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.

Prediction of Ligand Transport along Hydrophobic Enzyme Nanochannels.

Buried active sites of enzymes are connected to the bulk solvent through a network of hydrophobic channels. We developed a discretized model that can accurately predict ligand transport along hydrophobic channels up to six orders of magnitude faster than any other existing method. The non-dimensional nature of the model makes it applicable to any hydrophobic channel/ligand combination.

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.

Role of Water Hydrogen Bonding on Transport of Small Molecules inside Hydrophobic Channels.

We conducted a systematic analysis of water networking inside smooth hyperboloid hydrophobic structures (cylindrical, barrel, and hourglass shapes) to elucidate the role of water hydrogen bonding on the transport of small hydrophobic molecules (ligands). Through a series of molecular dynamics simulations, we established that a hydrogen-bonded network forming along the centerline resulted in a water exclusion zone adjacent to the walls. The size of the exclusion zone is a function of the geometry and the nonbonded interaction strength, defining the effective hydrophobicity of the structure. Exclusion of water molecules from this zone results in lower apparent viscosity, leading to acceleration of ligand transport up to 7 times faster than that measured in the bulk. Transport of ligands into and out of the hydrophobic structures was shown to be controlled by a single water molecule that capped the narrow regions in the structure. This mechanism provides physical insights into the behavior and role of water in the bottleneck regions of hydrophobic enzyme channels. These findings were then used in a sister publication [ Escalante , D. E. , Comput. Struct. Biotechnol. J. 2019 17 757 760 ] to develop a model that can accurately predict the transport of ligands along nanochannels of broad-substrate specificity enzymes.

Signal Disruption Leads to Changes in Bacterial Community Population.

The disruption of bacterial signaling (quorum quenching) has been proven to be an innovative approach to influence the behavior of bacteria. In particular, lactonase enzymes that are capable of hydrolyzing the N-acyl homoserine lactone (AHL) molecules used by numerous bacteria, were reported to inhibit biofilm formation, including those of freshwater microbial communities. However, insights and tools are currently lacking to characterize, understand and explain the effects of signal disruption on complex microbial communities. Here, we produced silica capsules containing an engineered lactonase that exhibits quorum quenching activity. Capsules were used to design a filtration cartridge to selectively degrade AHLs from a recirculating bioreactor. The growth of a complex microbial community in the bioreactor, in the presence or absence of lactonase, was monitored over a 3-week period. Dynamic population analysis revealed that signal disruption using a quorum quenching lactonase can effectively reduce biofilm formation in the recirculating bioreactor system and that biofilm inhibition is concomitant to drastic changes in the composition, diversity and abundance of soil bacterial communities within these biofilms. Effects of the quorum quenching lactonase on the suspension community also affected the microbial composition, suggesting that effects of signal disruption are not limited to biofilm populations. This unexpected finding is evidence for the importance of signaling in the competition between bacteria within communities. This study provides foundational tools and data for the investigation of the importance of AHL-based signaling in the context of complex microbial communities.

Enhancement of biocatalyst activity and protection against stressors using a microbial exoskeleton.

Whole cell biocatalysts can perform numerous industrially-relevant chemical reactions. While they are less expensive than purified enzymes, whole cells suffer from inherent reaction rate limitations due to transport resistance imposed by the cell membrane. Furthermore, it is desirable to immobilize the biocatalysts to enable ease of separation from the reaction mixture. In this study, we used a layer-by-layer (LbL) self-assembly process to create a microbial exoskeleton which, simultaneously immobilized, protected, and enhanced the reactivity of a whole cell biocatalyst. As a proof of concept, we used Escherichia coli expressing homoprotocatechuate 2,3-dioxygenase (HPCD) as a model biocatalyst and coated it with up to ten alternating layers of poly(diallyldimethylammonium chloride) (PDADMAC) and silica. The microbial exoskeleton also protected the biocatalyst against a variety of external stressors including: desiccation, freeze/thaw, exposure to high temperatures, osmotic shock, as well as against enzymatic attack by lysozyme, and predation by protozoa. While we observed increased permeability of the outer membrane after exoskeleton deposition, this had a moderate effect on the reaction rate (up to two-fold enhancement). When the exoskeleton construction was followed by detergent treatment to permeabilize the cytoplasmic membrane, up to 15-fold enhancement in the reaction rate was reached. With the exoskeleton, we increased in the reaction rate constants as much as 21-fold by running the biocatalyst at elevated temperatures ranging from 40 °C to 60 °C, a supraphysiologic temperature range not accessible by unprotected bacteria.

