- Email: [email protected]
Effects of corneal preservation conditions on human corneal endothelial cell culture
Accepted Manuscript Effects of corneal preservation conditions on human corneal endothelial cell culture Mohit Parekh, Gary Peh, Jodhbir S. Mehta, Sajjad Ahmad, Diego Ponzin, Stefano Ferrari PII:
S0014-4835(18)30614-6
DOI:
https://doi.org/10.1016/j.exer.2018.11.007
Reference:
YEXER 7520
To appear in:
Experimental Eye Research
Received Date: 16 August 2018 Revised Date:
7 October 2018
Accepted Date: 6 November 2018
Please cite this article as: Parekh, M., Peh, G., Mehta, J.S., Ahmad, S., Ponzin, D., Ferrari, S., Effects of corneal preservation conditions on human corneal endothelial cell culture, Experimental Eye Research (2018), doi: https://doi.org/10.1016/j.exer.2018.11.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Parekh M et al. Endothelial cell culture from preserved corneas
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EFFECTS OF CORNEAL PRESERVATION CONDITIONS ON HUMAN CORNEAL
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ENDOTHELIAL CELL CULTURE Authors
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Mohit Parekh1,2, Gary Peh3,4, Jodhbir S Mehta3,4,5,6, Sajjad Ahmad2,7, Diego Ponzin1,
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Stefano Ferrari1
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Affiliations
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Venice, Italy
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Singapore
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Aerospace Engineering, Nanyang Technological University, Singapore
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Correspondence
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Mohit Parekh MSc, PhD
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Institute of Ophthalmology, University College London,
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11-43 Bath Street, London, EC1V 9EL, UK
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Tel: 0044 7427652996
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Email: [email protected]
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Running title
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Endothelial cell culture from preserved corneas
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International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation,
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Institute of Ophthalmology, University College London, London, UK Tissue Engineering and Stem Cell group, Singapore Eye Research Institute,
Duke-NUS Graduate Medical School, Singapore
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Singapore National Eye Centre, Singapore
School of Material Science and Engineering and School of Mechanical and
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Moorfields Eye Hospital NHS Foundation Trust, London, UK
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Parekh M et al. Endothelial cell culture from preserved corneas
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Abstract
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The purpose of this study was to investigate the growth capacity of human corneal
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endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different
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storage conditions. The following conditions were evaluated, A) cold storage (CS)
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(Optisol GS) for 7 days at 4oC [n=6]; B) organ culture (OC) (Cornea Max) for 7 days
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at 31oC [n=6]; C) OC for 28 days at 31oC [n=6] and; D) CS for 7 days at 4oC followed
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by OC for 28 days at 31oC [n=6]. Following preservations, the Descemet membrane-
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endothelium complex was peeled and digested using Collagenase-Type1 and was
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subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-
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well chamber slide. Media was refreshed every alternate day. The confluence rate
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(%) was assessed, and overall viability was determined using Hoechst, Ethidium
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Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na+/K+-
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ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were
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used to analyse the cultures established from each condition. Donor tissues
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preserved in hypothermia (condition A) resulted in 9.3%±4.0% trypan-blue positive
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cells (TBPCs) hence lower number of HCEnCs was plated. There were <1% TBPCs
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observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and
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D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p=0.9836) at day 1. By day 9,
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HCEnCs established from all conditions became confluent except cells from
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condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and
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expressed HCEnC-associated markers. In conclusion, OC system has advantages
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over hypothermic media for the preservation of older donor corneas rejected for
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corneal transplant and deemed suitable for corneal endothelial cell expansion, with
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lower TBPCs before peeling and longer possible period of preservation over
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hypothermic storage system.
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Keywords
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Eye bank; cornea; preservation; endothelium; cell culture; storage
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1. Introduction
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The posterior monolayer of the human cornea comprises of endothelial cells that
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allows the transmission of soluble essential metabolites, and nutrients from the
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aqueous humour into the cornea (Srinivas, 2010), and is essential for maintaining
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corneal transparency (Joyce, 2003). The corneal endothelium must be maintained as
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the number of endothelial cells decrease throughout life (Bourne, 2003). If the
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endothelial cells are damaged to the stage that it impedes function, it can lead to
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oedema followed by opacity resulting in corneal blindness (Engelmann et al, 1999;
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Edelhauser, 2006). One of the main causes of endothelial failure is Fuch’s
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endothelial dystrophy, which can be treated by a corneal transplant using a healthy
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cadaveric donor cornea. As per a recent report, nearly 40% of the keratoplasties in
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the United States are for treating corneal endothelial dysfunction. It has been
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estimated that approximately 12.7 million patients are on the waiting list for a corneal
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transplant (Gain et al, 2016; Wong et al, 2017). Hence, to address this severe
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shortage of donor corneas, alternative approaches need to be identified to reduce
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the global burden of corneal tissues.
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Although the cells of the corneal endothelium are not known to regenerate within the
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eye (Joyce & Zhu, 2004), it has been shown that human corneal endothelial cells
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(HCEnCs) can be propagated using appropriate in vitro growth conditions, driving
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substantial research foci towards the development of alternative treatment options
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for corneal endothelial dysfunction through cell replacement therapeutics (Okumura
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et al, 2012). The development of a suitable graft alternative, through tissue
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engineering or injection of cultivated HCEnCs has already been proposed (Koizumi
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et al, 2012; Choi et al, 2010). The isolation and propagation techniques of HCEnCs
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in vitro have been described (Peh et al, 2011A, 2011B) and subsequently reviewed
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(Parekh et al, 2013; Parekh et al, 2016).
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Younger donors have advantages in terms of overall cell yield considering their high
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proliferative capability over older donor corneas, which tend to senesce at earlier
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passages (Peh et al, 2015). However, as most of the young donor corneas are
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usually suitable for transplantation, it is difficult to find such tissues for cellular
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expansion. If HCEnCs can be cultured from old donor corneas by modulating cell
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adhesion then it may potentially increase the donor pool for culturing HCEnCs
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(Parekh et al, 2017),
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Apart from donor characteristics, it is also important to evaluate the media used for
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corneal preservation, and its effects on culturing of the HCEnCs isolated from older
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donor, in a systemic donor-matched manner. If the cornea is to be preserved for a
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longer period of time (between 14 to 21 days), then the storage period, methodology,
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as well as conditions must be optimized, validated and described if the corneas are
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intended for subsequent cell culture and expansion. Hypothermic or cold storage
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(CS) (2-6o C) system is being used in the United States and majority of the Asian
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countries whereas most of the European eye banks prefer to use organ culture (OC)
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(31-37oC) method. The maximum storage time for CS has been limited normally to
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14 days whereas OC offers long-term storage for up to 4 weeks. OC has several
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advantages over CS in terms of microbiological checks, quality assurance of the
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tissues and preparation and planning time for surgery (Parekh et al, 2014; Parekh et
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al, 2015). Thus, this study intends to assess the culture capacity of HCEnCs when
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the corneas are preserved in different storage media, and in different conditions
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especially for those corneas that are excised from aged donors.
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2. Material and Methods
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2.1. Ethical Statement
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The corneas [n=24] were collected from the Veneto Eye Bank Foundation (FBOV)
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with written consent from the donor’s next-of-kin to be used for research. The
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methods were carried out in accordance with the declaration of Helsinki. The tissues
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were used under the laws of Centro Nazionale di Trapianti, Rome, Italy. The corneas
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were suitable for research and unsuitable for transplantation due to low endothelial
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cell count (<2200 cells/mm2). No other complications or indications like Diabetes,
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HIV or HBV were noted from the donor database. All the tissues with endothelial cell
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density (ECD) between 1500-2200 cells/mm2, with no mortality before preservation,
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age above 65 years without any previously known history of ocular surgery were
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included in the study.
