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...

<ol>
	<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. N., Billa, N., Rosli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+characterization+of+pDNA-loaded+alginate+microspheres+for+oral+administration+in+mice.">Formation and characterization of pDNA-loaded alginate microspheres for oral administration in mice.</a> Journal of Bioscience and Bioengineering. 113 (2), 133-140 (2012).</li><li>Moore, K., Amos, J., Davis, J., Gourdie, R., Potts, J. 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. Effects of formulations and process on morphology and particle size.</a> Journal of Microencapsulation. 23 (1), 69-78 (2006).</li><li>Gryshkov, O., 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=Process+engineering+of+high+voltage+alginate+encapsulation+of+mesenchymal+stem+cells.">Process engineering of high voltage alginate encapsulation of mesenchymal stem cells.</a> Materials Science and Engineering: C. 36, 77-83 (2014).</li><li>Thanos, C. G., 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=Sustained+secretion+of+ciliary+neurotrophic+factor+to+the+vitreous,+using+the+encapsulated+cell+therapy-based+NT-501+intraocular+device.">Sustained secretion of ciliary neurotrophic factor to the vitreous, using the encapsulated cell therapy-based NT-501 intraocular device.</a> Tissue Engineering. (11-12), 1617-1622 (2004).</li><li>Kauper, K., 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=Two-year+intraocular+delivery+of+ciliary+neurotrophic+factor+by+encapsulated+cell+technology+implants+in+patients+with+chronic+retinal+degenerative+diseases.">Two-year intraocular delivery of ciliary neurotrophic factor by encapsulated cell technology implants in patients with chronic retinal degenerative diseases.</a> Investigative Ophthalmology &amp; Visual Science. 53 (12), 7484-7491 (2012).</li><li>Kauper, K., 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=Long+term,+sustained+intraocular+delivery+of+a+VEGF+antagonist+using+encapsulated+cell+technology+implant+for+the+treatment+of+choroidal+neovascular+diseases.">Long term, sustained intraocular delivery of a VEGF antagonist using encapsulated cell technology implant for the treatment of choroidal neovascular diseases.</a> Investigative Ophthalmology &amp; Visual Science. 53, 455 (2012).</li><li>Annamalai, B., 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=Encapsulated+Cell+Technology-Based+Delivery+of+a+Complement+Inhibitor+Reduces+Choroidal+Neovascularization+in+a+Mouse+Model.">Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model.</a> Translational Visual Science Technology. 7 (2), 3 (2018).</li><li>Alge, C. 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=Retinal+Pigment+Epithelium+Is+Protected+Against+Apoptosis+by+&#945;B-Crystallin.">Retinal Pigment Epithelium Is Protected Against Apoptosis by &#945;B-Crystallin.</a> Investigative Ophthalmology &amp; Visual Science. 43 (11), 3575-3582 (2002).</li><li>Chiu, K., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreous+injection+for+establishing+ocular+diseases+model.">Intravitreous injection for establishing ocular diseases model.</a> Journal of Visualized Experiments. (8), 313 (2007).</li><li>Jove Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.+Anesthesia+Induction+and+Maintenance.">Lab Animal Research. Anesthesia Induction and Maintenance.</a> Journal of Visualized Experiments. , (2019).</li><li>Holz, F. G., 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=Efficacy+and+Safety+of+Lampalizumab+for+Geographic+Atrophy+Due+to+Age-Related+Macular+Degeneration:+Chroma+and+Spectri+Phase+3+Randomized+Clinical+Trials.">Efficacy and Safety of Lampalizumab for Geographic Atrophy Due to Age-Related Macular Degeneration: Chroma and Spectri Phase 3 Randomized Clinical Trials.</a> JAMA Ophthalmology. 136 (6), 666-677 (2018).</li><li>Kassa, E., Ciulla, T. A., Hussain, R. M., Dugel, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+inhibition+as+a+therapeutic+strategy+in+retinal+disorders.">Complement inhibition as a therapeutic strategy in retinal disorders.</a> Expert Opinion in Biological Therapy. 19 (4), 335-342 (2019).</li><li>Cashman, S. M., Ramo, K., Kumar-Singh, 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+Non+Membrane-Targeted+Human+Soluble+CD59+Attenuates+Choroidal+Neovascularization+in+a+Model+of+Age+Related+Macular+Degeneration.">A Non Membrane-Targeted Human Soluble CD59 Attenuates Choroidal Neovascularization in a Model of Age Related Macular Degeneration.</a> PLoS ONE. 6 (4), e19078 (2011).</li><li>Vincent, 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=Generation+of+combination+PDGF+/+VEGF-antagonist+ECT+devices.">Generation of combination PDGF / VEGF-antagonist ECT devices.</a> Investigative Ophthalmology &amp; Visual Science. 54, 3290 (2013).</li><li>Zhang, K., 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=Ciliary+neurotrophic+factor+delivered+by+encapsulated+cell+intraocular+implants+for+treatment+of+geographic+atrophy+in+age-related+macular+degeneration.">Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration.</a> Proc Natl Acad Sci USA. 108 (15), 6241-6245 (2011).</li><li>Chew, E. Y., 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=Ciliary+neurotrophic+factor+for+macular+telangiectasia+type+2:+results+from+a+phase+1+safety+trial.">Ciliary neurotrophic factor for macular telangiectasia type 2: results from a phase 1 safety trial.</a> American Journal of Ophthalmology. 159 (4), 659-666 (2015).</li><li>Birch, D. G., 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=Randomized+trial+of+ciliary+neurotrophic+factor+delivered+by+encapsulated+cell+intraocular+implants+for+retinitis+pigmentosa.">Randomized trial of ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for retinitis pigmentosa.</a> American Journal of Ophthalmology. 156 (2), 283-292 (2013).</li></ol>

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 main...

