This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1. Extraction of Sporopollenin Exine Capsules (SECs) from L. clavatum Spores
Note: The SEC extraction process involves a flammable powder (L. clavatum), hot corrosive acids, and flammable solvents, hence proper personal protective equipment (goggles, face mask, gloves, lab coat), approved risk assessment on usage, and disposal of chemicals by authorized laboratory personnel is essential.
<ol>
	<li>Spore Defatting
	<ol>
		<li>Prepare a reflux set-up in a fume hood by using a glass condenser, water circulation system, and water bath (Figure 1).</li>
		<li>Weigh 100 g L. clavatum spores without creating dust and away from any ignition source. 
		Note: The spores used here were commercially obtained (See Materials List).</li>
		<li>Transfer spores slowly to a 1 L polytetrafluoroethylene (PTFE) round bottom flask with a magnetic stirring rod.</li>
		<li>Add 500 ml acetone to the spores in the PTFE flask and shake the flask gently to form a homogeneous suspension.</li>
		<li>Place the PTFE flask in the water bath set at 50 &#176;C and connect to the reflux condenser.</li>
		<li>Perform refluxing in acetone for 6 hr with stirring at 200 rpm and then allow to cool at RT.</li>
		<li>Filter the suspension using filter paper under vacuum and collect the defatted spores in large (15 cm diameter) glass petri dishes.</li>
		<li>Dry the spores at room temperature (fume hood) by covering with aluminum foil with holes for solvent evaporation.</li>
	</ol>
	</li>
	<li>Acidolysis
	<ol>
		<li>Prepare 85% (v/v) phosphoric acid with deionized (DI) water and transfer defatted dry spores to a 1 L PTFE flask.</li>
		<li>Add 500 ml of phosphoric acid (85% v/v) to the defatted spores.</li>
		<li>Place the PTFE flask in a water bath set at 70 &#176;C and connect to the reflux condenser.</li>
		<li>Perform gentle refluxing (70 &#176;C) in phosphoric acid for 30 hr and then allow to cool at room temperature.</li>
		<li>Dilute the suspension by using 500 ml warm DI water and collect the SECs by vacuum filtration using filter paper.</li>
		<li>Transfer the SECs to a 3 L glass beaker to perform a series of washings.</li>
	</ol>
	</li>
	<li>SEC Washing
	<ol>
		<li>Place the 3 L glass beaker containing SECs in a fume hood.</li>
		<li>Add 800 ml hot (50 &#176;C) DI water to the SECs with gentle stirring for 10 min.</li>
		<li>Filter the suspension by using filter paper and a vacuum filtration set-up.</li>
		<li>Collect the SECs in a clean 3 L glass beaker.</li>
		<li>Repeat steps 1.3.1. to 1.3.4. five times.</li>
		<li>Add 600 ml hot (45 &#176;C) acetone to the SECs with gentle stirring for 10 min.</li>
		<li>Collect the SECs by filtration under vacuum and transfer the SECs to a clean 3 L glass beaker.</li>
		<li>Repeat steps 1.3.6. and 1.3.7. using 600 ml hot (50 &#176;C) 2 M hydrochloric acid.</li>
		<li>Repeat steps 1.3.6. and 1.3.7. using 600 ml hot (50 &#176;C) 2 M sodium hydroxide.</li>
		<li>Repeat steps 1.3.1 and 1.3.4 eight times.</li>
		<li>Repeat steps 1.3.6. and 1.3.7. using 600 ml hot (45 &#176;C) acetone.</li>
		<li>Repeat steps 1.3.6. and 1.3.7. using 600 ml hot (45 &#176;C) ethanol.</li>
