WO2010049181A1

WO2010049181A1 - Storage of conjuctival cells

Info

Publication number: WO2010049181A1
Application number: PCT/EP2009/007957
Authority: WO
Inventors: Darlene Ann Dartt; Marie Ann Shatos; Sten Raeder; Tor Paaske Utheim; Øygunn Aass UTHEIM; Ngoc-Trang-Thi Huynh
Original assignee: Oslo University Hospital; Schepens Eye Research Institute
Priority date: 2008-10-29
Filing date: 2009-10-28
Publication date: 2010-05-06

Abstract

A method of storing cultured conjunctival cells. The method comprises locating the cells in a liquid cell-storage medium at a temperature of between 3°C and 30°C, preferably between 22°C and 23°C, and storing the cells for a storage period of at least 24 hours.

Description

STORAGE OF CONJUCTIVAL CELLS

Field of the Invention

The present invention relates to a method of storing conjunctival cells and a method of preparing conjunctival cells suitable for implantation. The invention also relates to a cultured conjunctival cell storage container.

Background

The ocular surface is covered by conjunctival and corneal epithelia, each of which has a distinct cellular phenotype. They interact and are both crucial for maintaining a healthy ocular surface and preventing blindness. While the corneal epithelium preserves corneal transparency and is crucial for a clear visual image, the conjunctival epithelium plays an important role in maintaining the optical clarity of the cornea by providing lubrication to maintain a smooth and refractive surface. Conjunctiva also protects the eye against mechanical stress and infectious agents.

Conjunctival epithelium consists of two phenotypically distinct cell types, i.e. stratified squamous cells (90 - 95%) and goblet cells (5 - 10%). the latter cells secrete the gel forming mucins, which maintain a protective tear film.

Conjunctival and corneal epithelia undergo continuous replacement. Corneal stem cells are located in the limbal region, and conjunctival epithelial stem cells are in animal studies shown to be situated particularly in the forniceal regions (the folding of the eye lid), but also to some extent in the bulbar and palpebral regions.

Injuries and diseases can damage the ocular surface, leading to inflammation, scarring and depletion of goblet cells causing dry eye. In severe cases, a permanent eradication of the stem cell populations can occur, leading to pain and impaired vision/blindness. Examples of such injuries and diseases are shown in Table 1.

Table 1

Disorder Cause

Injuries Chemical burns

Thermal burns

Inflammatory: Stevens Johnson syndrome

Ocular cicatrical pemphigoid

Infectious: Trachoma

Viral papillomata

Post surgical conditions: Pterygium

Glaucoma bleb leakage

Tumor removal

Symblefaron

Eye lid and fornix reconstruction

Hereditary conditions: Aniridia

Many ocular surface disorders destroy both the conjunctiva and the cornea, and in these cases the conjunctival and the corneal epithelia need to be replaced. Another situation in which conjunctival epithelium needs to be replaced is where a patient has a large conjunctival lesion that requires surgical removal (e.g. recurrent papillomata, recurrent pterygium, and large nevi) and where the surgeon risks inducing conjunctival failure if the removed area is not covered with a conjunctival epithelial equivalent. Patients with glaucoma bleb leakage, symblefaron, and superior limbic keratoconjunctivitis will also be candidates for transplantation of conjunctival epithelial cells. Furthermore, recent studies demonstrate the feasibility of treating limbal stem cell deficiency with cultured conjunctival epithelial cells, which might reduce the need of an allogenic source of donor cells.^{1 '2}

US patent no. 7,049.139 discloses materials and methods for producing conjunctival tissue equivalents. It reports on the stages in the preparation of conjunctival cells, namely biopsy of the tissue: outgrowth of the cells; expansion of the number of cells (i.e. "culturing" the cells to use the terminology of this specification); and differentiation of the cells and formation of a tissue equivalent.

Despite the fact that millions of people worldwide suffer from ocular surface diseases, methods of transplanting cultured epithelial, especially conjunctival, sheets to the eyes of patients are not extensively used. This is because there are several practical problems that exist in the preparation of transplantable conjunctival cells.

Firstly, there is the problem that during the culturing process, the phenotype of cultured cells becomes more differentiated, whereas what is required for transplantation is undifferentiated conjunctival cells. This is coupled to the problem that conjunctival cells are generally harvested from recently-deceased individuals, which makes the timing of the transplantation surgery difficult because the surgery must take place shortly after the process of culturing the conjunctival cells, obtained from the donor, has been completed.

