Name: production of modified autologous conditioned serum and ex vivo assessment of its healing potential in murine corneal epithelium
Uploaded: 2023-03-24T14:15:00
Description: Watch this Scientific Journal Video about Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium at JoVE.com

The authors thank Ya-Lan Chien and Chia-Ying Lee for excellent technical assistance, and OnLine English company for the linguistic edition. This study was funded in part by Chang Gung Medical Research Project (Grant No. CMRPG3L1491).

The research was performed in compliance with institutional guidelines at the beginning of the protocol section. All protocols and procedures were carried out according to the Declaration of Helsinki and were reviewed and approved by the Chang Gung Medical Foundation Institutional Review Board. All volunteers were informed of the nature of this study and signed an informed consent form prior to their inclusion. The consumables required for the entire experimental procedure are presented in Figure 1</...

<ol>
	<li>Fox, R. I., Chan, R., Michelson, J. B., Belmont, J. B., Michelson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effect+of+artificial+tears+made+with+autologous+serum+in+patients+with+keratoconjunctivitis+sicca.">Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca.</a> Arthritis and Rheumatology. 27 (4), 459-461 (1984).</li><li>Noble, B. A., 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=Comparison+of+autologous+serum+eye+drops+with+conventional+therapy+in+a+randomised+controlled+crossover+trial+for+ocular+surface+disease.">Comparison of autologous serum eye drops with conventional therapy in a randomised controlled crossover trial for ocular surface disease.</a> The British Journal of Ophthalmology. 88 (5), 647-652 (2004).</li><li>Bradley, J. C., Bradley, R. H., McCartney, D. L., Mannis, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+growth+factor+analysis+in+dry+eye+syndrome.">Serum growth factor analysis in dry eye syndrome.</a> Clinical &amp; Experimental Ophthalmology. 36 (8), 717-720 (2008).</li><li>Alshammari, T. M., Al-Hassan, A. A., Hadda, T. B., Aljofan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+serum+sample+extraction+methods+and+their+suitability+for+mass+spectrometry+analysis.">Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis.</a> Saudi Pharmaceutical Journal. 23 (6), 689-697 (2015).</li><li>Tsubota, 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=Treatment+of+dry+eye+by+autologous+serum+application+in+Sj&#246;gren's+syndrome.">Treatment of dry eye by autologous serum application in Sj&#246;gren's syndrome.</a> The British Journal of Ophthalmology. 83 (4), 390-395 (1999).</li><li>Urzua, C. A., Vasquez, D. H., Huidobro, A., Hernandez, H., Alfaro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+double-blind+clinical+trial+of+autologous+serum+versus+artificial+tears+in+dry+eye+syndrome.">Randomized double-blind clinical trial of autologous serum versus artificial tears in dry eye syndrome.</a> Current Eye Research. 37 (8), 684-688 (2012).</li><li>Cui, D., Li, G., Akpek, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum+eye+drops+for+ocular+surface+disorders.">Autologous serum eye drops for ocular surface disorders.</a> Current Opinion in Allergy and Clinical Immunology. 21 (5), 493-499 (2021).</li><li>Jirsova, 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=The+application+of+autologous+serum+eye+drops+in+severe+dry+eye+patients;+subjective+and+objective+parameters+before+and+after+treatment.">The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment.</a> Current Eye Research. 39 (1), 21-30 (2014).</li><li>Pan, Q., Angelina, A., Marrone, M., Stark, W. J., Akpek, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum+eye+drops+for+dry+eye.">Autologous serum eye drops for dry eye.</a> Cochrane Database of Systematic Reviews. 2 (2), (2017).</li><li>Wang, 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=Autologous+serum+eye+drops+versus+artificial+tear+drops+for+dry+eye+disease:+a+systematic+review+and+meta-analysis+of+randomized+controlled+trials.">Autologous serum eye drops versus artificial tear drops for dry eye disease: a systematic review and meta-analysis of randomized controlled trials.</a> Ophthalmic Research. 63 (5), 443-451 (2020).</li><li>Soni, N. G., Jeng, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-derived+topical+therapy+for+ocular+surface+diseases.">Blood-derived topical therapy for ocular surface diseases.</a> The British Journal of Ophthalmology. 100 (1), 22-27 (2016).</li><li>Solomon, A., 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=anti-inflammatory+forms+of+interleukin-1+in+the+tear+fluid+and+conjunctiva+of+patients+with+dry-eye+disease.">anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease.</a> Investigative Ophthalmology and Visual Science. 42 (10), 2283-2292 (2001).</li><li>Meijer, H., Reinecke, J., Becker, C., Tholen, G., Wehling, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+anti-inflammatory+cytokines+in+whole+blood+by+physico-chemical+induction.">The production of anti-inflammatory cytokines in whole blood by physico-chemical induction.</a> Inflammation Research. 52 (10), 404-407 (2003).</li><li>Bielory, B. P., Shah, S. P., O'Brien, T. P., Perez, V. L., Bielory, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+therapeutics+for+ocular+surface+disease.">Emerging therapeutics for ocular surface disease.</a> Current Opinion in Allergy and Clinical Immunology. 16 (5), 477-486 (2016).</li><li>Stevenson, W., Chauhan, S. K., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+disease:+an+immune-mediated+ocular+surface+disorder.">Dry eye disease: an immune-mediated ocular surface disorder.</a> Archives of Ophthalmology. 130 (1), 90-100 (2012).</li><li>Yang, J., Guo, A., Li, Q., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+attenuates+interleukin-1&#946;-induced+apoptosis+and+inflammation+in+chondrocytes+through+targeting+hypoxia-inducible+factor-2&#945;.">Platelet-rich plasma attenuates interleukin-1&#946;-induced apoptosis and inflammation in chondrocytes through targeting hypoxia-inducible factor-2&#945;.