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2.2. Endothelial cell count and donor characteristics
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ECD and mortality [trypan blue positive cells (TBPCs)] before isolation was checked
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using trypan blue (TB) staining (0.25%) (Thermo Fisher Scientific (Rochester, NY,
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USA), which is routinely used in eye banks. Approximately 100 µL of TB was
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topically applied to stain the endothelial cells for 20 seconds followed by washing
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with phosphate buffered saline (PBS). TBPCs were recorded before isolation using
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an in-built eyepiece reticule for inverted microscope (Axiovert, Zeiss, Oberkochen,
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Germany). The reticule (10X10) was also used to check the number of cells before
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isolation and at confluence. An average of 5 readings were taken from different sites
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to lower the risk of biased data. The donor characteristics of 24 corneas were
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obtained retrospectively from FBOV database to determine the age, gender, post-
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mortem time, cause of death, ECD and mortality.
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2.3. Preservation media and conditions
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The tissues were preserved in conditions A) CS (Optisol GS – Bausch & Lomb, New
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York, USA) for 7 days at 4oC [n=6]; B) OC (Cornea Max – Eurobio, Les Ulis, France)
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for 7 days at 31oC [n=6]; C) OC for 28 days at 31oC [n=6] and D) CS for 7 days at
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4oC followed by OC for 28 days at 31oC [n=6]. For Conditions A and B, the corneas
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were obtained from the same donor (donor-matched study) with right eye in
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Condition A and left eye in Condition B. For Conditions C and D, random corneas
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were selected with endothelial cell count of 1600-2100 cells/mm2 with no baseline
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mortality.
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2.4. Peel and digest method
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The corneal tissues [n=24] were washed in sterile phosphate buffered saline (PBS)
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and the Descemet membrane-endothelial grafts were peeled in several pieces to
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ensure quick isolation, and a more even plating. The excised pieces were incubated
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in 2 mg/mL collagenase Type 1 (Thermo Fisher Scientific, Rochester, NY, USA)
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solution for 2-3 hours at 37oC and 5% CO2. The collagenase acts on the DM,
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degrading the membrane, allowing the corneal endothelium to form clusters of cells.
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These clusters were then centrifuged for 5 minutes at 1000 rpm, and re-suspended
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in TrypLE Express (1X), phenol red [Life Technologies, Monza, Italy] for 10 minutes
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at 37oC for another 5 minutes to further dissociate the endothelial cell clusters to
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smaller clumps and single cells. The supernatant was removed and the cells were
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re-suspended in 200 µL of the cell culture media. The culture media was composed
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of HamF12, M199, 5% FBS, 1% ascorbic acid, 0.5% Insulin Transferrin Selenium
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(ITS), Rec human FGF basic (10 ng/mL), 10 µM ROCK inhibitor (Y-27632) and
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antibiotics (Peh et al, 2011A, 2011B; Peh et al, 2015; Parekh et al, 2017; Peh et al.
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2013A, 2013B). The cells were re-suspended in a TB solution and counted using a
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haemocytometer slide. The TB positive cells were excluded and the number of
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plated cells was recorded for all the cultures.
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2.5. Plating cells
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Lab-Tek II chamber slides (8 chambers, 25X75 mm, 0.7 cm2 culture area) from
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Thermo Fisher Scientific (Rochester, NY, USA) was used for plating the cells. All the
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chambers were coated with 50 µL FNC coating mix [Cell attachment Reagent (FNC
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Coating mix) BRFF AF-10, US Biological Life Sciences, Salem, Massachusetts,
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USA] for at least 30-45 minutes at 37oC, 5% CO2. The residual coating was removed
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before plating the cells. Approximately 100 µL of the cell suspension was mixed well
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and added in each chamber to ensure equal distribution of cells from all the
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conditions. The cells from each donor cornea were isolated and plated, as
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mentioned above, in two wells. The cells were refreshed with media and were
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monitored every alternate day till confluence. The cells from all different conditions
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were isolated and plated as mentioned above.
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2.6. Morphological analysis of the cultured HCEnCs [n=24]
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The cells were visualized and the percentage of confluency was monitored every
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alternate day until confluence using a 10X10 reticule (0.1mm2) attached in the
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eyepiece of an inverted microscope (Axiovert, Zeiss, Germany) at 100X
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magnification. The confluence was evaluated by counting the number of blocks filled
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with cells observed in the eyepiece reticule per 10X10 mm2 field throughout the well.
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2.7. Hoechst, Ethidium homodimer and Calcein AM (HEC) staining to determine live
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and dead cells [n=2]
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Briefly, cultured cells were washed with PBS prior to the assay. 5 µL of Hoescht
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33342 (H) (Thermo Fisher Scientific, Rochester, NY, USA), 4 µL of Ethidium
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Homodimer EthD-1 (E), 2 µL Calcein AM (C) (Live/Dead viability/cytotoxicity kit,
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Thermo Fisher Scientific, Rochester, NY, USA) was mixed in 1 mL of PBS.
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Approximately 100 µL of the final solution was directly added on the cultured cells
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and the cells were incubated at room temperature in the dark for 45 minutes. With
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the solution still on the cells, the examination was carried out at 100X magnification
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with a Nikon Eclipse Ti-E (Nikon, Burgerweeshuispad, Amsterdam) using NIS
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Elements software (Nikon). Triple labelling showed the presence of Ethidium
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Homodimer stained in red representing the dead cells, Hoechst in blue representing
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the nuclei and Calcein AM in green marking the viable cells (Pipparelli et al, 2011).
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2.8. Immunostaining for tight junctions using ZO-1 [n=2], Na+/K+ ATPase [n=2], Ki-
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67 [n=2], 1A3 [n=2] and 2A12 [n=2]
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The cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) at room
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temperature (RT) for 20 minutes. The cells were permeabilized with 0.5% Triton X-
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100 in PBS for 30 minutes. [Note: the cells were not permeabilized with Triton X-100
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for 2A12]. After blocking with 5% goat serum for 1 hour at RT, the tissues were
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incubated overnight at 4oC with primary antibodies [anti-ZO-1, 1:200 (ZO1-1A12,
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Thermo Fisher Scientific, Rochester, NY, USA); anti-Na+/K+-ATPase, 1:50 (Na/K
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ATPase, Santacruz, Texas, USA) anti-Ki-67, 1:200 (MIB-1, Milan, Italy) and; anti-
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1A3, 1:100 (Tag-1A3) and anti-2A12, 1:100 (Tag-2A12) (Bioprocessing Technology
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Institute, Singapore) (Ding et al, 2014). The samples were incubated with goat anti-
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mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody in 10%
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goat serum for 2 hours at RT. 3 µL of Hoescht 33342 was mixed in 1mL PBS and
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100 µL of the solution was added on the top of cultured cells to stain the nucleus.
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After each step, the cells were washed 3 times with 1X PBS. After removing the wall
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of the Lab-Tek slide, the cells were covered with mounting medium and cover slips.
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Cells were examined with a Nikon Eclipse Ti-E (Nikon, Burgerweeshuispad,
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Amsterdam) using NIS Elements software (Nikon).
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2.9. Measurement and statistical analysis
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The confluence measurements were carried out as described earlier. ImageJ (FIJI)
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software (ImageJ bundled with Java 1.8.0_172, NIH, USA) was used for the
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measurement of endothelial cell numbers, as well as to assess the hexagonality and
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polymorphism of the cells based on ZO-1 expression. Percentage of Ki-67 positive
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cells as well as cell sizes based on Calcein AM staining of HCEnCs were also
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evaluated. The particles were analyzed using outline option and watershed was
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applied if necessary for Ki-67 positive cells. For ZO-1, the area was selected and
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using pre-defined commands in Macros for converting the image to overlay masks,
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the total number of cells was automatically counted whereas the hexagonal cells and
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polymorphic cells were counted based on the cell structure in the particular area
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(with 6 borders). The same Macros were used as previously described (Parekh et al,
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2017) to obtain results by simply inserting the algorithm in the ImageJ analysis. Cell
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surface area was determined using Calcein AM and analysed with ‘analyze particles’
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with size limits of 150-1000 µm2 to exclude background signals and large cell
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clusters. Data are expressed as mean ± SD.
