Name: microbiologically induced calcite precipitation mediated by sporosarcina pasteurii
Uploaded: 2016-04-16T14:00:00
Description: Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

We wish to acknowledge the partners in the Helmholtz-Alberta Initiative, the Helmholtz Association and the University of Alberta, for the support resulting from participation in this collaboration. Research funding is provided by the Helmholtz Association's Initiative and Networking Fund, the participating Helmholtz Centers and by the Government of Alberta through Alberta Environment's ecoTrust program.
Dr. Tanushree Ghosh is gratefully acknowledged for her critical inputs at a number of crucial stages.

NOTE: Perform the experimental protocols in the order described below. The bacterial culture protocol is discussed in Section 1 (Also see Figure 1). Section 2 describes the protocol for enriching the culture medium using external additives. Section 3 describes the protocols for multi-mode microscopy. Weights of all the individual components may be measured using an analytical balance. Volume of each solution may be measured using a volumetric cylinder.
NOTE: Proper biosafety p...

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Critical Steps: This manuscript describes in detail the protocols for culturing a viable sample of S. pasteurii. Once the culture has been readied, it must be suitably enriched. This is a key step vital to the success of the experiment because a failure to provide the proper chemical environment leads to either very long time scales of precipitation or a complete lack thereof. S. pasteurii is quite sensitive to several external agencies and must be cultured with a high degree of care an...

Sporosarcina pasteurii is a gram-positive bacterium able to survive in highly alkaline environments (pH~10)1 and is one of the bacterial species that can become a causative agent of a phenomenon called Microbiologically Induced Calcite Precipitation (MICP)2-4. MICP is a process wherein precipitation of calcium carbonate is induced by certain microbes under suitable environmental conditions. S. pasteurii has assumed importance in recent years due to its identification as a possible agent for inducing significant volumes of MICP under certain conditions. This possibility stems from the fact that S. pasteurii has the unique ability to secrete copious amounts of the enzyme urease. This enzyme acts as a catalyst, promoting an accelerated lysis of urea (a naturally occurring biochemical compound with widespread and abundant supply) in the presence of water molecules. Through a cascade of reactions, this process ultimately leads to the generation of negatively charged carbonate ions. These ions, in turn, react with positive metal ions like calcium to finally form precipitates of calcium carbonate (calcite); hence the label MICP5-9.
The process of MICP has been known and studied for several decades10,11. Over the past few years, MICP has been investigated for a wide range of engineering and environmental applications including bottom-up green construction12, enhancement of large-scale structures13,14 and carbon sequestration and storage15,16.
For example, Cunnigham17 et. al designed a high pressure moderate temperature flow reactor containing a Berea sandstone core. The reactor was inoculated with the bacteria S. fridgidimarina and under conditions of high-pressure supercritical carbon dioxide injection, a massive accumulation of biomass inside the pore volumes was observed, which led to more than 95% reduction in permeability. Jonkers and Schlangen18 studied the effect of certain special strains of bacteria on the self-healing process in concrete. External water transported into the pore network entering through the surface pores is expected to activate the dormant bacteria which in turn help structural strength via MICP. Tobler19 et al. have compared the ureolytic activity of S. pasteurii with an indigenous groundwater ureolytic microcosm under conditions favoring large-scale MCIP and found that S. pasteurii has a consistent capability to improve calcite precipitation even when the indigenous communities lacked prior urease activity. Mortensen20 et.al have studied the effects of external factors like soil type, concentration of ammonium chloride, salinity, oxygen concentration and lysis of cells on MICP. Their demonstration that the biological treatment process is very robust with respect to a wide variation in parameter space substantiates the fitness of this process for various large-scale remediation applications provided a proper enrichment process to reinforce the bacteria is undertaken. Phillips21 et. al designed experiments to study the changes in permeability and strength of a sand column and a sandstone core after being injected with S. pasteurii cultures. They found that while the permeability decreased 2 - 4 times while the fracture strength increased three times.
