This work is a continuation of a Montana Tech Research Assistant Mentorship Program project by Adolfo Martinez, Catherine Mulholland, Tyler Somerville, and Laurel Bitterman. Research was sponsored by the National Science Foundation under Grant No. OIA-1757351 and the Combat Capabilities Development Command Army Research Laboratory (Cooperative Agreement Number W911NF-15-2-0020). Any opinions, findings and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation or the Army Research Lab. We would also like to thank Amy Kuenzi, Lee Richards, Katelyn Alley, Chris Gammons, Max Wohlgenant, and Kris Bosch for their contributions.

1. Creation and maintenance of bacterial cellulose starter culture
<ol>
	<li>Obtain a starter culture of bacterial cellulose, approximately 50 g, in the form of a SCOBY. It can be purchased commercially (e.g., from Cultures for Health). Place the SCOBY into a 1 L beaker, covered with a paper towel.</li>
	<li>Boil 700 mL of deionized water, transfer it to a separate vessel from the one containing the SCOBY, and add 85 g of sucrose.</li>
	<li>Once the sucrose has dissolved, add 2 bags of black tea (4.87 g). Steep the tea for 1 h, then carefully remove the tea bags using a stir rod.</li>
	<li>Add 200 mL of distilled white vinegar to the tea. Let the mixture cool to 25 &#176;C. Once cooled, add 700 mL of the room temperature tea to the beaker containing the SCOBY. 
	&#8203;CAUTION: Adding the acidic tea while it is too hot may harm the organisms in the SCOBY.</li>
	<li>Cover the beaker with a paper towel and secure it with an elastic band, and place the beaker in a storage area that maintains a temperature of 25 &#176;C. This vessel is commonly referred to as stock culture or a hotel.</li>
	<li>To keep the SCOBY healthy, maintenance is required about 2 times a month.
	<ol>
		<li>Using gloved hands to hold back the SCOBY mats, drain the liquid from the hotel into a separate beaker. In the container with the liquid, add enough acidic tea for a total of 700 mL of solution.</li>
		<li>Dissolve 65 g of sucrose in the container with the acidic tea. While waiting for the sucrose to dissolve, carefully rinse the SCOBY mats in DI water.</li>
		<li>Once the sucrose is fully dissolved, the liquid can be added to the beaker containing the rinsed SCOBY mats. Cover the beaker and return it to the incubation area.</li>
	</ol>
	</li>
</ol>
2. Production of bacterial cellulose spheres
NOTE: Exercise caution when working with boiling water. Ensure glassware can withstand the boiling water temperatures, is free of defects, and is the appropriate size. A schematic describing the production of BC spheres is given in Figure 1.
<ol>
	<li>Boil 350 mL of deionized water using a tea kettle. Transfer the hot water to a 500 mL beaker. Dissolve 42.5 g of granulated sucrose into the hot water using a stir rod.</li>
