This work was supported by a Zhejiang Shuren University research project (2023R053 and 2023KJ237).

The blood used in the experiments was obtained from SPF-grade BALB/c female mice weighing 20-25 g and approximately 7 weeks old. The Animal Experimentation Ethics Committee of Zhejiang Shuren College approved all animal care and experimental procedures.
1. Solution preparation
<ol>
	<li>Weigh ALG and dissolve in ultrapure water by stirring in a water bath at 50 &#176;C to obtain 2% ALG solution.</li>
	<li>Separately prepare mass fraction with 5% (or molar mass concentration is 0.3 M) solutions of CaCl2, FeCl3, ZnSO4, and CuSO4 in ultrapure water. Pour each solution separately into a collecting dish.</li>
</ol>
2. Microfluidic electrospray device
<ol>
	<li>Take a glass capillary tube and place it over a blowtorch flame to soften, then stretch it to various diameters (50 &#181;m-500 &#181;m). Cut off the fine parts with a glass cutter and gently polish the tip port with high-quality silicon carbide sandpaper. After cleaning, connect the thick end of the capillary tube to a long tubing and the other end of the tubing to a dispensing needle and a 5 mL syringe (Supplementary Figure 1). 
	NOTE: Sharp edges of glass tube cuts are difficult to insert into the hose; thus, the break should be rounded at the flame edge.</li>
	<li>Attach the syringe to the microfluidic syringe pump and the capillary tubing at one end to the holder. Connect the red high-voltage end clip of the high-voltage power supply to the dispensing needle of the syringe and place the silver clip in the collection fluid. This configuration creates a simple microfluidic electrospray device (Figure 1A). 
	NOTE: Place the capillary tube perpendicular to the table.</li>
</ol>
3. ALG microspheres preparation
<ol>
	<li>Place the separate collection dishes containing different metal ions directly under the capillary tube. Turn on the microfluidic syringe pump and select Fast Forward to prime the tube with the ALG, then set the appropriate flow rate (2 mL/h).</li>
	<li>Switch on the high voltage power switch and turn the knob to set the appropriate voltage (5-7 kV).</li>
	<li>Add 20 mL solution to each of the 90 mm Petri dishes to collect ALG microspheres to form ALGMS cross-linked with different metal ions (Figure 1B).</li>
	<li>ALG forms microspheres under the action of electric field and gravity in different ion-collecting solutions within a few seconds. Aspirate the microspheres with a sterilized pipette and transfer them to individual 1.5 mL centrifuge tubes to obtain Zn2+, Ca2+, Cu2+, and Fe3+ -ALG hydrogel microspheres (Figure 1C). Label the Zn-ALGMS, Ca-ALGMS, Cu-ALGMS and Fe-ALGMS tubes. 
	NOTE: Microspheres of different diameters can be obtained by adjusting parameters such as voltage, tip diameter, flow rate, and solution concentration.</li>
	<li>Take a small amount of the prepared microspheres, disperse them as evenly as possible in PBS, and place them under a microscope for visualization.</li>
	<li>Randomly selected 10 observation fields in the same batch of microspheres, import the images to be measured into the ImageJ software, convert the images to the appropriate grayscale mode, and use the threshold segmentation technology to accurately define the area of the target particles and start the function of analyzing the particles so that ImageJ can identify and measure the target particles. Obtain data on the microspheres and then import the data into the Origin software for analysis and particle size mapping.</li>
</ol>
4. Antimicrobial performance test
<ol>
	<li>Prepare 5 mL suspensions of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) with an initial bacterial turbidity of 0.2 mcF using a liquid Luria-Bertani (LB) medium.</li>
	<li>Add 10 mL of each bacterial solution to 4 individual 15 mL sterile centrifuge tubes. Then, add 0.5 mL of Zn-ALGMS, Ca-ALGMS, Cu-ALGMS, and Fe-ALGMS ALG to each tube and place them in a constant-temperature shaker at 37 &#176;C and 200 rpm for 12 h. Add an equal amount of saline to the control group.</li>
	<li>Dilute each bacterial solution to 105 times with sterile water. Use a sterile glass bead to spread 200 &#181;L of bacterial solution on an LB solid medium.</li>