A second problem is that the harvesting and culturing of conjunctival cell explants requires specialist expertise and conditions and, generally speaking the number of centers capable of producing cultured conjunctival cells is limited. Nevertheless, the demand for cultured conjunctival cells is quite widespread and so cultured cells often need to be transported after being cultured. This is difficult because cultured cells of this type are regarded as being quite delicate and sensitive to movement. This problem is compounded by the fact that the culturing of conjunctival cells is typically carried out at 37⁰C (as reported, for example, in US 7,049,139) and thus there is a need for incubation of cells during transportation if these conditions are to be maintained.

A third problem is that the process of culturing conjunctival cells requires the exchange of cell medium on a regular basis (typically every two days). However, exchanging the medium in this way gives rise to the possibility of the cultured cells becoming infected and it is, of course, important that cultured cells instead remain sterile prior to implantation. Indeed, many countries require microbiological testing during culturing of cells so as to ensure quality control. The present invention arises from the finding that cultured conjunctival cells can be stored for several days under certain conditions, without significant differentiation of the conjunctival cells. Furthermore, the cells can be transported under these conditions. again without significant differentiation of the cells.

Summary of the Invention

According to a first aspect of the present invention, there is provided a method of storing cultured conjunctival cells comprising locating the cells in a liquid cell-storage medium.

In particular, the cultured conjunctival cells comprise or consist of stem cells. For example, at least 50% or at least 80% of cells may be stem cells.

Preferably, the cells are stored at a temperature of between 3⁰C and 3O⁰C for a storage period of at least 24 hours.

Conveniently, the liquid cell-storage medium is a serum free medium.

Preferably, the serum free medium comprises Optisol-GS.

Alternatively, the serum free medium comprises: a buffering agent and minimal essential medium. It is particularly preferred that the medium comprises MEM-HEPES, which is a relatively inexpensive medium.

Advantageously, the minimal essential medium comprises amino acids, salts, glucose and vitamins.

Alternatively, the liquid cell-storage medium comprises serum.

Conveniently, the liquid cell-storage medium comprises PAA Quantum 286. Advantageously, the cells are stored at a temperature of between 18 and 28⁰C. preferably 20 and 25⁰C, more preferably 22 and 24⁰C, more preferably 22 and 23⁰C.

Preferably, the storage period is at least two days, more preferably at least three days, more preferably at least four days, more preferably at least seven days.

Conveniently, the cells and the liquid cell-storage medium are stored in a sealed container, the cells and the liquid cell-storage medium filling the entire volume of the container such that substantially no gas is present above the surface of the liquid cell- storage medium.

Preferably, the cells are located in the same liquid cell-storage medium throughout the storage period.

Advantageously, the volume of the liquid cell-storage medium is less than 25ml, preferably less than 10ml, more preferably less than 5ml.

Conveniently, the cultured conjunctival cells form part of a tissue.

Preferably, there is substantially no increase in the differentiation of the conjunctival cells during the storage period.

Advantageously, the method further comprises the step of transporting the cells and the liquid cell-storage medium from a first location to a second location for a transportation period during the storage period.

Conveniently, the transportation period is at least 4 hours, preferably at least 6 hours, more preferably at least 4 days, more preferably at least 7 days.

According to another aspect of the present invention, there is provided a method of preparing conjunctival cells suitable for implantation comprising the steps of: (i) providing a conjunctival cell explant obtained from a donor: (ii) culturing the conjunctival cells in the explant in a liquid medium suitable for cell culturing in order to expand the number of cells; and

(iii) storing the cultured conjunctival cells in accordance with the first aspect of the present invention.

Conveniently, step (ii) comprises the step of maintaining the conjunctival cells at a temperature of between 35⁰C and 39⁰C, preferably 37⁰C.

Preferably, step (ii) comprises: maintaining the conjunctival cells submerged in the liquid medium suitable for cell culturing under an atmosphere comprising between 90% and 99% oxygen and between 10% and 1% carbon dioxide, more preferably 95% oxygen and 5% carbon dioxide.

Advantageously, the liquid medium suitable for cell culturing comprises pituitary extract.

Conveniently, the liquid medium suitable for cell culturing comprises calcium ions, preferably at a concentration of between 0.03 and 0.3mM. preferably between 0.12 and 0.17 mM, more preferably 0.15mM.

Preferably, step (ii) is carried out for a period of at least 7 days.

