Development of patient derived organoids for cancer drug screening applications

Cancer is a disease characterised by abnormal cell growth that can invade or spread to other regions of the body. Organoids are three-dimensional ex vivo tissue cultures made from embryonic stem cells, induced pluripotent stem cells, progenitor cells or tissue that serve as a physiological model for cancer research. These are designed to recapitulate the in vivo properties of tumours. Importantly, effective recapitulation of the structure of tissues and function is believed to predict patient response, allowing for the creation of personalised therapy in a timely manner that may be used in the clinic. This Review discusses the pre-clinical model and different types of human organoids as models for the development of high throughput drug screening and also aims to highlight how organoids are shaping the future of cancer research.     

骨髓间质干细胞体外分化为成骨细胞的实验研究

建立猪骨髓间质干细胞 (mesenchymalstemcells ,MSCs)体外分离培养方法。对猪MSCs体外分化为成骨细胞的能力进行研究。抽取猪骨髓 ,体外培养MSCs。取第二代MSCs ,以含有不同浓度的抗坏血酸、β -磷酸甘油、地塞米松及碱性成纤维细胞生长因子等条件培养基进行成骨细胞诱导分化。通过细胞形态变化 ,碱性磷酸酶染色及钙盐沉积对成骨细胞进行鉴定。结果表明MSC细胞形态由长梭形向多边形转变 ,ALP染色阳性 ,VonKossa染色阳性 ,经体外诱导分化后呈典型的成骨细胞样改变。猪骨髓MSCs可在体外长期、稳定培养 ,具有向成骨细胞分化的潜能 ,可以为骨组织工程研究提供较理想的细胞来源和动物模型。     

Malignant fibrous histiocytoma: an ultrastructural perspective

Malignant fibrous histiocytoma is a frequent diagnosis, but the relationship of the tumors to histologically similar soft tissue neoplasms is controversial. In this study, 157 examples representing the 4 main subtypes were reviewed by light microscopy and each tumor was studied with the electron microscope. Immunohistochemical stains were performed on 77 tumors. Electron micrographs on 100 fibrosarcomas were reviewed for comparison. Malignant fibrous histiocytomas often closely resemble fibrosarcomas at the ultrastructural level and differences between the two are generally of degree only. Evidence for true histiocytic differentiation was not found. The immunohistochemical results did not contradict the authors' impression from electron microscopy that malignant fibrous histiocytoma forms part of the histologic spectrum of tumors of fibroblasts.     

A probabilistic approach to measure the strength of bone cell adhesion to chemically modified surfaces

Patterned surfaces with alternating regions of amino silanes [N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS)] and alkyl silanes [dimethyldichlorosilane (DMS)] have been used to alter the kinetics of spatial distribution of cellsin vitro. In particular, we have previously observed the preferential spatial distribution of bone cells on the EDS regions of EDS/DMS patterned surfaces (10). In this study, we examined whether the mechanism of spatial distribution of cells on the EDS regions was adhesion mediated. Homogeneous layers of EDS and DMS were immobilized on quartz substrates and characterized by contact angle, X-ray photoelectron spectroscopy, and spectroscopic ellipsometry. The strength of bone cell attachment to the modified substrates was examined using a radial flow apparatus, within either 20 min or 2 hr of cell incubation in the presence of serum. A Weibull distribution was chosen to characterize the strength of cell-substratum adhesion. Within 20 min of cell exposure, the strength of adhesion was significantly larger on EDS and clean surfaces, compared with DMS surfaces (p<0.0001). Within 2 hr of cell incubation, there was no statistical difference between the strength of cell adhesion to EDS, DMS, and clean surfaces. The results of this study suggest that the surface chemistry mediates adhesion-based spatial cell arrangement through a layer of adsorbed serum proteins.     

胶原基真皮再生支架的微结构控制

综述了影响胶原基真皮再生支架微结构的各种因素及其控制方法。胶原基真皮支架的生物活性和修复创面的能力受到诸如除胶原外的其它主要材料 (第二组分 )、支架的孔径和孔隙率、支架厚度、生物活性因子以及交联度等多方面因素的综合影响。从材料学、生物学和医学的角度综合地应用物理、化学和生物学的手段探索各种影响因素的控制方法是组织工程皮肤研究的重点。     

Laboratory preparation of plasticware to support cell culture

Summary Many plastics, including polystyrene and poly(ethylene terephthalate), are unsuitable for cell culture applications as formed because they do not support cell growth. Although cells may attach to these materials, the attached cells typically round up and detach or die after a short time. However, plastics can be made to support normal cell attachment and growth through surface modification by glow discharge processes that produce ionized gas species which react with the surface of the plastic. This article describes radiofrequency glow discharge (RFGD) modification of plastics in the presence of organic vapors such as acetone, methanol and ethylene oxide. These treatments render laboratory plastics amenable to in vitro cell culture. Successful modification is a function of RFGD reaction parameters (position within the reactor, discharge power, system pressure, flow rate, and reaction time), and can be confirmed by electron spectroscopy for chemical analysis (ESCA). Identification by high resolution ESCA of functional groups introduced onto the surface by the RFGD process can be used to correlate cell growth with surface chemistry. A brief discussion of other processes thought to be used for preparation of commercial tissue culture ware is also provided.     