FTIR Analysis of Molecular Changes Associated with Warming Injury in Cryopreserved Leukocytes.

In this article, we explored the effects of cooling rate, dimethyl sulfoxide (DMSO) concentration, and thawing protocol on the post-thaw viability of frozen human white blood cells (WBCs). Different cooling rates (1, 2, 5, 10, 20, and 50 °C/min) at two DMSO concentrations (5 and 10% v/v) were tested as the samples were cooled to -120 °C. Frozen samples were thawed following either a fast (100 °C/min) or slow (2 °C/min) warming protocol applied in either a single stage or in two stages interrupted by a 6 min hold at -40, -50, -60, -70, or -80 °C. The highest post-thaw viability was obtained when WBCs were cooled at 2 °C/min in a 5% DMSO solution and warmed at the fastest rate (100 °C/min) without any interruption. Post-thaw viability decreased when the warming rate was reduced or when rapid warming was interrupted by a hold at a temperature below -60 °C. To elucidate the mechanisms of warming injury in addition to the biological response, several key interfacial and molecular phenomena require greater understanding; thus, we used Fourier transform infrared (FTIR) spectroscopy to investigate the roles of molecular structure and conformation in damage to cryopreserved WBCs during warming. During warming, FTIR spectra revealed the accumulation of cellular protein and lipid membrane damage below -60 °C if the samples were thawed slowly at 2 °C/min. The results presented here suggest that irreversible alterations of biomolecular structure are correlated with cell injury during warming; these deleterious effects appeared to be caused by one or more low-temperature kinetic processes, consistent with eutectic formation/melting and/or devitrification in the intracellular milieu.

Freeze/thaw of IGG solutions.

In this communication, the effect of mannitol and trehalose crystallization on the unfolding of IgG1, a monoclonal antibody, in the frozen state with repeat freeze/thaw under different pH conditions was explored. Formulations were annealed at -20 °C for 20 h five times (interrupted by freeze/thaw). This was done to induce excipient crystallization. Characterization of the frozen-thawed samples was performed by circular dichroism, particle analysis, and size exclusion chromatography. At a pH of 3, formation of insoluble and soluble aggregates was observed however, these could be reduced by the use of a surfactant. Cryoprotectant free formulations showed higher monomer content after freeze/thaw. At pH5, a single freeze/thaw cycle did not result in a significant increase in particle numbers. At pH range of 4-7 however, aggregate formation in the size range of 1-25 µm was observed after 5freeze/thaw cycles.

Enhanced biodegradation of atrazine by bacteria encapsulated in organically modified silica gels.

Biodegradation by cells encapsulated in silica gel is an economical and environmentally friendly method for the removal of toxic chemicals from the environment. In this work, recombinant E. coli expressing atrazine chlorohydrolase (AtzA) were encapsulated in organically modified silica (ORMOSIL) gels composed of TEOS, silica nanoparticles (SNPs), and either phenyltriethoxysilane (PTES) or methyltriethoxysilane (MTES). ORMOSIL gels adsorbed much higher amounts of atrazine than the hydrophilic TEOS gels. The highest amount of atrazine adsorbed by ORMOSIL gels was 48.91×10-3µmol/mlgel, compared to 8.71×10-3µmol/mlgel by the hydrophilic TEOS gels. Atrazine biodegradation rates were also higher in ORMOSIL gels than the TEOS gels, mainly due to co-localization of the hydrophobic substrate at high concentrations in close proximity of the encapsulated bacteria. A direct correlation between atrazine adsorption and biodegradation was observed unless biodegradation decreased due to severe phase separation. The optimized PTES and MTES gels had atrazine biodegradation rates of 0.041±0.003 and 0.047±0.004µmol/mlgel, respectively. These rates were approximately 80% higher than that measured in the TEOS gel. This study showed for the first time that optimized hydrophobic gel material design can be used to enhance both removal and biodegradation of hydrophobic chemicals.