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Non-parametric Wilcoxon test for paired data and one-way ANOVA test for
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independent measures using SAS statistical software was utilized to check for
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statistical significance between different conditions where p<0.05 was deemed to be
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statistically significant. A post-hoc correction to the significance was applied using
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Bonferroni.
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3. Results
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3.1. Donor characteristics and plating density
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Paired corneas were used from the same donor for conditions A and B therefore the
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donor characteristics were the same for both the conditions. ECD (cells/mm2) before
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the isolation of the cells is recorded in table 1. Trypan blue positive cells (TBPCs)
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(%) after preservation was found in condition A (Figure 1A) with limited TBPCs in
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conditions B (Figure 1B), C (Figure 1C) and D (Figure 1D). Approximately 9.3% ±
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4.0% TBPCs was found, mostly on the folds with a few scattered cells in Condition A
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(Figure 1A and 1E) before peeling. Very less amount of TBPCs was recorded before
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peeling in the other three conditions (Figure 1F). Number of cells plated per well of a
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Lab-Tek II chamber slide (8 chambers, 25X75 mm, 0.7 cm2 culture area) is half the
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amount of cells isolated from each cornea (table 1). All the results in table 1 were
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recorded for conditions A, B, C and D respectively with mean (±SD) [Range: Min-
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Max].
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3.2. Growth dynamics of HCEnCs cultured from different preservation conditions
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HCEnCs were observed at different days of culture (Figure 2) for morphology and
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confluency. At day 1, the growth rate (%) observed in Conditions A, B, C and D was
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14.0, 24.8, 23.4 and 25.4 respectively. At day 9, apart from Condition A, that showed
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94.2% confluency, all the other conditions showed 100% confluence (Figure 5A),
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suggesting that HCEnCs isolated following preservation in Condition A may have a
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slower propensity to propagate. However, the confluency rate was not statistically
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significantly different in either condition (p=0.9836). This may also be partly due to
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the presence of TBPCs, accounting to a lower overall cellular yield, and in turn,
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resulted in a lower initial number of plated cells observed in condition A.
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3.3. Overall cellular viability of HCEnCs cultured from different preservation
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conditions
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Cell viability was observed in all conditions (Figure 3). Cell area was determined on
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20 cells per condition using Calcein AM staining and ImageJ analysis. Cell area
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(µm2) was calculated and recorded in conditions A (Figure 3A), B (Figure 3B), C
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(Figure 3C) and D (Figure 3D) respectively without any statistical significant
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difference (p>0.05) (Figure 5B) (table 2).
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3.4. Characterization of HCEnCs cultured from different preservation conditions
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At the end of the culture, at day 9, ZO-1 (Figure 4) was expressed in all the
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conditions. Average value of hexagonality (%) and relative polymorphism (%)
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[polymegathism and pleomorphism] in the respective conditions are indicated in
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table 2 (Figure 5C). No significant difference was found in hexagonality and
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polymorphism. Na+/K+-ATPase, a function association marker for the corneal
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endothelial cells (Figure 4) and Tag-1A3 (Figure 4) was expressed in all the
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conditions suggesting the antigen target of CD166 and its presence on the cell
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surface of the cultured HCEnCs. Tag-2A12 (Figure 4) was also expressed in all the
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conditions showing the presence of cell surface epitopes PRDX-6 and confirming the
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cells to be HCEnCs. Ki-67, a proliferative marker showed Ki-67 positive cells (%)
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(Figure 4) that were counted using ImageJ and were recorded in conditions A, B, C,
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D as indicated in table 2 without any significant difference (Figure 5D).
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4. Discussion
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Alternative approaches for the treatment of corneal endothelial dysfunction have
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become possible with the ability to culture primary corneal endothelial cells (Peh et
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al, 2013A, 2013B; Shima et al, 2011; Kimoto et al, 2012; Zhu et al, 2012; Okumura
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et al, 2013), which enabled the development of cellular based therapy, as shown by
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the recent report of the corneal endothelial cell injection study (Kinoshita et al, 2018).
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However, the source of primary cells remains a challenge due to the limited
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expansion capacity of isolated primary HCEnCs, as well as the worldwide shortage
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of human donated corneas. It has been noted that cultivated HCEnCs derived from
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older donors have lower proliferative capability, where most derived HCEnCs formed
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cells of senescent morphology, indicating less overall cellular yield with potentially
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lesser functional capability than those derived from younger donors (Joyce et al,
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1995).
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Once the donor tissues are excised, they are sent to the eye bank and preserved in
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the storage media. Hypothermic preservation system offers short-term storage of
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corneal tissues (maximum 14 days). In Europe, many eye banks prefer OC storage
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method that includes the main storage medium comprising of serum and antibiotics
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that can preserve the corneal endothelium for up to 28 days at 31oC (Pels et al,
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2008). In this study, we investigated these different preservation conditions to
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evaluate the optimum storage method of the corneal tissues that are deemed for
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endothelial cell culture.
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In the donor-matched comparison between corneas stored in condition A and
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condition B, higher amount of TBPCs were observed in the corneas from condition
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A. The serum in OC is substantially ‘richer in nutrients’ which helps with the viability
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of the corneal endothelium, as observed in condition B. Condition A showed higher
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TBPCs before peeling, further indicating that the starting cell population may not be
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as healthy, leading to a lower cellular yield, and lower initial plating density, which
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may negatively impact the proliferative potential of the remaining isolated HCEnCs
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(Peh et al, 2013B). Preserved tissues in condition B were found to reach confluency
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faster than the corneas stored in condition A indicating that the endothelium may be
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better preserved in OC. In our observation, limited (<1%) endothelial cell death was
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observed in other conditions compared with condition A. Conditions C and D were a
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non donor-matched group, and although there may be potential donor variation,
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comparable results to condition B and superior outcomes to that of condition A
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strengthens the observation that corneas preserved in OC may be more suitable for
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HCEnC cultures in terms of general health of the corneal endothelium and overall
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cellular yield after cell isolation.
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When the tissues were preserved in only OC (condition B and C) or CS followed by
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OC (condition D), the cells were better preserved with high viability when broadly
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compared to tissues preserved in Condition A, in turn, yielding higher number of cells
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plated and reaching confluency faster. The non-viable cells could have dislodged
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and were replaced by healthy neighbouring cells through physical cellular spreading
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or potentially through proliferation (Joyce et al, 1995) in condition D. Condition D can
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potentially provide an added option to further extend the shelf life of corneas stored
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in hypothermic condition that may have become unsuitable for transplant, and
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effectively increase the storage time up to 35 days with minimal mortality. Thus
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increasing the time frame to organize these tissues that are downgraded from
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transplant-grade to research-grade corneas and can be used to establish cultures of
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HCEnCs.
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Varying factors such as health of the donor before death, the cause of death, as well
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as the duration from death to enucleation and the time from preservation to the
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establishment of culture (Peh et al, 2011A, 2011B; Beck et al, 1999; Miyata et al,
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2001; Parekh et al, 2017) have been noted to affect the cell culture. However, we did
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not find any significant deviation in establishing the culture of HCEnCs due to the
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above-mentioned factors.
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Live-dead cell staining showed no dead cells with no significant change in cell areas
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found in either conditions (figure 3). ZO-1 showed integrity of tight junction protein.
