Multiple approaches to reduce reconstitution time of lyophilized drug products with high protein concentration.

Background: Lyophilized drug products with high protein concentration often perform long reconstitution time, which is inconvenient for clinical use. The objective of this work is to achieve short reconstitution time with multiple and combined strategies. Methods: Here, we describe the following approaches that lead to reduction of reconstitution time, including adding annealing step, decreasing headspace pressure, decreasing protein concentration with reducing diluent volume, increasing high surface-area-to-height ratio of the cakes, increasing frequency of swirling and diluent temperature. Results: Among these strategies, reducing diluent volume to achieve high protein concentration and reducing headspace pressure show markedly reduction of reconstitution time. Moreover, we propose combined strategies to mitigate the reconstitution time, at the same time, to achieve same target dose in clinics. Conclusions: Therefore, this paper provides insights on the application of multiple strategies to accelerate the reconstitution of lyophilized drug products with high concentration, and facilitates their widespread clinical application.

Hypothermic Storage of Human Umbilical Vein Endothelial Cells and Their Hydrogel Constructs.

Human umbilical vein endothelial cells (HUVECs) have great potential in tissue engineering, regenerative medicine, and clinical applications. There is an ever-increasing demand to provide living HUVECs and HUVECs-hydrogel constructs to end users when needed in cell-based therapy and clinical applications. However, current methods to provide living cells and their constructs are mainly continuous culture and cryopreservation, which are high-cost, labor-intensive, time-consuming and inflexible. The research about hypothermic storage of HUVECs and their hydrogel constructs is still limited. Here, we studied the cell survival of HUVECs without encapsulation (W/O Encap) or with encapsulation (alginate, alginate with carboxymethyl chitosan [CMCH]) at 4°C and 25°C during 7 days, respectively. Also, we explored the optimal CMCH concentration for hypothermic storage, which is 0.5% (w/v) at 4°C and 25°C. Moreover, we evaluated the cell attachment after hypothermic storage. Our results enable the hypothermic storage of HUVECs and HUVEC-hydrogel constructs, and facilitate their application in tissue engineering and clinical medicine.

The Unusual Properties of Polytetrafluoroethylene Enable Massive-Volume Vitrification of Stem Cells with Low-Concentration Cryoprotectants.

Injectable stem cell-hydrogel constructs hold great potential for regenerative medicine and cell-based therapies. However, their clinical application is still challenging due to their short shelf-life at ambient temperature and the time-consuming fabrication procedure. Banking the constructs at cryogenic temperature may offer the possibility of "off-the-shelf" availability to end-users. However, ice formation during the cryopreservation process may compromise the construct quality and cell viability. Vitrification, cooling biological samples without apparent ice formation, has been explored to resolve the challenge. However, contemporary vitrification methods are limited to very small volume (up to ~0.25 ml) and/or need highly toxic and high concentration (up to ~8 M) of permeable cryoprotectants (pCPAs). Here, we show that polytetrafluoroethylene (PTFE, best known as Teflon for making non-stick cookware) capillary is flexible and unusually stable at a cryogenic temperature. By using the PTFE capillary as a flexible cryopreservation vessel together with alginate hydrogel microencapsulation and Fe3O4 nanoparticle-mediated nanowarming to suppress ice formation, massive-volume (10 ml) vitrification of cell-alginate hydrogel constructs with a low concentration (~2.5 M) of pCPA can be achieved. This may greatly facilitate the use of stem cell-based constructs for tissue regeneration and cell based therapies in the clinic.

DIS3L2 Promotes Progression of Hepatocellular Carcinoma via hnRNP U-Mediated Alternative Splicing.

DIS3-like 3'-5' exoribonuclease 2 (DIS3L2) degrades aberrant RNAs, however, its function in tumorigenesis remains largely unexplored. Here, aberrant DIS3L2 expression promoted human hepatocellular carcinoma (HCC) progression via heterogeneous nuclear ribonucleoproteins (hnRNP) U-mediated alternative splicing. DIS3L2 directly interacted with hnRNP U through its cold-shock domains and promoted inclusion of exon 3b during splicing of pre-Rac1 independent of its exonuclease activity, yielding an oncogenic splicing variant, Rac1b, which is known to stimulate cellular transformation and tumorigenesis. DIS3L2 regulated alternative splicing by recruiting hnRNP U to pre-Rac1. Rac1b was critical for DIS3L2 promotion of liver cancer development both in vitro and in vivo. Importantly, DIS3L2 and Rac1b expression highly correlated with HCC progression and patient survival. Taken together, our findings uncover an oncogenic role of DIS3L2, in which it promotes liver cancer progression through a previously unappreciated mechanism of regulating hnRNP U-mediated alterative splicing. SIGNIFICANCE: These findings establish the role and mechanism of the 3'-5' exoribonuclease DIS3L2 in hepatocellular carcinoma carcinogenesis.

Multifunctional Photo- and Magnetoresponsive Graphene Oxide-Fe3O4 Nanocomposite-Alginate Hydrogel Platform for Ice Recrystallization Inhibition.

