The study was supported in part by grants awarded to B. R. by the National Institutes of Health (R01EY019320), the Department of Veterans Affairs (RX000444 and BX003050), and the South Carolina SmartState Endowment.

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Medical University of South Carolina Animal Care and Use Committee under protocol ID 00399.
1. Cell Culture
<ol>
	<li>Generate human retinal pigment epithelial cells (ARPE-19) cell line stably expressing the gene of choice according to published protocols14,15.</li>
	<li>Maintain cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>Replace the medium every 2&#8211;3 days.</li>
	<li>Passage the cells after reaching 70%&#8211;80% confluence using standard tissue culture procedures.</li>
</ol>
2. Cell Encapsulation
<ol>
	<li>Mix sodium alginate with deionized (DI) water for a final concentration of 2% w/v and purify by filtration with a 0.2 &#181;m sterile syringe filter.</li>
	<li>Prepare the cells to be mixed with the alginate solution by trypsinizing, centrifuging, and washing them with 10 mM HEPES buffered saline solution (pH = 7.4). Using a hemocytometer, count the cells and adjust the final cell concentration to 1 x 106 in alginate solution. 
	NOTE: The encapsulation process should be run inside a sterilized hood.</li>
	<li>Load ~300 &#181;L aliquots of alginate and cells mixture into a 3 mL syringe and attach it to a syringe pump. The solution will be pumped through a 30 G blunt tip needle to a sterile gelling bath placed in a sterile 50 mL beaker below the syringe tip at 7 mm for a needle to bath spraying distance.</li>
	<li>The gelling bath contains a volume of 40 mL of 10 mM HEPES buffered saline containing 100 mM calcium chloride (CaCl2) and 0.5% w/v poly-L-ornithine (PLO). The PLO is a secondary polymer coating that can be omitted or changed according to the needs of the investigator.</li>
	<li>Adjust the voltage and flow rate and keep them both constant during the encapsulation process at 60 mm/h flow rate and 6.0 kV initial voltage to produce microcapsules size of ~150 &#181;m.</li>
	<li>Connect the clip of anode wire (red) of a high voltage generator needle tip to the needle and connect the ground clip (black) to the copper wire that is halfway submerged in the gelling bath. One batch of the alginate + cell mixture (1 mL) takes approximately 30 min to prepare the encapsulated cells (approximately 25,000 microcapsules).</li>
	<li>Wash the formed microcapsules containing cells with washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl2, pH = 7.4) twice. Do not use PBS for washing.</li>
	<li>Incubate the encapsulated cells with 10% FBS supplemented DMEM media in a humidified incubator at 37 &#176;C and 5% CO2. 
	NOTE: Encapsulation process instruments are depicted in Figure 1.</li>
</ol>
3. Confirmation that Encapsulation Does Not Affect Cell Viability
<ol>
	<li>After incubating the encapsulated cells for 24 h in media, prepare a small sample of 500 &#181;L (~30 microcapsules) for staining.</li>
	<li>Wash the microcapsules 2x using washing solution (10 mM HEPES buffered saline containing 1.5 mM CaCl2) and stain them for live-dead viability using a live/dead assay kit.</li>
	<li>Prepare a staining mixture of calcein AM (acetoxymethyl) and ethidium homodimer-1 at final concentrations of 2 &#181;M and 4 &#181;M, respectively.</li>
	<li>Add 2 mL of the staining mixture to the encapsulated cells and incubate for 30&#8211;45 min in the dark at room temperature (RT).</li>
	<li>With careful aspiration, aspirate the staining solution and wash the microcapsules 2x with the washing solution. Use a fluorescent microscope system to observe and image the encapsulated cells. 
	NOTE: Documentation of encapsulation is depicted in Figure 2.</li>
</ol>
4. Confirmation That Capsules Are the Appropriate Size for Delivery and Delivery of the Biologic
<ol>
	<li>Intravitreal injections in a mouse eye are typically performed with a 27 G blunt-tip needle (inner diameter of 210 &#181;m) attached to a 2.5 &#181;L Hamilton syringe.</li>
	<li>Generate capsules ranging from 100 to 200 &#181;m, dilute them in serum-free media, and carefully pull them up into the Hamilton syringe. PBS is not a suitable vehicle, as alginate capsules will dissolve in PBS.</li>
	<li>Slowly eject 1 &#181;L drops containing capsules onto a microscope slide and determine their integrity using an upright bright-field microscope.</li>
	<li>Adjust the capsule size (see step 2.3) by adjusting voltage and flow rate accordingly. Smaller microcapsules are produced in a nonlinear fashion by increasing voltage and slightly decreasing flow rate8. In our hands, capsules of 150 &#181;m in diameter proved to be most suitable. Adjustment of capsule size is also dependent on keeping the alginate concentration constant while changing the other parameters.</li>
	<li>Maintain a separate set of cells in capsules of the appropriate size in serum-free medium to determine the amount of secretion of the desired biologic. Use sensitive ELISAs or western blotting to determine the concentration of the biologics in the supernatant.</li>
	<li>Determine the required amount of capsules that need to be injected based on the known PK/PD (pharmacokinetics/pharmacodynamics) of the therapeutic. In our hands, 10 capsules per mouse eye proved to be most efficacious.</li>
</ol>
5. Capsule Delivery into Mouse Vitreous
<ol>
	<li>Perform intravitreal injections using a dissecting microscope. See Figure 3 for surgical set-up and materials used during the procedure. A detailed protocol for intravitreous injections can be found elsewhere16.</li>
	<li>Anesthetize mice by intraperitoneal injection of xylazine and ketamine (20 mg/kg and 80 mg/kg) or other preferred anesthetics approved by the specific institution&#8217;s Animal Care and Use Committee. Ensure the appropriate depth of anesthesia using a toe pinch17.</li>
	<li>Dilate the mouse pupils with phenylephrine HCL (2.5%) and atropine sulfate (1%) to allow for good visibility of the vitreous chamber and apply a lubricant eye gel to the eyes to keep them hydrated during the procedure.</li>
	<li>Puncture the sclera at the limbus with a 26 G needle as the guide hole, making sure that 1) the needle is at a 45&#176; angle with the eye and table and 2) the beveled tip is pointed upwards to avoid puncturing the lens.</li>
	<li>Carefully inject the capsules using a 27 G blunt-tip needle attached to a Hamilton syringe at a 45&#176; angle under visual inspection, making sure to avoid touching the lens with the needle. Injury of the lens will lead to cataract formation. The capsules should be visible in the vitreous using the dissecting microscope (Figure 4A).</li>
	<li>After retraction of the needle, treat the injection site with antibiotic ointments neomycin and polymyxin B sulfates in addition to dexamethasone ophthalmic antibiotic ointment.</li>
	<li>Apply goniotaire hypromellose demulcent ophthalmic solution (2.5%) to both eyes to prevent the corneas from drying out during the recovery period.</li>
	<li>Place the mouse on a heating pad held at 37&#176;C and monitor until fully awake.</li>
	<li>A successful injection of encapsulated ARPE-19 cells should reveal the presence of intact capsules in the vitreous chamber of the mouse with only minor amounts of debris when imaging the eye by optical coherence tomography or other methods (Figure 4B).</li>
	<li>The injected mouse eye, after a few days of recovery due to the surgery, is now ready for the experimental paradigm at hand. 
	NOTE: The surgical set-up and documentation of capsules in the eye are depicted in Figure 3 and Figure 4, respectively.</li>
</ol>