	<li>When the sucrose is fully dissolved, steep 1 bag of black tea (2.54 g) in the flask containing the sucrose and water for 1 h. After this, remove the tea bag with a stir rod, taking care to avoid breaking open the tea bag, and then dispose of it in the trash.</li>
	<li>Add 100 mL of distilled white vinegar to the beaker, and then thoroughly stir the mixture. Transfer 80 mL of the acidic tea mixture to a 250 mL baffled flask. Allow the tea mixture to cool to room temperature, 20 - 25 &#176;C. 
	NOTE: At this point, the mixture can be left to cool overnight or until prepared for the next step.</li>
	<li>Once the liquid temperature is at room temperature (20 - 25 &#176;C), add 20 mL of microbial starter culture liquid to the baffled flask. This liquid can be obtained from a SCOBY hotel. Cover the flask with parafilm.</li>
	<li>Place the baffled flask on an orbital shake table and set the speed to 125 rotations per minute (rpm). Allow the mixture to shake for 3 days in a room or incubator with a temperature from 20 - 25 &#176;C to produce BC spheres. 
	NOTE: If irregular shapes form in the flask contents or if cellulose clumps stick to the walls of the flask, they should be removed to prevent further irregular BC masses from forming. Use tweezers to remove unwanted BC masses, including thin strings, rings, tubular shapes, and other clearly non-spherical shapes.</li>
	<li>Once the spheres have formed, gently pour them from the flask and analyze, dispose, or use them in a way not outlined in this paper.</li>
</ol>
3. Using bacterial cellulose spheres to encapsulate particles or contaminants
<ol>
	<li>Follow steps 2.1-2.5 above.</li>
	<li>After shaking for 3 days, add about 0.01 g of finely ground particulate matter to the baffled flask. Appropriate solids include biochar (260 &#177; 140 &#181;m), mine waste (350 &#177; 140 &#181;m), and polystyrene microbeads (3 &#181;m). Data for these materials are outlined in the Representative Results section. Please see the attached Table of Materials for further descriptions of biochar, mine waste, and microbeads.</li>
	<li>Cover the flask again with the parafilm and place it back on an orbital shaker, using the same speed and ambient temperature (20 - 25 &#176;C) for 3 more days. Remove the BC encapsulated particles for analysis, disposal, or other uses.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62286/62286fig01.jpg" /> 
Figure 1. Bacterial cellulose sphere fabrication and encapsulation of solid particles. Step 1 involves combining bacterial stock culture with black tea, sugar, and vinegar media in a baffled flask. The disks in the stock culture represent BC mats. Then, the baffled flask is placed on an orbital shake table for 3 days. The middle step shows solids being added to the flask once BC spheres have formed. The flask is shaken for 3 more days. In the final step, BC spheres have continued increasing in size and encapsulated the solid particles. <a href="https://www.jove.com/files/ftp_upload/62286/62286fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

