John Frampton | University of Michigan (original) (raw)

Papers by John Frampton

Technology, May 30, 2014

Quantitative measurement of protein biomarkers is critical for biomarker validation and early dis... more Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and falsepositive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD).

Scientific Reports, May 2, 2014

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis ... more Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-pha... more Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-phase system droplets to a single phase was used to directly visualize Dextranase activity. DEX droplets were formed either by manual micropipetting or within a continuous PEG phase by computer controlled actuation of an orifice connecting rounded channels formed by backside diffused light lithography. The time required for the two-phase to one-phase transition was dependent on the Dextranase concentration, pH of the medium, and temperature. The apparent Michaelis constants for Dextranase were estimated based on previously reported catalytic constants, the binodal polymer concentration curves for PEG-DEX phase transition for each temperature, and pH condition. The combination of a microfluidic droplet system and phase transition observation provides a new method for label-free direct measurement of enzyme activity.

Analytical Chemistry, Apr 1, 2014

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of... more Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases.

Advanced Functional Materials

Ultrasound-driven microbubbles produce mechanical forces that can disrupt cell membranes (sonopor... more Ultrasound-driven microbubbles produce mechanical forces that can disrupt cell membranes (sonoporation). However, it is difﬁcult to control microbubble location with respect to cells. This lack of control leads to low sonoporation efficiencies and variable outcomes. In this study, aqueous two-phase system (ATPS) droplets are used to localize microbubbles in select micro-regions at the surface of living cells. This is achieved by stably partitioning microbubbles in dextran (DEX) droplets, deposited on living adherent cells in medium containing polyethylene glycol (PEG). The interfacial energy at the PEG-DEX interface overcomes microbubble buoyancy and prevents microbubbles from ﬂoating away from the cells. Spreading of the small DEX droplets retains microbubbles at the cell surface in deﬁned lateral positions without the need for antibody or cell-binding ligand conjugation. The patterned microbubbles are activated on a cell monolayer exposed to a broadly applied ultrasound ﬁeld (center frequency 1.25 MHz, active element diameter 0.6 cm, pulse duration 8 μ s or 30 s). This system enables efﬁcient testing of different ultrasound conditions for their effects on sonoporation-mediated membrane disruption and cell viability. Regions of cells without patterned microbubbles show no injury or membrane disruption. In microbubble patterned regions, 8 μ s ultrasound pulses (0.2-0.6 MPa) produce cell death that is primarily apoptotic. Ultrasound-induced apoptosis increases with higher extracellular calcium concentrations, with cells displaying all of the hallmarks of apoptosis including annexinV labeling, loss of mitochondrial membrane potential, caspase activation and changes in nuclear morphology.

Journal of Biophotonics

We utilize laser-generated focused ultrasound (LGFU) to create targeted mechanical disturbance on... more We utilize laser-generated focused ultrasound (LGFU) to create targeted mechanical disturbance on a few cells. The LGFU is transmitted through an optoacoustic lens that converts laser pulses into focused ultrasound. The tight focusing (<100 mm) and high peak pressure of the LGFU produces cavitational disturbances at a localized spot with micro-jetting and secondary shock-waves arising from micro-bubble collapse. We demonstrate that LGFU can be used as a non-contact, non-ionizing, high-precision tool to selectively detach a single cell from its culture substrate. Furthermore, we explore the possibility of biomolecule delivery in a small population of cells targeted by LGFU at pressure amplitudes below and above the cavitation threshold. We experimentally confirm that cavitational disruption is required for delivery of propidium iodide, a membrane-impermeable nucleic acid-binding dye, into cells.

Journal of Visualized Experiments

Cell patterning technologies that are fast, easy to use and affordable will be required for the f... more Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Tissue Engineering, Part C

The development of tools for patterning co-cultures of cells is a fundamental interest among cell... more The development of tools for patterning co-cultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection (ADE) and aqueous two-phase system (ATPS) exclusion patterning to introduce a method for patterning co-cultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters such as pulse, duration and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol (PEG) solution containing an additional cell type is then added to the culture dish to produce a two phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Furthermore, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell co-cultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding co-cultured cells.

Journal of Neuroscience Research

Deficiencies in protein degradation and proteolytic function within neurons are linked to a numbe... more Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3, and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, so it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons or be transported to cell bodies for degradation.

Biomedical Materials

Two-dimensional (2D) culture systems provide useful information about many biological processes. ... more Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

Annals of Biomedical Engineering, Jan 1, 2010

Neural prosthetic devices hold the potential to be used in the treatment of a variety of neurolog... more Neural prosthetic devices hold the potential to be used in the treatment of a variety of neurological disorders. However, their long-term clinical success is currently limited by the ability to achieve stable interfaces between devices and the CNS. Immunohistochemical analysis has shown that cellular responses occur in tissue surrounding implanted devices. These cellular responses have been correlated with the impedance measured from device electrodes, leading to the hypothesis that a possible mechanism resulting in inconsistent device performance is the formation of an electrically insulating glial sheath at the implantation site. However, little is known about what cellular and tissue changes affect impedance values and thus contribute to the decreases in electrode performance. We have designed an in vitro system in which cell conditions can be varied within an artificial tissue matrix surrounding a neural prosthetic device. In this study, high-density cultures of glial cells were analyzed by immunohistochemical methods and impedance spectroscopy. Astrocytes and microglia were cultured at various ratios within the matrix surrounding the probes, and were observed over a period of 2 weeks. Cell seeding conditions and confocal images were compared to impedance data to enable the effects of glial cell type on electrode impedance to be determined.