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	<li>Allen, T. M., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+systems:+entering+the+mainstream.">Drug delivery systems: entering the mainstream.</a> Science. 303 (5665), 1818-1822 (2004).</li><li>Tonnesen, H. H., Karlsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+in+drug+delivery+systems.">Alginate in drug delivery systems.</a> Drug Development and Industrial Pharmacy. 28 (6), 621-630 (2002).</li><li>Vilos, C., Velasquez, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+strategies+based+on+polymeric+microparticles.">Therapeutic strategies based on polymeric microparticles.</a> Journal of Biomedical Biotechnology. 672760, (2012).</li><li>Gasperini, L., Mano, J. F., Reis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+polymers+for+the+microencapsulation+of+cells.">Natural polymers for the microencapsulation of cells.</a> Journal of the Royal Society Interface. 11 (100), 20140817 (2014).</li><li>Gasper, D. P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Novel strategy to produce a drug delivery system for skin regeneration. Uma nova estrat&#233;gia para produzir um dispositivo para entrega de f&#225;rmacos que ser&#225; usado na regenera&#231;&#227;o da pele. , (2012).</li><li>Huang, S., Fu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+materials-based+cell+and+drug+delivery+systems+in+skin+regeneration.">Naturally derived materials-based cell and drug delivery systems in skin regeneration.</a> Journal of Controlled Release. 142 (2), 149-159 (2010).</li><li>Nograles, N., Abdullah, S., Shamsudin, M. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+polymeric+microcapsules+containing+a+low+molecular+weight+peptide+for+controlled+release.">Characterization of polymeric microcapsules containing a low molecular weight peptide for controlled release.</a> Microscopy and Microanalysis. 19 (1), 213-226 (2013).</li><li>Xu, Y., Skotak, M., Hanna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+encapsulation+of+water-soluble+protein+with+polylactide.+I.+Effects+of+formulations+and+process+on+morphology+and+particle+size.">Electrospray encapsulation of water-soluble protein with polylactide. I. 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The authors declare no competing financial interests.