This protocol outlines BC sphere production and encapsulation methods that are easy to conduct and cost effective. Through various adjustments to the original protocol, an adequate process has been identified. Critical steps must be followed to ensure viable spheres. All the ingredients involved in BC formation play a key role in the health and durability of the spheres. The sucrose feeds organisms, the tea provides nitrogen, and the vinegar lowers the pH to optimal conditions to prevent undesired contaminants<sup class=...

Bacterial cellulose (BC) has been noted for its potential industry use due to its mechanical strength, high purity and crystallinity, water retention abilities, and intricate fiber structure1,2,3,4. These characteristics make BC a favorable biomaterial for a variety of applications, including biomedical, food processing, and environmental remediation uses1. Formation of a BC film can be done with single organism cultures or mixed cultures like those used for kombucha5, a fermented tea beverage. Brewing kombucha relies on a &#34;Symbiotic Culture Of Bacteria and Yeast&#34;, commonly known as a SCOBY. Using this symbiotic culture of organisms, a similar technique is employed to create BC spheres. This biomaterial may be employed to help isolate environmental contaminants and anchor agricultural amendments like biochar to achieve more efficient crop production.
Previous literature has discussed how the characteristics of BC produced in agitated conditions compare to BC produced in a stationary culture. A stationary culture results in a film that forms at the liquid-air interface, while a shaken culture results in varying BC particles, strands, and spheres suspended within the liquid6. Many studies have referenced the claim that commercial production of BC is more feasible in the dynamic conditions6,7, providing rationale for applying this paper&#39;s method. Additionally, various investigations on the structure and properties of BC spheres have been conducted. Toyosaki et al.6 compared baffled and smooth-walled Erlenmeyer flasks in their agitated BC production. A study by Hu and Catchmark4 determined conditions for BC spheres that were used as guidelines for the current BC sphere production process, and their results indicate that the sphere size does not continue to increase after 60 hours. A review of BC production by Mohammad et al.1 indicates that shaking the BC culture ensures even oxygen supply and distribution, which is necessary for successful BC growth. Holland et al.8 have studied the crystallinity and chemical structure of BC using X-ray diffraction and Fourier transform infrared spectroscopy. It is assumed BC capsules will exhibit similar characteristics and future research will investigate structural properties. Studies have also explored the beneficial effects of using BC to produce improved biocomposites. Using epoxy-resin as a base, researchers have showed that the addition of BC improves material characteristics like fatigue life, fracture toughness, and tensile and flexural strength9,10. As shown by past and current research, many are interested in commercializing BC use.
Many researchers have investigated bacterial cellulose in controlled release systems, and the method described here generates capsules that could be utilized as controlled release systems. Much of this research focuses on controlled release in the biomedical field, as well as some exploration in controlled release fertilizer (CRF) administration. Based on the success of BC&#39;s controlled release of amoxicillin11, lidocaine12, and ibuprofen13, BC may exhibit similar delivery characteristics with other substances, such as a pelletized fertilizer. An overview of CRF&#39;s by Shaviv and Mikkelsen14 acknowledges that CRF&#39;s are more efficient, save labor, and generally cause less environmental degradation than conventional fertilizer application. Bacterial cellulose may work as a favorable encapsulating material for CRF&#39;s. Fertilizers may leach out of BC membranes or discharge as BC biodegrades15,16. BC&#39;s high water swelling capacity can also act as a beneficial soil amendment17,18,19 because both fertilizer nutrients and moisture may release into the ground through application of BC spheres. With these traits, a CRF formed by BC sphere encapsulation may have an advantage over other fertilizer coating materials that could have negative effects during their production and disposal stages. Adapting BC into a fertilizer coating may further improve CRF technologies. By lowering fertilizer release rate, crops will have sufficient time to uptake the fertilizer and prevent excess runoff into bodies of water, thereby reducing eutrophication and unoxygenated zones. Similar slow-release fertilizers have been prepared and piloted using polymer coatings20.
Unlike protocols outlined in previous research, this one focuses on uniform, cohesive sphere production rather than high cellulose yield. Moreover, BC encapsulation of other solids has been studied with cellulose films, but not spheres21. By expanding the research on bacterial cellulose spheres, further steps can be made to produce BC commercially, which is beneficial because of BC&#39;s environmentally safe features. This method of BC sphere fabrication utilizes inexpensive, readily available culinary ingredients. After the initial assembly, BC spheres begin to form within 2 days without interference. Producing BC spheres through this strategy requires little space and has an edible by-product, the fermented tea &#39;kombucha&#39;. Encapsulation techniques mentioned in other studies include coatings formed through the phase inversion technique22,23, matrix formation24, spray drying25, and direct encapsulation during synthesis26. The direct encapsulation method outlined in this manuscript is useful for those who desire an easy, inexpensive process that utilizes readily available materials.
Through this research, a successful protocol for BC sphere production and encapsulation was created. BC spheres can encapsulate solid particles of biochar, mine tailings, and polystyrene microbeads within their individual structures. Although not widely used in industry yet, BC is a practical, sustainably made, and naturally occurring material that could be used for future applications.

