Min Hu

Hydrodynamic spinning of hydrogel fibers.

Hydrogel scaffolds are highly hydrated polymer networks that allow cells to adhere, proliferate and differentiate in the treatment of diseased or injured tissues and organs. Using hydrodynamic shaping and in situ cross-linking of hydrogel precursors, we have developed a highly efficient hydrodynamic spinning approach for synthesizing hydrogel fibers of different diameters in a multiphase coaxial flow. A triple-orifice spinneret has been created, and three different types of hydrogel precursors have been examined. Without changing the spinning head, hollow and solid hydrogel fibers with different diameters have been spun by simply manipulating the ratio of input flow rates. Together with the ability of simultaneous cell-seeding in the hydrogel matrix, hydrodynamic spinning can be broadly applied to many hydrogel materials, providing a powerful technique in the preparation of fiber-like and tubule-like hydrogel constructs for tissue engineering.

Cell immobilization in gelatin-hydroxyphenylpropionic acid hydrogel fibers

Gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn-HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20 microm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering.

The performance of primary human renal cells in hollow fiber bioreactors for bioartificial kidneys

Bioartificial kidneys (BAKs) containing human primary renal proximal tubule cells (HPTCs) have been applied in clinical trials. The results were encouraging, but also showed that more research is required. Animal cells or cell lines are not suitable for clinical applications, but have been mainly used in studies on BAK development as large numbers of such cells could be easily obtained. It is difficult to predict HPTC performance based on data obtained with other cell types. To enable more extensive studies on HPTCs, we have developed a bioreactor containing single hollow fiber membranes that requires relatively small amounts of cells. Special hollow fiber membranes with the skin layer on the outer surface and consisting of polyethersulfone/polyvinylpyrrolidone were developed. The results suggested that such hollow fiber membranes were more suitable for the bioreactor unit of BAKs than membranes with an inner skin layer. An HPTC-compatible double coating was applied to the insides of the hollow fiber membranes, which sustained the formation of functional epithelia under bioreactor conditions. Nevertheless, the state of differentiation of the primary human cells remained a critical issue and should be further addressed. The bioreactor system described here will facilitate further studies on the relevant human cell type.

Achievements and challenges in bioartificial kidney development

Bioartificial kidneys (BAKs) combine a conventional hemofilter in series with a bioreactor unit containing renal epithelial cells. The epithelial cells derived from the renal tubule should provide transport, metabolic, endocrinologic and immunomodulatory functions. Currently, primary human renal proximal tubule cells are most relevant for clinical applications. However, the use of human primary cells is associated with many obstacles, and the development of alternatives and an unlimited cell source is one of the most urgent challenges. BAKs have been applied in Phase I/II and Phase II clinical trials for the treatment of critically ill patients with acute renal failure. Significant effects on cytokine concentrations and long-term survival were observed. A subsequent Phase IIb clinical trial was discontinued after an interim analysis, and these results showed that further intense research on BAK-based therapies for acute renal failure was required. Development of BAK-based therapies for the treatment of patients suffering from end-stage renal disease is even more challenging, and related problems and research approaches are discussed herein, along with the development of mobile, portable, wearable and implantable devices.

Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy

Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs). Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed. CTCs were captured by microsieve technology and enumerated, while three different methods of EBV cfDNA quantification were applied, including an in-house qPCR assay for BamHI-W fragment, a CE-IVD qPCR assay (Sentosa ®) and a dPCR (Clarity™) assay for Epstein-Barr nuclear antigen 1 (EBNA1). EBV cfDNA quantitation by all workflows showed stronger correlation with clinical stage, radiological response and overall survival in comparison with CTC enumeration. The highest detection rate of EBV cfDNA in pre-treatment samples was seen with the BamHI-W qPCR assay (89%), followed by EBNA1-dPCR (85%) and EBNA1-qPCR (67%) assays. Overall, we show that EBV cfDNA outperforms CTC enumeration in correlation with clinical outcomes of NPC patients undergoing treatment. Techniques such as dPCR and target selection of BamHI-W may improve sensitivity for EBV cfDNA detection.

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

A population of cell clusters circulating in cancer patients consist of tumor-derived endothelial cells reflecting features of the tumor vasculature. A new type of circulating tumor cluster Setting out to isolate circulating tumor cells (CTCs) in the blood of cancer patients, Cima et al. instead noticed that clusters, rather than single cells, were present in their sorting device. However, these clusters were not like others reported previously: The cells did not express the cell marker epithelial cell adhesion molecule (EpCAM), and they did not have the same mutations seen in the primary tumor—two features that are typical of CTCs. Instead, these clusters matched the expression profile and phenotype of endothelial cells, which are the cells lining the blood vessels. The authors first confirmed that these endothelial cells were shed by the tumor, through a series of experiments using patient-derived samples and in vivo studies in mice, and then demonstrated that the clusters did not form by coagulation after being shed; it was the tumor that was shedding intact clusters. Moving back to patients, Cima and colleagues also found that the presence of endothelial cell clusters correlated with early-stage disease, suggesting that these groups of cells might be a unique indicator of cancer, before treatment starts. Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.