		<li>Repeat steps 1.3.6. and 1.3.7. using 800 ml hot (50 &#176;C) DI water.</li>
		<li>Transfer the clean SECs to six large glass petri dishes and dry in a fume hood at RT for 24 hr to remove all water content.</li>
		<li>Dry the SECs in a vacuum oven at 60 &#176;C and 1 mbar vacuum for 10 hr or until constant weight.</li>
		<li>Collect the dried SECs and transfer to a polypropylene bottle.</li>
		<li>Store the SECs in a dry cabinet.</li>
	</ol>
	</li>
</ol>
2. Characterization of SECs
<ol>
	<li>Perform scanning electron microscopic analysis41 using spores and SECs produced at different stages.</li>
	<li>Perform elemental analysis41 using spores and SECs produced at different stages. Dry all samples at 60 &#176;C for at least 1 hr before elemental analysis and calculate the protein content using percent nitrogen with a multiplication factor of 6.25.11 Obtain results using triplicate measurements for each sample.</li>
	<li>Conduct micromeritic properties analysis using a dynamic image particle analyzer.41 </li>
	<li>Scan unprocessed, processed, and FITC-BSA-loaded SECs using confocal laser scanning microscopy.41 </li>
</ol>
3. Biomacromolecule Encapsulation by Vacuum Loading Technique
<ol>
	<li>Prepare 1.2 ml of 125 mg/ml bovine serum albumin (BSA) solution using DI water and transfer to a 50 ml polypropylene tube.</li>
	<li>Weigh 150 mg of SECs and transfer to the BSA solution.</li>
	<li>Vortex the tube for 10 min to form a homogeneous solution.</li>
	<li>Cover the tube using filter paper.</li>
	<li>Transfer the tube to a freeze dryer flask and dry for 4 hr with vacuum at 1 mbar.</li>
	<li>Perform steps 3.1 to 3.5. for three independent batches.</li>
	<li>Collect the BSA-loaded SECs and remove the agglomerates by using a mortar and pestle.</li>
	<li>Transfer the BSA-loaded SECs to a 50 ml tube and add 2 ml DI water to remove the residual BSA.</li>
	<li>Collect the SECs by centrifugation at 4,704 x g for 5 min and discard the supernatant.</li>
	<li>Repeat the washing with DI water once.</li>
	<li>Cover the tube containing BSA-loaded SECs using filter paper.</li>
	<li>Transfer the SECs to a freezer (-20 &#176;C) and freeze for 1 hr.</li>
	<li>Freeze dry the SECs for 24 hr and store in a freezer until further characterization.</li>
	<li>Prepare the placebo SECs without BSA using the same procedure as in steps 3.1. to 3.13.</li>
	<li>Prepare the FITC-BSA loaded SECs using the same procedure as in steps 3.1. to 3.13.</li>
</ol>
4. Determination of Encapsulation Efficiency
<ol>
	<li>Weigh 5 mg of BSA-loaded SECs in 2 ml polypropylene tubes.</li>
	<li>Add 1.4 ml phosphate buffered saline pH 7.4 (PBS) and vortex for 5 min.</li>
	<li>Probe sonicate the suspension at 40% amplitude for 3 cycles of 10 sec.</li>
	<li>Filter the solution using a 0.45 &#181;m Polyethersulfone (PES) filter.</li>
	<li>Perform the same procedure as in steps 4.1. to 4.4. using placebo SECs.</li>
	<li>Measure the absorbance at 280 nm using placebo as a blank.</li>
	<li>Quantify the amount of BSA encapsulated using a BSA standard curve (200 to 1,000 &#181;g/ml) in PBS.42</li>
</ol>