</a> Tissue and Cell. 73, 101646 (2021).</li><li>Shakouri, S. K., Dolati, S., Santhakumar, J., Thakor, A. S., Yarani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+conditioned+serum+for+degenerative+diseases+and+prospects.">Autologous conditioned serum for degenerative diseases and prospects.</a> Growth Factors. 39 (1-6), 59-70 (2021).</li><li>Gull, M., Pasek, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glycerol+and+its+derivatives+in+the+biochemistry+of+living+organisms,+and+their+prebiotic+origin+and+significance+in+the+evolution+of+life.">The role of glycerol and its derivatives in the biochemistry of living organisms, and their prebiotic origin and significance in the evolution of life.</a> Catalysts. 11 (1), 86 (2021).</li><li>Drew, V. J., Tseng, C. L., Seghatchian, J., Burnouf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+dry+eye+syndrome+treatment:+therapeutic+role+of+blood+products.">Reflections on dry eye syndrome treatment: therapeutic role of blood products.</a> Frontiers in Medicine. 5, 33 (2018).</li><li>Hung, K. H., Yeh, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+and+in+vivo+animal+models+for+mechanical+and+chemical+injuries+of+corneal+epithelium.">Ex vivo and in vivo animal models for mechanical and chemical injuries of corneal epithelium.</a> Journal of Visualized Experiments. (182), e63217 (2022).</li><li>Geerling, G., Maclennan, S., Hartwig, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum+eye+drops+for+ocular+surface+disorders.">Autologous serum eye drops for ocular surface disorders.</a> The British Journal of Ophthalmology. 88 (11), 1467-1474 (2004).</li><li>Rutgers, M., Saris, D. B., Dhert, W. J., Creemers, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+autologous+conditioned+serum+for+treatment+of+osteoarthritis,+in+vitro+effects+on+cartilage+metabolism+and+intra-articular+levels+after+injection.">Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection.</a> Arthritis Research &amp; Therapy. 12 (3), R114 (2010).</li><li>Antebi, 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=Short-term+physiological+hypoxia+potentiates+the+therapeutic+function+of+mesenchymal+stem+cells.">Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells.</a> Stem Cell Research &amp; Therapy. 9 (1), 265 (2018).</li><li>Chen, Y. M., Wang, W. Y., Lin, Y. C., Tsai, S. H., Lou, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+autologous+serum+eye+drops+with+contact+lenses+in+the+treatment+of+chemical+burn-induced+bilateral+corneal+persistent+epithelial+defects.">Use of autologous serum eye drops with contact lenses in the treatment of chemical burn-induced bilateral corneal persistent epithelial defects.</a> BioMed Research International. 2022, 6600788 (2022).</li><li>Diaz-Valle, 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=Comparison+of+the+efficacy+of+topical+insulin+with+autologous+serum+eye+drops+in+persistent+epithelial+defects+of+the+cornea.">Comparison of the efficacy of topical insulin with autologous serum eye drops in persistent epithelial defects of the cornea.</a> Acta Ophthalmologica. 100 (4), e912-e919 (2022).</li><li>Metheetrairut, C., 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=Comparison+of+epitheliotrophic+factors+in+platelet-rich+plasma+versus+autologous+serum+and+their+treatment+efficacy+in+dry+eye+disease.">Comparison of epitheliotrophic factors in platelet-rich plasma versus autologous serum and their treatment efficacy in dry eye disease.</a> Scientific Reports. 12 (1), 8906 (2022).</li><li>NaPier, E., Camacho, M., McDevitt, T. F., Sweeney, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+keratopathy:+current+challenges+and+future+prospects.">Neurotrophic keratopathy: current challenges and future prospects.</a> Annals of Medicine. 54 (1), 666-673 (2022).</li><li>Garcia-Conca, V., 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+treatment+of+hyposecretory+dry+eye+with+platelet-rich+plasma.">Efficacy and safety of treatment of hyposecretory dry eye with platelet-rich plasma.</a> Acta Ophthalmologica. 97 (2), e170-e178 (2019).</li><li>Gholian, 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=Use+of+autologous+conditioned+serum+dressings+in+hard-to-heal+wounds:+a+randomised+prospective+clinical+trial.">Use of autologous conditioned serum dressings in hard-to-heal wounds: a randomised prospective clinical trial.</a> Journal of Wound Care. 31 (1), 68-77 (2022).</li><li>Raeissadat, S. A., Rayegani, S. M., Jafarian, N., Heidari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+conditioned+serum+applications+in+the+treatment+of+musculoskeletal+diseases:+a+narrative+review.">Autologous conditioned serum applications in the treatment of musculoskeletal diseases: a narrative review.</a> Future Science OA. 8 (2), 776 (2022).</li><li>Tokawa, P. K. A., Brossi, P. M., Baccarin, R. Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+conditioned+serum+in+equine+and+human+orthopedic+therapy:+A+systematic+review.">Autologous conditioned serum in equine and human orthopedic therapy: A systematic review.</a> Research in Veterinary Science. 146, 34-52 (2022).</li><li>Evans, C. H., Chevalier, X., Wehling, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+conditioned+serum.+Physical+Medicine+and+Rehabilitation.">Autologous conditioned serum. Physical Medicine and Rehabilitation.</a> Clinics of North America. 27 (4), 893-908 (2016).</li><li>Coskun, H. S., Yurtbay, A., Say, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+rich+plasma+versus+autologous+conditioned+serum+in+osteoarthritis+of+the+knee:+clinical+results+of+a+five-year+retrospective+study.">Platelet rich plasma versus autologous conditioned serum in osteoarthritis of the knee: clinical results of a five-year retrospective study.</a> Cureus. 14 (4), e24500 (2022).</li></ol>

