Kyung Ho Roh

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.

Structurally Controlled Bio‐hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured bio-hybrid materials. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide–MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell–cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery.

The bone regenerative effect of chitosan microsphere-encapsulated growth hormone on bony consolidation in mandibular distraction osteogenesis in a dog model.

The purpose of this project was to study the effect of chitosan microsphere-encapsulated human growth hormone, which causes sustained release of chitosan and human growth hormone after implantation on early bony consolidation in distraction osteogenesis of a canine model. Forty-eight dogs were used for this study. An external distraction device was applied to the mandibular body after a vertical osteotomy, and the mandibular distraction was started 5 days after the operation at a rate of 1 mm/d up to a 10-mm distraction. The experimental group was divided into a control group (I), hyaluronic acid group (II), chitosan microsphere group (III), and chitosan microsphere-encapsulated human growth hormone group (IV). Normal saline was injected in group I. In group II, a 1-ml volume of hyaluronic acid solution was injected into the distracted area. In the group III, powder of chitosan microspheres and hGH were mixed with a 1-ml volume of hyaluronic acid to make an injectable form, and it was implanted into the distracted area. In group IV, powder of chitosan microsphere-encapsulated hGH was mixed with a 1-ml volume of hyaluronic acid. A total of 1-ml volume of the solution mix was implanted into the distracted area. Five dogs in each group (total of 20 dogs) were killed 3 weeks after completion of distraction. Twenty-eight dogs were killed at 6 weeks. Bone mineral density was 13.1% of the contralateral normal mandible at 3 weeks and 29.6% at 6 weeks in group I, 16.4% at 3 weeks and 40.4% at 6 weeks in group II, 16.6% at 3 weeks and 45.95% at 6 weeks in group III, and 29.6% at 3 weeks and 66.7% at 6 weeks in group IV. The mean three-point failure load was 16.1% in the control group, 34.7% in group II, 41.5% in group III, and 52.1% in group IV compared with the intact contralateral mandible, with statistical significance. In the histological findings, new bone was generated in all groups. In group IV, the formation of active woven bone was observed throughout the distracted area at 6 weeks. The amount of new bone formation in the distracted zone was in the order of group IV, group III, group II, and the control group. In conclusion, these findings suggest that chitosan microsphere-encapsulated hGH seems to be quite effective in early bone consolidation in distraction osteogenesis.

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

Significance Immune cell signaling is heavily associated with the spatial organization of molecules. Here, we examined the nanoscale organization of coreceptor CD4 and its relative spatial localization to the T-cell receptor and the active form of Src kinase p56lck (Lck), using two different superresolution microscopy techniques photoactivated localization microscopy and direct stochastic optical reconstruction microscopy in both living and fixed cells. With concurrent spatial analyses, we show that neither CD4/T-cell antigen receptor nor CD4/active Lck nanoclusters colocalize but only overlap at the interfaces. In activated T cells, the enhanced clustering of each kind results in increased seclusion from each other. Our observations here in molecular resolution may reveal the general roles that are played by nanoscale organization of critical components in immune cell signaling. CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

Effect of calcium sulfate-chitosan composite: pellet on bone formation in bone defect.

The purpose of this experiment was to study the effects of chitosan, calcium sulfate, and calcium sulfate-chitosan composite pellet on the osteogenesis of defective tibia in rabbits. Eighty New Zealand white rabbits, each weighing approximately 3 to 3.5 kg, were used for this study. A 1-cm ostectomy was made on the middle of the tibia of each rabbit with the periosteum preserved. Nothing was implanted in the control group (group 1), and five chitosan pellets (60 mg/pellet) were implanted in group 1, three OsteoSet® pellets (100 mg/pellet) in group 3, and four calcium sulfate-chitosan composite pellets (1 pellet, 80 mg; calcium sulfate 40 mg/pellet, chitosan 40 mg/pellet) in group 4. For each group, a radiographic study, bone mineral density test, three-point bending test, and histologic examination were performed in the second, fourth, and sixth weeks. In the radiologic study, in group 1, cortical bone was not formed even at 6 weeks. In group 2, it was observed at 6 weeks. In groups 3 and 4, cortical bone was partially seen around the fourth week. At 6 weeks, it was clearly observed on both sides, and the projection of the marrow cavity became distinctive, so bone consolidation was considered to be much progressed. The bone mineral density test and three-point bending test results appeared to be highly similar in groups 3 and 4 and in groups 2 and 1. Particularly at 6 weeks, the measures for groups 3 and 4 were statistically significant compared with those for groups 1 and 2 (P < 0.05). In histologic examination, new bone formation began to be seen at 2 weeks in all groups, but it was more active and faster in groups 3 and 4. At 6 weeks, fibrous connective tissue still remained at the center in groups 1 and 2; however, the fibrous connective tissue at the center was replaced with callus, the bony bridge was obvious, and lamellation of callus was observed more in groups 3 and 4. The results indicate that chitosan pellets, OsteoSet®, and chitosan-calcium sulfate composite pellets facilitate new bone formation on defected bone, and that particularly OsteoSet and chitosan-calcium sulfate composite pellets are more effective than chitosan.

Biomanufacturing of Therapeutic Cells: State of the Art, Current Challenges, and Future Perspectives

Stem cells and other functionally defined therapeutic cells (e.g., T cells) are promising to bring hope of a permanent cure for diseases and disorders that currently cannot be cured by conventional drugs or biological molecules. This paradigm shift in modern medicine of using cells as novel therapeutics can be realized only if suitable manufacturing technologies for large-scale, cost-effective, reproducible production of high-quality cells can be developed. Here we review the state of the art in therapeutic cell manufacturing, including cell purification and isolation, activation and differentiation, genetic modification, expansion, packaging, and preservation. We identify current challenges and discuss opportunities to overcome them such that cell therapies become highly effective, safe, and predictively reproducible while at the same time becoming affordable and widely available.

Engineering approaches for regeneration of T lymphopoiesis

T cells play a central role in immune-homeostasis; specifically in the induction of antigen-specific adaptive immunity against pathogens and mutated self with immunological memory. The thymus is the unique organ where T cells are generated. In this review, first the complex structures and functions of various thymic microcompartments are briefly discussed to identify critical engineering targets for regeneration of thymic functions in vitro and in vivo. Then the biomimetic regenerative engineering approaches are reviewed in three categories: 1) reconstruction of 3-D thymic architecture, 2) cellular engineering, and 3) biomaterials-based artificial presentation of critical biomolecules. For each engineering approach, remaining challenges and clinical opportunities are also identified and discussed.

Biocompatible Polymers: Structurally Controlled Bio-hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells (Adv. Mater. 48/2009).

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured biohybrid materials, report Joerg Lahann and co-workers on p. 4920. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.