骨髓基质细胞和壳聚糖/明胶共混材料生物相容性研究

目的:研究壳聚糖/明胶共混材料对离体培养的骨髓基质细胞粘附及增殖的影响,寻找骨髓基质细胞新的载体材料。方法:取2周龄幼兔的长骨采集骨髓,培养骨髓基质细胞,体外扩增1周后,种植于纯壳聚糖和壳聚糖/明胶共混材料的表面。在倒置光学显微镜、扫描电镜的辅助下,观察细胞的粘附和生长情况,种植7d后用透射电镜观察细胞功能状况,用MTT方法检测种植后2d、4d、6d、8d细胞的增殖情况。结果:壳聚糖/明胶共混材料和纯壳聚糖能促进骨髓基质细胞在材料表面粘附并保持其在机体内的形态。壳聚糖/明胶共混材料表面的骨髓基质细胞功能活跃。在材料表面和培养板表面培养的骨髓基质细胞均能持续增殖,而壳聚糖/明胶共混材料能显著促进骨髓基质细胞的增殖(P<0.01)。结论:壳聚糖/明胶共混材料保持了壳聚糖的某些生物活性,同时由于加入明胶,能促进骨髓基质细胞的增殖,可作为骨髓基质细胞的载体应用于组织工程。     

羟基磷灰石/胶原-聚乳酸三维多孔框架材料的制备成形及分析

目的：合成新型的复合生物材料框架作为骨组织工程研究的细胞外基质材料。方法：本研究采用材料学自组装技术的原理，以Ⅰ型胶原蛋白为分子模板，引导钙磷盐在液相中的矿化，制备具有天然骨基质层状结构的羟基磷灰石／胶原复合材料，并以热致分相法制备了羟基磷灰石／胶原-聚乳酸复合三维多孔框架。结果：羟基磷灰石／胶原复合材料具有与天然骨基质相似的成分与结构，加入聚乳酸制备成三维多孔框架，孔隙直径界于50um-300um。结论：羟基磷灰石／胶原-聚乳酸复合三维多孔框架可能作为骨组织工程良好的细胞外基质材料。     

Measurement of venous oxyhaemoglobin saturation in the adult human forearm by near infrared spectroscopy with venous occlusion

Measurement of the oxygenation of the peripheral tissues provides useful information about tissue perfusion. A method is described for the measurement of peripheral venous oxyhaemaglobin saturation (SvO₂) in the adult forearm by a non-invasive technique, near infrared spectroscopy (NIRS) with venous occlusion. A series of studies is performed on healthy adults to compare measurements of forearm SvO₂ made by NIRS with measurements of superficial venous SvO₂ made by co-oximetry, and to study the effect of different optode spacings. There is a significant correlation between forearm SvO₂ measured by NIRS and SvO₂ of superficial venous blood measured by cooximetry (n=19, r=0.7, p<0.0001). Higher values for SvO₂ were obtained using a 2.5 cm spacing than with a 4 cm spacing (mean difference=4.1% (95% Cl 1.4%–6.8%) n=16). This difference is likely to have been due to a more superficial volume of tissue being studied with the closer optode spacing. Peripheral SvO₂ can be measured non-invasively using NIRS with venous occlusion. It may prove to be a useful method to study circulatory disturbances.     

Blood coagulation and fibrinolysis after long-duration treadmill exercise controlled by individual anaerobic threshold

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60–120 min. Sixteen healthy male non-smokers underwent the TR test. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Extrinsic and intrinsic total (TTP_ex+in) and endogenous (ETP_ex+in) thrombin potential, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), plasmin-2-antiplasmin complex (PAP), D-dimer, tissue plasminogen activator antigen and activity (tPA-AG and tPA-ACT) and plasminogen activator inhibitor type 1 antigen and activity (PAI-1-AG and PAI-1-ACT) were measured. Immediately after TR, F1+2, TAT and TTP_ex+in were increased (P<0.05) while ETP_ex+in remained unchanged. In contrast, PAP, D-dimer, tPA-AG, tPA-ACT (P<0.05) were distinctly enhanced while PAI-1-ACT was decreased (P<0.05) immediately after exercise. The changes in tPA-AG, tPA-ACT, and PAI-1-ACT were reversed to nearly baseline while the enhancement in PAP and D-dimer was prolonged by more than 2 h after exercise. Long-duration exercise between 60 and 120 min controlled by IAT (90%) on a TR ergometer only implicates a small increase in thrombin generation markers and total (free and ₂-macroglubulin-bound thrombin), but not in endogenous (free) thrombin potential alone. In contrast, fibrinolysis is distinctly increased after this type of exercise. Endurance exercise with an intensity below 90% IAT and a duration below 2 h generates a more favourable condition for fibrinolysis than for blood coagulation in healthy young subjects. Data are given as mean (SD).