title: Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion.
abstract: 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.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Attayek
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine , Chapel Hill, North Carolina 27599, United States.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Hunsucker
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel HillNorth Carolina 27599, United States.
authors.forename: Yuli
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel HillNorth Carolina 27599, United States.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine , Chapel Hill, North Carolina 27599, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Armistead
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine , Chapel Hill, North Carolina 27599, United States.|Department of Chemistry, University of North Carolina , Chapel HillNorth Carolina 27599, United States.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D002448:Cell Adhesion; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D020014:K562 Cells; D046228:Microarray Analysis / Q000295:instrumentation; D009757:Nylons / Q000737:chemistry; D010316:Particle Size; D059010:Single-Cell Analysis / Q000295:instrumentation; D013499:Surface Properties; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004129:Dimethylpolysiloxanes; C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D009757:Nylons; D014764:Viral Proteins; C085707:poly(dimethylsiloxane)-polyamide copolymer