This cell encapsulation technique is relatively quick and easy to perform; however, certain points must be kept in mind to obtain accurate downstream results. Cells should be maintained in culture in a Petri dish prior to encapsulation and held at proper confluency. Encapsulation should be performed in a proper ventilation hood with regulated air flow, if possible. Too strong of an air current can affect capsule formation, especially in long-term experiments. Sterile utensils and solutions are critical for long-term maintenance of cells within the capsule.
At present, live-dead staining is used as a confirmatory tool to determine the viability of cells within the capsules. The number of cells per capsule and under the current condition (i.e., normally 12&#8211;20 cells per capsule) are also visually determined. The viability of each batch of encapsulated cell batches are monitored by this method. To further determine the viability of the cells, encapsulated cells are dissolved and re-cultured. This further demonstrates the viability and integrity of cells within the capsules, validating successful cellular encapsulation.
The parameters used for cellular encapsulation have been established for this particular cell type. The parameters stated above are those used for the encapsulation of ARPE 19 cells for these experiments. Flow rate, alginate concentration, voltage applied, and secondary coating of the capsules are all variables that can be adjusted for appropriate use of the capsules. Likewise, the amount of capsules required for a given experiment needs to be determined either empirically or based on the known PK/PD of the biologics. It is important to always perform appropriate control experiments, adding empty capsules to control for the presence of the capsules and capsules loaded with untransfected ARPE-19 cells to control for the presence of secreted factors. ARPE-19 cells can also be stably transfected with a control plasmid, as the presence of even a small number of capsules in the small vitreous of the mouse (&#60;10 &#181;L) appears to alter the normal physiology of the eye. Within this context, it is important to know the secretome of APRE-19 cells under encapsulation conditions (as well as in the presence of vitreous in a particular disease condition), as the secreted proteins may interfere with the efficacy of the biologic under investigation.
Finally, this technique was implemented to provide proof-of-principle for long-term delivery of complement inhibitors for the treatment of AMD and to improve the current method of intravitreal injections14. At the current stage of development, complement inhibitors are injected into the vitreous, typically using monthly injections. This includes injection of the complement factor D blocking antibody lampalizumab18, which failed in a phase III clinical trial to reduce the progression of geographic atrophy, or the complement factor 3 inhibitor APL-219, which is currently in a phase III clinical trial.
Intravitreal injection is hampered by side effects of the injection itself (i.e., risk of retinal detachment, rise in intraocular pressure, endophthalmitis, etc.). In addition, drug levels will vary significantly over the course of the month upon monthly intravitreal injections, and rebound reactions may be expected. As an alternative, gene therapy strategies are being developed, such as the soluble CD59 complement inhibitor20, which is currently in a phase I clinical trial. Encapsulated cells also allow for the continuous production of a biologic for extended periods of time and can be terminated (i.e., explanting of the capsule), if required.
To date, we have only tested for production of a biologic over the course of ~6 weeks14. It should be noted that the method described here should only be used for proof-of-principle in animal models and not for use in patients, as the alginate capsules are not sufficiently stable to completely prevent shredding during the injection and are not expected to last more than a few weeks. In contrast, a solid device such as that developed by Neurotech can last for years21 to deliver the required factors22,23,24. In addition, this new technique can also be combined with encapsulated drug delivery. Overall, it is expected that this emerging field will rapidly develop as an alternative strategy for repeated injections of therapeutics of gene therapy.