Conveniently, step (ii) comprises refreshing the liquid medium suitable for cell culturing, preferably between every 1 and 3 days.

According to a further aspect of the present invention, there is provided a cultured conjunctival cell storage container comprising: a sealable container: and a liquid cell- storage medium, wherein the respective volumes of the interior of the sealable container and the liquid cell-storage medium are such that when the container is sealed, it is completely filled with the liquid cell-storage medium.

Preferably, the interior volume of the sealable container is less than 25ml. more preferably less than 10ml. more preferably less than 5ml.

Advantageously, the liquid cell-storage medium is a serum free medium. Conveniently, the serum free medium comprises Optisol-GS.

Alternatively, the serum free medium comprises: a buffering agent and minimal essential medium.

Preferably, the minimal essential medium comprises amino acids, salts, glucose and vitamins.

Alternatively, the liquid cell-storage medium comprises serum.

Advantageously, the liquid cell-storage medium comprises PAA Quantum 286.

In some embodiments, "substantially no increase in the differentiation of cells" means that there are at least 80%, 90%, 95% or 99% of the number of undifferentiated cells at the end of storage as at the beginning.

There are a number of tests that can be used to determine whether there is substantially no increase in the differentiation of cells. For instance, it may be determined by analysing various immunohistochemical markers in the cells. For example the markers ABCG2, deltaNP63alfa_; Ki67_: PCNA, K3_; Integrin beta 4, and Alcian, can each be used alone or in combination to indicate that conjunctival cells are undifferentiated. In particular one test for the absence of differentiation is that the cells have low or negative expression of the marker K3. Further details of tests for determining the differentiation of conjunctival cells are provided in Example 1. Other markers for conjunctival cells are shown in Table 2. Table 2 - Semiquantitative immunohistochemical localization of stem cell markers in human ocular surface epithelia

-, undetectable; (+). weak positivity; +, moderate positivity; ++, strong positivity.

In this specification, the term "cultured" is used in relation to conjunctival cells and tissue to indicate that the cells or tissue have been subjected to "ex vivo expansion" and the terms can be used interchangeably. For example, typically tissue is removed from a donor at which point the tissue typically comprises only 2 or 3 stem cells (the remainder of the cells being differentiated cells). The explant is then subject to culturing at 37⁰C in hormonal epithelial medium under an atmosphere of 95% oxygen and 5% carbon dioxide. The medium is replaced every second day. The process of culturing, or ex vivo expansion, results in an increase in the number of undifferentiated cells in the explant due to replication of the stem cells. Cultured cells generally lack the connective tissue of uncultured cells and comprise fewer cell layers. Cultured cells are more suitable for transplantation than tissue taken directly from a donor. Figures

Figure 1 is a perspective view of a cultured conjunctival cell storage container in accordance with one embodiment of the present invention.

Figure 2 depicts laser confocal micrographs following viability staining of cultured human conjunctival cells prior to storage, after storage for four days and seven days in Optisol-GS, MEM-Hepes and Quantum 286. Live cells are CAM-positive and stain green (light grey in images), whereas dead cells are EH-I positive and stain red (dark grey in images). Magnification: x 250.

Figure 3 shows images of histological sections of cultured human conjunctival cells prior to storage, and after storage for four days and seven days in Optisol-GS, MEM- Hepes and Quantum 286. The blue staining (dark grey in images) on top of the superficial layer represents alcian blue and was used to visualise the epithelia before embedding in paraffin. Original magnification: x 400.

Figure 4 shows images of histological sections of cultured human conjunctival cells following storage at ambient temperature (23°C) for 58 hours in MEM-Hepes (pH 7.2) and after storage at ambient temperature (23 °C) for 40 hours in MEM-Hepes (pH 7.2), shaking at 200 rpm for 6 hours using a IKA MTS 2/4 digital microtiter shaker (IKA Werke GmbH & Co. KG, Staufen. Germany) and 12 hour storage (in total 58 hour storage). The blue staining (dark grey in images) on top of the superficial layer represents alcian blue and was used to visualise the epithelia before embedding in paraffin. Original magnification: x 400.

Figure 5 shows images of laser confocal micrographs following viability staining of cultured human conjunctival cells cultured in DMEM and RPMI medium prior to storage and after storage for four days in Optisol-GS, MEM-Hepes and Quantum 286. Live cells are CAM-positive and stain green (light grey in images), whereas dead cells are EH-I positive and stain red (dark grey in images). Magnification: x 250. Figure 6 shows images of histological sections of cultured human conjunctival cells cultured in DMEM and RPMI medium prior to storage and after storage for four days in Optisol-GS, MEM-Hepes and Quantum 286. The blue staining (dark grey in images) on top of the superficial layer represents alcian blue and was used to visualise the epithelia before embedding in paraffin. Original magnification: x 400.