S. pasteurii and its role in MICP are topics of active research and several issues relating to the mechanism of chemical precipitation are still not fully understood. In light of this, it is very important to have a set of consistent standardized protocols to accurately culture a suitably enriched stock of S. pasteurii to achieve MICP. Here, we outline a rigorous protocol that will ensure repeatability and reproducibility. This manuscript describes the detailed protocols for culturing S. pasteurii and suitably enriching the culture medium to induce precipitation. The process is investigated through various microscopic techniques such as optical and Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS). The focus of the manuscript is on the process of MICP. Procedures like SEM and SIMS, being well-established standard protocols, are not described separately.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Petridish</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:255px;">Petridishes being used as Agar plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:224px;">Pyrex Flasks</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S63268</td>
			<td style="width:255px;">Corning Erlenmeyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tris-Base</td>
			<td style="width:108px;">Promega</td>
			<td style="width:119px;">H5133</td>
			<td style="width:255px;">being used to make Tris-Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Hydrochloric Acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9892</td>
			<td style="width:255px;">1.0 N, Bioreagent, suitable for cell culture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Agar Powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td style="width:255px;">microbiology tested, plant cell culture tested, cell culture tested, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulphate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4418</td>
			<td style="width:255px;">for Molecular Biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Yeast extract powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">51475</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Measuring Cylinder</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">CP08559GC</td>
			<td style="width:255px;">Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC;1000ml,1/Pk</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Analytical Balance</td>
			<td style="width:108px;">OHAUS</td>
			<td style="width:119px;">AX124E</td>
			<td style="width:255px;">being used to measure weight of reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave</td>
			<td style="width:108px;">Brinkmann</td>
			<td style="width:119px;">58619000</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave Tape</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">52428864</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Aluminum Foil</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z185140</td>
			<td style="width:255px;">being used to seal the flask before placing it in Autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Bacterial Stock</td>
			<td style="width:108px;">Cedarlane</td>
			<td style="width:119px;">11859</td>
			<td style="width:255px;">-80&#176;C stock of S. pasteurii, ATCC No. is mentioned against Cat. No.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">Mline Single-Channel Mechanical Pipettors, Variable Volume</td>
			<td style="width:108px;">Biohit</td>
			<td style="width:119px;">725010</td>
			<td style="width:255px;">Marketed by VWR under catalog number 14005976</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Micropipette Tip</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">212772B</td>
			<td style="width:255px;">Used for scratching Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Incubator</td>
			<td style="width:108px;">Binder</td>
			<td style="width:119px;">80079098</td>
			<td style="width:255px;">Microbiology Incubator,BF Series</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Shaking Incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">14004300</td>
			<td style="width:255px;">VWR Signature Benchtop Shaking Incubators</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS)&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">BD Falcon Express Pipet-Aid Pipetting Device</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">357590</td>
			<td style="width:255px;">Marketed by VWR under catalog number 53106220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Parafilm</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793</td>
			<td style="width:255px;">Being used to seal Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Urea</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">U1250</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Sodium Bicarbonate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8875</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Calcium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
	</tbody>
</table>