334
Morphological analysis showed preservation of hexagonality (>65% in the central
335
zone) where <20% cells were noted as polymorphic. Na+/K+-ATPase marker showed
336
the presence of the ionic pumps in all the conditions. The expression of cell surface
337
CD166 and Prdx-6 have been monitored using TAG-1A3 and TAG-2A12 respectively
338
showing a good correlation with the current standard of morphological grading of
339
HCEnCs (Ding et al, 2014). We have optimized the number of cells plated in each
340
well and to reduce the bias of more proliferative cells in either condition, we
341
assessed the cells using Ki-67, which was not found to be significant between the
342
conditions.
343
In conclusion, the results indicate that HCEnCs can be established from corneas
344
preserved in all four conditions. However, to reduce further stress on the corneal
345
endothelium and maintain its viability during isolation of the cells, it is recommended
346
to transfer the tissues or preserve them in OC storage, as hypothermic media could
347
have potential limitations in terms of viable or low plating density, short-term
348
preservation leading to less amount of preparation time, and less optimal conditions
349
and growth factors to preserve these cells. HCEnCs from old donor corneas that are
350
readily available for research due to its less endothelial cell count and proliferative
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capability may ultimately provide clinicians with a new therapeutic modality in
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regenerative medicine and reduce the global shortage of the donor corneas for the
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treatment of endothelial disorders, if cultured.
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6. Figure Legends
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Figure 1: Human corneal endothelial cells on the cornea before peeling, viewed
498
using an inverted microscope under 40X magnification. A) Condition A, endothelium
499
of the corneas preserved in hypothermia for 7 days showing trypan blue positive
500
cells (marked with arrow), B) Condition B, endothelium of the corneas preserved in
501
OC for 7 days at 31oC showing limited TBPCs, C) Condition C, endothelium of the
502
corneas preserved in OC for 28 days at 31oC showing limited TBPCs with iatrogenic
503
folds developed due to the preservation and swelling of the tissue. D) Condition D,
504
endothelium of the corneas preserved for 7 days in hypothermia following by OC
505
preservation for 28 days at 31oC showing minimal mortality. E) Trypan blue positive
506
areas found at the periphery while peeling the tissue obtained from hypothermic
507
condition (marked in black arrow), yellow arrow indicates the already peeled area
508
after re-staining it with trypan blue stain. F) The tissues obtained from OC preserved
509
corneas. Yellow arrow indicates the area already peeled after re-staining with trypan
510
blue stain.
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Figure 2: Morphological analysis at different days of the culture in different culture
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conditions at 40X magnification. The confluence rate (%) was high in conditions B, C
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and D. The number of isolated cells was relatively low in Condition A.
515
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Figure 3: HEC staining to determine live/dead cells at 100X magnification in A)
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condition A, B) condition B, C) condition C and D) condition D respectively. The
518
presence of live (green) cells and nucleus (blue) were observed in different
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conditions without any dead cells. Confluent areas were observed at day 9 in all the
520
conditions.
521
Figure 4: ZO-1 staining for intracellular tight junctions and to determine the
523
hexagonality and polymorphic cells in different conditions at 200X magnification. The
524
cultured HCEnCs showed expression of ZO-1 in all conditions. Na+/K+-ATPase was
525
expressed in all the conditions showing hydration potential (200X magnification). 1A3
526
and 2A12 was observed in all the conditions (100X magnification). Expression of Ki-
527
67 was expressed in all four conditions (100X magnification).
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Figure 5: Graphical representation of the conditions and relative values. A)
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Confluence rate representing confluency (%) of the cells obtained from different
531
conditions at different time points. A slow growth rate was observed when the
532
corneas were preserved in Condition A i.e. 7 days of preservation in cold storage
533
against the other conditions that showed similar growth trend. B) Cell area
534
determined by counting the cell surface area of 20 cells per condition showed no
535
significant difference between the samples. C) Hexagonality and polymorphism was
536
monitored and calculated after ImageJ analysis however, no significant difference
537
was found between any conditions. D) No significant difference was found in Ki-67
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expression.
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Number of cells counted from each cornea. The number of cells plated is half of the value below, per well 160,841.26 (±22,181.25) [136,778.4 – 180,471.5] 177,305.33 (±21,935.85) [151,976 – 189,970] 191,553.08 (±7150.21) [180,471.50 – 199,468.5] 193,136.16 (±4905.00) [189970 – 199,468.50]
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Age (years)
M:F
PM (hours)
ECD before isolation (cells/mm2)
C-A
74.33 (± 1.15) [73-75]
4:2
14.00 (±6.97) [6.15-19.45]
1866.67 (±230.94) [1600-2000]
C-B
74.33 (± 1.15) [73-75]
4:2
14.00 (±6.97) [6.15-19.45]
1866.67 (±230.94) [1600-2000]
C-C
73.00 (± 4.08) [68-78]
4:2
7.72 (±5.16) [3.5-17.12]
2016.67 (±75.28) [1900-2100]
0.52 (±0.28)
C-D
72.00 (±4.77) [65-76]
5:1
7.45 (±1.32) [5.2-8.35]
2033.33 (±51.64) [2000-2100]
0.47 (±0.28)
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0.46 (±0.35)
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Table 1: Donor characteristics and the number of cells plated from one donor cornea preserved in respective preservation medium, divided in two wells of an 8 well chamber slide. C-A: Condition A - CS (Optisol GS – Bausch & Lomb, USA) for 7 days at 4oC C-B: Condition B - OC (Cornea Max – Eurobio, France) for 7 days at 31oC C-C: Condition C - OC for 28 days at 31oC C-D: Condition D - CS for 7 days at 4oC followed by OC for 28 days at 31oC (CS=Cold Storage and OC=Organ Culture) PM=Post Mortem time; ECD = Endothelial Cell Density; TBPC = Trypan Blue Positive Cells; M:F = Male:Female The results are indicated as Mean (SD) [Range].
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Day 9 ECD (cells/mm2)
Cell area (μm2)
Hexagonality (%)
Polymorphism (%)
Ki-67 positive cells (%)
2050 (±132.29) [1900-2150]
421.1 (±383.3) [159-892] 68.4(±2.70) [65-72] 16.6(±3.13) [12-20]
17.55 (±3.12) [13.5-20.8]
C-B
2050 (±180.27) [1850-2200]
383.3 (±211.5) [152-931] 67.0(±5.15) [61-72] 17.4(±4.39) [13-23]
17.58 (±2.47) [15.2-21.8]
C-C
2141 (±49.16) [2100-2200]
360.8 (±200.4) [154-828] 67.4(±4.45) [62-73] 17.0(±3.67) [12-20]
17.23 (±3.34) [12.1-20.9]
C-D
2175 (±147.48) [2150-2300]
407.6 (±207.1) [167-784] 66.8(±3.96) [62-72] 17.6(±4.56) [13-23]
pValue
0.5473
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C-A
0.8312
0.8818
0.9630
17.93 (±3.07) [13.4-21.0] 0.9645
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Table 2: End-stage analysis of cultured human donor corneal endothelial cells including number of cells, cell area, hexagonality, polymorphism and number of proliferative cells in each studied condition as below. C-A: Condition A - CS (Optisol GS – Bausch & Lomb, USA) for 7 days at 4oC C-B: Condition B - OC (Cornea Max – Eurobio, France) for 7 days at 31oC C-C: Condition C - OC for 28 days at 31oC C-D: Condition D - CS for 7 days at 4oC followed by OC for 28 days at 31oC (CS=Cold Storage and OC=Organ Culture) The results are indicated as Mean (SD) [Range]
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1. Mortality before isolation was observed in tissues from hypothermia. 2. Tissues for HCEnC culture cannot be preserved for a long time in hypothermia. 3. Old age donor tissues from organ culture (OC) can increase the donor pool. 4. OC preserved tissues are suitable for HCEnC culture.