In this study, a protocol for the preparation of mACS is described and the benefit of mACS eye drops in the wound healing of animal models is further shown. The crucial modification of this mACS protocol is the addition of approximately 0.5 mL of 10% glycerol solution in each test tube, which creates suitable hypoxic conditions during the 4 h incubation at 37 &#176;C. This setting provides the AS with proper stress and prompt cells to secrete the necessary growth factors that help wound healing. The 0.22 &#181;m filter c...

The therapeutic effects of autologous serum (AS) in dry eye diseases were first reported in the 1980s by Fox et al.1. It is believed that both the lubricating property and the essential epitheliotropic biochemical components in AS, mimicking natural tears, benefit the proliferation of corneal epithelial cells. Over the past decades, several studies have been performed on this basis. Trophic components include epidermal growth factor (EGF), vitamin A, transforming growth factor &#946; (TGF- &#946;), and other cytokines. Interestingly, the serum is rich in TGF- &#946; and vitamin A, which are believed to play a pivotal role in epidermal proliferation2,3,4,5. In addition, when treating patients with ocular surface diseases, several studies have showed some advantages of AS eye drops in patient-reported outcomes, other objective dry eye parameters6,7, and microscopic findings such as cell density8. Meta-analysis studies revealed that there might be some benefits in improving patient&#39;s syndromes with AS eye drops treatment, but long-term results and observations are still lacking9,10.
Unlike AS, platelet-rich plasma (PRP) is derived from adding an anticoagulant during preparation, with further differential centrifugation and chemical activation of the platelets. Compared with AS, numerous chemicals and growth factors, such as TGF- &#946;, vascular endothelial growth factor (VEGF), and EGF, are present in PRP. It has also been applied to ocular surface diseases with clinical benefits in symptom relief11.
The cross-link between epithelial defects and inflammation is complex. Notably, immunopathophysiology is another important issue in ocular surface diseases. Pro-inflammatory cytokines, such as IL-1&#946; and IFN-&#947;, are believed to be pivotal mediators in inflammatory cascades12. New avenues of treatment are thus opened based on understanding the immune mechanism. Strategies to stop this inflammatory process, including the production of interleukin-1 receptor antagonist (IL-1Ra) and other anti-inflammatory cytokines, may also play an important role in ocular surface diseases13,14,15.
Since 1998, Orthokine, a commercialized autologous conditioned serum (ACS), has been used clinically in orthopedic patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), and spinal disorders13. Compared with AS and PRP, treatment with chemically coated glass beads and hypoxic incubation to activate monocytes are the specific features of ACS16. Theoretically, more anti-inflammatory factors can be secreted by adding survival stress to the cells, resulting in a higher concentration of essential immune-modulating components, including IL-1Ra. The improved therapeutic benefits of ACS in OA, compared with AS, have also been reported17. Ocular surface diseases share similar immune backgrounds with orthopedic inflammatory diseases in some respects. Therefore, based on the successful results of human blood-derived therapy in the orthopedic field, ACS might have advantages over conventional treatments in clinical practice by epitheliotropic and immune-modulating properties. Although ACS has been widely used in orthopedic inflammatory diseases, its clinical applications in ophthalmology still need to be explored, which may be hindered by its high cost, lack of literature support, and lack of standardization of the preparation process, resulting in diverse performance.
In this video article, a novel, cost-effective, and convenient method was demonstrated to generate the modified ACS (mACS), or plasma rich in growth factors (PRGF), producing an eye drop solution with a comparable practical value to commercialized ACSs. The key ideas of adding anticoagulants and triggering the blood cells to secrete anti-inflammatory cytokines by stressed incubation were retained, but unlike the chemically-induced methods, such as those based on CrSO4-coated glass beads and commercial kits, the critical stress status is physically induced by hypoxic incubation in this method. Moreover, glycerol was added to provide extra benefits, including an increase in the stability of the membrane of blood cells, maintenance of a proper osmotic extracellular fluid pressure18, and an appropriate source of nutrients in hypoxic conditions that avoid&#160;overstressing the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;96-well culture plate</td>
