Experimental design of a culture approach for corneal endothelial cells

Experimental design of a culture approach for corneal endothelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Experimental design of a culture approach for corneal endothelial cells Maria Dolores Montalvo-Parra, Isaac Alejandro Vidal-Paredes, Cesar E. Calzada-Rodriguez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-25005/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The harvesting of corneal endothelial cells (CECs) has received special attention given its potential as therapy for corneal blindness. The main challenges to overcome for this purpose are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CECs can retain their functional characteristics. The alternance of different media formulations to harvest CECs has an impact on the cellular yield and morphology. We herein analyzed eight different sequences of growth factor-supplemented proliferative (P) and non-supplemented resting (R) media upon passages to find the optimal P/R culture media sequence in regards of cell yield, morphology, procollagen I production, ATPase function, and the expression of ZO-1 and ATPase. Results PRPR and PRRR sequences showed the higher cell yield and hexagonal morphology rate. CECs cultured in the PRRR sequence produced procollagen I, showed Na/K-ATPase function, and expression of ZO-1 and Na/K-ATPase by immunocytochemistry. Our study sets a culture approach to guarantee CECs expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Conclusions Alternation of P and R culture media improves CECs culture. PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation. Stem Cell & Developmental Cell Biology Corneal endothelium ATPase Collagen Circularity Experimental design Cell culture Figures Figure 1 Figure 3 Figure 5 Figure 7 Figure 9 Background Corneal endothelial cells (CECs) are the cells of the inner layer of the cornea that functions as a barrier and pump for the maintenance of the optimal hydration level for the eye to perform its vision function [ 1 , 2 ]. The clarity and nutrition of the cornea rely on these functions, given that it is an avascular tissue [ 3 ]. The damage to the corneal endothelium can lead to blindness because CECs are arrested in the G 1 cell cycle phase[ 4 – 6 ]. Corneal blindness can only be treated through transplantation, however, this procedure faces the shortage of tissue donors [ 3 , 7 – 10 ]. The expansion of CECs through culture has received special attention among the efforts to develop an alternative to donor tissue. Several experimental approaches aim to develop a method to isolate and harvest CECs for corneal blindness cell therapy and tissue engineering [ 3 , 11 ]. For these purposes, challenges such as culture media[ 12 – 16 ] formulation cellular density [ 17 ], and number of passages that assures functionality [ 16 , 18 ] need to be overcome. The factors that affect the overall yield of CECs are donor-to-donor variation and the use of different serum-supplemented culture media[ 19 , 20 ]. Although a robust and clearly described culture methodology is still lacking, a great amount of clinical interest has been generated for the development of alternative approaches in the treatment for corneal endothelium damage-associated corneal blindness using cultured CECs. We previously reported a two-phase CECs culture system that allows cellular expansion and retains specific molecular markers, hexagonal morphology and monolayer arrangement [ 21 ]. Now, in order to set the best culture media combination, we aimed to analyze the CECs obtained from different approaches in terms of cell yield, morphological analysis, pro-collagen production, ATPase function, and the expression of ZO-1 and ATPase. Methods 1. Experimental Design A two-phase culture system was established to be used in CECs culture. Two-phase refers to the alternation of two media of different compositions: Proliferative (P) and resting (R) media. Our modified formula [ 21 ] of previously reported P media [ 14 ] contained Opti-MEM I (Gibco®; Thermo Fisher Scientific, Waltham, MA) supplemented with 8% fetal bovine serum (FBS; Cellgro, Manassas, VA) and 1% penicillin/streptomycin (Pen/Strep; Thermo Fisher Scientific), 20 ng/ml of nerve growth factor (NGF; Sigma-Aldrich Co.), 5 ng/ml of epidermal growth factor (EGF; Sigma-Aldrich Co.), 200 mg/l of calcium chloride (Sigma-Aldrich Co.), 20 µg/ml of ascorbic acid (Sigma-Aldrich Co.), 0.08% chondroitin sulfate (Sigma-Aldrich Co.). R media formulation was Opti-MEM I supplemented with 8% FBS and 1% Pen/Strep. Experimental design consisted of alternating media through several passages to evaluate combinations of the phases of our culture system as shown in Fig. 1. Eight sequences of alternating media were evaluated. The objective was to produce a maximum cell yield while conserving CECs characteristics. Passage 0 consisted in recently isolated CECs cultured in a 12-well plate with P media until hexagonal morphology was observed (confluence beyond 80%). All passages herein mentioned followed a hexagonal morphology and > 80% confluence rule. At passage 1, CECs were splitted 1:2 and the medium was changed using R media. The culture continued up to passage 3. An Axiovert 40 CFL contrast microscope (CFL; Carl Zeiss AG, Oberkochen, Germany) featuring a PowerShot A640 digital camera (Canon Inc., Tokyo, Japan) was used to register the cell morphology. 2. Isolation And Culture Of CEC This study was approved by the Institutional Ethics Committee (School of Medicine of Tecnologico de Monterrey), number 2017-005. All animals were treated according to the Guide for the Care and Use of Laboratory Animals. The corneas were obtained from four 3-month-old New Zealand rabbits weighing ~ 3 kg. The rabbits were euthanized under general anesthesia with a combination of xylazine 5 mg/kg and 30 mg/kg of ketamine [WU2] (Pisa Farmaceutica, Guadalajara, México); followed by a lethal intraperitoneal injection of sodic pentobarbital (Pets Pharma, Estado de Mexico, Mexico). The corneas were excised, rinsed with 37 °C Phosphate Buffered Saline (PBS) pH 7.4 (Gibco, Thermo Fisher Scientific, Waltham, MA) with 1% streptomycin/penicillin antibiotics (Thermo Fisher Scientific), and placed in a sterile tissue culture dish. All rabbit CECs were isolated using the “peel-and-digest” approach. Excised whole corneas were placed in a petri dish with PBS, all inside the Biosafety cabinet. Then, Trypan blue (0.04%, Milipore Sigma, Merck KGaA, Darmstadt, Germany) was poured on the endothelial side of the cornea for 1 min to stain CE borders. Trypan blue was then rinsed using PBS. The border of CE was gently pushed around the whole circumference. Descemet’s membrane with the intact endothelium (DM/CE) was carefully peeled from the corneal stroma and rinsed several times with 37° C PBS with 1% antibiotics. DM/CE complexes were incubated in Opti-MEM I 8% FBS and the 1% antibiotic combination overnight to stabilize the cells before culture. CE were incubated, while shaking at 170, rpm with 1 mg/ml of collagenase type I (Sigma-Aldrich Co., St. Louis, MO) at 37° C for 1 h. Cells were collected following centrifugation at 375 × g for 10 min and seeded into a 12-well culture dish. 3. Cell Yield Analysis Cell yield was calculated to produce evidence of morphology changes when cells were continuously cultured in P media and when cultured occurred in alternation with R media. For this analysis, the quotient of cellular concentration (cells/ml) at the end of passage 2 divided by the cellular concentration at the end of passage 1 was calculated for each media sequence. The average and the standard error were calculated. A t test was used to analyze statistically significant differences between the calculated yields. 