Tuning ice recrystallization (IR) has attracted tremendous interest in fundamental research and a variety of practical applications, including food and pharmaceutical engineering, fabrication of anti-icing coating and porous materials, and cryopreservation of biological cells and tissues. Although great efforts have been devoted to modulation of IR for better microstructure control of various materials, it still remains a challenge, especially in cryopreservation, where insufficient suppression of IR during warming is fatal to the cells. Herein, we report an all-in-one platform, combining the external physical fields and the functional materials for both active and passive suppression of IR, where the photo- and magnetothermal dual-modal heating of GO-Fe3O4 nanocomposites (NCs) can be used to suppress IR with both enhanced global warming and microscale thermal disturbance. Moreover, the materials alginate hydrogels and GO-Fe3O4 NCs can act as IR inhibitors for further suppression of the IR effect. As a typical application, we show that this GO-Fe3O4 nanocomposite-alginate hydrogel platform can successfully enable low-cryoprotectant, high-quality vitrification of stem cell-laden hydrogels. We believe that the versatile ice recrystallization inhibition platform will have a profound influence on cryopreservation and tremendously facilitate stem cell-based medicine to meet its ever-increasing demand in clinical settings.

Vitrification of stem cell-laden core-shell microfibers with unusually low concentrations of cryoprotective agents.

Cell-laden alginate hydrogel microfibers are particularly useful for building and repairing complex tissues because they are long, thin, and flexible. Therefore, they have important application value in regenerative medicine and clinical treatments. Cryopreservation is indispensable in order to ensure their "off-the-shelf" ready availability. Ice-free vitrification is considered an ideal method to preserve stem cell constructs (from cells to the overall ultrastructure of hydrogel). However, the vitrification process for preserving cell constructs requires highly toxic and cell membrane permeable cryoprotective agents (pCPA) and even requires the assistance of complex physical field based space warming technology. Therefore, a simple and feasible method is urgently needed. In addition, there are no reports about microfiber vitrification, as reports are limited to microcapsules. In this study, a novel device with nylon mesh for vitreous cryopreservation of hydrogel microfibers is developed to achieve ultra-rapid heat transfer by effectively suppressing film boiling during cooling. This may provide a low-toxic and cost-effective method for vitrification of cell-laden hydrogel microfibers with ultra-low concentrations of pCPA, facilitating their application in regenerative medicine.

Magnetothermal heating facilitates the cryogenic recovery of stem cell-laden alginate-Fe3O4 nanocomposite hydrogels.

Constructs of magnetic nanocomposite hydrogels microencapsulated with stem cells are of great interest as smart materials for tissue engineering and regenerative medicine. Due to the short shelf life of such biocomposites at an ambient temperature, their long-term storage and banking at cryogenic temperatures are essential for the "off-the-shelf" availability of such biocomposites for widespread clinical applications. However, high-quality cryogenic recovery of stem cell-nanocomposite hydrogel constructs has not yet been achieved due to the damage to cells and/or microstructures of hydrogel constructs caused by ice formation, particularly during warming from cryogenic temperatures. Herein, stem cell-magnetic nanocomposite hydrogel constructs, which have an inherent magnetothermal property provided by embedded magnetic nanoparticles, are explored to achieve ultra-rapid cryogenic warming. The binding of water molecules by the hydrogel combined with the magnetothermal heating greatly suppressed ice formation during both cryogenic cooling and warming. Thus, the cryogenic recovery of nanocomposite hydrogel constructs with intact microstructures and fully functional stem cells from ultra-low temperatures was successfully achieved. We further demonstrated that magnetic nanocomposite hydrogels microencapsulated with stem cells could be conveniently manipulated for a self-assembled 3D culture. Together, we have developed a highly efficient and easy-to-perform approach for the cryogenic recovery of stem cell-encapsulated magnetic nanocomposite hydrogel constructs. Our results will facilitate the applications of such stem cell-magnetic nanocomposite hydrogels in regenerative medicine and tissue engineering.

Calculation of the virtual current in an electromagnetic flow meter with one bubble using 3D model.

Based on the theory of electromagnetic induction flow measurement, the Laplace equation in a complicated three-dimensional (3D) domain is solved by an alternating method. Virtual current potentials are obtained for an electromagnetic flow meter with one spherical bubble inside. The solutions are used to investigate the effects of bubble size and bubble position on the virtual current. Comparisons are done among the cases of 2D and 3D models, and of point electrode and large electrode. The results show that the 2D model overestimates the effect, while large electrodes are least sensitive to the bubble. This paper offers fundamentals for the study of the behavior of an electromagnetic flow meter in multiphase flow. For application, the results provide a possible way to estimate errors of the flow meter caused by multiphase flow.

Calculation and measurement of the magnetic field in a large diameter electromagnetic flow meter.

An idealized magnet model previously used in a simple coil configuration is applied to a more complicated engineering design problem, where an electromagnetic flow meter in diameter of 500 mm is used with five pairs of coils. The numerical results are compared with measurement data. The difference between theory and experiment and the possible causes of errors are discussed. It is shown that the idealized magnet model is practical for the design and the analysis of the flow meter. The work also shows that the traditional uniform field design based on two dimensional (2D) analysis is not suitable for this kind of flow meter. Thus nonuniform field and 3D analysis is needed.