Label-free enrichment of human adipose-derived stem cells using a continuous microfluidic sorting cascade.

Human adipose tissue is a rich source of mesenchymal stem cells (MSCs). Human adipose-derived stem cells (ADSCs) are first prepared by tissue digestion of lipoaspirate. The remaining constituent contains a mixture of ADSCs, other cell types and lysed fragments. We have developed a scalable microfluidic sorter cascade which enabled high-throughput and label-free enrichment of ADSCs prepared from tissue-digested human adipose samples to improve the quality of purified stem cell product. The continuous microfluidic sorter cascade was composed of spiral-shaped inertial and deterministic lateral displacement (DLD) sorters which separated cells based on size difference. The cell count characterization results showed >90% separation efficiency. We also demonstrated that the enriched ADSC sub-population by the microfluidic sorter cascade yielded 6× enhancement of expansion capacity in tissue culture. The incorporation of this microfluidic sorter cascade into ADSC preparation workflow facilitates the generation of transplantation-scale stem cell product. We anticipate our stem cell microfluidic sorter cascade will find a variety of research and clinical applications in tissue engineering and regeneration medicine.

Critical Considerations for Regeneration of Vascularized Composite Tissues.

Effective vascularization is vital for survival and functionality of complex tissue-engineered organs. The formation of the microvasculature, composed of endothelial cells (ECs) alone, has been mostly used to restore the vascular networks in organs. However, recent heterocellular studies demonstrate that co-culturing is a more effective approach in revascularization of engineered organs. This review presents key considerations for manufacturing of artificial vascularized composite tissues. We summarize the importance of co-cultures and the multicellular interactions with ECs, as well as design and use of bioreactors, as critical considerations for tissue vascularization. In addition, as an emerging scaffolding technique, this review also highlights the current caveats and hurdles associated with three-dimensional bioprinting and discusses recent developments in bioprinting strategies such as four-dimensional bioprinting and its future outlook for manufacturing of vascularized tissue constructs. Finally, the review concludes with addressing the critical challenges in the regulatory pathway and clinical translation of artificial composite tissue grafts. Impact statement Regeneration of composite tissues is critical as biophysical and biochemical characteristics differ between various types of tissues. Engineering a vascularized composite tissue has remained unresolved and requires additional evaluations along with optimization of methodologies and standard operating procedures. To this end, the main hurdle is creating a viable vascular endothelium that remains functional for a longer duration postimplantation, and can be manufactured using clinically appropriate source of cell lines that are scalable in vitro for the fabrication of human-scale organs. This review presents key considerations for regeneration and manufacturing of vascularized composite tissues as the field advances.

Bridging the gap in peripheral nerve repair with 3D printed and bioprinted conduits.

Over the past two decades, a number of fabrication methods, including 3D printing and bioprinting, have emerged as promising technologies to bioengineer nerve conduits that closely replicate features of the native peripheral nerve, with the aim of augmenting or supplanting autologous nerve grafts. 3D printing and bioprinting offer the added advantage of rapidly creating composite peripheral nerve matrices from micron-scaled units, using an assortment of synthetic, natural and biologic materials. In this review, we explore the evolution of automated 3D manufacturing technologies for the development of peripheral nerve conduits and discuss aspects of conduit design, based on microarchitecture, material selection, cell and protein inclusion, and mechanical properties, as they are adaptable to 3D printing. Additionally, we highlight advancements in the application of bio-imaging modalities toward the fabrication of patient-specific nerve conduits. Lastly, we outline regulatory as well as clinical challenges that must be surmounted for the translation of 3D printing and bioprinting technology to the clinic. As a whole, this review addresses topics that may situate 3D manufacturing at the forefront of fabrication technologies that are exploited for the generation of future revolutionary therapies like in situ printing of peripheral nerves.

Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (ßTCP) via Fusion to a High Affinity, Multimeric ßTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors.

Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant ßTCP scaffolds and to demonstrate the bioactivity of EGF tethered to ßTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against ßTCP and composites of ßTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for ßTCP. When a single copy of this ßTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for ßTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to ßTCP, with only 25% of the protein released after 7 days at 37oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance the proliferation of hBMSC when presented in tethered form on commercial ßTCP bone regeneration scaffolds.