Journal of Neural Engineering

One limitation to the use of neuroprosthestic devices for chronic application, in the treatment o... more One limitation to the use of neuroprosthestic devices for chronic application, in the treatment of disease, is the reactive cell responses that occur surrounding the device after insertion. These cell and tissue responses result in increases in device impedance and failure of the device to interact with target populations of neurons. However, few tools are available to assess which components of the reactive response contribute most to changes in tissue impedance. An in vitro culture system has been developed that is capable of assessing individual components of the reactive response. The system utilizes alginate cell encapsulation to construct three-dimensional architectures that approach the cell densities found in rat cortex. The system was constructed around neuroNexus acute probes with on-board circuitry capable of monitoring the electrical properties of the surrounding tissue. This study demonstrates the utility of the system by demonstrating that differences in cell density within the three-dimensional alginate constructs result in differences in resistance and capacitance as measured by electrochemical impedance spectroscopy. We propose that this system can be used to model components of the reactive responses in brain tissue, and that the measurements recorded in vitro are comparable to measurements recorded in vivo.

Biomedical Microdevices

Laminar and pulsatile flow of aqueous solutions in microfluidic channels can be useful for contro... more Laminar and pulsatile flow of aqueous solutions in microfluidic channels can be useful for controlled delivery of cells and molecules. Dispersion effects resulting from diffusion and convective disturbances, however, result in reagent delivery profiles becoming blurred over the length of the channels. This issue is addressed partially by using oil-in-water phase systems. However, there are limitations in terms of the biocompatibility of these systems for adherent cell culture. Here we present a fully biocompatible aqueous two-phase flow system that can be used to pattern cells within simple microfluidic channel designs, as well as to deliver biochemical treatments to cells according to discrete boundaries. We demonstrate that aqueous two-phase systems are capable of precisely delivering cells as laminar patterns, or as islands by way of forced droplet formation. We also demonstrate that these systems can be used to precisely control chemical delivery to preformed monolayers of cells growing within channels. Treatments containing trypsin were localized more reliably using aqueous two-phase delivery than using conventional delivery in aqueous medium.

Lab on a Chip

Exposure of a negative photoresist-coated glass slide with diffused light from the backside throu... more Exposure of a negative photoresist-coated glass slide with diffused light from the backside through a mask with disconnected features provides multi-level rounded channels with narrow orifices in one exposure. Using these structures, we construct microfluidic systems capable of creating aqueous two-phase system droplets where one aqueous phase forms droplets and the other aqueous phase forms the surrounding matrix. Unlike water-in-oil droplet systems, aqueous two-phase systems can have very low interfacial tensions that prevent spontaneous droplet formation. The multi-level channels fabricated by backside lithography satisfy two conflicting needs: (i) the requirement to have narrowed channels for efficient valve closure by channel deformation and (ii) the need to have wide channels to reduce the flow velocity, thus reducing the capillary number and enhancing droplet formation.

Cell Transplantation

Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell tr... more Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology, and can strongly influence stem cell fate. Here we demonstrate that severe-low O ₂ concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs), and that MSCs induced under severe hypoxic conditions function as normal MSCs; i.e., they repopulate after cloning, express specific markers (Vimentin, CD29, CD90, CD105 and CD140a) and differentiate into adipocytes, osteoblasts and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.

Biotechnology Journal

Conventional culture systems are often limited in their ability to regulate the growth and differ... more Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user-controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insight into the implications of microfluidics on pluripotent stem cell research.

Stem Cells and Development

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulati... more In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection (ICSI) followed by in vitro culture. Although only weak expression of POU5f1 was observed in the ICMs of in vitro cultured follicle-derived embryos, repeated careful cloning enabled establishment of three stable ESC lines. These ESC lines displayed the morphological characteristics of primed-pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1 and Sox2. Furthermore, these ESCs could be differentiated into each of the three different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

Reproductive Medicine and Biology

Despite recent advances in reproductive medicine, there are still no effective treatments for sev... more Despite recent advances in reproductive medicine, there are still no effective treatments for severe infertility caused by congenital absence of germ cells or gonadotoxic treatments during prepubertal childhood. However, the development of technologies for germ cell formation from stem cells in vitro, induction of pluripotency from somatic cells, and production of patient-specific pluripotent stem cells may provide new solutions for treating these severe fertility problems. It may be possible to produce germ cells in vitro from our own somatic cells that can be used to restore fertility. In addition, these technologies may also bring about novel therapies by helping to elucidate the mechanisms of human germ cell development. In this review, we describe the current approaches for obtaining germ cells from pluripotent stem cells, and provide basic information about induction of pluripotency and germ cell development.

Biochemical surface modification has been used to direct cell attachment and growth on a biocompa... more Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72h in vitro, and remained viable on protein-stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.

Journal of Biomedical Materials Research Part A, Jan 1, 2007

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple sign... more Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth.