A Model for Mechanical Stress Limited Bacterial Growth and Resporulation in Confinement.

In this study, we propose a self-limiting growth model forBacillus subtilisspores confined within porous polyacrylamide (PA) hydrogels. We observed thatB. subtilisspores germinate into vegetative cells within the hydrogel matrix, forming spherical colonies. These colonies expand until the mechanical stress they exert on their environment surpasses the yield stress of the hydrogel, leading to formation of a nonpermeable layer that halts nutrient diffusion and forces the bacteria to resporulate. These novel observations suggest a model to explain why bacterial growth in confined environments and material interfaces may be limited, providing insight for natural phenomena and biotechnological applications involving bacterial encapsulation.

Physicochemical Mechanisms of Protection Offered by Agarose Encapsulation during Cryopreservation of Mammalian Cells in the Absence of Membrane-Penetrating Cryoprotectants.

During freeze/thaw, cells are exposed to mechanical, thermal, chemical, and osmotic stresses, which cause loss of viability and function. Cryopreservation agents such as dimethyl sulfoxide (DMSO) are deployed to minimize freeze/thaw damage. However, there is a pressing need to eliminate DMSO from cryopreservation solutions due to its adverse effects. This is of the highest priority especially for cryopreservation of infusible/transplantable cell therapy products. In order to address this issue, we introduce reversible encapsulation in agarose hydrogels in the presence of the membrane-impermeable cryoprotectant, trehalose, as a viable, safe, and effective cryopreservation method. Our findings, which are supported by IR spectroscopy and differential scanning calorimetry analyses, demonstrate that encapsulation in 0.75% agarose hydrogels containing 10-20% trehalose inhibits mechanical damage induced by eutectic phase change, devitrification, and recrystallization, resulting in post-thaw viability comparable to the gold standard 10% DMSO.

Core-shell encapsulation formulations to stabilize desiccated Bradyrhizobium against high environmental temperature and humidity.

Engineered materials to improve the shelf-life of desiccated microbial strains are needed for cost-effective bioaugmentation strategies. High temperatures and humidity of legume-growing regions challenge long-term cell stabilization at the desiccated state. A thermostable xeroprotectant core and hydrophobic water vapour barrier shell encapsulation technique was developed to protect desiccated cells from the environment. A trehalose core matrix increased the stability of desiccated Bradyrhizobium by three orders of magnitude over 20 days at 32°C and 50% relative humidity (RH) compared to buffer alone; however, the improvement was not deemed sufficient for a shelf-stable bioproduct. We tested common additives (skim milk, albumin, gelatin and dextran) to increase the glass transition temperature of the desiccated product to provide further stabilization. Albumin increased the glass transition temperature of the trehalose-based core by 40°C and stabilized desiccated Bradyrhizobium for 4 months during storage at high temperature (32°C) and moderate humidity (50% RH) with only 1 log loss of viability. Although the albumin-trehalose core provided exceptional protection against high temperature, it was ineffective at higher humidity conditions (75%). We therefore incorporated a paraffin shell, which protected desiccated cells against 75% RH providing proof of concept that core and shell encapsulation is an effective strategy to stabilize desiccated cells.

Method to Isolate Dormant Cancer Cells from Heterogeneous Populations.

Cancer recurrence is responsible for a high percentage of cancer-related deaths. Primary tumor removal, chemotherapy, and radiotherapy often leave behind cancer cells that are clinically undetectable. Recent evidence has shown that subpopulations of these residual cancer cells enter into a prolonged dormant state, remaining quiescent for months to years, and eventually lead to metastases and relapse (Sosa et al. Nat Rev Cancer 14:611-622, 2014). Identifying the presence of and isolating these dormancy-capable cells (DCCs) from resected tumors or bodily fluids may therefore provide an opportunity to understand their biology and develop personalized treatments for patients at risk for relapse. Physical confinement in a stiff and porous 3D matrix, which inhibits proliferation, migration, and growth of the immobilized cells, has been shown to isolate DCC populations (Preciado et al. Technology 05:1-10, 2017; Reátegui et al. J Mater Chem B 2:7440-7448, 2014). Isolated DCCs can then be recovered from the gel and analyzed. Here we describe this immobilization method that can be used to isolate DCCs from heterogeneous cell populations that may also include dormancy-incapable cancer cells and host cells.

Engineering Bacillus subtilis for the formation of a durable living biocomposite material.

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.

Corrigendum: Signal Disruption Leads to Changes in Bacterial Community Population.

[This corrects the article DOI: 10.3389/fmicb.2019.00611.].

Induction of dormancy by confinement: An agarose-silica biomaterial for isolating and analyzing dormant cancer cells.