			<td>Merck KGaA, Germany</td>
			<td>CLS3997</td>
			<td></td>
		</tr>
		<tr>
			<td>Barraquer lid speculum</td>
			<td>katena</td>
			<td>K1-5355</td>
			<td>15 mm</td>
		</tr>
		<tr>
			<td>Barraquer needle holder</td>
			<td>Katena</td>
			<td>K6-3310</td>
			<td>without lock&#160;</td>
		</tr>
		<tr>
			<td>Barron Vacuum Punch 8.0 mm</td>
			<td>katena</td>
			<td>K20-2108</td>
			<td>for cutting filter paper</td>
		</tr>
		<tr>
			<td>BD 10.0 mL vacutainer tubes containing heparin 158 USP units</td>
			<td>Becton,Dickinson and Company, US</td>
			<td>367880</td>
			<td>At least 6 tubes, necessary to collect blood for subsequent experiments and to avoid blood agglutination</td>
		</tr>
		<tr>
			<td>BD 21 G butterfly-winged infusion set</td>
			<td>Becton,Dickinson and Company, US</td>
			<td>367281</td>
			<td>For even distribution of glycerol solution</td>
		</tr>
		<tr>
			<td>C57BL/6 mice&#160;</td>
			<td>National Laboratory Animal Center</td>
			<td>RMRC11005</td>
			<td>for mouse model</td>
		</tr>
		<tr>
			<td>Castroviejo forceps 0.12 mm</td>
			<td>katena&#160;</td>
			<td>K5-2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td>5811000428</td>
			<td>3,500 x g for 10 min</td>
		</tr>
		<tr>
			<td>Cheng Yi 10.0 mL sterilized eye dropper bottle</td>
			<td>Cheng Yi Chemical, Taiwan</td>
			<td>CP405141</td>
			<td>Must be sterile and as the storage container for the final product</td>
		</tr>
		<tr>
			<td>Corneal rust ring remover with 0.5 mm burr</td>
			<td>Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX</td>
			<td>CHI-675</td>
			<td>for debridement of the corneal epithelium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified minimal essential medium</td>
			<td>Merck KGaA, Germany</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper&#160;</td>
			<td>Toyo Roshi Kaisha,Ltd.</td>
			<td>1.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium ophthalmic strips U.S.P</td>
			<td>OPTITECH</td>
			<td>OPTFL100</td>
			<td>staining for corneal epithelial defect&#160;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Firstek, Taiwan</td>
			<td>S300S</td>
			<td>37 &#176;C for 4 h</td>
		</tr>
		<tr>
			<td>Kanam sterile gloves</td>
			<td>Kanam Latex Industries, India</td>
			<td>EN455</td>
			<td>For aseptic operation</td>
		</tr>
		<tr>
			<td>Merck 0.22 &#181;m filter</td>
			<td>Merck KGaA, Germany</td>
			<td>PR05359</td>
			<td>At least 2 filters for mACS filtration</td>
		</tr>
		<tr>
			<td>Nang Kuang 250 mL 10% glycerol solution</td>
			<td>Nang Kuang Pharmaceutical, Taiwan</td>
			<td>19496</td>
			<td>To offer suitable membrane stabilization effect and extracellular osmotic pressure for blood cells</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>TAIWAN BIOTECH CO., LTD.</td>
			<td>100-120-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin biopsy punch 2mm</td>
			<td>STIEFEL</td>
			<td>22650</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss Meditec, Dublin, CA</td>
			<td>SV11</td>
			<td>microscope for surgery</td>
		</tr>
		<tr>
			<td>Terumo 18 G needle</td>
			<td>Terumo, Taiwan</td>
			<td>SMACF0120-18BX</td>
			<td>3.0 mL syringe with 18 G needle to extract the supernatant after centrifugation</td>
		</tr>
		<tr>
			<td>Terumo 20.0 mL syringe</td>
			<td>Terumo, Taiwan</td>
			<td>MDSS20ES</td>
			<td>Could be used to collect serum after initial centrifugation and use it for secondary centrifugation.</td>
		</tr>
		<tr>
			<td>Terumo 3.0 mL syringe with the 23 G needle</td>
			<td>Terumo, Taiwan</td>
			<td>MDSS03S2325</td>
			<td>3.0 mL syringe is used to extract the supernatant after centrifugation. Then connect the filter and the 23 G needle for injection into the eye drop bottles.</td>
		</tr>
		<tr>
			<td>Westcott Tenotomy Scissors Medium</td>
			<td>katena</td>
			<td>K4-3004</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of modified autologous conditioned serum and ex vivo assessment of its healing potential in murine corneal epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Figure 1&#160;and Figure 2 show the materials needed for the experiment, and Figure 3 displays the sequential steps and the successful mid-products during the preparation of mACS. First, 0.5 mL of 10% glycerol solution was added into each 10 mL sterile test tube (Figure 3A). Then, 60-70 mL of venous blood was obtained from the patient, and 10 mL of blood was injected into each tube (<strong class="xfig"...