4. Morphological Analysis CECs were observed (Axioivert 40CFL inverted microscope, Zeizz, Germany) and photo documented daily. Scale was set on each photograph and 40 cells per daily image were delimited with free shape ROI tool from Image J. Perimeter (P), area (A), circularity (C), aspect radio (AR) and roundness (R) indexes were obtained using Measure (Analyze menu). Basal parameters were also measured from cells observed in pictures obtained from recently isolated Descemet’s membrane. Statistical analysis was performed through a one-way ANOVA complemented with an all pairwise multiple comparison procedure by Holm-Sidak method (P = 0.001). Microsoft Excel (2007, Redmond, WA) was used for data processing. Statistical analysis and graph creation were carried out using Systat Sigma Plot (V. 11, San Jose, CA). 5. Protein Production Analysis Cultured cells were detached using trypsin and then and centrifuged at 500 x g for 5 min at 4 °C to obtain a cell lysate for total protein analysis. The pellet was washed three times with 1X PBS buffer and incubated 20 min with extraction buffer (Abcam ab193970) on ice. The solution was centrifuged at 18,000 x g for 20 minutes at 4 °C. The supernatant was used for the total protein concentration analysis. Colorimetric bicinchoninic acid assay (BCA Pierce 23225) was used. A standard curve was prepared using albumin provided in the kit. The concentration of total protein was measured at 562 nm in a spectrophotometer. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t -test using Microsoft Excel (2007, Redmond, WA). 6. Procollagen I Alpha 1 Analysis Pro-collagen I-α1 was determined as a measure of the ability of the cells to produce collagen 1-α1, ELISA Kit (Abcam ab210966) was used with the cell lysates described before. The procedure was followed in accordance with the kit manufacturer and the absorbance was measured at 562 nm in a microplate reader. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t -test using Microsoft Excel (2007, Redmond, WA). 7. Biomarker Immunodetection (ZO1 And ATPase) Immunocytochemistry was performed on basal whole cornea and on CECs passage 2 cultured with PRR media sequence combination to analyze the presence ZO-1 (Thermo Fisher, 61–7300, Waltham, MA), and Na/K-ATPase (Abcam, ab176163, Cambridge, UK). The procedure consisted of overnight cell stabilization over coverslips with poly-D lysine (Sigma-Aldrich, P7280), fixation with 4% paraformaldehyde, nonspecific bonding blockage with 5% bovine serum albumin (BSA; Sigma-Aldrich, A-7030), overnight 4 °C incubation with primary antibodies (ZO-1 5 µg/ml and Na/K-ATPase 1:100), and incubation with Alexa Fluor 488 secondary antibody (Abcam, ab150077) for 1 h at room temperature. Fluroshield Mounting Medium with 4′,6-Diamidino-2′-phenylindole dihydrochloride (DAPI; Abcam, ab104139) counterstain was used. Epifluorescence was registered with a widefield fluorescence microscope (Zeiss Imager Z1) with an AxioCam HRm (Zeiss) camera (Göttingen, Germany). Whole width cornea sections were used as controls [ 15 ]. 8. ATPase Activity Analysis A colorimetric enzymatic assay was used to measure the ATPase activity (Mybiosource MBS8243226). Cells (CEC and dermal fibroblasts) were harvested when confluence was reached and sonicated for the detection in accordance with the manufacturer procedure. The final absorbance was registered at 660 nm. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t -test using Microsoft Excel (2007, Redmond, WA). Results Cell yield Cell yield analysis was carried out to select the media sequence with higher yield (Table 1 ). The expansion folds were calculated for the sequences with the higher cell concentration at the end of passage 3. Briefly, sequence 1 produced a 12-fold, sequence 2 a 6.5-fold, sequence 3 a 4.6-fold, and sequence 4 produced a 7.5-fold expansion. Time to confluence varied for the different media sequences as shown in Fig. 2. Moreover, differences in the time to reach confluence were observed on the same sequence as seen for combinations 4–7. Table 1 Media sequence selection based on cellular expansion yield. Higher cell yields were reached with sequences 1–7. Cellular size was observed to be smaller at higher yields. Selection of optimum sequence also considered morphology. Thus, sequence 4 (PRRR) was selected. Expansion yield was calculated for each media sequence as the average of the difference between the initial seeded cells and the final cellular concentration when trypsinized for each passage. Cell counts were performed with a Neubauer chamber by duplicate. Results were compared with one way ANOVA (p = 0.001). Proliferative media (P); resting media (R). Cell concentration (cell number × 10 3 ) / mm 3 Sequence Passage 1 Passage 2 Passage 3 Initial Final Initial Final Initial Final 1) P-R-P-P-R 15 60 126 190 2) P-R-P-P-R 23 73 142 150 3) P-R-P-R 30 80 133 140 4) P-R-R-R 17 63 80 128 5) P-R-R-R 13 58 83 50 6) P-R-R-R 19 62 82 97 7) P-R-R-R 22 26 80 28 8) R-R-R-R 26 40 52 68 Cell Morphology We previously reported circularity values for all-proliferative media combination P-P-P-P (0.41 ± 0.19 SD), for P-R-R-R combination (sequences 4-7; 0.6 ± 0.18 SD), and circularity control values of primary isolated rabbit CECs (0.77 ± 0.063 SD)[21]. In that study, the P-P-P-P combination was discarded because it was detrimental for molecular expression. Our result on circularity evaluation of all-resting media combination, sequence 8 R-R-R-R, was 0.69 ± 0.11 SD. Sequences 1-3 with P-R-P-R combination showed a 0.60 ± 0.14 SD circularity value. P-R-R-R media combination observed in Sequence 4 (Figures 1,2) was selected as the balance between circularity and a good proliferation rate. Because of that, we further evaluated roundness, aspect ratio, area and perimeter through three passages in order to assess morphological changes as shown in Figure 3 . There is a change in area and perimeter upon confluence. The observed drop in these values indicates that cell size is affected by contact inhibition. Protein Concentration, Pro-collagen I α1 Production, And Na/K-ATPase Analysis Total protein concentration for CECs was 1,511.02 ± 443.19 µg/1 × 10 5 and 490 ± 70.26 