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Directed neural differentiation of embryonic stem cells (ESCs) has been studied extensively to improve the treatment of neurodegenerative disorders. This can be done through stromal-cell derived inducing activity (SDIA), by culturing ESCs directly on top of a layer of feeder stromal cells. However, the stem cells usually become mixed with the feeder cells during the differentiation process, making it difficult to obtain a pure population of the differentiated cells for further use. To address this issue, a non-planar microfluidic device is used here to encapsulate murine ESCs (mESCs) in the 3D liquid core of microcapsules with an alginate hydrogel shell of different sizes for early neural differentiation through SDIA, by culturing mESC-laden microcapsules over a feeder layer of PA6 cells. Furthermore, the alginate hydrogel shell of the microcapsules is modified via oxidation or RGD peptide conjugation to examine the mechanical and chemical effects on neural differentiation of the encapsulated mESC aggregates. A higher expression of Nestin is observed in the aggregates encapsulated in small (∼300 μm) microcapsules and cultured over the PA6 cell feeder layer. Furthermore, the modification of the alginate with RGD facilitates early neurite extension within the microcapsules. This study demonstrates that the presence of the RGD peptide, the SDIA effect of the PA6 cells, and the absence of leukemia inhibition factor from the medium can lead to the early differentiation of mESCs with extensive neurites within the 3D microenvironment of the small microcapsules. This is the first study to investigate the effects of cell adhesion and degradation of the encapsulation materials for directed neural differentiation of mESCs. The simple modifications (i.e., oxidation and RGD incorporation) of the miniaturized 3D environment for improved early neural differentiation of mESCs may potentially enhance further downstream differentiation of the mESCs into more specialized neurons for therapeutic use and drug screening.

Astrocyte is the most abundant cells in brain and plays critical roles in brain homeostasis and functions. Although hyperthermia (or fever) is a common symptom in patients, its influence on astrocyte viability, morphology, and functions remains elusive. Here we developed an in vitro astrocyte culture system capable of precisely controlling culture temperature to study astrocyte responses under clinically-relevant hyperthermic temperatures (38 ∼ 41°C). We found that hyperthermia in this temperature range does not alter cell morphology, but significantly affects cell viability, activation and functions. Specifically, high-hyperthermia (40°C and 41°C) causes irreversible and permanent damages to astrocytes and compromises their normal viability and functionalities repairing damaged neural tissue, recycling neurotransmitters, and promoting brain development, while mild-hyperthermia (38°C and 39°C) induces astrocyte activation and cytokine secretion without significant decreases in cell viability. This study sheds new insights into our understanding of various fever-associated symptoms, enabling the future development of astrocyte-targeted therapy to treat brain diseases via hyperthermia.

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.

Hydrogel microcapsules provide miniaturized and biocompatible niches for three-dimensional (3D) in vitro cell culture. They can be easily generated by droplet-based microfluidics with tunable size, morphology, and biochemical properties. Therefore, microfluidic generation and manipulation of cell-laden microcapsules can be used for 3D cell culture to mimic the in vivo environment towards applications in tissue engineering and high throughput drug screening. In this review of recent advances mainly since 2010, we will first introduce general characteristics of droplet-based microfluidic devices for cell encapsulation with an emphasis on the fluid dynamics of droplet breakup and internal mixing as they directly influence microcapsule’s size and structure. We will then discuss two on-chip manipulation strategies: sorting and extraction from oil into aqueous phase, which can be integrated into droplet-based microfluidics and significantly improve the qualities of cell-laden hydrogel microcapsules. Finally, we will review various applications of hydrogel microencapsulation for 3D in vitro culture on cell growth and proliferation, stem cell differentiation, tissue development, and co-culture of different types of cells.

The three classical core technologies for the preservation of live mammalian biospecimens—slow freezing, vitrification and hypothermic storage—limit the biomedical applications of biospecimens. In this Review, we summarize the principles and procedures of these three technologies, highlight how their limitations are being addressed via the combination of microfabrication and nanofabrication, materials science and thermal-fluid engineering and discuss the remaining challenges. This Review examines classical methods for the preservation of mammalian biospecimens and the associated mechanisms of cryoinjury and discusses how the methods’ applicability limitations are being addressed.

Cell preservation is an enabling technology for widespread distribution and applications of mammalian cells. Traditional cryopreservation via slow-freezing or vitrification provides long-term storage but requires cytotoxic cryoprotectants (CPA) and tedious CPA loading/unloading, cooling, and recovering procedures. Hypothermic storage around 0–4 °C is an alternative method but only works for a short period due to its high storage temperatures. Here, we report on the deep-supercooling (DSC) preservation of human adipose-derived stem cells at deep subzero temperatures without freezing for extended storage. Enabled by surface sealing with an immiscible oil phase, cell suspension can be preserved in a liquid state at −13 °C and −16 °C for 7 days with high cell viability, retention of stemness, attachment, and multilineage differentiation capacities. These results demonstrate that DSC is an improved short-term preservation approach to provide off-the-shelf cell sources for booming cell-based medicine and bioengineering.