microbiologically induced calcite precipitation mediated by sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

S. pasteurii being an alkaliphile24 can survive relatively harsh conditions. When the above mentioned culture protocol is followed, and S. pasteurii is grown inside a chamber, the bacteria leads to the precipitation of calcium carbonate over time (Figure 2A). Figure 2 (b) shows a phase-contrast optical microscopic image of the bacterial cell population within the culture medium. Individual cells can be clearly distinguished, w...

Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

The overall goal of this protocol is to induce calcite precipitation in a stagnant culture of Sporosarcina pasteurii. This method can help us answer key biophysical questions about Sporosarcina pasteurii-mediated calcite precipitation, like the pertinent length and time scales. The main advantage of this technique is the fact that it standardizes the complex steps involved in suitably culturing and enriching Sporosarcina pasteurii that ensures reliable calcite precipitation.To begin, assemble Petri dishes, flasks, Tris base, HCl, agar, Millipore water, and a pH meter. Sterilize all containers by autoclaving at 121 degrees Celsius before use. Prepare one liter of a 0.13 molar aqueous solution of Tris buffer by mixing 15.75 grams of Tris base with one liter of Millipore water.Add approximately 2.8 milliliters of 50%HCl to adjust the buffer to a pH of nine. Once the culture is ready, it must be suitably enriched with the proper buffer. This step is critical to the success of the experiment, because a failure to provide the proper chemical environment can lead to very long time scales of precipitation or even a complete lack thereof.Next, divide the one liter of buffer into two parts as follows. In one 400 milliliter aliquot, dissolve eight grams of ammonium sulfate. In a second 400 milliliter aliquot, dissolve 16 grams of yeast extract.In 100 milliliters of the buffer, mix two grams of ammonium sulfate, and in the last 100 milliliters of buffer, add four grams of yeast extract and four grams of agar. After wrapping the flasks in aluminum foil, autoclave the four solutions. Immediately after autoclaving, set the two 400 milliliter solutions aside.Combine the two 100 milliliter solutions, mix well, and divide between 10 to 12 Petri dishes. To culture bacterial samples, remove the bacterial stock from the minus 80 degree Celsius freezer and allow it to thaw properly. Then place it along with an agar plate in the biosafety hood.Select a micropipette of the lowest possible volume and dip a micropipette tip into the Sporosarcina pasteurii stock before using the tip to streak an agar plate. Incubate the plate at 31 degrees Celsius for 48 hours, then visually examine for the presence of single colonies. If none are present, incubate for an additional 24 hours.Repeat the check and incubations until single colonies are detected. To prepare the final bacterial culture samples, mix together the two 400 milliliter solutions autoclaved earlier in a sterilized beaker, and transfer 125 milliliters into a flask. Perform a visual examination of the agar plate to identify regions with high concentrations of single colonies.With a micropipette tip, gently nudge and break one of the colonies. Then dip the same tip into the 125 milliliter flask and stir it thoroughly to ensure that a sufficient number of cells are transferred. Place the flask in a shaking incubator at 30 degrees Celsius and 150 rpm for two to three days.To determine a final cell count, draw seven parallel equidistant bold lines on the bottom of one of the agar plates so that they are visible from the top. Place three small drops of about ten microliters of non-diluted culture into one segment. Add one milliliter of non-diluted culture to nine milliliters of sterilized PBS to obtain a one-to-ten dilution.Then place three small drops of the dilution onto a second segment of the plate. Transfer the diluted culture to a new flask, and use PBS to dilute an additional ten times to ten to the minus two. Repeat the plating and dilution process until generating a dilution of at least ten to the minus seven to ensure single colonies will be obtained.Incubate the plate at 31 degrees Celsius for one to two days before counting the colonies in each section of the plate. Transfer nine milliliters of the prepared liquid culture into several sterilized ten milliliter centrifuge tubes. Prepare 100 milliliters of the external enrichment stock solution by using an analytical balance to carefully measure out two grams per liter of urea, one gram per liter of ammonium chloride, 212 milligrams per liter of sodium bicarbonate, and 280 milligrams per liter of calcium chloride.Into a beaker with fresh medium, combine all the ingredients except the urea, and autoclave. After autoclaving, mix the urea with one milliliter of fresh medium and pass the solution through a syringe fitted with a 0.2 micrometer filter before adding to the enrichment medium. Add one milliliter of the enrichment medium with additives to the sterilized centrifuge tubes containing nine milliliters of the prepared culture.Vortex each tube and incubate in a non-shaking incubator at 30 degrees Celsius. Monitor all the units regularly for initiation of precipitation. Once precipitation is detected with the naked eye, transfer a small volume of fluid to a high magnification microscopy chambered cover glass system.Initially view under 4X magnification to observe a wide area of coverage before progressively increasing the magnification. Perform staining and SEM experiments according to the text protocol. When S.pasteurii is cultured following the protocol demonstrated in this video, the bacteria lead to the precipitation of calcium carbonate that settles to the bottom of the tube over time.This figure shows phase contrast images of a clearly distinguished, rod-like bacterial population within the medium. Here, the well-defined cleavage lines, a hallmark of crystallinity, can be seen. This X-ray photoelectron spectroscopy graph for the same sample contains peaks corresponding to carbonate groups that pinpoint the existence of calcium carbonate.Shown in this panel is an untreated sponge that easily compresses when subjected to a one kilogram weight. When a sponge was immersed in a calcite precipitant solution with S.pasteurii for seven days and then dried, it hardened and displayed enhanced compressive strength due to pore clogging from calcite precipitation. Once mastered, this technique can be completed in a few hours if all the steps are done properly.However, although the individual steps take just a few minutes each, there are longer idle periods in between where the sample must be incubated for hours or even days. Following this procedure, several other techniques like additional characterization can be performed to answer other questions about, for example, the statistical distribution of polymorphous forms in calcium carbonate. After watching this video, you should have a proper and clear understanding of how to suitably culture Sporosarcina pasteurii to enrich it properly that ensures calcite precipitation, and also how to observe the process through multi-mode microscopy.Don't forget that working with high pressure autoclaves can be extremely hazardous and hence, all precautions like complete venting of the chamber and wearing of PPEs must be ensured before performing this procedure.

microbiologically-induced-calcite-precipitation-mediated-sporosarcina

Research

JoVE Journal

Bioengineering

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...