µg/1 × 10 5 for dermal fibroblasts. Figure 4 shows the total protein concentration µg per 1 × 10 5 cells. A significant difference with a P-value < 0.05 was found between the CECs and control cells. Procollagen I determination showed significant difference between CECs and control cells with a P-value < 0.01 (Figure XB). Procollagen I was also found to represent less than 1% of the total protein of corneal endothelial cell lysates (9.94 × 10 − 6%) and was found to be 6 times higher compared to control cells. Na/K-ATPase concentration was 1.52 × 10 − 6 U/10 4 in CEC and 4.42 × 10 − 7 U/10 4 in HDF (Fig. 4). Biomarker Immunodetection (ZO1 And ATPase) Whole cornea immunofluorescence showed a basal state control to compare specific marker expression and location, as well as cell morphology (Fig. 3 ). CECs cultured with sequence 4 expressed ZO-1 and Na/K-ATPase. Also, ZO-1 in basal CECs and those in passage 2 showed the expected hexagonal shape and expressed the location of the protein on the apical side [ 16 ]. Nevertheless, Na/K-ATPase basolateral location could not be assessed completely due to the swelling of stroma in whole cornea basal samples. Mitotic figures were observed at passage two; the implications will be discussed further on. Discussion Cell yield All sequences showed a fold decrease with each passage as shown in Table 2 . Nevertheless, sequences 4 and 6 maintained the fold number at passages 1 and 2. Also, our results on all resting media combinations RRRR (sequence 8) are in accordance with previous reports of low proliferation and loss of circularity within passages [ 19 ]. Moreover, days to confluence increase with each passage, regardless of the media combination sequence as shown in Fig. 2. This means media composition beyond the first passage is irrelevant. Table 2 Passage fold change analysis. A fold change ratio from passage to passage was calculated to ease the comparison of cell yield within passages. Passage fold change (∆) Sequence Passage 1 Passage 2 Passage 3 1) P-R-P-P-R 4 2.1 1.58 2) P-R-P-P-R 3.2 1.9 1.05 3) P-R-P-R 2.6 1.9 1.05 4) P-R-R-R 3.7 1.3 1.6 5) P-R-R-R 4.46 1.43 0.6 6) P-R-R-R 3.26 1.32 1.8 7) P-R-R-R 0.18 1.15 0.93 8) R-R-R-R 1.53 1.3 1.3 Cell Morphology Several authors have evaluated the effect of growth factor supplementation of culture media to harvest CECs maintaining the hexagonal morphology and molecular characteristics [ 19 , 21 , 23 – 25 ]. Our results in rabbit cells cultured in a two-phase culture system agree with the described in both, rabbit and human CECs [ 17 , 19 , 26 ]. Though, the main difference in the composition of our media and that of the other authors is that we do not use ROCK inhibitors. Moreover, this study has allowed us to understand that the modulation of cellular proliferation signaling produced by a two-phase culture system is only a prevention method for mesenchymal transition [ 21 ]. Shape descriptors Circularity, Roundness and Aspect Ratio tended to 1 at the end of passage 1 and 3. This means that shape descriptors tend to hexagonallity when confluence is reached. Also, we compared obtained circularity values with previously reported results on primary isolated rabbit CECs (0.77 ± 0.063 SD) and found that all PM combinations showed the lowest circularity values. On the contrary all RM combinations at sequence 8 showed the nearest circularity value. Though sequences pertaining to a two phase cultures system exhibit an intermediate value and proliferation is favored too. Thus, our two phase culture system reaches a balance between morphology and cell yield [ 27 ]. In regards of area and perimeter drop upon confluence, further studies required to determine if a stabilization or compaction process occurs as observed with human CECs [ 4 , 28 , 29 ] Total Protein, Pro-collagen I And Na/K-ATPase Concentration Total protein, pro-collagen I, and NA/K-ATPase concentrations were higher in CECs. A heterogeneous amount of CECs per group were evaluated due to their challenging isolation and culture. Nevertheless, in all protein determinations concentration resulted to be higher on CECs. Also, normalization to single cell production was carried out for total protein and pro-collagen I in order to validate the comparison. CECs showed 3.2-fold higher protein concentration than control cells. Collagen I is one of the most abundant proteins produced by the corneal endothelium [ 30 ] whose arrangement is involved in corneal clarity. Procollagen I concentration was six times higher in CECs than in the control cells. This is in accordance with a previous study that reports CECs higher ability of proline hydroxylation than most of the fibroblastic cell lines [ 31 ]. The Na/K-ATPase colorimetric analysis for CECs is a low cost and feasible methodology that results useful to screen testing. The results support those of the immunocytochemistry analysis performed herein. Moreover, the functionality of the Na/K-ATPase is related to CECs hexagonal morphology procured with the PRRR media combination and corroborated with the morphological analyses we provide [ 32 ]. Conclusions With the alternations of sequences of PM and RM we gathered evidence in favor of the use of dual culture media systems to improve CECs yield maintaining the hexagonal morphology. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation. Also, the PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences with similar results. Finally, the findings on total protein, procollagen I and Na/K-ATPase concentrationscorroborates the immunocytochemistry evidence on cellular identity of a highly organized and active metabolic monolayer tissue. Sketching the limits of cultured CECs expansion is an unavoidable safety issue to be addressed in order to advance onto an in-vivo model. Our study allows guaranteeing cellular and tissular quality, as well as functionality for transplantation so further efforts might focus on improving surgery practices [ 33 ] and suggesting further monitoring techniques. Declarations Ethics approval: This study was approved by the institutional local ethics committee (School of Medicine of Tecnologico de Monterrey), number 2019-021. All animals were treated according to the Guide for the Care and Use of Laboratory Animals adhering to the guidelines for the human treatment and ethical use of animals for vision research stated by the Association for Research in Vision and Ophthalmology. Consent for publication: Not applicable. Availability of data and materials: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: Funding from Tecnologico de Monterrey and CONACYT PN 6558 was used for this project. Authors' contributions: MPMD gathered cell yield, morphology and immunocytochemistry data; created the experimental design, analyzed data, created figures and was a major contributor in writing and reviewing the manuscript. VPIA analyzed and improved experimental design figures, and was a major contributor in writing and reviewing the manuscript. CRCE and CRIT carried out Image J morphology analysis. TGGF contributed in the gathering data for the cell yield. GED acquired Na/K ATPase data. LMM acquired protein production data. ZJ analyzed protein production and Na/K ATPase data, created corresponding figures, was a major reviewed the manuscript and procured CONACYT funding. VGJE is the leader of our research group, performed major revisions to this manuscript and provided funding from Tecnologico de Monterrey. All authors read and approved the final manuscript. Acknowledgements: Not applicable References Peh GSL, Toh K-P, Wu F-Y, Tan DT, Mehta JS. Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas. Mohan RR, editor. PLoS One [Internet]. 2011 [cited 2019 Nov 25];6:e28310. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22194824 . DelMonte DW, Kim T. Anatomy and physiology of the cornea. J Cataract Refract Surg [Internet]. Elsevier; 2011 [cited 2019 Feb 25];37:588–98. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21333881 . Brunck MEG, Nielsen LK. Concise Review: Next-Generation Cell Therapies to Prevent Infections in Neutropenic Patients. 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Available from: http://www.nature.com/articles/srep29047 . Javadi M, Feizi S, Jafari R, Hosseini S, Safapour S. Factors influencing graft endothelial cell density after descemet stripping automated endothelial keratoplasty. J Ophthalmic Vis Res [Internet]. 2018;13:10. Available from: http://www.jovr.org/text.asp?2018/13/1/10/222909 . Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-25005","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":520264,"identity":"9bcdf0fc-1296-4b6b-9570-5d8e8323aa6f","order_by":1,"name":"Maria Dolores Montalvo-Parra","email":"","orcid":"https://orcid.org/0000-0002-4789-5134","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Dolores","lastName":"Montalvo-Parra","suffix":""},{"id":520265,"identity":"7ad20797-6b59-46c6-b13e-89dfbb1129b3","order_by":2,"name":"Isaac Alejandro Vidal-Paredes","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"Alejandro","lastName":"Vidal-Paredes","suffix":""},{"id":520266,"identity":"57da4313-33d0-4a5b-996b-7f08a27ac0bd","order_by":3,"name":"Cesar E. Calzada-Rodriguez","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cesar","middleName":"E.","lastName":"Calzada-Rodriguez","suffix":""},{"id":520267,"identity":"44189bf6-078e-43f0-8250-afde96fb7d52","order_by":4,"name":"Italia Tatnaí Cárdenas-Rodríguez","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Italia","middleName":"Tatnaí","lastName":"Cárdenas-Rodríguez","suffix":""},{"id":520268,"identity":"0b89fe44-a02b-4bd3-9688-a402b3f1aae7","order_by":5,"name":"Guiomar Farid Torres Guerrero","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guiomar","middleName":"Farid Torres","lastName":"Guerrero","suffix":""},{"id":520269,"identity":"a4dd25f9-5345-4686-afd3-ca9fcceab827","order_by":6,"name":"Daniela Gomez Elizondo","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"Gomez","lastName":"Elizondo","suffix":""},{"id":520270,"identity":"aad92ae1-6aa1-412c-84d8-cbece8867742","order_by":7,"name":"Mariana Lopez Martínez","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"Lopez","lastName":"Martínez","suffix":""},{"id":520271,"identity":"04fcbb62-fffc-4f87-bf32-55f43f77de5c","order_by":8,"name":"Judith Zavala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACxgYGgwNAAsw+wFBBrJaDEC0MBxjOGBBlkQEDXAtjGxFamGckbzz8cQeDPf+M5AcHfs77I8ffwJ0mgddhM9IKDhw8w8AscSPN4GDvNgNjiQO8m/HaxTgjB+iXNgY2hjMHDA7wbjNI3MDAu/EBMVp45M8c/3Dw7xyDeqCWDQeI0SJhcLzH4DBvg0GCAUFbep4VHDjbJmFgeLyn4LDMMWPDGYcJ+MWwPXnzh8o2G3u5w+wbH76pkZPnb+/dhjfEDBvAFLIaZnzqgUCegPwoGAWjYBSMAgYGAHGNUbElgiEZAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Judith","middleName":"","lastName":"Zavala","suffix":""},{"id":520272,"identity":"bca7d432-f7e6-48b3-a11c-e2c6d080cba6","order_by":9,"name":"Jorge E. Valdez-García","email":"","orcid":"","institution":"Instituto Tecnologico y de Estudios Superiores de Monterrey","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"E.","lastName":"Valdez-García","suffix":""}],"badges":[],"createdAt":"2020-04-24 10:37:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-25005/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-25005/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1002585,"identity":"e54aa528-97e0-4373-b60e-f6cfcfeace2f","added_by":"auto","created_at":"2020-04-30 15:24:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82431,"visible":true,"origin":"","legend":"Experimental design. Eight sequences of alternating proliferative (P) and resting (R) were evaluated. All cultures were stabilized with proliferative media until confluence at passage 0; sequence 8 was the exception. Passages are marked with dashed red lines.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/fig1.png"},{"id":1002586,"identity":"29cef8bd-276d-401d-a5dc-1b6742369200","added_by":"auto","created_at":"2020-04-30 15:24:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119253,"visible":true,"origin":"","legend":"Media alternation sequence design. The first approach to optimum media sequence was determined through this experimental design. Passage number was totally experimental as in a screening study to corroborate literature. Proliferative media (P); resting media (R).","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/fig2.png"},{"id":1002587,"identity":"a13b0140-e882-488b-92d9-e5aa4f286c5d","added_by":"auto","created_at":"2020-04-30 15:24:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278775,"visible":true,"origin":"","legend":"Culture of rabbit corneal endothelium cells with a PRR combination. a) Changes in shape descriptors: circularity, roundness and aspect ratio; b) changes in cell area and perimeter; c) rabbit corneal endothelium cells at passage 0, day 3; d) rabbit corneal endothelium cells at passage 2; e) human corneal endothelium from the Visual Atlas of Eye Bank Specular Microscopy – Donor Corneal Endothelium Imaging [22].","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/fig3.png"},{"id":1002588,"identity":"c69fb592-86eb-4b7a-b4a6-c0273522be40","added_by":"auto","created_at":"2020-04-30 15:24:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":45769,"visible":true,"origin":"","legend":"Protein production analysis. Total protein concentration a), pro-collagen I b), and Na/K-ATPase c) in HDFA and CECs cultured with the media sequence 4 PRRR. (*p\u003c0.05, **p\u003c0.01).","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/fig4.png"},{"id":1002589,"identity":"3c4f1fa1-a78c-4b69-b8fb-1ccd477d0d92","added_by":"auto","created_at":"2020-04-30 15:24:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":539602,"visible":true,"origin":"","legend":"Immunocytochemistry of specific molecular markers of basal and cultured rabbit corneal endothelium cells. Basal corneal endothelium epifluorescence images of a whole cornea exhibit Zonula Occludens tight junctions (ZO-1) and Sodium-Potassium ATPase (Na/K-ATPase) expression. Also, cultured corneal endothelium cells exhibit the specific molecular markers.