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 (, 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.

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.

This article describes a biomimetic core-shell platform with a collagen-based core and an alginate hydrogel shell for cell and tissue culture. With this system, chemical and physical properties of extracellular matrix (ECM) in the core microenvironment can be controlled to regulate proliferation and development of cells/tissues under miniaturized three-dimensional (3D) culture.

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.

Microdroplets and microcapsules have been widely produced using microfluidic flow-focusing junctions for biomedical and chemical applications. However, the multiphase microfluidic flow at the flow-focusing junction has not been well investigated. In this study, the displacement of two (core and shell) aqueous fluids that disperse into droplets altogether in a carrier oil emulsion was investigated both numerically and experimentally. It was found that extensive displacement of the two aqueous fluids within the droplet during its formation could occur as a result of the shear effect of the carrier fluid and the capillary effect of interfacial tension. We further identified that the two mechanisms of fluid displacement can be evaluated by two dimensionless parameters. The quantitative relationship between the degree of fluid displacement and these two dimensionless parameters was determined experimentally. Finally, we demonstrated that the degree of fluid displacement could be controlled to generate hydrogel microparticles of different morphologies using planar or nonplanar flow-focusing junctions. These findings should provide useful guidance to the microfluidic production of microscale droplets or capsules for various biomedical and chemical applications.

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.

Advancements in tissue engineering require the development of new technologies to study cell behavior in vitro. This study focuses on stem cell behavior within various miniaturized three-dimensional (3D) culture conditions of alginate biomaterials modified with the Arg-Gly-Asp (RGD) peptide known for its role in cell adhesion/attachment. Human embryonic palatal mesenchyme (HEPM) cells, bone marrow derived mesenchymal stem cells (MSCs), and human adipose derived stem cells (ADSCs) were cultured on a flat hydrogel of different concentrations of alginate-RGD, and in the miniaturized 3D core of microcapsules with either a 2% alginate or 2% alginate-RGD shell. The core was made of 0%, 0.5%, or 2% alginate-RGD. Cell spreading was observed in all systems containing the RGD peptide, and the cell morphology was quantified by measuring the cell surface area and circularity. In all types of stem cells, there was a significant increase in the cell surface area (p < 0.05) and a significant decrease in cell circularity (p < 0.01) in alginate-RGD conditions, indicating that cells spread much more readily in environments containing the peptide. This control over the cell spreading within a 3D microenvironment can help to create the ideal biomimetic condition in which to conduct further studies on cell behavior.

Regenerative therapies require availability of an abundant healthy cell source which can be achieved by efficient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly efficient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64-modified mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell specific functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9-VP64 pretreated cells ([Formula: see text]) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively affect the post-thaw viability. Co-delivering chemically modified dCas9-VP64 mRNA with gRNAs is an efficient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet effective way to accelerate a wide array of cellular research and translational medical applications.

Cryopreservation of stem cells is important to meet their ever-increasing demand by the burgeoning cell-based medicine. The conventional slow freezing for stem cell cryopreservation suffers from inevitable cell injury associated with ice formation and the vitrification (i.e., no visible ice formation) approach is emerging as a new strategy for cell cryopreservation. A major challenge to cell vitrification is intracellular ice formation (IIF, a lethal event to cells) induced by devitrification (i.e., formation of visible ice in previously vitrified solution) during warming the vitrified cells at cryogenic temperature back to super-zero temperatures. Consequently, high and toxic concentrations of penetrating cryoprotectants (i.e., high CPAs, up to ~8 M) and/or limited sample volumes (up to ~2.5 μl) have been used to minimize IIF during vitrification. We reveal that alginate hydrogel microencapsulation can effectively inhibit devitrification during warming. Our data show that if ice formation were minimized during cooling, IIF is negligible in alginate hydrogel-microencapsulated cells during the entire cooling and warming procedure of vitrification. This enables vitrification of pluripotent and multipotent stem cells with up to ~4 times lower concentration of penetrating CPAs (up to 2 M, low CPA) in up to ~100 times larger sample volume (up to ~250 μl, large volume).

A dielectrophoresis (DEP)-based method is reported to achieve 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 (IFT) forces with no trapped oil while the encapsulated cells are free from the electrical damages due to the Faraday cage effect.

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.

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.

Microfluidics involving two immiscible fluids (oil and water) has been increasingly used to produce hydrogel microparticles with wide applications. However, it is difficult to extract the microparticles out of the microfluidic Stokes flows of oil that have a Reynolds number (the ratio of inertia to viscous force) much less than one, where the dominant viscous force tends to drive the microparticles to move together with the surrounding oil. Here, we present a passive method for extracting hydrogel microparticles in microfluidic Stokes flow from oil into aqueous extracting solution on-chip by utilizing the intrinsic interfacial tension between oil and the microparticles. We further reveal that the thickness of an “extended confining layer” of oil next to the interface between oil and aqueous extracting solution must be smaller than the radius of microparticles for effective extraction. This method uses a simple planar merging microchannel design that can be readily fabricated and further integrated into a fluidic system to extract microparticles for wide applications.