The principal cause of cancer deaths is the residual disease, which eventually results in metastases. Certain metastases are induced by disseminated dormancy-capable single cancer cells that can reside within the body undetected for months to years. Awakening of the dormant cells starts a cascade resulting in the patient's demise. Despite its established clinical significance, dormancy research and its clinical translation have been hindered by lack of in vitro models that can identify, isolate, and analyze dormancy-capable cells. We have previously shown that immobilization of cells in a stiff microenvironment induces dormancy in dormancy-capable cell lines. In this communication, we present a novel biomaterial and an in vitro immobilization method to isolate, analyze, and efficiently recover dormancy-capable cancer cells. MCF-7, MDA-MB-231, and MDA-MB-468 cells were individually coated with agarose using a microfluidic flow-focusing device. Coated cells were then immobilized in a rigid and porous silica gel. Dormancy induction by this process was validated by decreased Ki-67 expression, increased p38/ERK activity ratio, and reduced expression of CDK-2, cyclins D1, and E1. We showed that we can reliably and repeatedly induce dormancy in dormancy-capable MCF-7 cells and enhance the dormancy-capable sub-population in MDA-MB-231 cells. As expected, dormancy-resistant MDA-MB-468 cells did not survive immobilization. The dormant cells could be awakened on demand, by digesting the agarose gel in situ, and efficiently recovered by magnetically separating the silica gel, making the cells available for downstream analysis and testing. The awakened cells were shown to regain motility immediately, proliferating, and migrating normally.

Immobilization rapidly selects for chemoresistant ovarian cancer cells with enhanced ability to enter dormancy.

Around 20-30% of ovarian cancer patients exhibit chemoresistance, but there are currently no methods to predict whether a patient will respond to chemotherapy. Here, we discovered that chemoresistant ovarian cancer cells exhibit enhanced survival in a quiescent state upon experiencing the stress of physical confinement. When immobilized in stiff silica gels, most ovarian cancer cells die within days, but surviving cells exhibit hallmarks of single-cell dormancy. Upon extraction from gels, the cells resume proliferation but demonstrate enhanced viability upon reimmobilization, indicating that initial immobilization selects for cells with a higher propensity to enter dormancy. RNA-seq analysis of the extracted cells shows they have signaling responses similar to cells surviving cisplatin treatment, and in comparison to chemoresistant patient cohorts, they share differentially expressed genes that are associated with platinum-resistance pathways. Furthermore, these extracted cells demonstrate greater resistance to cisplatin and paclitaxel, despite being proliferative. In contrast, serum starvation and hypoxia could not effectively select for chemoresistant cells upon removal of the environmental stress. These findings demonstrate that ovarian cancer chemoresistance and the ability to enter dormancy are linked, and immobilization rapidly distinguishes chemoresistant cells. This platform could be suitable for mechanistic studies, drug development, or as a clinical diagnostic tool.

Dextranol: An inert xeroprotectant.

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like the polysaccharide dextran are critical for preserving dried protein samples by forming a rigid glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum as well as maintaining activity of biologic drug compounds. However, we found that dextran reacts with both dried serum proteins and lyophilized purified proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran's anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and matrix-metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextranol-based xeroprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when stored for a period of two weeks at 45°C. We also found that switching from dextran to dextranol in a lyophilization formulation eliminates this unwanted reaction, even at elevated temperatures. Dextranol offers a small and easy modification to dextran that significantly improves the molecule's function as a xeroprotectant by eliminating the potential for damaging protein-polysaccharide conjugation.

Stability of lyophilized albumin formulations: Role of excipient crystallinity and molecular mobility.

In freeze-dried protein formulations, the composition governs the physical forms of the excipients and hence their functionality. It is also necessary to understand the effect of composition on the molecular relaxation behavior, a key factor influencing protein stability. Mannitol (bulking agent) - trehalose (lyoprotectant) - bovine serum albumin (BSA) lyophiles with varying trehalose to BSA mass ratios were investigated. The crystalline phases were characterized by X-ray diffractometry. The secondary structure of albumin in lyophiles and reconstituted solutions was evaluated by IR spectroscopy and circular dichroism, respectively. Dielectric spectroscopy was used to obtain the relaxation time of freeze-dried samples. When trehalose to BSA ratio was 0.2, while mannitol crystallized predominantly as the δ-anhydrous polymorph, trehalose remained amorphous. At lower concentrations of BSA, mannitol crystallized in both hemihydrate and anhydrous forms, and trehalose as dihydrate. The extent of dehydration during subsequent drying was dictated by the trehalose to BSA ratio in the formulation. A gradual increase in the Johari-Goldstein relaxation time was observed as the concentration of trehalose increased in the formulation. BSA was more susceptible to stresses from thawing than drying.