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/fig5.png"},{"id":13500963,"identity":"fc420f2e-4dca-4944-84dc-eb6168af4ff5","added_by":"auto","created_at":"2021-09-16 23:08:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1711892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-25005/v1/a20d158c-0ec5-4053-ac9e-2a5ae1946214.pdf"}],"financialInterests":"","formattedTitle":"Experimental design of a culture approach for corneal endothelial cells","fulltext":[{"header":"Background","content":" \u003cp\u003eCorneal endothelial cells (CECs) are the cells of the inner layer of the cornea that functions as a barrier and pump for the maintenance of the optimal hydration level for the eye to perform its vision function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The clarity and nutrition of the cornea rely on these functions, given that it is an avascular tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The damage to the corneal endothelium can lead to blindness because CECs are arrested in the G\u003csub\u003e1\u003c/sub\u003e cell cycle phase[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Corneal blindness can only be treated through transplantation, however, this procedure faces the shortage of tissue donors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe expansion of CECs through culture has received special attention among the efforts to develop an alternative to donor tissue. Several experimental approaches aim to develop a method to isolate and harvest CECs for corneal blindness cell therapy and tissue engineering [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor these purposes, challenges such as culture media[\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] formulation cellular density [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and number of passages that assures functionality [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] need to be overcome. The factors that affect the overall yield of CECs are donor-to-donor variation and the use of different serum-supplemented culture media[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although a robust and clearly described culture methodology is still lacking, a great amount of clinical interest has been generated for the development of alternative approaches in the treatment for corneal endothelium damage-associated corneal blindness using cultured CECs.\u003c/p\u003e \u003cp\u003eWe previously reported a two-phase CECs culture system that allows cellular expansion and retains specific molecular markers, hexagonal morphology and monolayer arrangement [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Now, in order to set the best culture media combination, we aimed to analyze the CECs obtained from different approaches in terms of cell yield, morphological analysis, pro-collagen production, ATPase function, and the expression of ZO-1 and ATPase.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003e1. Experimental Design\u003c/h2\u003e\n \u003cp\u003eA two-phase culture system was established to be used in CECs culture. Two-phase refers to the alternation of two media of different compositions: Proliferative (P) and resting (R) media. Our modified formula [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] of previously reported P media [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] contained Opti-MEM I (Gibco\u0026reg;; Thermo Fisher Scientific, Waltham, MA) supplemented with 8% fetal bovine serum (FBS; Cellgro, Manassas, VA) and 1% penicillin/streptomycin (Pen/Strep; Thermo Fisher Scientific), 20\u0026nbsp;ng/ml of nerve growth factor (NGF; Sigma-Aldrich Co.), 5\u0026nbsp;ng/ml of epidermal growth factor (EGF; Sigma-Aldrich Co.), 200\u0026nbsp;mg/l of calcium chloride (Sigma-Aldrich Co.), 20\u0026nbsp;\u0026micro;g/ml of ascorbic acid (Sigma-Aldrich Co.), 0.08% chondroitin sulfate (Sigma-Aldrich Co.). R media formulation was Opti-MEM I supplemented with 8% FBS and 1% Pen/Strep. Experimental design consisted of alternating media through several passages to evaluate combinations of the phases of our culture system as shown in Fig.\u0026nbsp;1. Eight sequences of alternating media were evaluated. The objective was to produce a maximum cell yield while conserving CECs characteristics.\u003c/p\u003e \u003cp\u003ePassage 0 consisted in recently isolated CECs cultured in a 12-well plate with P media until hexagonal morphology was observed (confluence beyond 80%). All passages herein mentioned followed a hexagonal morphology and \u0026gt;\u0026thinsp;80% confluence rule. At passage 1, CECs were splitted 1:2 and the medium was changed using R media. The culture continued up to passage 3. An Axiovert 40 CFL contrast microscope (CFL; Carl Zeiss AG, Oberkochen, Germany) featuring a PowerShot A640 digital camera (Canon Inc., Tokyo, Japan) was used to register the cell morphology.\u003c/p\u003e \n\u003ch2\u003e2. Isolation And Culture Of CEC\u003c/h2\u003e\n \u003cp\u003eThis study was approved by the Institutional Ethics Committee (School of Medicine of Tecnologico de Monterrey), number 2017-005. All animals were treated according to the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003cp\u003eThe corneas were obtained from four 3-month-old New Zealand rabbits weighing\u0026thinsp;~\u0026thinsp;3\u0026nbsp;kg. The rabbits were euthanized under general anesthesia with a combination of xylazine 5\u0026nbsp;mg/kg and 30\u0026nbsp;mg/kg of ketamine [WU2] (Pisa Farmaceutica, Guadalajara, M\u0026eacute;xico); followed by a lethal intraperitoneal injection of sodic pentobarbital (Pets Pharma, Estado de Mexico, Mexico). The corneas were excised, rinsed with 37\u0026nbsp;\u0026deg;C Phosphate Buffered Saline (PBS) pH 7.4 (Gibco, Thermo Fisher Scientific, Waltham, MA) with 1% streptomycin/penicillin antibiotics (Thermo Fisher Scientific), and placed in a sterile tissue culture dish.\u003c/p\u003e \u003cp\u003eAll rabbit CECs were isolated using the \u0026ldquo;peel-and-digest\u0026rdquo; approach. Excised whole corneas were placed in a petri dish with PBS, all inside the Biosafety cabinet. Then, Trypan blue (0.04%, Milipore Sigma, Merck KGaA, Darmstadt, Germany) was poured on the endothelial side of the cornea for 1\u0026nbsp;min to stain CE borders. Trypan blue was then rinsed using PBS. The border of CE was gently pushed around the whole circumference. Descemet\u0026rsquo;s membrane with the intact endothelium (DM/CE) was carefully peeled from the corneal stroma and rinsed several times with 37\u0026deg; C PBS with 1% antibiotics. DM/CE complexes were incubated in Opti-MEM I 8% FBS and the 1% antibiotic combination overnight to stabilize the cells before culture. CE were incubated, while shaking at 170, rpm with 1\u0026nbsp;mg/ml of collagenase type I (Sigma-Aldrich Co., St. Louis, MO) at 37\u0026deg; C for 1\u0026nbsp;h. Cells were collected following centrifugation at 375\u0026thinsp;\u0026times;\u0026thinsp;g for 10\u0026nbsp;min and seeded into a 12-well culture dish.