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The authors have nothing to disclose.

In this work, a systematic analysis of SEC extraction from L. clavatum spores is presented and this report shows that it is possible to produce higher quality capsules while also achieving a significant streamlining of the pre-existing commonly used protocol.11 In contrast to the existing protocol requiring a high process temperature (180 &#176;C) and a long process duration (7 days),11 the current SEC extraction processing optimization was primarily focused on reducing the temperature and ...

There is significant interest in natural plant-based capsules obtained from plant spores and pollens for use in microencapsulation applications.1-15 In nature, spores and pollen provide protection for sensitive genetic materials against harsh environmental conditions. The basic structure of plant spores and pollen typically comprises an outer shell layer (exine), an inner shell layer (intine), and the internal cytoplasmic material. The exine is comprised of a chemically robust biopolymer1,9,10,13,16 referred to as sporopollenin and the intine is comprised primarily of cellulosic materials.16-18 Empty capsules can be isolated by various processes7,9 for removing cytoplasmic material, proteins, and the intine layer.2,12,16 These sporopollenin exine capsules (SECs) provide a compelling alternative to synthetic encapsulants due to their narrow size distribution and uniform morphology.7,9,13,19,20 The development of standardized processes to obtain SECs from various plant species, such as Lycopodium clavatum, opens the potential for a wide range of microencapsulation applications in the fields of drug delivery, foods, and cosmetics.6,10-13,21
In order to obtain SECs, researchers first treated spores and pollen with organic solvents and refluxed in alkaline solutions to remove cytoplasmic contents.22-25 However, the remaining capsule structure was determined to still contain the cellulosic intine layer. In order to remove this, researchers explored the use of prolonged acidic hydrolysis processing using hydrochloric acid, hot sulfuric acid, or hot phosphoric acid over several days,22-25 with phosphoric acid becoming the preferred method of SEC intine removal.2 However, ongoing research over the years has shown that various spores and pollens have differing degrees of resilience to the harsh processing methods commonly used.26,27 Some spores and pollens are completely degraded and lose all structural integrity in strong alkaline solutions, or become heavily damaged in strong acidic solutions.16 The variability in SEC response to treatment conditions is due to subtle variations in the chemical structure and exine morphology of the sporopollenin exine material between species.28 Due to the variability in the robustness of sporopollenin exine capsules (SECs), it is necessary to optimize the processing conditions for each species of spore and pollen.
Plant spores from the species L. clavatum have become the most widely studied single source of SECs. It is proposed that this is primarily due to its widespread availability, low cost, monodispersity, and chemical robustness.9,29 The spores can be easily harvested and contain sporoplasmic contents in the form of groupings of 1 - 2 &#181;m cellular organelles and biomolecules.11 L. clavatum spores have been used as a natural powder lubricant,30,31 a base for cosmetics,30 and in herbal medicine32-36 for a wide range of therapeutic applications. The SECs obtained from L. clavatum have been shown to be more resilient to processing than SECs from other species of spores and pollen.2 After processing, the resulting SECs have been shown to retain their intricate microridge structures and high morphological uniformity while providing a large internal cavity for encapsulation.7 Studies indicate that L. clavatum SECs can be used for the encapsulation of drugs,10,13 vaccines,11 proteins,7,14 cells,8 oils,5-7,9 and food supplements.5,15 Observed SEC loading efficiencies are relatively high in comparison to conventional encapsulation materials.7 There are also a number of reported benefits to SEC encapsulation such as the ability to mask tastes,6,10 and to provide some degree of natural protection against oxidation.12 In the existing studies, the most commonly used SEC extraction method for L. clavatum is based on four main steps. Step one is solvent refluxing in acetone for up to 12 hr at 50 &#176;C to defat the spores.11 Step two is alkaline refluxing in 6% potassium hydroxide for up to 12 hr at 120 &#176;C to remove cytoplasmic and proteinaceous materials.11 Step three is acid refluxing in 85% phosphoric acid for up to 7 days at 180 &#176;C to remove the cellulosic intine material.11 Step four is a comprehensive washing process using water, solvents, acids, and bases to remove all remaining non-exine material and chemical residues.
The main goals of SEC extraction in relation to encapsulation applications are to produce capsules which are empty of cytoplasmic material, free from potentially allergenic proteins, and morphologically intact.2,37 However, from an industrial manufacturing perspective, it is also important to consider additional economic and environmental factors, such as, energy efficiency, production duration, safety, and resulting waste. With regards to energy efficiency, both high temperatures and long processing times affect production costs as well as environmental footprint. Production duration and turnaround time are key factors influencing processing profitability. Of particular concern is that high temperature phosphoric acid processing increases safety issues and is known to result in corrosive scaling which leads to significant increases in infrastructure maintenance and delays in batch turnaround times.38-40 Where possible, minimizing the number of steps required may lead to significantly reducing the waste produced. However, the commonly used four-step process of L. clavatum SEC extraction has simply evolved from decades of research and has had little actual process optimization. Recently, Mundargi et al.,41 made a significant contribution to the ongoing work in this field by systematically evaluating and optimizing one of the most commonly reported SEC extraction techniques.
In the first section of this study: spore defatting is demonstrated utilizing acetone processing at 50 &#176;C for 6 hr; sporoplasm and intine removal procedures are demonstrated utilizing 85% phosphoric acid processing at 70 &#176;C for 30 hr; extensive washing with water, solvents, acid, and base is used to demonstrate the removal of residual sporoplasmic contents; and SEC drying is demonstrated utilizing convection drying and vacuum oven drying. In the third section, SEC vacuum loading is demonstrated utilizing vacuum loading of a model protein, bovine serum albumin (BSA), followed by BSA-loaded-SEC washing and lyophilization. In the fourth section, the determination of the BSA encapsulation efficiency is demonstrated utilizing centrifugation, probe sonication, and UV/Vis spectrometry.