Cell-based therapies represent revolutionary biological techniques that have been applied widely in medicine. Recently, they have been successfully applied in the treatment of neurodegenerative diseases, eye diseases, and cancer. Cell therapies cover a wide range of fields from cell replacement to drug delivery, and this protocol focuses on the latter. Biodegradable alginate microcapsules (MC) have shown effectiveness as a delivery system, and they are becoming widely used in the biomedical field. Alginate has been used in microencapsulation due to its simple gelling process, biodegradability, excellent biocompatibility, and stability under in vivo conditions1,2,3,4.
The electrospray method, as a microencapsulation technique, has been successfully utilized to encapsulate peptides and proteins using alginate (base polymer) and poly-l-ornithine (secondary coating polymer). Both polymers are naturally found and used for their biocompatibility5,6,7. However, the main challenge in cell-based therapies is suppression of the host immune system to avoid side effects caused by immunosuppressive drugs. The permeability of alginate microcapsules is considered a suitable property for cell encapsulation, which allows diffusion of nutrients, waste, and therapeutic factors into and out of cells while masking them from the host immune response8,9,10.
In the eye, encapsulated cells have been used in clinical trials for the constant delivery of biologics (i.e., growth factors11,12 and growth factor antagonists13) for the treatment of retinitis pigmentosa or age-related macular degeneration. Other targets such as complement inhibitors14 are also currently being explored in preclinical settings.

<table>
	<tbody>
		<tr>
			<td>3 mL Syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 1&#34; Blunt needle</td>
			<td>SAI Infusion technology</td>
			<td>B30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt, from brown algae</td>
			<td>Sigma</td>
			<td>A0682</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulfate Ophthalmolic solution (1%)</td>
			<td>Akorn</td>
			<td>NDC 17478-215-15</td>
			<td>for pupil dilation</td>
		</tr>
		<tr>
			<td>BD 1 mL Syringe 26 G x 3/8 (0.45 mm x 10 mm)</td>
			<td>Becton, Dickinson and Company</td>
			<td>DG518105 500029609 REF 309625</td>
			<td>to generate the guide hole</td>
		</tr>
		<tr>
			<td>Calcium chloride, Anhydrous, granular</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal Tears</td>
			<td>Alcon</td>
			<td>NDC 0078-0429-47</td>
			<td>to lubricate the eyes during anesthesia</td>
		</tr>
		<tr>
			<td>Goniotaire: Hypromellose (2.5%) Ophthalmolic Demulcent Solution (Sterile)</td>
			<td>Altaire Pharmaceuticals Inc.</td>
			<td>NDC 59390-182-13</td>
			<td>to lubricate the eyes during anesthesia</td>
		</tr>
		<tr>
			<td>Hamilton Needle/syringe Tip: 27 Gauge, Small Hub RN NDL, custum length (12mm), point style 3, 6/PK</td>
			<td>Hamilton</td>
			<td>7803-01</td>
			<td>for intravitreal delivery of capsules</td>
		</tr>
		<tr>
			<td>Hamilton Syringe: 2.5 &#181;L, Model 62 RN SYR, NDL Sold Separately</td>
			<td>Hamilton</td>
			<td>7632-01</td>
			<td>for intravitreal delivery of capsules</td>
		</tr>
		<tr>
			<td>HEPES buffer, 1M</td>
			<td>Fisher Bioreagents</td>
			<td>BP299100</td>
			<td></td>
		</tr>
		<tr>
			<td>High voltage generator</td>
			<td>ESD EMC Technology</td>
			<td>ES813-D20</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Thermofisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ornithine hydrochloride, 99%</td>
			<td>Alfa Aesar</td>
			<td>A12111</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin and Polymyxin B Sulfates and Dexamethasone Ophthalmolic Ointment</td>
			<td>SANDOZ</td>
			<td>NDC 61314-631-36</td>
			<td>antibiotic to prevent infection after intravitreal injection</td>
		</tr>
		<tr>
			<td>Phenolephrine Hydrochloride Ophthalmolic Solution (2.5%)</td>
			<td>Akorn</td>
			<td>NDC 17478-201-15</td>
			<td>for pupil dilation</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filters, 0.2 um</td>
			<td>VWR</td>
			<td>28143-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>GRASEBY</td>
			<td>MS16A</td>
			<td></td>
		</tr>
	</tbody>
</table>

encapsulated cell technology for the delivery of biologics to the mouse eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