\u003c/p\u003e \n\u003ch2\u003e3. Cell Yield Analysis\u003c/h2\u003e\n\u003cp\u003eCell yield was calculated to produce evidence of morphology changes when cells were continuously cultured in P media and when cultured occurred in alternation with R media. For this analysis, the quotient of cellular concentration (cells/ml) at the end of passage 2 divided by the cellular concentration at the end of passage 1 was calculated for each media sequence. The average and the standard error were calculated. A \u003cem\u003et\u003c/em\u003e test was used to analyze statistically significant differences between the calculated yields.\u003c/p\u003e \n\u003ch2\u003e4. Morphological Analysis\u003c/h2\u003e\n \u003cp\u003eCECs were observed (Axioivert 40CFL inverted microscope, Zeizz, Germany) and photo documented daily. Scale was set on each photograph and 40 cells per daily image were delimited with free shape ROI tool from Image J. Perimeter (P), area (A), circularity (C), aspect radio (AR) and roundness (R) indexes were obtained using Measure (Analyze menu). Basal parameters were also measured from cells observed in pictures obtained from recently isolated Descemet\u0026rsquo;s membrane. Statistical analysis was performed through a one-way ANOVA complemented with an all pairwise multiple comparison procedure by Holm-Sidak method (P\u0026thinsp;=\u0026thinsp;0.001). Microsoft Excel (2007, Redmond, WA) was used for data processing. Statistical analysis and graph creation were carried out using Systat Sigma Plot (V. 11, San Jose, CA).\u003c/p\u003e \n\u003ch2\u003e5. Protein Production Analysis\u003c/h2\u003e\n \u003cp\u003eCultured cells were detached using trypsin and then and centrifuged at 500 x g for 5\u0026nbsp;min at 4\u0026nbsp;\u0026deg;C to obtain a cell lysate for total protein analysis. The pellet was washed three times with 1X PBS buffer and incubated 20\u0026nbsp;min with extraction buffer (Abcam ab193970) on ice. The solution was centrifuged at 18,000 x g for 20 minutes at 4\u0026nbsp;\u0026deg;C. The supernatant was used for the total protein concentration analysis. Colorimetric bicinchoninic acid assay (BCA Pierce 23225) was used. A standard curve was prepared using albumin provided in the kit. The concentration of total protein was measured at 562\u0026nbsp;nm in a spectrophotometer. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a \u003cem\u003et\u003c/em\u003e-test using Microsoft Excel (2007, Redmond, WA).\u003c/p\u003e \n\u003ch2\u003e6. Procollagen I Alpha 1 Analysis\u003c/h2\u003e\n \u003cp\u003ePro-collagen I-α1 was determined as a measure of the ability of the cells to produce collagen 1-α1, ELISA Kit (Abcam ab210966) was used with the cell lysates described before. The procedure was followed in accordance with the kit manufacturer and the absorbance was measured at 562\u0026nbsp;nm in a microplate reader. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a \u003cem\u003et\u003c/em\u003e-test using Microsoft Excel (2007, Redmond, WA).\u003c/p\u003e \n\u003ch2\u003e7. Biomarker Immunodetection (ZO1 And ATPase)\u003c/h2\u003e\n \u003cp\u003eImmunocytochemistry was performed on basal whole cornea and on CECs passage 2 cultured with PRR media sequence combination to analyze the presence ZO-1 (Thermo Fisher, 61\u0026ndash;7300, Waltham, MA), and Na/K-ATPase (Abcam, ab176163, Cambridge, UK). The procedure consisted of overnight cell stabilization over coverslips with poly-D lysine (Sigma-Aldrich, P7280), fixation with 4% paraformaldehyde, nonspecific bonding blockage with 5% bovine serum albumin (BSA; Sigma-Aldrich, A-7030), overnight 4\u0026nbsp;\u0026deg;C incubation with primary antibodies (ZO-1 5\u0026nbsp;\u0026micro;g/ml and Na/K-ATPase 1:100), and incubation with Alexa Fluor 488 secondary antibody (Abcam, ab150077) for 1\u0026nbsp;h at room temperature. Fluroshield Mounting Medium with 4\u0026prime;,6-Diamidino-2\u0026prime;-phenylindole dihydrochloride (DAPI; Abcam, ab104139) counterstain was used. Epifluorescence was registered with a widefield fluorescence microscope (Zeiss Imager Z1) with an AxioCam HRm (Zeiss) camera (G\u0026ouml;ttingen, Germany). Whole width cornea sections were used as controls [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \n\u003ch2\u003e8. ATPase Activity Analysis\u003c/h2\u003e\n \u003cp\u003eA colorimetric enzymatic assay was used to measure the ATPase activity (Mybiosource MBS8243226). Cells (CEC and dermal fibroblasts) were harvested when confluence was reached and sonicated for the detection in accordance with the manufacturer procedure. The final absorbance was registered at 660\u0026nbsp;nm. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a \u003cem\u003et\u003c/em\u003e-test using Microsoft Excel (2007, Redmond, WA).\u003c/p\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell yield\u003c/h2\u003e \u003cp\u003eCell yield analysis was carried out to select the media sequence with higher yield (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The expansion folds were calculated for the sequences with the higher cell concentration at the end of passage 3. Briefly, sequence 1 produced a 12-fold, sequence 2 a 6.5-fold, sequence 3 a 4.6-fold, and sequence 4 produced a 7.5-fold expansion. Time to confluence varied for the different media sequences as shown in Fig.\u0026nbsp;2. Moreover, differences in the time to reach confluence were observed on the same sequence as seen for combinations 4\u0026ndash;7.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMedia sequence selection based on cellular expansion yield. Higher cell yields were reached with sequences 1\u0026ndash;7. Cellular size was observed to be smaller at higher yields. Selection of optimum sequence also considered morphology. Thus, sequence 4 (PRRR) was selected. Expansion yield was calculated for each media sequence as the average of the difference between the initial seeded cells and the final cellular concentration when trypsinized for each passage. Cell counts were performed with a Neubauer chamber by duplicate. Results were compared with one way ANOVA (p\u0026thinsp;=\u0026thinsp;0.001). Proliferative media (P); resting media (R).