	<li>Spread the solution quickly and evenly on the coating plate, avoiding back and forth in the same area. Then, place the plates in an incubator at 37 &#176;C for 12 h. Observe the colonies of different groups. 
	NOTE: All aseptic procedures should be performed near an alcohol lamp. Sterilize microbial culture vessels, inoculation utensils, and culture media.</li>
</ol>
5. Drug release testing
<ol>
	<li>To evaluate the drug release ability of different microspheres, use bovine serum albumin (BSA) as a loaded model drug. Add 500 &#181;L of each microsphere (Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS) to 1 mg/mL BSA suspension for 24 h.</li>
	<li>For the drug release assay, add each saturated sample to 5.0 M of phosphate-buffered saline (PBS; pH 7.4), followed by agitation for 15 min at 37 &#176;C with continuous shaking at 80 rpm.</li>
	<li>Use 100 &#181;L of the solution and replace the medium with a fresh one at 1, 3, 6, 12, 24, 36, and 48 h.</li>
	<li>Quantify the supernatant protein concentrations at specific times using a BCA kit per the manufacturer&#39;s instructions. Quantitatively measure the released drug by enzyme marker to obtain the corresponding absorbance and convert the measured value to actual drug concentration according to the standard curve. Calculate the rate of drug release using the following formula: 
	Percentage of drug release = (concentration of drug released at a specific time point / initial drug concentration) x 100%</li>
	<li>At regular intervals, remove an equal volume of PBS from each well and replace it with an equal volume of fresh medium.</li>
</ol>
6. Hemolysis test
<ol>
	<li>Place the prepared Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS in PBS and incubate them at 37 &#176;C for 24 h to formulate the incubation solution.</li>
	<li>Obtain whole blood from healthy BALB/c mice by ocular phlebotomy, collected in anticoagulated tubes containing sodium citrate. Vortex and centrifuge at 1509 x g for 15 min to obtain red blood cells.</li>
	<li>Flush the red blood cells 2x-3x with PBS and prepare a 10% red blood cell solution with PBS.</li>
	<li>Preparation of various ALGMS: Take 500 &#181;L of each microsphere for the experimental group, 1 mL ultrapure water (ddH2O) for the positive control group, and 1 mL of PBS solution for the negative control group and mix each with 20 &#181;L of blood cell solution.</li>
	<li>Incubate the samples at 37 &#176;C for 4 h, centrifuge at 1509 x g, 15 min. Keep the supernatant aside and photograph the red blood cells in each group after hemolysis.</li>
	<li>Take the hemolysis rate of the positive control group as 100% and the negative control group as 0%. Measure the absorbance values of the supernatants in each group and compute the hemolysis rate by using the formula: 
	Hemolysis (%) = (Absorbance values of the supernatants of each group- 
	Absorbance values of distilled water alone) / (Absorbance values of the positive control group- Absorbance values of distilled water alone) x 100</li>
</ol>
7. Cytobiocompatibility test
<ol>
	<li>Wash the different ALGMS 2x with PBS and place in a 5 mL Petri dish for 15 min and discard the supernatant.</li>
	<li>Separately add different ALGMS at 0.2 mL in complete culture medium (DMEM) containing 10% FBS, incubate at 37 &#176;C for 24 h, and filter the solution to obtain the leachate.</li>
	<li>Culture NIH3T3 cells to approximately 70% cell density in 24-well plates, treat the NIH3T3 cells with microspheres leaching solution, and continue to culture with complete medium for another 24 h.</li>
	<li>Remove the cell culture medium and wash the cells with PBS.</li>
	<li>Assess cell viability using Calcein-AM/PI assay. Take 2.5 &#181;L of Calcein-AM solution (4 mM) and 12.5 &#181;L of PI solution (2 mM) and add them to 5 mL of 1x PBS and mix well to obtain a working solution with 2 &#181;M Calcein-AM and 5 &#181;M PI.</li>
	<li>Add the working solution to previously seeded cell culture plates at 500 &#181;L per well, incubate at 37 &#176;C under light protection for 15 min, and observe and photograph under an inverted fluorescence microscope. Set up three replicates for each group.</li>
	<li>Calculate cell viability according to the following formula: 
	Cell viability (%) = number of viable cells / (number of viable cells + number of dead cells)</li>
</ol>

