Ying-Chih Chang

Type I Collagen-Functionalized Supported Lipid Bilayer as a Cell Culture Platform

The supported phospholipid bilayer serves as an important biomimetic model for the cell membrane in both basic and applied scientific research. We have constructed a biomimetic platform based on a supported phospholipid bilayer that is functionalized with type I collagen to serve as a substrate for cell culture. To create the type I collagen-functionalized lipid bilayer assembly, a simple chemical approach was employed: lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (DP-NGPE), a carboxylic acid-functionalized phospholipid, were prepared and then fused onto an SiO(2) substrate to form a supported lipid bilayer. Subsequently, type I collagen molecules were introduced to form stable collagen-lipid conjugates via amide linkages with activated DP-NGPE lipids. The binding kinetics of the conjugation process and the resultant changes in film thickness and viscoelasticity were followed using the quartz crystal microbalance with dissipation (QCM-D) monitoring. The morphology of the conjugated collagen adlayer was investigated with atomic force microscopy (AFM). We observed that the adsorbed collagen molecules tended to self-assemble into fibrillar structures. Fluorescence recovery after photobleaching (FRAP) was utilized to estimate lateral lipid mobility, which was reduced by up to 20% after the coupling of type I collagen to the underlying lipid bilayer. As a cell culture platform, the collagen-conjugated supported lipid bilayer showed promising results. Smooth muscle cells (A10) retained normal growth behavior on the collagen-functionalized platform, unlike the bare POPC lipid bilayer and the POPC/DG-NGPE bilayer without collagen. The biomimetic functionalized lipid system presented here is a simple, yet effective approach for constructing a cell culture platform to explore the interactions between extracellular matrix components and cells.

Circulating Tumor Cell Count Correlates with Colorectal Neoplasm Progression and Is a Prognostic Marker for Distant Metastasis in Non-Metastatic Patients

Enumeration of circulating tumor cells (CTCs) has been proven as a prognostic marker for metastatic colorectal cancer (m-CRC) patients. However, the currently available techniques for capturing and enumerating CTCs lack of required sensitivity to be applicable as a prognostic marker for non-metastatic patients as CTCs are even more rare. We have developed a microfluidic device utilizing antibody-conjugated non-fouling coating to eliminate nonspecific binding and to promote the multivalent binding of target cells. We then established the correlation of CTC counts and neoplasm progression through applying this platform to capture and enumerate CTCs in 2 mL of peripheral blood from healthy (n = 27), benign (n = 21), non-metastatic (n = 95), and m-CRC (n = 15) patients. The results showed that the CTC counts progressed from 0, 1, 5, to 36. Importantly, after 2-year follow-up on the non-metastatic CRC patients, we found that those who had ≥5 CTCs were 8 times more likely to develop distant metastasis within one year after curable surgery than those who had <5. In conclusion, by employing a sensitive device, CTC counts show good correlation with colorectal neoplasm, thus CTC may be as a simple, independent prognostic marker for the non-metastatic CRC patients who are at high risk of early recurrence.

Antibody conjugated supported lipid bilayer for capturing and purification of viable tumor cells in blood for subsequent cell culture

Interest in the identification and isolation of circulating tumor cells (CTCs) has been growing since the introduction of CTCs as an alternative to the tumor tissue biopsy, which can potentially be important indices for prognosis and cancer treatment. However, the contamination of non-specific binding of normal hematologic cells makes high purity CTCs detection problematic. Furthermore, preserving the viability of CTCs remains a challenge. In this study, we proposed to construct an anti-EpCAM functionalized supported lipid bilayer (SLB), a biomimetic and non-fouling membrane coating, for CTCs capturing, purification and maintaining the viability. Healthy human blood spiked with pre-stained colorectal cancer cell lines, HCT116 and colo205, were used to investigate interaction of cells with the anti-EpCAM functionalized SLB surfaces. Over 97% of HCT116, and 72% of colo205 were captured and adhered by the surface anti-EpCAM; conversely, the majority of blood cells were easily removed by gentle buffer exchange, with the overall purity of cancer cells exceeding 95%. The bound cancer cells were subsequently detached for cell culture. Both HCT116 and colo205 continued to proliferate over 2-week observation period, indicating that the anti-EpCAM functionalized SLB platform providing a simple strategy for capturing, purifying, and releasing viable targeted rare cells.