S0014-4835(18)30614-6
DOI:
https://doi.org/10.1016/j.exer.2018.11.007
Reference:
YEXER 7520
To appear in:
Experimental Eye Research
Received Date: 16 August 2018 Revised Date:
7 October 2018
Accepted Date: 6 November 2018
Please cite this article as: Parekh, M., Peh, G., Mehta, J.S., Ahmad, S., Ponzin, D., Ferrari, S., Effects of corneal preservation conditions on human corneal endothelial cell culture, Experimental Eye Research (2018), doi: https://doi.org/10.1016/j.exer.2018.11.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Parekh M et al. Endothelial cell culture from preserved corneas
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EFFECTS OF CORNEAL PRESERVATION CONDITIONS ON HUMAN CORNEAL
2
ENDOTHELIAL CELL CULTURE Authors
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Mohit Parekh1,2, Gary Peh3,4, Jodhbir S Mehta3,4,5,6, Sajjad Ahmad2,7, Diego Ponzin1,
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Stefano Ferrari1
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Affiliations
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1
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Venice, Italy
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2
10
3
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Singapore
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4
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5
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6
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Aerospace Engineering, Nanyang Technological University, Singapore
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7
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Correspondence
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Mohit Parekh MSc, PhD
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Institute of Ophthalmology, University College London,
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11-43 Bath Street, London, EC1V 9EL, UK
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Tel: 0044 7427652996
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Email: [email protected]
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Running title
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Endothelial cell culture from preserved corneas
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International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation,
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Institute of Ophthalmology, University College London, London, UK Tissue Engineering and Stem Cell group, Singapore Eye Research Institute,
Duke-NUS Graduate Medical School, Singapore
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Singapore National Eye Centre, Singapore
School of Material Science and Engineering and School of Mechanical and
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Moorfields Eye Hospital NHS Foundation Trust, London, UK
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Parekh M et al. Endothelial cell culture from preserved corneas
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3433
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Abstract
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The purpose of this study was to investigate the growth capacity of human corneal
29
endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different
30
storage conditions. The following conditions were evaluated, A) cold storage (CS)
31
(Optisol GS) for 7 days at 4oC [n=6]; B) organ culture (OC) (Cornea Max) for 7 days
32
at 31oC [n=6]; C) OC for 28 days at 31oC [n=6] and; D) CS for 7 days at 4oC followed
33
by OC for 28 days at 31oC [n=6]. Following preservations, the Descemet membrane-
34
endothelium complex was peeled and digested using Collagenase-Type1 and was
35
subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-
36
well chamber slide. Media was refreshed every alternate day. The confluence rate
37
(%) was assessed, and overall viability was determined using Hoechst, Ethidium
38
Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na+/K+-
39
ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were
40
used to analyse the cultures established from each condition. Donor tissues
41
preserved in hypothermia (condition A) resulted in 9.3%±4.0% trypan-blue positive
42
cells (TBPCs) hence lower number of HCEnCs was plated. There were <1% TBPCs
43
observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and
44
D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p=0.9836) at day 1. By day 9,
45
HCEnCs established from all conditions became confluent except cells from
46
condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and
47
expressed HCEnC-associated markers. In conclusion, OC system has advantages
48
over hypothermic media for the preservation of older donor corneas rejected for
49
corneal transplant and deemed suitable for corneal endothelial cell expansion, with
50
lower TBPCs before peeling and longer possible period of preservation over
51
hypothermic storage system.
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Keywords
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Eye bank; cornea; preservation; endothelium; cell culture; storage
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1. Introduction
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The posterior monolayer of the human cornea comprises of endothelial cells that
56
allows the transmission of soluble essential metabolites, and nutrients from the
57
aqueous humour into the cornea (Srinivas, 2010), and is essential for maintaining
58
corneal transparency (Joyce, 2003). The corneal endothelium must be maintained as
59
the number of endothelial cells decrease throughout life (Bourne, 2003). If the
60
endothelial cells are damaged to the stage that it impedes function, it can lead to
61
oedema followed by opacity resulting in corneal blindness (Engelmann et al, 1999;
62
Edelhauser, 2006). One of the main causes of endothelial failure is Fuch’s
63
endothelial dystrophy, which can be treated by a corneal transplant using a healthy
64
cadaveric donor cornea. As per a recent report, nearly 40% of the keratoplasties in
65
the United States are for treating corneal endothelial dysfunction. It has been
66
estimated that approximately 12.7 million patients are on the waiting list for a corneal
67
transplant (Gain et al, 2016; Wong et al, 2017). Hence, to address this severe
68
shortage of donor corneas, alternative approaches need to be identified to reduce
69
the global burden of corneal tissues.
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Although the cells of the corneal endothelium are not known to regenerate within the
71
eye (Joyce & Zhu, 2004), it has been shown that human corneal endothelial cells
72
(HCEnCs) can be propagated using appropriate in vitro growth conditions, driving
73
substantial research foci towards the development of alternative treatment options
74
for corneal endothelial dysfunction through cell replacement therapeutics (Okumura
75
et al, 2012). The development of a suitable graft alternative, through tissue
76
engineering or injection of cultivated HCEnCs has already been proposed (Koizumi
77
et al, 2012; Choi et al, 2010). The isolation and propagation techniques of HCEnCs
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in vitro have been described (Peh et al, 2011A, 2011B) and subsequently reviewed
79
(Parekh et al, 2013; Parekh et al, 2016).
80
Younger donors have advantages in terms of overall cell yield considering their high
81
proliferative capability over older donor corneas, which tend to senesce at earlier
82
passages (Peh et al, 2015). However, as most of the young donor corneas are
83
usually suitable for transplantation, it is difficult to find such tissues for cellular
84
expansion. If HCEnCs can be cultured from old donor corneas by modulating cell
85
adhesion then it may potentially increase the donor pool for culturing HCEnCs
86
(Parekh et al, 2017),
87
Apart from donor characteristics, it is also important to evaluate the media used for
88
corneal preservation, and its effects on culturing of the HCEnCs isolated from older
89
donor, in a systemic donor-matched manner. If the cornea is to be preserved for a
90
longer period of time (between 14 to 21 days), then the storage period, methodology,
91
as well as conditions must be optimized, validated and described if the corneas are
92
intended for subsequent cell culture and expansion. Hypothermic or cold storage
93
(CS) (2-6o C) system is being used in the United States and majority of the Asian
94
countries whereas most of the European eye banks prefer to use organ culture (OC)
95
(31-37oC) method. The maximum storage time for CS has been limited normally to
96
14 days whereas OC offers long-term storage for up to 4 weeks. OC has several
97
advantages over CS in terms of microbiological checks, quality assurance of the
98
tissues and preparation and planning time for surgery (Parekh et al, 2014; Parekh et
99
al, 2015). Thus, this study intends to assess the culture capacity of HCEnCs when
100
the corneas are preserved in different storage media, and in different conditions
101
especially for those corneas that are excised from aged donors.
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2. Material and Methods
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2.1. Ethical Statement
104
The corneas [n=24] were collected from the Veneto Eye Bank Foundation (FBOV)
105
with written consent from the donor’s next-of-kin to be used for research. The
106
methods were carried out in accordance with the declaration of Helsinki. The tissues
107
were used under the laws of Centro Nazionale di Trapianti, Rome, Italy. The corneas
108
were suitable for research and unsuitable for transplantation due to low endothelial
109
cell count (<2200 cells/mm2). No other complications or indications like Diabetes,
110
HIV or HBV were noted from the donor database. All the tissues with endothelial cell
111
density (ECD) between 1500-2200 cells/mm2, with no mortality before preservation,
112
age above 65 years without any previously known history of ocular surgery were
113
included in the study.
