Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system

The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation

Isolation and in Vitro Culture of Rare Cancer Stem Cells from Patient-Derived Xenografts of Pancreatic Ductal Adenocarcinoma

Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 minutes using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:104 to 1:106 on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly(dimethylsiloxane (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%) and viability (94 – 100%) for rare cell isolation. As a demonstration of the technology’s value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44 and CD326; surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44 and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90 ± 8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm2) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 μL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm2) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices

Isolation and in Vitro Culture of Rare Cancer Stem Cells from Patient-Derived Xenografts of Pancreatic Ductal Adenocarcinoma

Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10 000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.1 cm array of micropallets (50 × 50 × 75 μm structures and 25 μm micropallet gap) was fabricated on a large glass substrate, providing an array of approximately 1.3 million releasable microstructures. Image analysis algorithms were developed to permit array screening for identification of fluorescently labeled cells in less than 15 min using an epifluorescent wide-field microscope with a computer controlled translational stage. Device operation was tested by culturing HeLa cells transfected with green fluorescent protein (GFP) admixed with wild-type HeLa cells at ratios of 1:10⁴ to 1:10⁶ on the array followed by screening to identify flourescent cells. Micropallets containing cells of interest were then selectively released by a focused laser pulse and collected on a numbered poly­(dimethylsiloxane) (PDMS) substrate with high viability. A direct comparison of this technology with fluorescence-activated cell sorting (FACS) demonstrated that micropallet arrays offered enhanced post sorting purity (100%), yield (100%), and viability (94–100%) for rare cell isolation. As a demonstration of the technology’s value, pancreatic tumor cells from Panc-1 cell lines and patient-derived xenografts were screened for the presence of CD24, CD44, and CD326: surface markers of pancreatic CSCs. Following cell isolation and culture, 63 ± 23% of the isolated Panc-1 cells and 35% of sorted human xenograft cells formed tumor spheroids retaining high expression levels of CD24, CD44, and CD326. The ability to isolate rare cells from relatively small sample sizes will facilitate our understanding of cell biology and the development of new therapeutic strategies

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

Abstract Background The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. Results The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. Conclusions The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function. Graphical abstrac

Array-Based Platform To Select, Release, and Capture Epstein–Barr Virus-Infected Cells Based on Intercellular Adhesion

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein–Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes

<i>In Vitro</i> Polarization of Colonoids to Create an Intestinal Stem Cell Compartment

<div><p>The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.</p></div

Incorporation of EdU into colonoids after a 2 h pulse in the absence of a gradient.

<p>(A,C) Brightfield (left) and overlaid red/blue fluorescence (right) images of colonoids cultured within a multiwell plate (A) or microchannel (C) for 5 days then labeled with EdU (red) and the Hoechst 33342 (blue) (n = 18 colonoids in 5 microchannels and n = 16 colonoids in 3 multiwells). Scale bars represent 50 μm. (B, D) Compass plots displaying the EDU polarization magnitude and angle for individual colonoids (blue) cultured in the multiwell plate (B) or microchannel (C) for 5 days and pulsed with EdU. The average magnitude and angle of the vector can be seen in red (poorly visualized due to the near-zero magnitude).</p