Watch this Scientific Journal Video about Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium at JoVE.com

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

This process presents an innovative blood derived product called mACS, which does not require any complex technical processing. It also accelerates wound healing to a greater extent than conventional autologous serum. In addition to providing a standardized preparation process, this technique can also reduce the cost of serum eyedrops, improving patient accessibility to a more affordable treatment option.This technique may be applied in fields that require promoting wound or tissue regeneration, repair, and anti-inflammation. This technique will be a great advantage for difficult to heal wounds and chronic inflammatory diseases such as severe ocular surface diseases. To begin, inject 10 milliliters of the previously drawn human venous blood into each of the six vacutainer tubes.Place the tubes in an incubator at 37 degrees Celsius for four hours and then centrifuge the tubes at 3, 500 G for 10 minutes at room temperature. Decap the tubes after placing them on the rack. After putting on sterile gloves, use a three milliliter syringe equipped with an 18 gauge needle to draw out the mACS from each tube.Pull out the needle and attach a 0.22 micrometer filter. Connect the 23 gauge, 1.5 inch blood collection needle with the original three milliliter syringe to the outlet of the filter. Push the needle gently through the filter into the prepared sterile eyedrop bottles for immediate use, store the eyedrops at four degrees Celsius.In the ex-vivo surface healing model, subjects treated with the mACS eyedrops displayed superior corneal healing relative to those treated with the autologous serum drops. At 16 hours, the corneal wounds of all groups healed slower than the one treated with 0.5%mACS. The murine cornea of the DMEM groups and autologous serum at 24 hours demonstrated comparable healing with mACSS at 32 hours except for the normal saline group.All other groups showed close to full recovery. The most important step is to follow aseptic procedures during the process. Serum products are rich in nutrients so using contaminated products on the cornea can cause severe corneal ulcers.This product can be used to assess the healing of wounds and the improvement of ocular surface disease symptoms. Analyzing the cytokine component and conducting larger randomized controlled trials of mACS are essential for further research.

production-modified-autologous-conditioned-serum-ex-vivo-assessment

Research

JoVE Journal

Medicine

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...