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2.2. Endothelial cell count and donor characteristics
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ECD and mortality [trypan blue positive cells (TBPCs)] before isolation was checked
117
using trypan blue (TB) staining (0.25%) (Thermo Fisher Scientific (Rochester, NY,
118
USA), which is routinely used in eye banks. Approximately 100 µL of TB was
119
topically applied to stain the endothelial cells for 20 seconds followed by washing
120
with phosphate buffered saline (PBS). TBPCs were recorded before isolation using
121
an in-built eyepiece reticule for inverted microscope (Axiovert, Zeiss, Oberkochen,
122
Germany). The reticule (10X10) was also used to check the number of cells before
123
isolation and at confluence. An average of 5 readings were taken from different sites
124
to lower the risk of biased data. The donor characteristics of 24 corneas were
125
obtained retrospectively from FBOV database to determine the age, gender, post-
126
mortem time, cause of death, ECD and mortality.
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2.3. Preservation media and conditions
128
The tissues were preserved in conditions A) CS (Optisol GS – Bausch & Lomb, New
129
York, USA) for 7 days at 4oC [n=6]; B) OC (Cornea Max – Eurobio, Les Ulis, France)
130
for 7 days at 31oC [n=6]; C) OC for 28 days at 31oC [n=6] and D) CS for 7 days at
131
4oC followed by OC for 28 days at 31oC [n=6]. For Conditions A and B, the corneas
132
were obtained from the same donor (donor-matched study) with right eye in
133
Condition A and left eye in Condition B. For Conditions C and D, random corneas
134
were selected with endothelial cell count of 1600-2100 cells/mm2 with no baseline
135
mortality.
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2.4. Peel and digest method
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The corneal tissues [n=24] were washed in sterile phosphate buffered saline (PBS)
139
and the Descemet membrane-endothelial grafts were peeled in several pieces to
140
ensure quick isolation, and a more even plating. The excised pieces were incubated
141
in 2 mg/mL collagenase Type 1 (Thermo Fisher Scientific, Rochester, NY, USA)
142
solution for 2-3 hours at 37oC and 5% CO2. The collagenase acts on the DM,
143
degrading the membrane, allowing the corneal endothelium to form clusters of cells.
144
These clusters were then centrifuged for 5 minutes at 1000 rpm, and re-suspended
145
in TrypLE Express (1X), phenol red [Life Technologies, Monza, Italy] for 10 minutes
146
at 37oC for another 5 minutes to further dissociate the endothelial cell clusters to
147
smaller clumps and single cells. The supernatant was removed and the cells were
148
re-suspended in 200 µL of the cell culture media. The culture media was composed
149
of HamF12, M199, 5% FBS, 1% ascorbic acid, 0.5% Insulin Transferrin Selenium
150
(ITS), Rec human FGF basic (10 ng/mL), 10 µM ROCK inhibitor (Y-27632) and
151
antibiotics (Peh et al, 2011A, 2011B; Peh et al, 2015; Parekh et al, 2017; Peh et al.
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152
2013A, 2013B). The cells were re-suspended in a TB solution and counted using a
153
haemocytometer slide. The TB positive cells were excluded and the number of
154
plated cells was recorded for all the cultures.
155
2.5. Plating cells
157
Lab-Tek II chamber slides (8 chambers, 25X75 mm, 0.7 cm2 culture area) from
158
Thermo Fisher Scientific (Rochester, NY, USA) was used for plating the cells. All the
159
chambers were coated with 50 µL FNC coating mix [Cell attachment Reagent (FNC
160
Coating mix) BRFF AF-10, US Biological Life Sciences, Salem, Massachusetts,
161
USA] for at least 30-45 minutes at 37oC, 5% CO2. The residual coating was removed
162
before plating the cells. Approximately 100 µL of the cell suspension was mixed well
163
and added in each chamber to ensure equal distribution of cells from all the
164
conditions. The cells from each donor cornea were isolated and plated, as
165
mentioned above, in two wells. The cells were refreshed with media and were
166
monitored every alternate day till confluence. The cells from all different conditions
167
were isolated and plated as mentioned above.
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2.6. Morphological analysis of the cultured HCEnCs [n=24]
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The cells were visualized and the percentage of confluency was monitored every
171
alternate day until confluence using a 10X10 reticule (0.1mm2) attached in the
172
eyepiece of an inverted microscope (Axiovert, Zeiss, Germany) at 100X
173
magnification. The confluence was evaluated by counting the number of blocks filled
174
with cells observed in the eyepiece reticule per 10X10 mm2 field throughout the well.
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2.7. Hoechst, Ethidium homodimer and Calcein AM (HEC) staining to determine live
177
and dead cells [n=2]
178
Briefly, cultured cells were washed with PBS prior to the assay. 5 µL of Hoescht
179
33342 (H) (Thermo Fisher Scientific, Rochester, NY, USA), 4 µL of Ethidium
180
Homodimer EthD-1 (E), 2 µL Calcein AM (C) (Live/Dead viability/cytotoxicity kit,
181
Thermo Fisher Scientific, Rochester, NY, USA) was mixed in 1 mL of PBS.
182
Approximately 100 µL of the final solution was directly added on the cultured cells
183
and the cells were incubated at room temperature in the dark for 45 minutes. With
184
the solution still on the cells, the examination was carried out at 100X magnification
185
with a Nikon Eclipse Ti-E (Nikon, Burgerweeshuispad, Amsterdam) using NIS
186
Elements software (Nikon). Triple labelling showed the presence of Ethidium
187
Homodimer stained in red representing the dead cells, Hoechst in blue representing
188
the nuclei and Calcein AM in green marking the viable cells (Pipparelli et al, 2011).
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2.8. Immunostaining for tight junctions using ZO-1 [n=2], Na+/K+ ATPase [n=2], Ki-
191
67 [n=2], 1A3 [n=2] and 2A12 [n=2]
192
The cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) at room
193
temperature (RT) for 20 minutes. The cells were permeabilized with 0.5% Triton X-
194
100 in PBS for 30 minutes. [Note: the cells were not permeabilized with Triton X-100
195
for 2A12]. After blocking with 5% goat serum for 1 hour at RT, the tissues were
196
incubated overnight at 4oC with primary antibodies [anti-ZO-1, 1:200 (ZO1-1A12,
197
Thermo Fisher Scientific, Rochester, NY, USA); anti-Na+/K+-ATPase, 1:50 (Na/K
198
ATPase, Santacruz, Texas, USA) anti-Ki-67, 1:200 (MIB-1, Milan, Italy) and; anti-
199
1A3, 1:100 (Tag-1A3) and anti-2A12, 1:100 (Tag-2A12) (Bioprocessing Technology
200
Institute, Singapore) (Ding et al, 2014). The samples were incubated with goat anti-
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mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody in 10%
202
goat serum for 2 hours at RT. 3 µL of Hoescht 33342 was mixed in 1mL PBS and
203
100 µL of the solution was added on the top of cultured cells to stain the nucleus.
204
After each step, the cells were washed 3 times with 1X PBS. After removing the wall
205
of the Lab-Tek slide, the cells were covered with mounting medium and cover slips.
206
Cells were examined with a Nikon Eclipse Ti-E (Nikon, Burgerweeshuispad,
207
Amsterdam) using NIS Elements software (Nikon).
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2.9. Measurement and statistical analysis
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The confluence measurements were carried out as described earlier. ImageJ (FIJI)
211
software (ImageJ bundled with Java 1.8.0_172, NIH, USA) was used for the
212
measurement of endothelial cell numbers, as well as to assess the hexagonality and
213
polymorphism of the cells based on ZO-1 expression. Percentage of Ki-67 positive
214
cells as well as cell sizes based on Calcein AM staining of HCEnCs were also
215
evaluated. The particles were analyzed using outline option and watershed was
216
applied if necessary for Ki-67 positive cells. For ZO-1, the area was selected and
217
using pre-defined commands in Macros for converting the image to overlay masks,
218
the total number of cells was automatically counted whereas the hexagonal cells and
219
polymorphic cells were counted based on the cell structure in the particular area
220
(with 6 borders). The same Macros were used as previously described (Parekh et al,
221
2017) to obtain results by simply inserting the algorithm in the ImageJ analysis. Cell
222
surface area was determined using Calcein AM and analysed with ‘analyze particles’
223
with size limits of 150-1000 µm2 to exclude background signals and large cell
224
clusters. Data are expressed as mean ± SD.