<table><tbody><tr><td>&lt;em&gt;Lycopodium clavatum&lt;/em&gt; spores (s-type)</td><td>Sigma</td><td>19108-500G-F</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153-50G</td><td></td></tr><tr><td>FITC-conjugated BSA</td><td>Sigma</td><td>A9771-250MG</td><td></td></tr><tr><td>Phosphoric acid (85% w/v)</td><td>Sigma</td><td>438081-2.5L</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>V800202&amp;nbsp;</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881-1KG&amp;nbsp;</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>V800022</td><td></td></tr><tr><td>Ethanol</td><td>AcME&amp;nbsp;</td><td>C000356</td><td></td></tr><tr><td>Deionized water</td><td>Millipore purified water&amp;nbsp;</td><td></td><td></td></tr><tr><td>Qualitative filter paper (grade No. 1, cotton cellulose)</td><td></td><td></td><td></td></tr><tr><td>Polystyrene microspheres (50 &amp;plusmn; 1 &amp;micro;m)&amp;nbsp;</td><td>Thermoscientific (CA, USA)</td><td>4250A</td><td></td></tr><tr><td>Vectashield&amp;nbsp;</td><td>Vector labs (CA, USA)</td><td>H-1000</td><td></td></tr><tr><td>Sticky-slides, D 263 M Schott glass, No.1.5H (170 &amp;mu;m, 25 mm x 75 mm) unsterile glass slide</td><td>Ibidi GmbH (Munich, Germany)</td><td>10812</td><td></td></tr><tr><td>Commercial Lycopodium SECs (L-type)</td><td>Polysciences, Inc. (PA, USA)</td><td>16867-1</td><td></td></tr><tr><td>Heating plates</td><td>IKA, Germany</td><td></td><td></td></tr><tr><td>Scanning electron microscope</td><td>Jeol, Japan</td><td>&amp;nbsp;JFC-1600</td><td></td></tr><tr><td>Elemental analyzer&amp;nbsp;</td><td>Elementar, Germany</td><td>VarioEL III</td><td></td></tr><tr><td>FlowCam: The benchtop system</td><td>Fluid Imaging Technologies, USA</td><td>FlowCamVS</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss, Germany</td><td>LSM710</td><td></td></tr><tr><td>Freeze dryer</td><td>Labconco, USA&amp;nbsp;</td><td></td><td></td></tr><tr><td>UV Spectrometer</td><td>Boeco, Germany</td><td>S220</td><td></td></tr></tbody></table>

extraction of plant-based capsules for microencapsulation applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

Streamlined Extraction Process for Sporopollenin Exine Capsules 
The L. clavatum SEC extraction was achieved by three main steps: (1) Defatting using acetone; (2) Acidolysis using phosphoric acid 85% (v/v); and (3) Extensive SEC washing using solvents. The flow of the streamlined SEC extraction process is presented in Figure 1 A - I. Briefly, the process involves a defatting step with a...

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Extraction of Plant-based Capsules for Microencapsulation Applications

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

extraction-of-plant-based-capsules-for-microencapsulation-applications

Research

JoVE Journal

Bioengineering

12.0K Views.  Nanyang Technological University.  Dit protocol / manuscript beschrijft een gestroomlijnde werkwijze voor de productie van sporopollenin exine capsules (SEC) van Lycopodium clavatum sporen en voor het laden van hydrofiele stoffen in deze SEC. 

Video: Extraction of Plant-based Capsules for Microencapsulation Applications

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

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 ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

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.

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 ...

Engineering

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 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Medicine

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Chemistry

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

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.

Extraction of Plant-based Capsules for Microencapsulation Applications

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