Developing Sodium Alginate Hydrogel Microspheres for Biomedical Use

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S., Hashem, A. H., Khattab, T. A., Kamel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antibacterial+hydrogels+based+on+sodium+alginate.">New antibacterial hydrogels based on sodium alginate.</a> Int. J Biol Macromol. 248, 125872 (2023).</li><li>Olukman &#350;ahin, M., &#350;anl&#305;, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+5-fluorouracil+release+properties+investigation+from+pH+sensitive+sodium+alginate+coated+and+uncoated+methyl+cellulose/chitosan+microspheres.">In vitro 5-fluorouracil release properties investigation from pH sensitive sodium alginate coated and uncoated methyl cellulose/chitosan microspheres.</a> Int J Biol Macromol. 258 (1), 128895 (2024).</li><li>Chi, H., Qiu, Y., Ye, X., Shi, J., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+strategy+of+hydrogel+microsphere+and+its+application+in+skin+repair.">Preparation strategy of hydrogel microsphere and its application in skin repair.</a> Front Bioeng Biotechnol. 11, 1239183 (2023).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+dual-adhesive+particles+from+microfluidic+electrospray+for+bone+regeneration.">Bio-inspired dual-adhesive particles from microfluidic electrospray for bone regeneration.</a> Nano Res. 16 (4), 5292-5299 (2022).</li><li>Zheng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol-encapsulated+microspheres+prepared+by+microfluidic+electrospray+for+alleviating+inflammatory+pain.">Celastrol-encapsulated microspheres prepared by microfluidic electrospray for alleviating inflammatory pain.</a> Biomater Adv. 149, 213398 (2023).</li><li>Yang, L., Yang, W., Xu, W., Zhao, Y., Shang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+Janus+microcarriers+with+sequential+actives+release+for+bone+regeneration.">Bio-inspired Janus microcarriers with sequential actives release for bone regeneration.</a> Chem Eng J. 476, 146797 (2023).</li><li>Yang, L., Sun, L., Zhang, H., Bian, F., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-inspired+lubricated+drug+delivery+particles+from+microfluidic+electrospray+for+osteoarthritis+treatment.">Ice-inspired lubricated drug delivery particles from microfluidic electrospray for osteoarthritis treatment.</a> ACS Nano. 15 (12), 20600-20606 (2021).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-flexible+stretchable+liquid+metal+circuits+with+antimicrobial+properties+through+selective+laser+activation+for+health+monitoring.">Ultra-flexible stretchable liquid metal circuits with antimicrobial properties through selective laser activation for health monitoring.</a> Chem Eng J. 482, 149173 (2024).</li><li>Sukhodub, L., Kumeda, M., Sukhodub, L., Bielai, V., Lyndin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+doping+effect+on+the+physicochemical,+antimicrobial,+and+wound+healing+profiles+of+alginate-based+composite.">Metal ions doping effect on the physicochemical, antimicrobial, and wound healing profiles of alginate-based composite.</a> Carbohydr Polym. 304, 120486 (2023).</li><li>Daly, A. C., Riley, L., Segura, T., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microparticles+for+biomedical+applications.">Hydrogel microparticles for biomedical applications.</a> Nat Rev Mater. 5 (1), 20-43 (2020).</li><li>Bertsch, P., Diba, M., Mooney, D. J., Leeuwenburgh, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-healing+injectable+hydrogels+for+tissue+regeneration.">Self-healing injectable hydrogels for tissue regeneration.</a> Chem Rev. 123 (2), 834-873 (2023).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomass+microcapsules+with+stem+cell+encapsulation+for+bone+repair.">Biomass microcapsules with stem cell encapsulation for bone repair.</a> Nanomicro Lett. 14 (1), 4 (2021).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK-Cell-Encapsulated+porous+microspheres+via+microfluidic+electrospray+for+tumor+immunotherapy.">NK-Cell-Encapsulated porous microspheres via microfluidic electrospray for tumor immunotherapy.</a> ACS Appl Mater. 11 (37), 33716-33724 (2019).</li><li>Nan, L., Zhang, H., Weitz, D. A., Shum, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+future+of+droplet+microfluidics.">Development and future of droplet microfluidics.</a> LOC. 24 (5), 1135-1153 (2024).</li></ol>

No conflicts of interest are to be disclosed.

In this protocol, we present a method for preparing ALGMS based on microfluidic electrospray technology. The method is simple to operate and yields a large number of microspheres with uniform roundness and controllable diameter. This approach offers convenience to researchers and can promote the research and application of hydrogel microspheres. In addition, by cross-linking with different metal ions, the stability and bioactivity of the ALGMS were improved. In the antimicrobial experiments, Cu-ALGMS and Zn-ALGMS exhibit...