<table><tbody><tr><td>100 mL graduated cylinder</td><td></td><td></td><td></td></tr><tr><td>1000 mL beaker</td><td></td><td></td><td></td></tr><tr><td>25 mL graduated cylinder</td><td></td><td></td><td></td></tr><tr><td>250 mL Erlenmeyer baffled flask</td><td>Chemglass</td><td>CLS-2040-02</td><td></td></tr><tr><td>500 mL beaker</td><td></td><td></td><td></td></tr><tr><td>Balance</td><td></td><td></td><td></td></tr><tr><td>Biochar</td><td></td><td></td><td>Ponderosa pine heat treated under argon gas, heated at 15 &amp;deg;C per minute to 800 &amp;deg;C</td></tr><tr><td>Black tea</td><td></td><td></td><td></td></tr><tr><td>Deionized water</td><td></td><td></td><td></td></tr><tr><td>Distilled white vinegar</td><td></td><td></td><td></td></tr><tr><td>Elastic band</td><td></td><td></td><td></td></tr><tr><td>Microbial starter culture</td><td>Cultures for Health</td><td></td><td></td></tr><tr><td>Mine waste</td><td></td><td></td><td>Collected from Butte, MT: 46.001978,-112.582465. Mine waste contains soil and metals originating from past copper mining. Mn, Si, Ca, Al, and Fe were the five most prevalent elements measured in the mine waste through x-ray diffraction.</td></tr><tr><td>Mortar and pestle</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker</td><td></td><td></td><td>Used various brands</td></tr><tr><td>Paper towel</td><td></td><td></td><td></td></tr><tr><td>Polystyrene microbeads</td><td>Polybead</td><td>17138</td><td>3 micron diameter</td></tr><tr><td>Stir rod</td><td></td><td></td><td></td></tr><tr><td>Sucrose</td><td></td><td></td><td></td></tr><tr><td>Tea kettle</td><td></td><td></td><td></td></tr><tr><td>TGA</td><td>TA Instruments</td><td>TA Q500</td><td>400 &amp;deg;C/min to 800 &amp;deg;C, 100 mL/min N2</td></tr><tr><td>Thermometer</td><td></td><td></td><td></td></tr><tr><td>XRF Analyzer</td><td>ThermoFisher Scientific</td><td>10131166</td><td></td></tr></tbody></table>

bacterial cellulose spheres that encapsulate solid materials

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an affordable and simple method for BC sphere production. In addition to producing these spheres, an encapsulation method for solid particles has also been identified. To produce BC spheres, water, black tea, sugar, vinegar, and bacterial culture are combined in a baffled flask and the contents are agitated. After determining the proper culture conditions for BC sphere formation, their ability to encapsulate solid particles was tested using biochar, polymer beads, and mine waste. Spheres were characterized using ImageJ software and thermal gravimetric analysis (TGA). Results indicate that spheres with 7.5 mm diameters can be made in 7 days. Adding various particles increases the average size range of the BC capsules. The spheres encapsulated 10 - 20% of their dry mass. This method shows low-cost sphere production and encapsulation that is possible with easily obtainable materials. BC spheres may be used in the future as a contaminant removal aid, controlled release fertilizer coating, or soil amendment.

BC spheres have the fastest growth rate during the first 48 h of culture (Figure 2). Figure 2 also shows how the spheres tend to reach a maximum average size and then remain constant. In this experiment, the spheres reached an average diameter of 7.5 &#177; 0.2 mm. Although the BC spheres never completely deteriorate within the 10 day growth period, they did start to form tendrils that extend off the main body of the sphere around the eighth day. This can be see...

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Bacterial Cellulose Spheres that Encapsulate Solid Materials

This protocol presents an easy, inexpensive method of forming bacterial cellulose (BC) spheres. This biomaterial can function as an encapsulation medium for solid materials, including biochar, polymer spheres, and mine waste.

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an ...

bacterial-cellulose-spheres-that-encapsulate-solid-materials

Research

JoVE Journal

Engineering

4.3K Views.  Montana Technological University. Ons protocol is belangrijk, omdat het zich uitbreidt op de huidige methoden voor het produceren van een biologisch afbreekbaar, natuurlijk voorkomend biomateriaal. Onze methode legt de nadruk op de productie van reproduceerbare bacteriële cellulosebollen. Het belangrijkste voordeel van deze techniek is het gemak van toegang tot de benodigde materialen: suiker, water, thee, azijn, bacterieel kweekstarter, een bafflekolf en een orbitale shaker.Iemand die deze techniek voor het eerst uit...

Video: Bacterial Cellulose Spheres that Encapsulate Solid Materials

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Developmental Biology

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bioengineering

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Cancer Research

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides&#39; corresponding nanoparticles&#8212;the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)&#8212;the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the ...

Chemistry

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biology

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Bacterial Cellulose Spheres that Encapsulate Solid Materials

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