Continuous-flow sorting of stem cells and differentiation products based on dielectrophoresis.

This paper presents a continuous-flow microfluidic device for sorting stem cells and their differentiation progenies. The principle of the device is based on the accumulation of multiple dielectrophoresis (DEP) forces to deflect cells laterally in conjunction with the alternating on/off electric field to manipulate the cell trajectories. The microfluidic device containing a large array of oblique interdigitated electrodes was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with human mesenchymal stem cells (hMSC) and their differentiation progenies (osteoblasts) was carried out at different flow rates, and clear separation of the two populations was achieved. Most of the osteoblasts experiencing stronger DEP forces were deflected laterally and continuously, following zig-zag trajectories, and moved towards the desired collection outlet, whereas most of the hMSCs remained on the original trajectory due to weaker DEP forces. The experimental measurements were characterized and evaluated quantitatively, and consistent performance was demonstrated. Collection efficiency up to 92% and 67% for hMSCs and osteoblasts, respectively, along with purity up to 84% and 87% was obtained. The experimental results established the feasibility of our microfluidic DEP sorting device for continuous, label-free sorting of stem cells and their differentiation progenies.

An engineered bivalent neuregulin protects against doxorubicin-induced cardiotoxicity with reduced proneoplastic potential.

BACKGROUND: Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1ß is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1ß (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor-like domain of neuregulin-1ß (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1ß. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. METHODS AND RESULTS: NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). CONCLUSIONS: NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

Multilayer thin film coatings capable of extended programmable drug release: application to human mesenchymal stem cell differentiation.

The promise of cellular therapy lies in healing damaged tissues and organs in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Adult stem cells are ideally suited for cellular therapies due to their pulripotency and the ease with which they can be cultured on novel functionalized substrates. Creating environments to control and successively driving their differentiation toward a lineage of choice is one of the most important challenges of current cell-based engineering strategies. In recent years, a variety of biomedical platforms have been prepared for stem cell cultures, primarily to provide efficient delivery of growth or survival factors to cells and a conducive microenvironment for their growth. Here, we demonstrate that repeating tetralayer structures composed of biocompatible poly(methacrylic acid) (PMAA)/poly(acryl amide) (PAAm)/poly(methacrylic acid) (PMAA)/poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) micelles arrayed in layer-by-layer (LbL) films can serve as a payload region for dexamethasone (dex) delivery to human mesenchymal stem cells (MSCs). This architecture can induce MSC differentiation into osteoblasts in a dose-dependent manner. The amount of dex loaded in the films is controlled by varying the deposition conditions and the film thickness. Furthermore, release of dex is also controlled by changing the amount of covalent crosslinking of multilayers via thermal treatments. The multilayer architecture including payload and cell-adhesion region introduced here are well suited for extended cell culture thus affording the important and protective effect of both dex release and immobilization. These films may find applications in the local delivery of immobilized therapeutics for biomedical applications, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.

Engineered bivalent ligands to bias ErbB receptor-mediated signaling and phenotypes.

The ErbB receptor family is dysregulated in many cancers, and its therapeutic manipulation by targeted antibodies and kinase inhibitors has resulted in effective chemotherapies. However, many malignancies remain refractory to current interventions. We describe a new approach that directs ErbB receptor interactions, resulting in biased signaling and phenotypes. Due to known receptor-ligand affinities and the necessity of ErbB receptors to dimerize to signal, bivalent ligands, formed by the synthetic linkage of two neuregulin-1ß (NRG) moieties, two epidermal growth factor (EGF) moieties, or an EGF and a NRG moiety, can potentially drive homotypic receptor interactions and diminish formation of HER2-containing heterodimers, which are implicated in many malignancies and are a prevalent outcome of stimulation by native, monovalent EGF, or NRG. We demonstrate the therapeutic potential of this approach by showing that bivalent NRG (NN) can bias signaling in HER3-expressing cancer cells, resulting in some cases in decreased migration, inhibited proliferation, and increased apoptosis, whereas native NRG stimulation increased the malignant potential of the same cells. Hence, this new approach may have therapeutic relevance in ovarian, breast, lung, and other cancers in which HER3 has been implicated.

Use of sibutramine in obese mexican adolescents: a 6-month, randomized, double-blind, placebo-controlled, parallel-group trial.