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Non-parametric Wilcoxon test for paired data and one-way ANOVA test for
226
independent measures using SAS statistical software was utilized to check for
227
statistical significance between different conditions where p<0.05 was deemed to be
228
statistically significant. A post-hoc correction to the significance was applied using
229
Bonferroni.
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3. Results
231
3.1. Donor characteristics and plating density
232
Paired corneas were used from the same donor for conditions A and B therefore the
233
donor characteristics were the same for both the conditions. ECD (cells/mm2) before
234
the isolation of the cells is recorded in table 1. Trypan blue positive cells (TBPCs)
235
(%) after preservation was found in condition A (Figure 1A) with limited TBPCs in
236
conditions B (Figure 1B), C (Figure 1C) and D (Figure 1D). Approximately 9.3% ±
237
4.0% TBPCs was found, mostly on the folds with a few scattered cells in Condition A
238
(Figure 1A and 1E) before peeling. Very less amount of TBPCs was recorded before
239
peeling in the other three conditions (Figure 1F). Number of cells plated per well of a
240
Lab-Tek II chamber slide (8 chambers, 25X75 mm, 0.7 cm2 culture area) is half the
241
amount of cells isolated from each cornea (table 1). All the results in table 1 were
242
recorded for conditions A, B, C and D respectively with mean (±SD) [Range: Min-
243
Max].
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3.2. Growth dynamics of HCEnCs cultured from different preservation conditions
246
HCEnCs were observed at different days of culture (Figure 2) for morphology and
247
confluency. At day 1, the growth rate (%) observed in Conditions A, B, C and D was
248
14.0, 24.8, 23.4 and 25.4 respectively. At day 9, apart from Condition A, that showed
249
94.2% confluency, all the other conditions showed 100% confluence (Figure 5A),
250
suggesting that HCEnCs isolated following preservation in Condition A may have a
251
slower propensity to propagate. However, the confluency rate was not statistically
252
significantly different in either condition (p=0.9836). This may also be partly due to
253
the presence of TBPCs, accounting to a lower overall cellular yield, and in turn,
254
resulted in a lower initial number of plated cells observed in condition A.
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3.3. Overall cellular viability of HCEnCs cultured from different preservation
256
conditions
257
Cell viability was observed in all conditions (Figure 3). Cell area was determined on
258
20 cells per condition using Calcein AM staining and ImageJ analysis. Cell area
259
(µm2) was calculated and recorded in conditions A (Figure 3A), B (Figure 3B), C
260
(Figure 3C) and D (Figure 3D) respectively without any statistical significant
261
difference (p>0.05) (Figure 5B) (table 2).
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3.4. Characterization of HCEnCs cultured from different preservation conditions
264
At the end of the culture, at day 9, ZO-1 (Figure 4) was expressed in all the
265
conditions. Average value of hexagonality (%) and relative polymorphism (%)
266
[polymegathism and pleomorphism] in the respective conditions are indicated in
267
table 2 (Figure 5C). No significant difference was found in hexagonality and
268
polymorphism. Na+/K+-ATPase, a function association marker for the corneal
269
endothelial cells (Figure 4) and Tag-1A3 (Figure 4) was expressed in all the
270
conditions suggesting the antigen target of CD166 and its presence on the cell
271
surface of the cultured HCEnCs. Tag-2A12 (Figure 4) was also expressed in all the
272
conditions showing the presence of cell surface epitopes PRDX-6 and confirming the
273
cells to be HCEnCs. Ki-67, a proliferative marker showed Ki-67 positive cells (%)
274
(Figure 4) that were counted using ImageJ and were recorded in conditions A, B, C,
275
D as indicated in table 2 without any significant difference (Figure 5D).
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4. Discussion
278
Alternative approaches for the treatment of corneal endothelial dysfunction have
279
become possible with the ability to culture primary corneal endothelial cells (Peh et
280
al, 2013A, 2013B; Shima et al, 2011; Kimoto et al, 2012; Zhu et al, 2012; Okumura
281
et al, 2013), which enabled the development of cellular based therapy, as shown by
282
the recent report of the corneal endothelial cell injection study (Kinoshita et al, 2018).
283
However, the source of primary cells remains a challenge due to the limited
284
expansion capacity of isolated primary HCEnCs, as well as the worldwide shortage
285
of human donated corneas. It has been noted that cultivated HCEnCs derived from
286
older donors have lower proliferative capability, where most derived HCEnCs formed
287
cells of senescent morphology, indicating less overall cellular yield with potentially
288
lesser functional capability than those derived from younger donors (Joyce et al,
289
1995).
290
Once the donor tissues are excised, they are sent to the eye bank and preserved in
291
the storage media. Hypothermic preservation system offers short-term storage of
292
corneal tissues (maximum 14 days). In Europe, many eye banks prefer OC storage
293
method that includes the main storage medium comprising of serum and antibiotics
294
that can preserve the corneal endothelium for up to 28 days at 31oC (Pels et al,
295
2008). In this study, we investigated these different preservation conditions to
296
evaluate the optimum storage method of the corneal tissues that are deemed for
297
endothelial cell culture.
298
In the donor-matched comparison between corneas stored in condition A and
299
condition B, higher amount of TBPCs were observed in the corneas from condition
300
A. The serum in OC is substantially ‘richer in nutrients’ which helps with the viability
301
of the corneal endothelium, as observed in condition B. Condition A showed higher
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TBPCs before peeling, further indicating that the starting cell population may not be
303
as healthy, leading to a lower cellular yield, and lower initial plating density, which
304
may negatively impact the proliferative potential of the remaining isolated HCEnCs
305
(Peh et al, 2013B). Preserved tissues in condition B were found to reach confluency
306
faster than the corneas stored in condition A indicating that the endothelium may be
307
better preserved in OC. In our observation, limited (<1%) endothelial cell death was
308
observed in other conditions compared with condition A. Conditions C and D were a
309
non donor-matched group, and although there may be potential donor variation,
310
comparable results to condition B and superior outcomes to that of condition A
311
strengthens the observation that corneas preserved in OC may be more suitable for
312
HCEnC cultures in terms of general health of the corneal endothelium and overall
313
cellular yield after cell isolation.
314
When the tissues were preserved in only OC (condition B and C) or CS followed by
315
OC (condition D), the cells were better preserved with high viability when broadly
316
compared to tissues preserved in Condition A, in turn, yielding higher number of cells
317
plated and reaching confluency faster. The non-viable cells could have dislodged
318
and were replaced by healthy neighbouring cells through physical cellular spreading
319
or potentially through proliferation (Joyce et al, 1995) in condition D. Condition D can
320
potentially provide an added option to further extend the shelf life of corneas stored
321
in hypothermic condition that may have become unsuitable for transplant, and
322
effectively increase the storage time up to 35 days with minimal mortality. Thus
323
increasing the time frame to organize these tissues that are downgraded from
324
transplant-grade to research-grade corneas and can be used to establish cultures of
325
HCEnCs.
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Varying factors such as health of the donor before death, the cause of death, as well
327
as the duration from death to enucleation and the time from preservation to the
328
establishment of culture (Peh et al, 2011A, 2011B; Beck et al, 1999; Miyata et al,
329
2001; Parekh et al, 2017) have been noted to affect the cell culture. However, we did
330
not find any significant deviation in establishing the culture of HCEnCs due to the
331
above-mentioned factors.
