High-density adherent culture of CHO cells using rolled scaffold bioreactor.

Rapid expansion of biopharmaceutical market calls for more efficient and reliable platforms to culture mammalian cells on a large scale. Stirred-tank bioreactors have been widely used for large-scale cell culture. However, it requires months of trials and errors to optimize culture conditions for each cell line. In this article, we extend our earlier studies on rolled scaffold (RS) bioreactors for high-density adherent cell culture and report two new implementations of RSs with greatly enhanced mass-manufacturability, termed as Mesh-RS and Fiber-RS. CHO-K1 cells were successfully expanded in Mesh-RS and Fiber-RS bioreactors with an average growth rate of 1.09 ± 0.04 1/day and 0.95 ± 0.07 1/day, which were higher than those reported in similar studies. Fiber-RS bioreactor exhibited a very high cell density of 72.8 × 106 cells/ml. Besides, a dialyzer was integrated into the RS bioreactor to remove cellular waste and to replenish nutrients without disturbing the cells. By collecting the dialyzed media separately, the dialysis efficiency was significantly improved. In conclusion, the developed RS bioreactor has a strong potential to provide a highly reliable and easily scalable platform for large-scale cell culture in the biopharmaceutical industry.

Calf circumference: cutoff values from the NHANES 1999-2006.

BACKGROUND: Calf circumference (CC) is used in geriatric studies as a simple and practical skeletal muscle (SM) marker for diagnosing low SM and sarcopenia. Currently applied CC cutoff points were developed in samples including older participants; values representative of the full adult lifespan are lacking. OBJECTIVES: We aimed to develop CC cutoff points and to identify relevant confounding factors from the large and diverse NHANES 1999-2006 population sample. METHODS: Demographic, anthropometric, and imaging data (DXA, appendicular lean mass) from the adult (age ≥18 y) NHANES sample were partitioned into subgroups according to sex, age, ethnicity, and race. Adults aged 18-39 y and BMI (in kg/m2) 18.5-24.9 were set as a reference population; CC cutoff points were derived at 1 and 2 SDs below the mean. RESULTS: The sample included 17,789 participants, 51.3% males and 48.7% females, with respective ages (mean ± SD) of 43.3 ± 16.1 y and 45.5 ± 16.9 y. CC was strongly correlated with appendicular lean mass, r = 0.84 and 0.86 for males and females (both P < 0.001), respectively. Significant differences in mean CC were present across sex, ethnic, self-reported race, and BMI groups. Adjusting CC for adiposity using BMI revealed a decrease in CC beginning after the second decade in males and third decade in females. Rounded CC cutoff values for moderately and severely low CC were 34 cm and 32 cm (males), and 33 cm and 31 cm (females), respectively. Our findings support the use of BMI-adjusted CC values for participants outside the normal-weight BMI range (18-24.9). CONCLUSIONS: This study defined CC values in a diverse population sample along with a BMI-adjustment approach that helps to remove the confounding effects of adiposity and thereby improves CC as a useful clinical estimate of SM mass.

Digital anthropometric volumes: Toward the development and validation of a universal software.

PURPOSE: Anthropometry is a method for quantifying body size and shape often used to derive body composition and health risk prediction models. Recent technology advancements led to development of three-dimensional (3D) optical scanners that can overcome most of the limitations associated with manual anthropometric data collection. However, each of the currently available devices offers proprietary measurements that do not match conventional anthropometric definitions. The aim of the current study was to develop and then evaluate the precision and accuracy of new "universal" 3D optical analysis software that calculates digital anthropometric volumes using identical standard landmarks across scanners. METHODS: Dual-energy x-ray absorptiometry (DXA) and air displacement plethysmography (ADP) total body and regional volume and fat mass reference measurements and 3D optical scans from two proprietary devices were collected from 356 participants to evaluate the robustness of total body and regional volume and fat mass measurements calculated by the developed software. Linear regression modeling with threefold cross validation was used to evaluate total body and regional fat masses from 3D scans. RESULTS: Total body and regional volumes measured by DXA and ADP had strong associations with corresponding estimates from the commercial 3D optical scanners coupled with the universal software (e.g., R2  = 0.98 for Styku and R2  = 1.00 for SS20, for both DXA and ADP comparisons). Regional body volumes also had strong correlation between DXA and the 3DO scanners (e.g., for arm, leg and trunk, respective R2 s of 0.75, 0.86, and 0.97 for Styku and 0.79, 0.89, and 0.98 for SS20). Similarly, there were strong associations between DXA- measured total body and regional fat mass and 3D optical estimates calculated by the universal software (e.g., for total body, arm, leg and trunk, respective R2 s of 0.86, 0.72, 0.77, and 0.88 for Styku and 0.84, 0.76, 0.78, and 0.85 for SS20). Absolute differences in volumes and fat mass between the reference methods and the universal software values revealed underlying proprietary scanner differences that can be improved when designing future devices. CONCLUSIONS: The current study suggests that, when compared against values calculated using DXA and ADP, the universal software was able to measure total and regional body volumes reliably from scans obtained by two different scanners. The universal software, with future refinements, combined with potential optical scanner design improvements, creates new opportunities for developing large multicenter anthropometric databases with uniformly defined body dimensions that can be used for modeling health risks. CLINICAL TRIAL REGISTRATION ID: Shape Up! Adults Study, NCT0363785.

