Biomedical subjects

Alexander Revzin

Publications and source records attributed to Alexander Revzin.

6 recordsLinked to original sources

PubMed2006

Analysis of local tissue-specific gene expression in cellular micropatterns.

While cellular micropatterning approaches are employed extensively in cell biology and tissue engineering, only a limited number of methods for analysis of local function in the context of a complex, microfabricated environment are currently available. The present study develops a novel strategy for analysis of local tissue-specific function in cellular micropatterns. Model hepatocytes (HepG2 cells) were seeded onto silane-modified glass slides containing robotically printed arrays of collagen type I. These model hepatocytes formed cell arrays with individual cell cluster dimensions (150 or 500 microm) corresponding in size to the printed collagen spots. Non-parenchymal cells (3T3 fibroblasts) were added to hepatocellular micropatterns to create heterotypic cocultures. Expression of hepatic phenotype in HepG2 cells was first verified by traditional techniques including intracellular staining and ELISA for albumin. In order to evaluate local liver function in the cellular microarray, individual array members composed of approximately 400 hepatocytes were retrieved using laser capture microdissection and analyzed with real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). Hepatic function was assessed based on expression of four genes associated with differentiated liver phenotype: albumin, transferrin, alpha-fetoprotein, and alpha1-antitrypsin. "Titration" experiments, carried out to identify the smallest population of HepG2 cells yielding detectable mRNA levels and RT-PCR signals, showed that extraction area of 12,500 microm2 (corresponding to approximately 70 cells) provided detectable gene expression signals. All four liver-specific genes were routinely evaluated after extraction of approximately 400 HepG2 from the micropatterned surfaces. Significantly, selective retrieval and subsequent analysis of tissue-specific function was demonstrated for hepatic cells micropatterned alone and in coculture with non-parenchymal cells. In the future, methods described in this study will offer the possibility to investigate dynamic and reciprocal interactions between two or more cell types positioned on a microfabricated cell culture surface. We also envision the proposed approaches to be ideally suited for cell analysis in the context of combinatorial microenvironment.

3T3 Cells↗

PubMed2006

Panning of multiple subsets of leukocytes on antibody-decorated poly(ethylene) glycol-coated glass slides.

The antibody (Ab) array format provides a unique opportunity to pan and characterize multiple leukocyte subsets in parallel. However, the questions of reproducibility and robustness of leukocyte panning on Ab arrays need to be answered for this technology to become an immunophenotyping tool. The present study sought to address several of these questions, including: (1) purity of leukocyte subsets captured on Ab regions, (2) dynamics of leukocyte binding, (3) elimination of non-specific cell adhesion, and (4) standardization of cell washing conditions. Abs for CD4 T-cells, CD8 T-cells, CD36 monocytes, and CD16b neutrophils were dispensed onto standard glass slides containing a thin film of poly(ethylene glycol) (PEG) hydrogel. PEG gel coating was highly effective in eliminated non-specific cell adhesion on the surface. Incubation of the Ab arrays with red blood cell (RBC) depleted whole blood resulting in antigen-specific panning of leukocyte subsets on the respective Ab domains. A flow through chamber was employed to determine optimal shear stress conditions for removal of non-specifically attached cells. The purity of the four subsets remaining on the surface after washing was determined by Wright staining and immunofluorescence, and was found to be as follows: CD4 T-cells (99.2+/-0.3%), CD8 T-cells (98.7+/-0.3%), CD36 monocytes (97.2+/-0.9%), and CD16b neutrophils (99.1+/-0.6%). In conclusion, the methods described in this study allow to separate whole blood into pure leukocyte subsets with minimal sample preparation and handling. These approaches will be valuable in the future development of Ab arrays as tools for quantitative immunophenotyping of leukocytes.

Antibodies↗

PubMed2005

Enrichment using antibody-coated microfluidic chambers in shear flow: model mixtures of human lymphocytes.

Isolation of phenotypically-pure cell subpopulations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated microfluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm2 was found to provide most efficient separation. Cell adhesion was shown to follow pseudo-first order kinetics, and an anti-CD19 coated (Raji-depletion) device with approximately 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally determined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time.

Antibodies↗

PubMed2004

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes.

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.

B-Lymphocytes↗

PubMed2004

Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

3T3 Cells↗

PubMed2002

Poly(ethylene glycol) hydrogel microstructures encapsulating living cells.

We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.

Animals↗