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eCell concentration (cell number\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e) / mm\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePassage 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePassage 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003ePassage 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eInitial\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eFinal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eInitial\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eFinal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eInitial\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eFinal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1) P-R-P-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2) P-R-P-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3) P-R-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8) R-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eCell Morphology\u003c/h2\u003e\n\u003cp\u003eWe previously reported circularity values for all-proliferative media combination P-P-P-P (0.41 \u0026plusmn; 0.19 SD), for P-R-R-R combination (sequences 4-7; 0.6 \u0026plusmn; 0.18 SD), and circularity control values of primary isolated rabbit CECs (0.77 \u0026plusmn; 0.063 SD)[21]. In that study, the P-P-P-P combination was discarded because it was detrimental for molecular expression. Our result on circularity evaluation of all-resting media combination, sequence 8 R-R-R-R, was 0.69 \u0026plusmn; 0.11 SD.\u0026nbsp; Sequences 1-3 with P-R-P-R combination showed a 0.60 \u0026plusmn; 0.14 SD circularity value. P-R-R-R media combination observed in Sequence 4 (Figures 1,2) was selected as the balance between circularity and a good proliferation rate. Because of that, we further evaluated roundness, aspect ratio, area and perimeter through three passages in order to assess morphological changes as shown in Figure 3\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is a change in area and perimeter upon confluence. The observed drop in these values indicates that cell size is affected by contact inhibition.\u003c/p\u003e\n\u003ch2\u003eProtein Concentration, Pro-collagen I α1 Production, And Na/K-ATPase Analysis\u003c/h2\u003e\n \u003cp\u003eTotal protein concentration for CECs was 1,511.02\u0026thinsp;\u0026plusmn;\u0026thinsp;443.19\u0026nbsp;\u0026micro;g/1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e and 490\u0026thinsp;\u0026plusmn;\u0026thinsp;70.26\u0026nbsp;\u0026micro;g/1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e for dermal fibroblasts. Figure\u0026nbsp;4 shows the total protein concentration \u0026micro;g per 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells. A significant difference with a P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was found between the CECs and control cells. Procollagen I determination showed significant difference between CECs and control cells with a P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (Figure XB). Procollagen I was also found to represent less than 1% of the total protein of corneal endothelial cell lysates (9.94\u0026thinsp;\u0026times;\u0026thinsp;10\u0026thinsp;\u0026minus;\u0026thinsp;6%) and was found to be 6 times higher compared to control cells. Na/K-ATPase concentration was 1.52\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e U/10\u003csup\u003e4\u003c/sup\u003e in CEC and 4.42\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e U/10\u003csup\u003e4\u003c/sup\u003e in HDF (Fig.\u0026nbsp;4).\u003c/p\u003e\n\u003ch2\u003eBiomarker Immunodetection (ZO1 And ATPase)\u003c/h2\u003e\n \u003cp\u003eWhole cornea immunofluorescence showed a basal state control to compare specific marker expression and location, as well as cell morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). CECs cultured with sequence 4 expressed ZO-1 and Na/K-ATPase. Also, ZO-1 in basal CECs and those in passage 2 showed the expected hexagonal shape and expressed the location of the protein on the apical side [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nevertheless, Na/K-ATPase basolateral location could not be assessed completely due to the swelling of stroma in whole cornea basal samples. Mitotic figures were observed at passage two; the implications will be discussed further on.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell yield\u003c/h2\u003e \u003cp\u003eAll sequences showed a fold decrease with each passage as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Nevertheless, sequences 4 and 6 maintained the fold number at passages 1 and 2. Also, our results on all resting media combinations RRRR (sequence 8) are in accordance with previous reports of low proliferation and loss of circularity within passages [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, days to confluence increase with each passage, regardless of the media combination sequence as shown in Fig.\u0026nbsp;2. This means media composition beyond the first passage is irrelevant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePassage fold change analysis. A fold change ratio from passage to passage was calculated to ease the comparison of cell yield within passages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003ePassage fold change (∆)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSequence\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePassage 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePassage 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePassage 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1) P-R-P-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2) P-R-P-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3) P-R-P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7) P-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8) R-R-R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eCell Morphology\u003c/h2\u003e\n \u003cp\u003eSeveral authors have evaluated the effect of growth factor supplementation of culture media to harvest CECs maintaining the hexagonal morphology and molecular characteristics [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our results in\u003c/p\u003e \u003cp\u003erabbit cells cultured in a two-phase culture system agree with the described in both, rabbit and human CECs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Though, the main difference in the composition of our media and that of the other authors is that we do not use ROCK inhibitors. Moreover, this study has allowed us to understand that the modulation of cellular proliferation signaling produced by a two-phase culture system is only a prevention method for mesenchymal transition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eShape descriptors Circularity, Roundness and Aspect Ratio tended to 1\u0026nbsp;at the end of passage 1 and 3. This means that shape descriptors tend to hexagonallity when confluence is reached. Also, we compared obtained circularity values with previously reported results on primary isolated rabbit CECs (0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.063 SD) and found that all PM combinations showed the lowest circularity values. On the contrary all RM combinations at sequence 8 showed the nearest circularity value. Though sequences pertaining to a two phase cultures system exhibit an intermediate value and proliferation is favored too. Thus, our two phase culture system reaches a balance between morphology and cell yield [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn regards of area and perimeter drop upon confluence, further studies required to determine if a stabilization