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.

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