14.275和2.374倍。结论发现了与CD34+细胞体外增殖相关的一些重要基因,为从基因水平调控CD34+细胞体外增殖提供依据。     

视网膜色素上皮细胞培养及其超微结构的观察

目的 体外培养视网膜色索上皮(retinal pigment epithelium RPE)细胞并对其进行超微结构观察,为RPE细胞在细胞和分子水平研究奠定基础。方法 采用酶消化法对RPE细胞进行取材和培养,利用光镜和电镜进行观察及分析。结果 培养的细胞贴壁生长,融合后呈单层、镶嵌式排列;电镜下可见到细胞表面绒毛、细胞之间复合体、细胞间质少及细胞排列紧密等上皮组织的特征性结构,细胞内可直接观察到大量黑色素颗粒。结论 为各种损伤致RPE细胞形态学改变的研究奠定了基础。     

Hormonal regulation of medium chain fatty acid synthesis by mouse mammary gland explants

Explants of pregnant mouse mammary tissue were cultured in media supplemented with various hormones. During the last few hours of culture, explants were labeled with [¹⁴C] acetate. Fatty acid synthesis by the tissue was analyzed using reverse phase thin layer chromatography, and incorporation of radioactivity into the medium chain fatty acid (MCFA) fraction was calculated as total MCFA per mg tissue fresh weight and as a percentage of the total fatty acid radioactivity (%MCFA). After 48 hr of culture, explants had an elevated %MCFA synthesis only when exposed to media containing insulin, cortisol and prolactin, confirming previous reports. Increasing doses of prolactin (maximal response at 300 mg/ml) caused a coordinate rise in both %MCFA synthesis and total MCFA synthesis per mg fresh weight. Epithelial cells isolated from explants after culture for 48 hr with insulin, cortisol and prolactin showed an elevated %MCFA synthesis compared to cells from explants cultured with insulin and cortisol alone, indicating that hormonal stimulation of mammary explants causes a change in the chain length of fatty acids synthesized by mammary epithelial cells. The specificity of the explant response to prolactin was tested with other hormones: FSH and calcitonin had no effect, whereas bovine growth hormone showed activity only at high concentration. Progesterone and 17β-estradiol also had no effect. The analysis of MCFA synthesis provides another means of (a) assessing hormonal action upon mammary tissue, and (b) evaluating the biological activity of prolactin.     

Differences in the Central Energy Metabolism of Cancer Cells between Conventional 2D and Novel 3D Culture Systems

The conventional two-dimensional (2D) culture is available as an in vitro experimental model. However, the culture system reportedly does not recapitulate the in vivo cancer microenvironment. We recently developed a tissueoid cell culture system using Cellbed, which resembles the loose connective tissue in living organisms. The present study performed 2D and three-dimensional (3D) culture using prostate and bladder cancer cell lines and a comprehensive metabolome analysis. Compared to 3D, the 2D culture had significantly lower levels of most metabolites. The 3D culture system did not impair mitochondrial function in the cancer cells and produce energy through the mitochondria simultaneously with aerobic glycolysis. Conversely, ATP production, biomass (nucleotides, amino acids, lipids and NADPH) synthesis and redox balance maintenance were conducted in 3D culture. In contrast, in 2D culture, biomass production was delayed due to the suppression of metabolic activity. The 3D metabolome analysis using the tissueoid cell culture system capable of in vivo cancer cell culture yielded results consistent with previously reported cancer metabolism theories. This system is expected to be an essential experimental tool in a wide range of cancer research fields, especially in preclinical stages while transitioning from in vitro to in vivo.     

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.     

Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.     

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture

Serum albumin physically interacts with fatty acids, small molecules, metal ions, and several other proteins. Binding with a plethora of bioactive substances makes it a critical transport molecule. Albumin also scavenges the reactive oxygen species that are harmful to cell survival. These properties make albumin an excellent choice to promote cell growth and maintain a variety of eukaryotic cells under in vitro culture environment. Furthermore, purified recombinant human serum albumin is mostly free from impurities and modifications, providing a perfect choice as an additive in cell and tissue culture media while avoiding any regulatory constraints. This review discusses key features of human serum albumin implicated in cell growth and survival under in vitro conditions.     

Hypoxia and flow perfusion modulate proliferation and gene expression of articular chondrocytes on porous scaffolds

The combination of reduced oxygen tension and flow perfusion bioreactor culture is investigated for its effect on the proliferation, glycosaminoglycan production, and chondrogenic gene expression of bovine articular chondrocytes on porous polymer scaffolds. It was hypothesized that the combination of such factors would more closely replicate the in situ environment of these cells, leading to improvements in the cell phenotype. Chondrocytes were seeded onto electrospun poly(ε‐caprolactone) scaffolds and cultured in static or perfusion culture in either normoxic or hypoxic conditions for 6days. Results demonstrated that the combination of hypoxic and perfusion culture led to an increase in chondrocyte proliferation and glycosaminoglycan production, as well as an improvement in the ratio of collagen II/I gene expression over perfusion culture alone. The results demonstrate the need to combine multiple signals in vitro, in order to improve tissue growth by more closely replicating the native environment of cells. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3158–3166, 2013     

