Haishui Huang

The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.

Coaxial electrospray of liquid core-hydrogel shell microcapsules for encapsulation and miniaturized 3D culture of pluripotent stem cells

A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on the 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic.

Nanoparticle-mediated intracellular delivery enables cryopreservation of human adipose-derived stem cells using trehalose as the sole cryoprotectant.

In this study, pH responsive genipin-cross-linked Pluronic F127-chitosan nanoparticles (GNPs) was synthesized to encapsulate trehalose for intracellular delivery to cryopreserve primary human adipose-derived stem cells (hADSCs). Trehalose is a disaccharide of glucose used by lower organisms to survive extreme cold in nature and has been used to cryopreserve various biomacromolecules. However, it does not enter mammalian cells because of its highly hydrophilic nature, and has only been used in combination with other cell-penetrating cryoprotectants (such as dimethyl sulfoxide, DMSO) to cryopreserve mammalian cells. Our data show that trehalose can be efficiently encapsulated in our GNPs for intracellular delivery, which enables cryopreservation of primary hADSCs using the nontoxic sugar as the sole cryoprotectant. This capability is important because the conventional approach of cryopreserving mammalian cells using highly toxic (at body temperature) cell-penetrating cryoprotectants requires multistep washing of the cryopreserved cells to remove the toxic cryoprotectant for further use, which is time-consuming and associated with significant cell loss (∼10% during each washing step). By contrast, the trehalose-cryopreserved cells can be used without washing, which should greatly facilitate the wide application of the burgeoning cell-based medicine.

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary ☆

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

Stiffness-Independent Highly Efficient On-Chip Extraction of Cell-Laden Hydrogel Microcapsules from Oil Emulsion into Aqueous Solution by Dielectrophoresis

A dielectrophoresis (DEP)-based method achieves highly efficient on-chip extraction of cell-laden microcapsules of any stiffness from oil into aqueous solution. The hydrogel microcapsules can be extracted into the aqueous solution by DEP and interfacial tension forces with no trapped oil, while the encapsulated cells are free from electrical damage due to the Faraday cage effect.

The crucial role of zona pellucida in cryopreservation of oocytes by vitrification

Mammalian oocytes have a proteinaceous hydrogel-like outer shell known as the zona pellucida (ZP) that semi-encloses their plasma membrane and cytoplasm. In this study, we cryopreserved mouse oocytes either with or without ZP by vitrification. Our results show that the presence of an intact ZP could significantly improve the post-vitrification survival of oocytes to 92.1% from 13.3% for oocytes without ZP. Moreover, there was no significant difference in embryonic development between fresh and cryopreserved oocytes with ZP after in vitro fertilization (IVF). Further atomic force microscopy (AFM) analysis showed that the intact oocytes with ZP have an elastic modulus that is more than 85 times higher than that of oocytes without ZP. This may partially explain the important role of ZP in protecting the oocytes by resisting the mechanical stress due to possible ice formation during cryopreservation by vitrification. Collectively, this study reveals a new biophysical role of ZP during vitrification of oocytes and suggests microencapsulation of the many mammalian cells without a ZP in ZP-like hydrogel is an effective strategy to improve their survival post cryopreservation by vitrification.

Predehydration and Ice Seeding in the Presence of Trehalose Enable Cell Cryopreservation

Conventional approaches for cell cryopreservation require the use of toxic membrane-penetrating cryoprotective agents (pCPA), which limits the clinical application of cryopreserved cells. Here, we show intentionally induced ice formation at a high subzero temperature (> -10 °C) during cryopreservation, which is often referred to as ice seeding, could result in significant cell injury in the absence of any pCPA. This issue can be mitigated by predehydrating cells using extracellular trehalose to their minimal volume with minimized osmotically active water before ice seeding. We further observe that ice seeding can minimize the interfacial free energy that drives the devastating ice recrystallization-induced cell injury during warming cryopreserved samples. Indeed, by combining predehydration using extracellular trehalose with ice seeding at high subzero temperatures, high cell viability or recovery is achieved for fibroblasts, adult stem cells, and red blood cells after cryopreservation without using any pCPA. The pCPA-free technology developed in this study may greatly facilitate the long-term storage and ready availability of living cells, tissues, and organs that are of high demand by modern cell-based medicine.

Long-term deep-supercooling of large-volume water and red cell suspensions via surface sealing with immiscible liquids

Supercooling of aqueous solutions is a fundamentally and practically important physical phenomenon with numerous applications in biopreservation and beyond. Under normal conditions, heterogeneous nucleation mechanisms critically prohibit the simultaneous long-term (> 1 week), large volume (> 1 ml), and low temperatures (< −10 °C) supercooling of aqueous solutions. Here, we report on the use of surface sealing of water by an oil phase to significantly diminish the primary heterogeneous nucleation at the water/air interface. We achieve deep supercooling (down to −20 °C) of large volumes of water (up to 100 ml) for long periods (up to 100 days) simultaneously via this approach. Since oils are mixtures of various hydrocarbons we also report on the use of pure alkanes and primary alcohols of various lengths to achieve the same. Furthermore, we demonstrate the utility of deep supercooling via preliminary studies on extended (100 days) preservation of human red blood cells.Supercooled water is susceptible to spontaneous freezing, and preventing this process is a challenge. Here, the authors use surface sealing with immiscible liquids to eliminate primary ice nucleation at the water/air interface, enabling deep supercooling of large volumes of water and red cell suspensions for long time periods.