Комбинаторной функциональный метод скрининга для получения понимание воздействия молекулярного состава микросреды на клеточные функции описаны. Метод использует существующие микрочипов на основе технологии для создания массивов определены комбинаторной микросреды, которые поддерживают клеточной адгезии и функционального анализа.
The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).
MEArray метод, представленный здесь дает функциональный анализ клеточных взаимодействиях и комбинаторной микроокружения 4. MEArray анализ сочетает в себе использование базовых технологий micropatterning, клеточной биологии, и микрочипов печати роботов и анализ устройств, которые доступны на…
The authors have nothing to disclose.
ML поддерживается NIA (R00AG033176 и R01AG040081) и лаборатории, руководимой исследований и развития, Министерство энергетики США контракту № DE-AC02-05CH11231.
Name of the reagent | Company | Catalog number | Comments (optional) |
Glass slides 25 mm x 75 mm | VWR | 48311-600 | |
Glass coverslips (no.1) 24 mm x 50 mm | VWR | 48393-241 | |
Staining dish (or Coplan jar) | VWR | 25461-003 | |
Petri dishes (15 cm) | BD Falcon | 351058 | |
NaOH (1.0N) | Sigma-Aldrich | S2567 | |
APES (>98% (3-Aminopropyl)triethoxysilane) | Sigma-Aldrich | A3648 | |
Glutaraldehyde | Sigma-Aldrich | G7651 | 50% in water |
APS (>98% Ammonium Persulfate) | Sigma-Aldrich | A3678 | Prepare 10% working solution with ddH2O |
TEMED (N,N,N’,N’-Tetramethylethylenediamine) | Sigma-Aldrich | T9281 | |
Acrylamide (40%) | Sigma-Aldrich | A4058 | |
Bis-Acrylamide (2% w/v) | Fisher BioReagents | BP1404-250 | |
0.45 μm Syringe filter 4-mm nylon | Nalgene | 176-0045 | |
FITC | Sigma-Aldrich | F4274 | |
PDMS (polydimethylsiloxane) | Dow Corning | 3097358-1004 | Sylgard 184 Elastomer kit via Ellsworth Adhesives |
2-chamber slides | NUNC | 177380 | |
Pluronic F108 | BASF | 30089186 | |
Aquarium sealant | Dow Corning | DAP 00688 | |
Fluormount-G | Southern Biotech | 0100-01 | |
Disposable plastic cups | |||
Tongue depressors | |||
Nitrile gloves | |||
Plastic microscope slide boxes | |||
Spin coater | WS-400B-6NPP/LITE | Laurell Technologies Corporation | |
Oven | |||
Digital hotplate | |||
384-well plates | A brand appropriate for the microarray robot | ||
Microarray printing robot | |||
Inverted phase and fluorescence microscope | |||
Axon microarray scanners | Molecular Devices | Multiple configurations exist |