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,...

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, ...

This technique is a dependable way to deliver any content such as drugs, proteins, or cells to specific locations in tissues or animals. You can track the delivery and tune the release of the contents to fit the experiment you desire. It can be used to treat numerous diseases from wound healing to ocular and cardiac diseases.The main advantage of this method is the ability to deliver therapeutic cells using alginate microcapsules to the target sites without causing any immune reactions and maintain a sustained delivery of fresh secreted therapeutics for a longer period of time. Begin by culturing retinal epithelial cells according to manuscript directions. Passage the cells when they reach 70 to 80%confluency using standard tissue culture procedures.To encapsulate the cells, mix sodium alginate with deionized water for a final concentration of 2%weight to volume and purify the mixture via filtration with a 0.2 micrometer sterile syringe filter. Trypsinize, centrifuge, and wash the cells with 10 millimolar HEPES buffered saline solution. Use a hemocytometer to count the cells and adjust the final cell concentration to one million per one milliliter of alginate solution.Load 300 microliter aliquots of the alginate and cells mixture into a three milliliter syringe and attach it to a syringe pump. Prepare a gelling bath according to manuscript directions and place it in a sterile 50 milliliter beaker below the syringe tip at a seven millimeter needle to bath spraying distance. Adjust the voltage to eight kilovolts and the flow rate to 30 millimeters per hour to produce microcapsules that are about 150 micrometers in size.Connect the black ground clip to the copper wire that is halfway submerged in the gelling bath and connect the clip of the red anode wire to the needle. It takes approximately 30 minutes to prepare encapsulated cells out of each one milliliter batch of the alginate and cell mixture. When formed, wash the microcapsules with washing solution twice and incubate them with 10%FBS supplemented DMEM media in a humidified incubator at 37 degrees Celsius and 5%CO2.After a 24-hour incubation, prepare a 500 microliter sample of microcapsules for staining by washing them twice in washing solution. Use a LIVE/DEAD assay kit to stain them for viability. To ensure that the microcapsules are appropriately sized, dilute them in serum-free media.Carefully aspirate them with a 2.5 microliter Hamilton syringe. Slowly eject one microliter drops onto a microscope slide and determine the integrity of the microcapsules using an upright Brightfield microscope. In an appropriately anesthetized mouse, apply ocular lubricant to maintain eye moisture and use a 26 gauge 3/8 inch beveled syringe needle to establish an initial eye puncture just below the limbus.Make sure to approach the eye from a nasal direction at a 45 degree angle keeping the beveled tip upward to avoid lens perforation. Next, ensure that the entire needle tip is inserted into the eye for guide hole generation, but be careful not to travel too far and puncture the temporal retina. Once this is complete, slowly retract the needle.Within the micromanipulator, place a 27 gauge blunt tip 2.5 microliter Hamilton syringe that's been filled with capsule solution and line the needle up with the initial guide hole. Hold the animal's head in a stable position and deliberately maneuver the needle towards the eye. To ensure a correct needle position within the vitreous, insert the syringe until the needle tip can be visualized behind the lens.Then inject the capsule solution slowly over the course of 25 to 30 seconds. And once completed, gradually retract the blunt tip syringe in order to minimize injury to the eye. If necessary, adjust the capsule size by adjusting the voltage and flow rate according to manuscript directions.The encapsulation of the cells in alginate was confirmed with Brightfield imaging and a LIVE/DEAD assay demonstrated that 90%of cells were viable post-encapsulation. Long-term viability of the cells was ensured by dissolving the capsules in sodium citrate and replating. Intravitreal injection was performed under visual inspection which allowed for the visualization of capsules in the vitreous.When attempting this procedure, do not use PBS for any washing steps because PBS dissolves formed alginate microcapsules. After its development, our lab is used as a delivery system to treat forms of heart failure and hypertension. This technique is considered a promising tool for the future treatment of different cardiovascular and neurological diseases.

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Research

JoVE Journal

Bioengineering

5.9K Views.  University of South Carolina. This technique is a dependable way to deliver any content such as drugs, proteins, or cells to specific locations in tissues or animals. You can track the delivery and tune the release of the contents to fit the experiment you desire. It can be used to treat numerous diseases from wound healing to ocular and cardiac diseases.The main advantage of this method is the ability to deliver therapeutic cells using alginate microcapsules to the target sites without causing any immune reactions...