Development of rolled scaffold for high-density adherent cell culture.

Bioreactors for large-scale culture of mammalian cells are playing vital roles in biotechnology and bioengineering. Various bioreactors have been developed, but their capacity and efficiency are often limited by insufficient mass transfer rate and high shear stress. A rolled scaffold (RS) is a fully defined scaffold for high-density adherent culture of mammalian cells. The RS is a polymer film with spacers, that is rolled into a cylinder with a pre-determined gap between each turn. Cells are cultured on its inner surfaces, while media flows through the gap. The RS exhibits high surface-area-to-volume ratio over 100 cm2/mL and can transport nutrients and gases with significantly reduced shear stress via convection in a unidirectional laminar flow, rather than diffusion and random turbulent flow as in stirred-tank bioreactors. In this paper, we expanded Chinese Hamster Ovary cells with RS bioreactors and demonstrated cell culture density over 60 million cells/mL with a growth rate higher than conventional suspension culture. Besides, murine embryonic stem cells were successfully expanded without losing their pluripotency. The RS will provide an affordable, scalable, and reliable platform for large-scale culture of recombinant cells in biopharmaceutical industries and shear-sensitive stem cells for tissue engineering.

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth.

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. SIGNIFICANCE: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099.

Multifrequency Optomechanical Stiffness Measurement of Single Adherent Cells on a Solid Substrate with High Throughput.

Mechanical properties of a cell reflect its biological and pathological conditions and there have been active research efforts to develop high-throughput platforms to mechanically characterize single cells. Yet, many of these research efforts are focused on suspended cells and use a flow-through configuration. In this paper, the stiffness of single adherent cells are optomechanically characterized using the vibration-induced phase shift (VIPS) without detaching them from the substrate. With the VIPS measurements, the frequency and amplitude dependency of the cell stiffness is investigated and statistically significant difference in the cell stiffness is confirmed after exposure to various drugs affecting cytoskeleton network. Furthermore, a 3-dimensional finite element model of a cell on a vibrating substrate is developed to extract the mechanical property from the measured VIPS. The developed technique can characterize the mechanical properties of single adherent cells at multiple frequencies with high throughput and will provide valuable clues in understanding cell mechanics.

Fabrication and characterization of self-folding thermoplastic sheets using unbalanced thermal shrinkage.

Self-folding or micro-origami technologies are actively investigated as a novel manufacturing process to fabricate three-dimensional macro/micro-structures. In this paper, we present a simple process to produce a self-folding structure with a biaxially oriented polystyrene sheet (BOPS) or Shrinky Dinks. A BOPS sheet is known to shrink to one-third of its original size in plane, when it is heated above 160 °C. A grid pattern is engraved on one side of the BOPS film with a laser engraver to decrease the thermal shrinkage of the engraved side. The thermal shrinkage of the non-engraved side remains the same and this unbalanced thermal shrinkage causes folding of the structure as the structure shrinks at high temperature. We investigated the self-folding mechanism and characterized how the grid geometry, the grid size, and the power of the laser engraver affect the bending curvature. The developed fabrication process to locally modulate thermomechanical properties of the material by engraving the grid pattern and the demonstrated design methodology to harness the unbalanced thermal shrinkage can be applied to develop complicated self-folding macro/micro structures.

Optomechanical measurement of the stiffness of single adherent cells.

Recent advances in mechanobiology have accumulated strong evidence showing close correlations between the physiological conditions and mechanical properties of cells. In this paper, a novel optomechanical technique to characterize the stiffness of single adherent cells attached on a substrate is reported. The oscillation in a cell's height on a vertically vibrating reflective substrate is measured with a laser Doppler vibrometer as apparent changes in the phase of the measured velocity. This apparent phase shift and the height oscillation are shown to be affected by the mechanical properties of human colorectal adenocarcinoma cells (HT-29). The reported optomechanical technique can provide high-throughput stiffness measurement of single adherent cells over time with minimal perturbation.