Figure 7 shows images of Immunofluorescent staining of keratin 4 (K4) of cultured human conjunctival cells cultured in DMEM and RPMI medium prior to storage and after storage for four days in Optisol-GS, MEM-Hepes and Quantum 286. Original magnification: * 400.

Figure 8 shows images of viability staining and histological sections of cultured human conjunctival cells after storage for seven days in Optisol-GS and MEM-HEPES. Original magnification: x 400.

Figure 9 shows images of viability staining, histological sections, and immunofluorescent staining for K4, p75 and HPA in cultured human conjunctival cells expanded in Quantum medium prior to storage and following storage at ambient temperature (23 °C) for 4 days in MEM-Hepes (pH 7,2) and after shaking at 50 rpm for 7 hours (in total 4 days storage). Original magnification: x 400.

Detailed Description

Embodiments of the present invention will now be described.

In order to prepare conjunctival cells suitable for implantation into a recipient individual, a conjunctival cell explant is obtained from a donor individual and the cells are cultured as is known in the art (see. for example, US7,049,139, which is incorporated herein by reference). In brief, a human amniotic membrane is first prepared on which the explant may be cultured. The human amniotic membrane is sutured, with the epithelial side face up, to a polyester membrane of a culture insert plate (e.g. of the type available from Costar, Corning, New York. New York, USA). The conjunctival cell explant is then located on the human amniotic membrane and submerged under a medium suitable for culturing of the conjunctival cells. A typical medium is supplemented hormonal epithelial medium (e.g. comprising HEPES-buffered DMEM containing sodium bicarbonate and Ham's Fl 2 (1 :1) supplemented with 5% FBS. 0.5% dimethyl sulfoxide. 2 ng/mL human epidermal growth factor. 5 μg/mL insulin. 5 μg/mL transferrin. 5 ng/mL selenium. 3 ng/mL hydrocortisone. 30 ng/mL cholera toxin, 50 μg/mL gentamycin, and 1.25 μg/mL amphotericin B). The explant is incubated for 14 days at 37⁰C under an atmosphere of 5% carbon dioxide and 95% oxygen. The medium is changed (refreshed) every 2 to 3 days.

After the conjunctival cells have been cultured, they are stored for a period of time. Referring to Figure 1 , there is provided a container 1 , which is sealable. for example by means of screw cap 2. In order to store the conjunctival cells, the container 1 is filled with a liquid cell-storage medium 3. In some embodiments, the liquid cell-storage medium 3 is a serum-free medium such as Optisol-GS or MEM-HEPES. The advantage of a serum-free medium is that it is much less likely to carry infection than a serum- based medium. Furthermore, serum-free media are more consistently produced so it is easier to produce more consistent and reliable results with a serum-free medium. However, in some other embodiments, the liquid cell-storage medium 3 comprises serum, for example, in one embodiment the medium 3 is PAA Quantum 286.

The cultured conjunctiva] cells 4, located on the amniotic membrane 5 which is in turn sutured to the polyester membrane of a culture insert plate 6, are then removed from the culture medium and transferred, under sterile conditions, to the container 1. The container 1 is then sealed by closing of the screw cap 2. It is to be appreciated that since the container 1 was filled with the liquid cell-storage medium 3, transferring of the cultured conjunctival cells 4, amniotic membrane 5 and culture insert plate 6, may result in some overflow of the liquid cell-storage medium 3 from the container 1. In any case, after sealing of the container 1 , the liquid cell-storage medium 3 completely fills the sealed container 1 such that there is no, or substantially no (less than 1% by volume) gas in the container 1 above the surface of the liquid cell-storage medium 3.

The sealed container 1 is stored at ambient temperature (23 ⁰C) for a period of time of at least 24 hours and in some embodiments at least 4 or 7 days. In some embodiments, the container 1 is provided with a septum (not shown) through which samples of the liquid cell-storage medium may be taken, using a hypodermic needle, in order to carry out microbiological testing of the liquid cell-storage medium 3.