or compaction process occurs as observed with human CECs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e\n\u003ch2\u003eTotal Protein, Pro-collagen I And Na/K-ATPase Concentration\u003c/h2\u003e\n \u003cp\u003eTotal protein, pro-collagen I, and NA/K-ATPase concentrations were higher in CECs. A heterogeneous amount of CECs per group were evaluated due to their challenging isolation and culture. Nevertheless, in all protein determinations concentration resulted to be higher on CECs. Also, normalization to single cell production was carried out for total protein and pro-collagen I in order to validate the comparison. CECs showed 3.2-fold higher protein concentration than control cells. Collagen I is one of the most abundant proteins produced by the corneal endothelium [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] whose arrangement is involved in corneal clarity. Procollagen I concentration was six times higher in CECs than in the control cells. This is in accordance with a previous study that reports CECs higher ability of proline hydroxylation than most of the fibroblastic cell lines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The Na/K-ATPase colorimetric analysis for CECs is a low cost and feasible methodology that results useful to screen testing. The results support those of the immunocytochemistry analysis performed herein. Moreover, the functionality of the Na/K-ATPase is related to CECs hexagonal morphology procured with the PRRR media combination and corroborated with the morphological analyses we provide [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eWith the alternations of sequences of PM and RM we gathered evidence in favor of the use of dual culture media systems to improve CECs yield maintaining the hexagonal morphology. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation. Also, the PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences with similar results. Finally, the findings on total protein, procollagen I and Na/K-ATPase concentrationscorroborates the immunocytochemistry evidence on cellular identity of a highly organized and active metabolic monolayer tissue.\u003c/p\u003e \u003cp\u003eSketching the limits of cultured CECs expansion is an unavoidable safety issue to be addressed in order to advance onto an \u003cem\u003ein-vivo\u003c/em\u003e model. Our study allows guaranteeing cellular and tissular quality, as well as functionality for transplantation so further efforts might focus on improving surgery practices [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and suggesting further monitoring techniques.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval: \u003cem\u003eThis study was approved by the institutional local ethics committee (School of Medicine of Tecnologico de Monterrey), number 2019-021. All animals were treated according to the Guide for the Care and Use of Laboratory Animals adhering to the guidelines for the human treatment and ethical use of animals for vision research stated by the Association for Research in Vision and Ophthalmology.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication: \u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: \u003cem\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests:\u0026nbsp; \u003cem\u003eThe authors declare that they have no competing interests.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFunding: \u003cem\u003eFunding from Tecnologico de Monterrey and CONACYT PN 6558 was used for this project.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAuthors' contributions: \u003cem\u003eMPMD gathered cell yield, morphology and immunocytochemistry data; created the experimental design, analyzed data, created figures and was a major contributor in writing and reviewing the manuscript. VPIA analyzed and improved experimental design figures, and was a major contributor in writing and reviewing the manuscript. CRCE and CRIT carried out Image J morphology analysis. TGGF contributed in the gathering data for the cell yield. GED acquired Na/K ATPase data. LMM acquired protein production data. ZJ analyzed protein production and Na/K ATPase data, created corresponding figures, was a major reviewed the manuscript and procured CONACYT funding. VGJE is the leader of our research group, performed major revisions to this manuscript and provided funding from Tecnologico de Monterrey. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAcknowledgements: \u003cem\u003eNot applicable\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003ePeh GSL, Toh K-P, Wu F-Y, Tan DT, Mehta JS. Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas. Mohan RR, editor. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.jovr.org/text.asp?2018/13/1/10/222909\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Corneal endothelium, ATPase, Collagen, Circularity, Experimental design, Cell culture","lastPublishedDoi":"10.21203/rs.3.rs-25005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-25005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe harvesting of corneal endothelial cells (CECs) has received special attention given its potential as therapy for corneal blindness. The main challenges to overcome for this purpose are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CECs can retain their functional characteristics. The alternance of different media formulations to harvest CECs has an impact on the cellular yield and morphology. We herein analyzed eight different sequences of growth factor-supplemented proliferative (P) and non-supplemented resting (R) media upon passages to find the optimal P/R culture media sequence in regards of cell yield, morphology, procollagen I production, ATPase function, and the expression of ZO-1 and ATPase.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePRPR and PRRR sequences showed the higher cell yield and hexagonal morphology rate. CECs cultured in the PRRR sequence produced procollagen I, showed Na/K-ATPase function, and expression of ZO-1 and Na/K-ATPase by immunocytochemistry. Our study sets a culture approach to guarantee CECs expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAlternation of P and R culture media improves CECs culture. PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation.\u003c/p\u003e","manuscriptTitle":"Experimental design of a culture approach for corneal endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-04-30 15:24:32","doi":"10.21203/rs.3.rs-25005/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"043f4414-bebb-47d6-81fb-537552dcf3d8","owner":[],"postedDate":"April 30th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":91864,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2020-04-30T15:24:33+00:00","versionOfRecord":[],"versionCreatedAt":"2020-04-30 15:24:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-25005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-25005","identity":"rs-25005","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}