5/5/2023 0 Comments Cellprofiler custom script![]() 1A) utility was implemented in the Python programming language (Python Software Foundation, ) with a user interface (Fig. M aterials and M ethods Automation of Array ScanningĪn automated array scanning (process flow shown in Fig. Cell selection was based on fluorescent staining of human EpCAM (CD326), which is expressed in colorectal cancers 14 and many pancreatic tumors 15. The utility of the platform was demonstrated by sorting cells from very small samples (less than 10 4 cells) obtained from PDX models of a pancreatic ductal adenocarcinoma and a liver metastasis of a colorectal carcinoma. A software package was developed that supports rapid user initialization and automated multichannel and timelapse imaging of uniform arrays of microstructures including, but not limited to, micropallet arrays, microraft arrays, and microwell arrays. Here, we present an integrated platform for the automated imaging, analysis and release of micropallets from arrays. ![]() Previous applications of micropallet array technology depended on time-consuming manual inspection of the array to identify cells of interest followed by the manually triggered release of individual micropallets, limiting the throughput and accessibility of the technology 12, 13. Individual micropallets are released by focusing a low energy laser pulse at the photoresist-glass interface at high magnification and numerical aperture (20×, 0.7 NA) 11. The micropallets serve as culture sites for cells and function as cell carriers on release from the array. ![]() Micropallets can be fabricated of various dimensions and array sizes optimized for sorting samples of varying size from 10 2 to 10 6 cells 9, 10. They consist of arrays of transparent and individually releasable pedestals fabricated by single-step photolithography on a glass substrate 9. Micropallet arrays are an attractive and flexible platform for sorting and isolating cells from mixed samples 8. These approaches are laborious, time-consuming and, in the case of limiting dilution, can consume significant quantities of reagents. As a consequence, manual cell picking and limiting dilution remain the most commonly used techniques for sorting such exceedingly small primary cell samples. Damage from the UV and IR lasers used in these approaches remains a concern 7. Several variants of laser capture microdissection (LCM) have been demonstrated for sorting small samples of live cells including gravity assisted microdissection and laser pressure catapult microdissection 6. ![]() The initialization of sort gates requires that a large number of cells be analyzed before a sort is possible, rendering the sorting of very small samples (<10 6 cells) impossible 5. Such throughput hampers the ability of FACS to sort exceedingly small samples, such as those produced by patient biopsies and from animal models. Current generation FACS instruments are capable of measuring more than 20 parameters simultaneously 1 and achieve sort throughputs of greater than 50,000 cells per second 2 but are typically operated on the order of 1,000's of cells per second 3, 4. © 2014 International Society for Advancement of Cytometryįluorescence-activated cell sorting (FACS) remains the gold standard for high throughput cell sorting. These data demonstrate the ability to automate and efficiently separate samples with very low number of cells. We developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM (CD326) as a selection criteria. For very small tissue samples (on the order of 10 3 cells) purification by fluorescence-activated cell sorting (FACS) is not feasible while magnetic activated cell sorting (MACS) of small samples results in very low purity, low yield, and poor viability. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size. Patient-derived xenografts (PDXs) comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size.
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