Developing Antifouling Biointerfaces Based on Bioinspired Zwitterionic Dopamine through pH-Modulated Assembly

The use of synthetic biomaterials as implantable devices typically is accompanied by considerable nonspecific adsorption of proteins, cells, and bacteria. These may eventually induce adverse pathogenic problems in clinical practice, such as thrombosis and biomaterial-associated infection. Thus, an effective surface coating for medical devices has been pursued to repel nonspecific adsorption from surfaces. In this study, we employ an adhesive dopamine molecule conjugated with zwitterionic sulfobetaine moiety (SB-DA), developed based on natural mussels, as a surface ligand for the modification of TiO2. The electrochemical study shows that the SB-DA exhibits fully reversible reduction-oxidation behavior at pH 3, but it is irreversible at pH 8. A contact angle goniometer and X-ray photoelectron spectroscopy were utilized to explore the surface hydration, chemical states, and bonding mechanism of SB-DA. The results indicate that the binding between hydroxyl groups of SB-DA and TiO2 converts from hydrogen bonds to bidentate binding upon the pH transition from pH 3 to 8. In order to examine the antifouling properties of SB-DA thin films, the modified substrates were brought into contact with bovine serum albumin and bacteria solutions. The fouling levels were monitored using a quartz crystal microbalance with dissipation sensor and fluorescence optical microscope. Tests showed that the sample prepared via the pH transition approach provides the best resistance to nonspecific adsorption due to the high coverage and stability of the SB-DA films. These findings support the mechanism of the pH-modulated assembly of SB-DA molecules, and for the first time we demonstrate the antifouling properties of the SB-DA to be comparable with traditional thiol-based zwitterionic self-assemblies. The success of modification with SB-DA opens an avenue for developing a biologically inspired surface chemistry and can have applications over a wide spectrum of bioapplications. The strategy of the pH transition can also be applied to other functional dopamine derivatives.

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

Effects of extracellular matrix protein functionalized fluid membrane on cell adhesion and matrix remodeling

In order to study cellular responses and extracellular matrix protein remodeling mediated by biomaterials coating, we proposed a biomimetic construct containing protein-conjugated supported lipid bilayers (SLBs) as a cell culture platform. Single or multi-component proteins-bound SLBs were fabricated by conjugating type I collagen and/or fibronectin on the N-hydroxysulfosuccinimide-functionalized SLBs. The proposed protein-conjugated systems were quantitatively characterized by the quartz crystal microbalance with dissipation. NIH 3T3 fibroblasts were cultured on the model constructs and on oxygen plasma pretreated polystyrene (PSo) for parallel comparison. The retards of mobility of SLB after protein conjugation and cell culture were estimated by fluorescence recovery after photobleaching. The resulting cell morphology, adsorption kinetics and somatic dynamics were examined microscopically. We found that, on the SLB based cultures, the largest spreading size and cell number counts of 3T3 fibroblasts were found on the fibronectin containing surfaces. However, on the protein-coated PSo surfaces, no such distinguishable differences can be observed on all protein contents. Immunofluorescent staining results revealed that adsorption of endogenously produced fibronectin by 3T3 cells on PSo based surfaces is significantly more than that on SLB based surfaces. This suggests that the anti-fouling nature of underneath SLBs have played an important role in preventing 3T3 cells from effectively remodeling their microenvironment, whereas cells can easily remodel the nonspecific adsorption prone surfaces such as PSo based platforms. In summary, the protein conjugated SLB surfaces can serve as a platform for determining and regulating cell specific binding and subsequent signaling events with extracellular environments.

Effect of solvents and temperature on the conformation of poly(beta-benzyl-L-aspartate) brushes.

We report on the synthesis and characterization of end-tethering polypeptide monolayers based on poly(beta-benzyl-L-aspartate) (PBLA) homopolymer and PBLA-b-poly(gamma-benzyl-L-glutamate) block copolymer. The homopolypeptide and copolypeptide brushes were fabricated by the sequential, surface-initiated vapor deposition polymerization of the N-carboxyanhydride of beta-benzyl-L-aspartate or gamma-benzyl-L-glutamate, yielding 80-nm-thick, chemically grafted films after 30 min of reaction time. Both Fourier transform infrared spectrometry and circular dichroism showed that the polypeptide brushes could be reversibly and repeatedly switched between left-handed and right-handed alpha-helical structures in response to solvent vapor exposure or permanently converted to a beta-sheet structure when heated to 160 degrees C in air. The facile, in vacuo manufacturability and the robustness of the films of PBLA-based brushes could allow them to be incorporated as active components for biosensing and nanofabricated devices.