332
Live-dead cell staining showed no dead cells with no significant change in cell areas
333
found in either conditions (figure 3). ZO-1 showed integrity of tight junction protein.
334
Morphological analysis showed preservation of hexagonality (>65% in the central
335
zone) where <20% cells were noted as polymorphic. Na+/K+-ATPase marker showed
336
the presence of the ionic pumps in all the conditions. The expression of cell surface
337
CD166 and Prdx-6 have been monitored using TAG-1A3 and TAG-2A12 respectively
338
showing a good correlation with the current standard of morphological grading of
339
HCEnCs (Ding et al, 2014). We have optimized the number of cells plated in each
340
well and to reduce the bias of more proliferative cells in either condition, we
341
assessed the cells using Ki-67, which was not found to be significant between the
342
conditions.
343
In conclusion, the results indicate that HCEnCs can be established from corneas
344
preserved in all four conditions. However, to reduce further stress on the corneal
345
endothelium and maintain its viability during isolation of the cells, it is recommended
346
to transfer the tissues or preserve them in OC storage, as hypothermic media could
347
have potential limitations in terms of viable or low plating density, short-term
348
preservation leading to less amount of preparation time, and less optimal conditions
349
and growth factors to preserve these cells. HCEnCs from old donor corneas that are
350
readily available for research due to its less endothelial cell count and proliferative
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capability may ultimately provide clinicians with a new therapeutic modality in
352
regenerative medicine and reduce the global shortage of the donor corneas for the
353
treatment of endothelial disorders, if cultured.
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6. Figure Legends
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Figure 1: Human corneal endothelial cells on the cornea before peeling, viewed
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using an inverted microscope under 40X magnification. A) Condition A, endothelium
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of the corneas preserved in hypothermia for 7 days showing trypan blue positive
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cells (marked with arrow), B) Condition B, endothelium of the corneas preserved in
501
OC for 7 days at 31oC showing limited TBPCs, C) Condition C, endothelium of the
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corneas preserved in OC for 28 days at 31oC showing limited TBPCs with iatrogenic
503
folds developed due to the preservation and swelling of the tissue. D) Condition D,
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endothelium of the corneas preserved for 7 days in hypothermia following by OC
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preservation for 28 days at 31oC showing minimal mortality. E) Trypan blue positive
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areas found at the periphery while peeling the tissue obtained from hypothermic
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condition (marked in black arrow), yellow arrow indicates the already peeled area
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after re-staining it with trypan blue stain. F) The tissues obtained from OC preserved
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corneas. Yellow arrow indicates the area already peeled after re-staining with trypan
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blue stain.
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Figure 2: Morphological analysis at different days of the culture in different culture
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conditions at 40X magnification. The confluence rate (%) was high in conditions B, C
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and D. The number of isolated cells was relatively low in Condition A.
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Figure 3: HEC staining to determine live/dead cells at 100X magnification in A)
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condition A, B) condition B, C) condition C and D) condition D respectively. The
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presence of live (green) cells and nucleus (blue) were observed in different
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conditions without any dead cells. Confluent areas were observed at day 9 in all the
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conditions.
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Figure 4: ZO-1 staining for intracellular tight junctions and to determine the
523
hexagonality and polymorphic cells in different conditions at 200X magnification. The
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cultured HCEnCs showed expression of ZO-1 in all conditions. Na+/K+-ATPase was
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expressed in all the conditions showing hydration potential (200X magnification). 1A3
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and 2A12 was observed in all the conditions (100X magnification). Expression of Ki-
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67 was expressed in all four conditions (100X magnification).
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Figure 5: Graphical representation of the conditions and relative values. A)
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Confluence rate representing confluency (%) of the cells obtained from different
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conditions at different time points. A slow growth rate was observed when the
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corneas were preserved in Condition A i.e. 7 days of preservation in cold storage
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against the other conditions that showed similar growth trend. B) Cell area
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determined by counting the cell surface area of 20 cells per condition showed no
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significant difference between the samples. C) Hexagonality and polymorphism was
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monitored and calculated after ImageJ analysis however, no significant difference
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was found between any conditions. D) No significant difference was found in Ki-67
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Number of cells counted from each cornea. The number of cells plated is half of the value below, per well 160,841.26 (±22,181.25) [136,778.4 – 180,471.5] 177,305.33 (±21,935.85) [151,976 – 189,970] 191,553.08 (±7150.21) [180,471.50 – 199,468.5] 193,136.16 (±4905.00) [189970 – 199,468.50]
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Age (years)
M:F
PM (hours)
ECD before isolation (cells/mm2)
C-A
74.33 (± 1.15) [73-75]
4:2
14.00 (±6.97) [6.15-19.45]
1866.67 (±230.94) [1600-2000]
C-B
74.33 (± 1.15) [73-75]
4:2
14.00 (±6.97) [6.15-19.45]
1866.67 (±230.94) [1600-2000]
C-C
73.00 (± 4.08) [68-78]
4:2
7.72 (±5.16) [3.5-17.12]
2016.67 (±75.28) [1900-2100]
0.52 (±0.28)
C-D
72.00 (±4.77) [65-76]
5:1
7.45 (±1.32) [5.2-8.35]
2033.33 (±51.64) [2000-2100]
0.47 (±0.28)
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Table 1: Donor characteristics and the number of cells plated from one donor cornea preserved in respective preservation medium, divided in two wells of an 8 well chamber slide. C-A: Condition A - CS (Optisol GS – Bausch & Lomb, USA) for 7 days at 4oC C-B: Condition B - OC (Cornea Max – Eurobio, France) for 7 days at 31oC C-C: Condition C - OC for 28 days at 31oC C-D: Condition D - CS for 7 days at 4oC followed by OC for 28 days at 31oC (CS=Cold Storage and OC=Organ Culture) PM=Post Mortem time; ECD = Endothelial Cell Density; TBPC = Trypan Blue Positive Cells; M:F = Male:Female The results are indicated as Mean (SD) [Range].
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Day 9 ECD (cells/mm2)
Cell area (μm2)
Hexagonality (%)
Polymorphism (%)
Ki-67 positive cells (%)
2050 (±132.29) [1900-2150]
421.1 (±383.3) [159-892] 68.4(±2.70) [65-72] 16.6(±3.13) [12-20]
17.55 (±3.12) [13.5-20.8]
C-B
2050 (±180.27) [1850-2200]
383.3 (±211.5) [152-931] 67.0(±5.15) [61-72] 17.4(±4.39) [13-23]
17.58 (±2.47) [15.2-21.8]
C-C
2141 (±49.16) [2100-2200]
360.8 (±200.4) [154-828] 67.4(±4.45) [62-73] 17.0(±3.67) [12-20]
17.23 (±3.34) [12.1-20.9]
C-D
2175 (±147.48) [2150-2300]
407.6 (±207.1) [167-784] 66.8(±3.96) [62-72] 17.6(±4.56) [13-23]
pValue
0.5473
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0.8818
0.9630
17.93 (±3.07) [13.4-21.0] 0.9645
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Table 2: End-stage analysis of cultured human donor corneal endothelial cells including number of cells, cell area, hexagonality, polymorphism and number of proliferative cells in each studied condition as below. C-A: Condition A - CS (Optisol GS – Bausch & Lomb, USA) for 7 days at 4oC C-B: Condition B - OC (Cornea Max – Eurobio, France) for 7 days at 31oC C-C: Condition C - OC for 28 days at 31oC C-D: Condition D - CS for 7 days at 4oC followed by OC for 28 days at 31oC (CS=Cold Storage and OC=Organ Culture) The results are indicated as Mean (SD) [Range]
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1. Mortality before isolation was observed in tissues from hypothermia. 2. Tissues for HCEnC culture cannot be preserved for a long time in hypothermia. 3. Old age donor tissues from organ culture (OC) can increase the donor pool. 4. OC preserved tissues are suitable for HCEnC culture.