Drug delivery systems are a research hotspot in the field of bio-tissue engineering, aiming to improve drug delivery efficiency and efficacy and reduce adverse reactions and side effects1. Among these systems, hydrogel microspheres, characterized by good biocompatibility, tunable mechanical properties, and functional plasticity, are one of the most commonly used vehicles for drug loading and delivery2. They can be used for both slow and controlled release of drugs, provide good protective effects for drugs, avoid or minimize non-specific effects of drugs in other tissues, and target drug delivery to specific tissue structures3. Therefore, hydrogel microspheres have become a new and efficient drug delivery system, with research in this field gradually emerging4.
Hydrogel microspheres are typically synthesized from biodegradable materials, including polysaccharides, proteins, and natural polymers5. Among them, ALG is a biocompatible, biodegradable polysaccharide extracted from marine brown algae6. Its molecular chain contains free hydroxyl and carboxyl groups that can crosslink with most divalent or multivalent cations to form a water-insoluble hydrogel structure with a three-dimensional network5. The hydrogel microspheres formed by ALG can be converted into negatively charged polyelectrolytes in neutral and alkaline solutions. This repulsion between negative charges causes the microspheres to swell, allowing the release of the encapsulated active ingredient or drug. These properties have led to the consideration of ALG microspheres as promising drug carriers widely used for drug loading and controlled release7.
Various methods exist for the preparation of hydrogel microspheres. Traditional ALGMS preparation methods usually include the sol-gel method or the emulsion-sol method. These methods involve steps such as precipitation, co-precipitation, and gelation reactions to obtain the target microspheres8. In recent years, with the continuous development of microfluidic technology, the microfluidic electrospray method has gradually become an efficient and precise microsphere preparation method9. This method utilizes microfluidic technology to electrospray a polymer solution through a microfine nozzle to form micrometer-sized droplets and microspheres during the subsequent curing or cross-linking process10. Compared to the traditional method, microfluidic electrospray offers precise control of microsphere particle size and morphology by adjusting parameters such as solution flow rate, voltage, and fine nozzle size11. It also enables high-speed continuous preparation of microspheres, improving preparation efficiency and maintaining mild reaction conditions. In addition, ALGMS can be prepared to possess various functions, such as controlled-release drugs and loaded catalysts, enabling their easy application in various fields.
Here, we present a protocol for the preparation of ALG microspheres using the microfluidic electrospray method. The process involves passing an ALG solution through a microfluidic device and subjecting it to electrospray. The resulting droplets were collected in the solution containing different metal ions (Ca2+, Cu2+, Zn2+, and Fe3+) to initiate the cross-linking reaction. This reaction improves the stability and adhesion of the microspheres and endows them with different functionalities. This method is easy to perform, and the synthesized microspheres exhibit good size uniformity in their morphology. In addition, we investigated their antimicrobial properties, slow drug-release ability, and biocompatibility. This protocol will be useful for further drug development and production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>120 mesh screen</td>
			<td>Solarbio,China</td>
			<td>YA0946</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol burner</td>
			<td>Solarbio,China</td>
			<td>YA2320</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Wukong Biotechnology,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic Acid Assay reagent</td>
			<td>Meilunbio,China</td>
			<td>MA0082</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Lablead,China</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#160; powder</td>
			<td>Aladdin,China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM/PI</td>
			<td>Biosharp,China</td>
			<td>BL130A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Corning,America</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4&#160; powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7758-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibicol,China</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#160; powder</td>
			<td>Aladdin,China</td>
			<td>7705-08-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HAKATA,China</td>
			<td>HN-FBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>Sartorius,Germany</td>
			<td>CC0028</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscopy</td>
			<td>Evidentscientific,Japan</td>
			<td>BX53(LED)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic syringe pump</td>
			<td>Longerpump,England</td>
			<td>LSP01-3A</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH3T3</td>
			<td>HyGyte,China</td>
			<td>TCM-C752</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermofisher,America</td>
			<td>150464</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Thermofisher,America</td>
			<td>3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Thermofisher,America</td>
			<td>Axia ChemiSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate powder&#160;</td>
			<td>Bjbalb,China</td>
			<td>Y13095</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4 powder</td>
			<td>Jnxinyuehuagong,China</td>
			<td>7733-02-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of cross-linked sodium alginate microspheres with different metal ions using the microfluidic electrospray technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

Characterization of ALGMS cross-linked with different metal ions 
The optical morphology of Ca-ALGMS, Cu-ALGMS, Zn-ALGMS, and Fe-ALGMS is shown in Figure 2, exhibiting good sphericity, smooth surface, uniform particle size distribution (Supplementary Figure 2) and excellent monodispersity. We further performed microscopic characterization using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. As shown in <strong clas...

Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

preparation-cross-linked-sodium-alginate-microspheres-with-different

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1.4K Views.  Zhejiang Shuren University. The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Video: Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...