Jane Ru Choi

Impact of Low Oxygen Tension on Stemness, Proliferation and Differentiation Potential of Human Adipose-derived Stem Cells.

Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O2 tension on their functional properties has not been well determined. In this study, we investigated the effects of O2 tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O2) and hypoxia (2% O2). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O2 tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.

Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics

Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future.

Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

Sensitive biomolecule detection in lateral flow assay with a portable temperature–humidity control device

Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature-humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55-60°C and 37-40°C produced optimum nucleic acid hybridization and antigen-antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25°C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings.

In Situ Normoxia Enhances Survival and Proliferation Rate of Human Adipose Tissue-Derived Stromal Cells without Increasing the Risk of Tumourigenesis

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.

Improved sensitivity of lateral flow assay using paper-based sample concentration technique

Lateral flow assays (LFAs) hold great promise for point-of-care testing, especially in resource-poor settings. However, the poor sensitivity of LFAs limits their widespread applications. To address this, we developed a novel device by integrating dialysis-based concentration method into LFAs. The device successfully achieved 10-fold signal enhancement in Human Immunodeficiency Virus (HIV) nucleic acid detection with a detection limit of 0.1 nM and 4-fold signal enhancement in myoglobin (MYO) detection with a detection limit of 1.56 ng/mL in less than 25 min. This simple, low-cost and portable integrated device holds great potential for highly sensitive detection of various target analytes for medical diagnostics, food safety analysis and environmental monitoring.

Polydimethylsiloxane-Paper Hybrid Lateral Flow Assay for Highly Sensitive Point-of-Care Nucleic Acid Testing

In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future.

Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications

Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR.

Advances in paper-based sample pretreatment for point-of-care testing

Abstract In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.

Paper-based point-of-care testing for diagnosis of dengue infections

Abstract Dengue endemic is a serious healthcare concern in tropical and subtropical countries. Although well-established laboratory tests can provide early diagnosis of acute dengue infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Point-of-care (POC) devices, particularly paper-based POC devices, are typically rapid, cost-effective and user-friendly, and they can be used as diagnostic tools for the prompt diagnosis of dengue at POC settings. Here, we review the importance of rapid dengue diagnosis, current dengue diagnostic methods, and the development of paper-based POC devices for diagnosis of dengue infections at the POC.