Hydroxyapatite cages with aligned pores for bone grafting – Seeding of human osteoblast-like cells in vitro and their response in dynamic culture mode

Hydroxyapatite (HA) is a highly regarded synthetic bone graft material. Porous HA ceramics blocks are used to substitute harvested natural bone grafts. Being similar to bone mineral, HA material integrates with the host bone through surface osteointegration and slowly resorb along with the natural bone remodeling process. The blocks in use currently have random and tortuous pore structures. The present work explores the usefulness of cage-like HA ceramic design with end-to-end open pores, with the help of in vitro cell culture methods. Such a structure, on implantation, will take up the blood factors and cells and host the bone remodeling process inside the bulk of the cage, leading to early healing. In the study, HA samples with aligned through-pores were prepared and explored in vitro, with a focus on how the pores host the cells inside and to what level the cells maintain their activity. Human osteoblast-like cells (HOS) were used, at different seeding and culturing approaches. Cell seeding was done through (i) conventional large volume cell suspension, (ii) a confined mini chamber with a limited volume of cell suspension, and (iii) placing a concentrated drop of cell suspension directly on top of the scaffold. The third approach gave the best cell adhesion and proliferation, and hence used for further explorations. A dynamic culture system was designed in-house by bifurcating the cell culture wells using vertical inserts, holding the samples horizontally with their ends open to both sides, and making the media flow across using a rocker platform. The HOS cell adhesion, viability and proliferation were tested in the HA cages, in static and dynamic culture conditions, with conventional porous ceramics as the control. The cell infiltration was deeper and the cell viability over a period of 7 days was significantly higher in dynamic culture conditions in the test samples.     

Evaluation of MC3T3-E1 Cell Osteogenesis in Different Cell Culture Media

Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.     

新生小鼠表皮细胞的培养——从胎盘组织提取液对细胞生理作用的评价

实验以DMEM为基础培养基，分别用5%小牛血清，5%小牛血清、10%人胎盘组织提取液和5%小牛血清、20ug/L胰岛素、400ug/L氢化可的松三种方法培养新生昆明小鼠的表面皮基底细胞，经对比实验，确认人胎盘组织提取液具有的营养和生长因子的作用，能促进细胞增殖和新陈代谢，是一种较好的化妆品营养和活性添加剂。     

基因重组细胞在大规模长期培养中培养条件对HBsAg表达的影响

作者利用15升转瓶培养CHO-B_(43)工程细胞,观察各种培养条件对细胞形态及生物学特性的影响,发现在一定范围内,增加旋转机转数,提高细胞维持液量和维持液pH、降低培养温度,均能使细胞维持在良好状态,并且细胞分泌表达的HBsAg滴度较高。CHO工程细胞在培养中产生大量酸性代谢产物,因而耐碱性强。     

Crosstalk of Diabetic Conditions with Static Versus Dynamic Flow Environment—Impact on Aortic Valve Remodeling

Type 2 diabetes mellitus (T2D) is one of the prominent risk factors for the development and progression of calcific aortic valve disease. Nevertheless, little is known about molecular mechanisms of how T2D affects aortic valve (AV) remodeling. In this study, the influence of hyperinsulinemia and hyperglycemia on degenerative processes in valvular tissue is analyzed in intact AV exposed to an either static or dynamic 3D environment, respectively. The complex native dynamic environment of AV is simulated using a software-governed bioreactor system with controlled pulsatile flow. Dynamic cultivation resulted in significantly stronger fibrosis in AV tissue compared to static cultivation, while hyperinsulinemia and hyperglycemia had no impact on fibrosis. The expression of key differentiation markers and proteoglycans were altered by diabetic conditions in an environment-dependent manner. Furthermore, hyperinsulinemia and hyperglycemia affect insulin-signaling pathways. Western blot analysis showed increased phosphorylation level of protein kinase B (AKT) after acute insulin stimulation, which was lost in AV under hyperinsulinemia, indicating acquired insulin resistance of the AV tissue in response to elevated insulin levels. These data underline a complex interplay of diabetic conditions on one hand and biomechanical 3D environment on the other hand that possesses an impact on AV tissue remodeling.     

抗rhTNFα杂交瘤细胞的体外培养及抗体分泌的研究

采用非CO2依赖培养基（CO2-IM）在无CO2、37℃条件下培养抗rhTNFα杂交瘤细胞，培养1周PH无显著变化，细胞生长趋势和抗体分泌显著超过DMEM。将CO2－IM与SFPF－HM混合后添加ITSE可以培养抗rhTNF杂交瘤细胞，其细胞生长速率、密度、活力和抗体产量均达到或超过含血清培养。     

兔骨髓基质干细胞的分离培养及鉴定

目的探讨兔骨髓基质干细胞(MSC)体外分离培养及鉴定。方法自兔髂骨抽取骨髓,采用密度梯度离心法分离纯化出MSC,并增殖。观察MSC的生长情况及形态学特点,流式细胞仪检测第3代MSC表面抗原的表达情况。结果体外培养的兔MSC贴壁生长,呈长梭形,可增殖形成克隆;MSC阳性表达CD29,CD90,但CD34,CD45呈阴性。结论利用密度梯度离心法获取的MSC具有大量增殖的能力,表达CD29,CD90,不表达CD34,CD45。