BACKGROUND: The prevalence of overweight and obesity in children and adolescents is increasing in both the United States and Mexico. OBJECTIVE: The goal of this article was to assess the efficacy and safety of sibutramine in obese Mexican adolescents. METHODS: This was a 6-month, randomized, double-blind, placebo-controlled, prospective clinical trial of sibutramine QD. Male and female patients aged 14 to 18 years with sex-specific body mass index (BMI) for age and sex >85th percentile were eligible. The primary end points for the trial were the baseline versus end point absolute values for body weight, BMI, and percentage of the initial BMI (%BMI); secondary end points were waist circumference and percentage of the initial waist circumference (%waist). These were measured at days -15, 0, 30, 60, 90, 120, 150, and 180 of the study. Quality of life was assessed at the study start and end using the 36-Item Short-Form Health Survey (SF-36) questionnaire. Blood pressure and heart rate were assessed, and adverse events (AEs) were recorded. Both groups received individually tailored diet and exercise programs. RESULTS: Forty-six patients (age range, 14-18 years) with a BMI >95th percentile for age were included (sibutramine group, n = 23 [14 females, 9 males]; placebo group, n = 23 [12 females, 11 males]). Twenty-one patients in the sibutramine group and 19 patients in the placebo group completed the 6-month trial. Using the intent-to-treat data, weight (mean [SD]) in the sibutramine group changed from 92.5 (14.6) kg to 85.7 (14.4) kg, for a net weight loss of 7.3 kg (95% CI 4.6-9.9), a waist circumference loss of 8.0 cm (95% CI, 4.7-11.3), and a % BMI loss of 9.2% (95% CI, 6.9-11.6). In the placebo group, weight changed from 98.9 (22.7) kg to 94.6 (22.5) kg, a weight loss of 4.3 kg (95% CI, 1.7-6.9), a waist circumference loss of 3.8 cm (95% CI, 0.7-7.0), and a %BMI loss of 5.2% (95% CI, 2.4-7.9) (P < 0.05 for all intragroup comparisons; P > 0.05 for the intergroup comparisons). Mean (SD) scores on the SF-36 scale in the sibutramine group changed from 78.0 (13.3) at baseline to 84.8 (7.4) at study end (P < 0.05); the respective values in the placebo group were 82.8 (10.3) and 87.3 (7.6) (P < 0.05). At base-line, systolic blood pressure (SBP) was 116.7 (5.9) mm Hg in the sibutramine group and 118.3 (7.6) mm Hg in the placebo group; at end point, the respective SBPs were 112.4 (9.6) mm Hg and 112.6 (6.5) mm Hg. At baseline, diastolic blood pressure (DBP) was 78.9 (4.5) mm Hg in the sibutramine group and 79.5 (5.2) mm Hg in the placebo group; at end point, the respective DBPs were 73.5 (6.3) mm Hg and 76.6 (6.2) mm Hg. At baseline, heart rate was 76.3 (6.4) beats/min in the sibutramine group and 81.1 (9.5) beats/min in the placebo group; at end point, the respective findings were 79.8 (8.8) beats/min and 77.6 (8.6) beats/min (P > 0.05 for all preceding intergroup comparisons). One patient in the sibutramine group had increased blood pressure (at month 3) and 3 had increased heart rate (at months 1, 2, and 4); 2 patients receiving placebo had increased blood pressure (month 3) and 2 had increased heart rate (at months 1 and 3). These changes disappeared in 1 week and did not require treatment or trial suspension. Additionally, in the sibutramine group, 3 patients experienced 4 mild AEs: headache, dry mouth, headache with nausea, and headache with weakness and paleness (P > 0.05). In the placebo group, 3 patients experienced 4 mild AEs: 2 cases of headache, as well as 1 case of headache with somnolence and 1 case of headache with dry mouth (P > 0.05). CONCLUSION: Sibutramine 10 mg QD in addition to diet and exercise was effective and generally well tolerated in this population of obese Mexican adolescents.

Continuous desulfurization of dibenzothiophene with Rhodococcusrhodochrous IGTS8 (ATCC 53968).

Desulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrous IGTS8 was demonstrated in a 2-L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L(-1) h(-1)) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first-order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h(-1). Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off-gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.