ARPE-19 cells are a spontaneously immortalized human RPE cell line that has been shown to be amenable to encapsulation and long-term survival upon implantation of capsules into the eye. The tools for alginate encapsulation are shown in Figure 1. In this study, it was demonstrated that upon encapsulation in alginate, the cells in alginate capsules were confirmed by bright-field imaging (Figure 2A). Live-dead assays were performed on the cells inside the capsules, demonstrating 90% viability post-encapsulation (Figure 2B). To ensure long-term viability of the cells inside capsules, capsules were dissolved in sodium citrate and cells were then re-plated (Figure 2C). After confirming in independent experiments that the stable transfected ARPE-19 cells expressed and secreted the biologic that was desired14, and after establishing the parameters for safely encapsulating cells, capsules were produced for intraocular injection.
Intravitreal injections into the mouse eye requires the use of a 27 G blunt-tip needle (inner diameter of 210 &#181;m) and accurate delivery system to consistently inject the required small volume of 1 &#181;L. The size of capsules that were not damaged during ejection through the 27 G needle was confirmed by microscopy (Figure 2A). An optimal size of 150 &#181;m was identified. Intravitreal injection was performed under visual inspection, which allowed the visualization of capsules in the vitreous (Figure 4A). Likewise, OCT imaging was performed, which confirmed that a majority of capsules in the vitreous chamber of the mouse were intact with relatively minor amounts of debris (Figure 4B). In follow-up published experiments, it was confirmed that the desired biologic was present in the eye at therapeutically relevant doses, inhibiting the disease process under investigation14. These results confirmed that encapsulated cells can safely be injected into mouse eyes for the long-term delivery of biologics.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60162/60162fig01.jpg" /> 
Figure 1: Encapsulation process instruments.&#160;The set-up includes (A) a 3 mL syringe, (B) 0.2 &#181;m filters, (C) a 30 G, &#189; inch blunt-tip needle, (D) an electronic syringe driver, (E) a high voltage generator, (F) the system set-up, and (G) a 7 mm fixed distance between the needle tip and gelling solution surface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60162/60162fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60162/60162fig02.jpg" /> 
Figure 2: Cell encapsulation.&#160;(A) Microcapsules filled with ARPE 19 cells. (B) Live/ dead assay: [a] red fluorescent color indicates dead cells, and [b] green fluorescent color indicates live cells. (C) Cultured cells after recovery from microcapsules beginning with a small cluster growing to confluency. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60162/60162fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60162/60162fig03.jpg" /> 
Figure 3: Surgical set-up.&#160;A dissecting microscope (1) with a video camera (2) is used to visualize the procedure. The mouse is weighed (3) to administer the appropriate amount of anesthetic and placed on a small platform (4) to ease handling and injection. Eyes are dilated with mydriatics atropine and phenylepinephrin (5) and are kept hydrated with lubricant (6). A guide hole is punctured just outside the limbus using a 26 G needle (7). The glass syringe (8), loaded with appropriately diluted capsules (9), is placed at a 45&#176; angle to the mouse eye and advanced through the guide hole, using a micromanipulator (10). The needle track as well as the capsules being released can be visualized [(11); see Figure 4]. Upon retraction of the needle, the cornea is moistened with hypromellose (12) and antibiotic ointment (13) applied to the injection site to avoid infection. Following the procedure, the mouse will be placed on a 37 &#176;C heating pad and monitored until fully awake. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60162/60162fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60162/60162fig4v2.jpg" /> 
Figure 4: Encapsulated cell technology to deliver ARPE-19 into the eye.&#160;(A) Injections are performed using a blunt 27 G needle attached to a Hamilton syringe. The needle track can be followed as the tip of the needle enters the eye, avoiding the lens and the capsules (arrowhead) can been visualized, magnified by the optical system of the mouse eye. It should be noted that the circular reflection of the light source. (B) Capsules (arrowhead) can be imaged in the vitreous using optical coherence tomography. Scale bars = 200 &#181;m. Panel B was reprinted with permission from Annamalai et al.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60162/60162fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye at JoVE.com

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

encapsulated-cell-technology-for-delivery-biologics-to-mouse

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.2K Views.  University of South Carolina. Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Video: Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.