The sealed container may be transported to another location during this period of storage, for example to a hospital where the recipient individual is awaiting the explant. Such transportation can take up to several hours or even several days. Since storage takes place at ambient temperature, no specialised incubation or cooling/freezing equipment is required for the sealed container 1 which makes transportation relatively- straightforward. It has been found that even after several hours of transportation, the phenotype of the cultured conjunctival cells remains substantially unchanged, that is to say there is substantially no increase in the differentiation of the conjunctival cells during transportation. In this regard, the absence of gas from the sealed container 1 is particular advantageous during transportation since turbulence within the liquid cell- storage medium 3 is minimised and the liquid cell-storage medium in fact acts physically to protect and cushion the conjunctival cells 4 during movement, vibration etc. Furthermore, the absence of gas from the sealed container 1 ensures that the conjunctival cells 4 remain submerged in the liquid cell-storage medium 3 irrespective of movement of the sealed container 1 during transportation.

The volume of the interior of the sealed container 1 , which thus dictates the volume of liquid cell-storage medium 3 and other components (the conjunctival cells 4, amniotic membrane 5 and culture insert plate 6) present in the container 1. is quite low in some embodiments. For example in one embodiment the volume is 25 ml, in another embodiment the volume is 10 ml and in a further embodiment, the volume is 5ml. Accordingly, the size of the container and the amount of liquid cell-storage medium 3 required for storage can be reduced in order to make transportation of the conjunctival cells 4 even easier and less expensive.

Following storage and any necessary transportation of the cultured conjunctival cells, the container 1 is unsealed and the culture insert plate 6 is removed from the container 1. The cultured conjunctival cells 4 are then removed from the amniotic membrane 5 and may be surgically implanted in an eye of the recipient individual. EXAMPLES

Example 1

Experimental design

Conjunctival biopsies were cultured on intact human amniotic membrane (HAM) in Dulbecco's modified Eagle's medium (DMEM) medium. The conjunctival epithelial equivalent explants were stored in a closed container in Optisol-GS (serum free medium), MEM-Hepes (serum free medium), and Quantum 286 (serum based medium) at ambient temperature (23°C) for four of seven days.

Materials

Dulbecco's modified Eagle's medium (DMEM), HEPES (N-2-hydroxyethylpiperazine- N'-ethane-sulphonic acid) -buffered DMEM containing sodium bicarbonate and Ham's F12 (1 : 1), Minimal essential medium (MEM), Hepes, Hanks' balanced salt solution, fetal bovine serum (FBS), insulin-transferrin-sodium selenite media supplement, human epidermal growth factor, dimethyl sulfoxide, hydrocortisone, gentamycin, amphotericin B, and mouse anti-ABCG2 antibody (clone bxp21) were purchased from Sigma-Aldrich (St. Louis, MO). Optisol-GS was from Bausch & Lomb (Irvine. CA) whereas Quantum 286 was from PAA Laboratories GmbH (Pasching, Austria). Dispase II was obtained from Roche Diagnostics (Basel, Switzerland), cholera toxin A subunit from Biomol (Exeter, UK), 6 mm biopsy punches from Kai Industries (Gifu, Japan), 6-0 C-2 monofilament sutures (Ethicon Ethilon) from Johnson & Johnson (New Brunswick, NJ), 24 mm culture plate inserts (Netwell, 74 μm mesh size polyester membrane) from Costar Corning (New York, NY), and vancomycin from Abbott Laboratories (Abbott Park, IL). Anti-PCNA antibody (clone PClO) were obtained from Dako (Glostrup. Denmark), rabbit polyclonal anti-del taNp63α antibody from Primm (Milano, Italy), mouse anti-Ki67 antibody (clone SP6) from LabVision Corporation (Fremont, CA), anti-integrin β4 (rabbit polyclonal) and anti-p75 (clone PCl O) from Abeam (Cambridge, MA), and mouse anti-K3 antibody (clone AE5) from ImmuQuest (Cleveland, UK). Medium description

Optisol-GS

Optisol-GS consists of chondroitin sulfate, dextran. Optisol base powder mixture, sodium bicarbonate, gentamycin. streptomycin sulfate, amino acids mixture, sodium pyruvate. L-Glutamine. 2-Mercaptoethanol. and water.

MEM-Hepes See Table 1.