Selection, Enrichment, and Maintenance of Self-Renewal Liver Stem/Progenitor Cells Utilizing Polypeptide Polyelectrolyte Multilayer Films

Recent progress has led to the identification of liver stem/progenitor cells as suitable sources for generating transplantable liver cells. However, the great variability in methods utilized to isolate liver stem/progenitor cells is a considerable challenge for clinical applications. The polyelectrolyte-multilayer technique can constitute a useful method for selective cell adhesion. Whether enrichment of liver stem/progenitor cells can be achieved utilizing polypeptide polyelectrolyte-multilayer films was investigated in current work. Fetal liver cells isolated from E13.5 mouse embryos were seeded on the poly-l-glutamic acid/poly-l-lysine alternating films, and we revealed that fetal liver stem/progenitor cells were selected and formed colonies. These undifferentiated colonies were maintained on the films composed of four alternating layers, with the topmost poly-l-glutamic acid layer judged by the constitutive expression of stem-cell markers such as Dlk-1, CD49f, and CD133 and self-renew marker-beta-catenin. Our work has demonstrated that highly tunable polyelectrolyte-multilayer films were suitable for selective enrichment of liver stem/progenitor cells in vitro.

Differentiation of Neural Stem/Progenitor Cells Using Low-Intensity Ultrasound

Herein, we report the evaluation of apoptosis, cell differentiation, neurite outgrowth and differentiation of neural stem/progenitor cells (NSPCs) in response to low-intensity ultrasound (LIUS) exposure. NSPCs were cultured under different conditions, with and without LIUS exposure, to evaluate the single and complex effects of LIUS. A lactic dehydrogenase assay revealed that the cell viability of NSPCs was maintained with LIUS exposure at an intensity range from 100 to 500 mW/cm(2). Additionally, in comparison with no LIUS exposure, the cell survival rate was improved with the combination of medium supplemented with nerve growth factor and LIUS exposure. Our results indicate that LIUS exposure promoted NSPC attachment and differentiation on a glass substrate. Neurite outgrowth assays revealed the generation of longer, thicker neurites after LIUS exposure. Furthermore, LIUS stimulation substantially increased the percentage of differentiating neural cells in NSPCs treated with nerve growth factor in comparison with the unstimulated group. The high percentage of differentiated neural cells indicated that LIUS induced neuronal networks denser than those observed in the unstimulated groups. Furthermore, the release of nitric oxide, an important small-molecule neurotransmitter, was significantly upregulated after LIUS exposure. It is therefore reasonable to suggest that LIUS promotes the differentiation of NSPCs into neural cells, induces neurite outgrowth and regulates nitric oxide production; thus, LIUS may be a potential candidate for NSPC induction and neural cell therapy.

A High Circulating Tumor Cell Count in Portal Vein Predicts Liver Metastasis From Periampullary or Pancreatic Cancer: A High Portal Venous CTC Count Predicts Liver Metastases

AbstractCirculating tumor cells (CTCs) released from a periampullary or pancreatic cancer can be more frequently detected in the portal than the systemic circulation and potentially can be used to identify patients with liver micrometastases. Aims of this study is to determine if CTCs count in portal venous blood of patients with nonmetastatic periampullary or pancreatic adenocarcinoma can be used as a predictor for subsequent liver metastases. CTCs were quantified in portal and peripheral venous blood samples collected simultaneously during pancreaticoduodenectomy in patients with presumed periampullary or pancreatic adenocarcinoma without image-discernible metastasis. Postoperatively patients were monitored for liver metastasis by abdominal magnetic resonance imaging or computed tomography every 3 months for 1 year. Sixty patients with a pathological diagnosis of periampullary or pancreatic adenocarcinoma were included in the study. Multivariate analysis indicated that portal CTC count was a significant predictor for liver metastases within 6 months after surgery. Eleven of 13 patients with a high portal CTCs count (defined as >112 CMx Platform estimated CTCs in 2 mL blood) developed liver metastases within 6 months after surgery. In contrast, only 6 of 47 patients with a low portal CTC count developed liver metastases (P < 0.0001). A value of 112 CMx Platform estimated CTCs had 64.7% sensitivity and 95.4% specificity to predict liver metastases within 6 months after surgery. We concluded that a high CTC count in portal venous blood collected during pancreaticoduodenectomy in patients with periampullary or pancreatic adenocarcinoma without metastases detected by currently available imaging tools is a significant predictor for liver metastases within 6 months after surgery.