Adsorption and Biodegradation of Aromatic Chemicals by Bacteria Encapsulated in a Hydrophobic Silica Gel.

An adsorbent silica biogel material was developed via silica gel encapsulation of Pseudomonas sp. NCIB 9816-4, a bacterium that degrades a broad spectrum of aromatic pollutants. The adsorbent matrix was synthesized using silica precursors methyltrimethoxysilane and tetramethoxysilane to maximize the adsorption capacity of the matrix while maintaining a highly networked and porous microstructure. The encapsulated bacteria enhanced the removal rate and capacity of the matrix for an aromatic chemical mixture. Repeated use of the material over four cycles was conducted to demonstrate that the removal capacity could be maintained with combined adsorption and biodegradation. The silica biogel can thus be used extensively without the need for disposal, as a result of continuous biodegradation by the encapsulated bacteria. However, an inverse trend was observed with the ratio of biodegradation to adsorption as a function of log Kow, suggesting increasing mass-transport limitation for the most hydrophobic chemicals used (log Kow > 4).

Simulation of the Bottleneck Controlling Access into a Rieske Active Site: Predicting Substrates of Naphthalene 1,2-Dioxygenase.

Naphthalene 1,2-dioxygenase (NDO) has been computationally understudied despite the extensive experimental knowledge obtained for this enzyme, including numerous crystal structures and over 100 demonstrated substrates. In this study, we have developed a substrate prediction model that moves away from the traditional active-site-centric approach to include the energetics of substrate entry into the active site. By comparison with experimental data, the accuracy of the model for predicting substrate oxidation is 92%, with a positive predictive value of 93% and a negative predictive value of 98%. Also, the present analysis has revealed that the amino acid residues that provided the largest energetic barrier for compounds entering the active site are residues F224, L227, P234, and L235. In addition, F224 is proposed to play a role in controlling ligand entrance via π-π stacking stabilization as well as providing stabilization via T-shaped π-π interactions once the ligand has reached the active-site cavity. Overall, we present a method capable of being scaled to computationally discover thousands of substrates of NDO, and we present parameters to be used for expanding the prediction method to other members of the Rieske non-heme iron oxygenase family.

In Silico Identification of Bioremediation Potential: Carbamazepine and Other Recalcitrant Personal Care Products.

Emerging contaminants are principally personal care products not readily removed by conventional wastewater treatment and, with an increasing reliance on water recycling, become disseminated in drinking water supplies. Carbamazepine, a widely used neuroactive pharmaceutical, increasingly escapes wastewater treatment and is found in potable water. In this study, a mechanism is proposed by which carbamazepine resists biodegradation, and a previously unknown microbial biodegradation was predicted computationally. The prediction identified biphenyl dioxygenase from Paraburkholderia xenovorans LB400 as the best candidate enzyme for metabolizing carbamazepine. The rate of degradation described here is 40 times greater than the best reported rates. The metabolites cis-10,11-dihydroxy-10,11-dihydrocarbamazepine and cis-2,3-dihydroxy-2,3-dihydrocarbamazepine were demonstrated with the native organism and a recombinant host. The metabolites are considered nonharmful and mitigate the generation of carcinogenic acridine products known to form when advanced oxidation methods are used in water treatment. Other recalcitrant personal care products were subjected to prediction by the Pathway Prediction System and tested experimentally with P. xenovorans LB400. It was shown to biodegrade structurally diverse compounds. Predictions indicated hydrolase or oxygenase enzymes catalyzed the initial reactions. This study highlights the potential for using the growing body of enzyme-structural and genomic information with computational methods to rapidly identify enzymes and microorganisms that biodegrade emerging contaminants.

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability.

PURPOSE: The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported. METHODS: The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively. RESULTS: Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D2O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing. CONCLUSIONS: The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.