Table 1 - MEM (Minimum Essential Medium)

Components mg/L

Inorganic Salts

Calcium Chloride anhydrous 200.00

Potassium Chloride 400.00

Magnesium Sulphate anhydrous 97.70

Sodium Chloride 6800.00 Sodium Dihydrogen Phosphate • H₂O 140.00

Sodium Hydrogen Carbonate 2200.00

Amino Acids

L-Arginine • HCl 126.00

L-Cystine 24.00

L-Glutamine in El 5-825 292.00

L-Histidine • HCl • H₂O 42.00

L-Isoleucine 52.00

L-Leucine 52.00

L-Lysine • HCl 72.50

L-Methionine 15.00

L-Phenylalanine 32.00

L-Threonine 48.00

L-Tryptophan 10.00

L-Tyrosine 36.00

L-Valine 46.00

Vitamins

D-Calcium Pantothenate 1.00

Cholin Chloride 1.00

Folic Acid 1.00 Myo-Inositol 2.00

Nicotinamide 1.00

Pyridoxal • HCl 1.00

Riboflavin 0.10

Thiamine • HCl 1.00

Other Components

D-Glucose anhydrous 1000.00

Phenol Red 11.00

Quantum 286

See Table 3

Table 3 - Quantum 286

Harvesting of human conjunctival tissue

Oriented cadaveric human conjunctival biopsies were obtained from one donor with research consent from the Centro de Oftalmologia Barraquer (Barcelona, Spain). The samples were stored in organ culture medium and shipped in ambient temperature to Centre for clinical research, Ulleval university hospital (Oslo, Norway).

Human conjunctival explant cultures on intact amniotic membranes Human amniotic membranes (AM) were preserved according to a method described by Lee & Tseng.³ After thawing at room temperature, devitalized intact AM was fastened, with the epithelial side facing up, to the polyester membrane of a culture plate insert using monofilament sutures, as previously reported.⁴'⁵ The conjunctival tissue was rinsed with DMEM containing 50 μg/mL gentamicin and 1.25 μg/mL amphotericin B, was placed in a culture dish and exposed for ten minutes to Dispase II (1.2 U/mL) in Mg2+ and Ca2+ free Hanks' balanced salt solution at 37⁰C under humidified 5% carbon dioxide. Following one rinse with DMEM containing 10% FBS, the tissue was divided, using a steel blade, into explants measuring 1x1 mm. In the center of each AM insert, explants were cultured in supplemented hormonal epithelial medium (HEPES -buffered DMEM containing sodium bicarbonate and Ham's F12 (1 :1) supplemented with 5% FBS. 0.5% dimethyl sulfoxide. 2 ng/mL human epidermal growth factor. 5 μg/mL insulin. 5 μg/mL transferrin. 5 ng/mL selenium, 3 ng/mL hydrocortisone, 30 ng/mL cholera toxin. 50 μg/mL gentamycin. and 1.25 μg/mL amphotericin B). Cultures were incubated for 14 days at 37⁰C in humidified 5% carbon dioxide, and the medium was changed every 2 to 3 days.

Eye bank storage of conjunctival explant cultures

Preparation for eye bank storage was performed in a class II safety cabinet. Eye bank storage medium (Optisol-GS, MEM-HEPES (pH 7.2), and PAA-Quantum) was preheated at 31⁰C. 40 mL of each medium was added to 50 mL plastic containers. Conjunctival explant cultures in polyester culture plate inserts were transferred by disposable forceps to the storage containers. The cap was closed and secured with parafilm to establish a closed tissue storage system without gas above the medium, and the containers were stored for 4 days (n=2 in each experimental group) and 7 days (n=2) at 23 °C in a heating cabinet.

Live-dead viability assays

Viability staining was performed using a calcein-acetoxymethyl ester (CAM)/ethidium homodimer 1 (EH-I) (Invitrogen, Carlsbad, CA) assay⁶ with some modifications. Discs of cultured epithelium and AM on polyester membranes from cultures following zero (n = 3), four (n = 2 in each experimental group) and seven (n = 2 in each experimental group) days of storage were trephinated using a 6 mm Kai biopsy punch. The disks were incubated in phosphate-buffered saline (PBS) containing 2 μM CAM and 2 μM EH-I (23°C and 45 minutes), washed with PBS, and mounted on cover-slipped glass slides. Fluorescent images of the basal layer were photographed using an Axiovert 100 LSM 510 laser scanning confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany). The numbers of live and dead cells (green and red fluorescence, respectively) were counted in 5 fields at x 250 magnification, and the percentage of live cells calculated.

Histology and immunostaining

Cultures after zero (n = 3), four (n = 2 in each experimental group) and seven (n = 2 in each experimental group) days of storage were fixed in neutral buffered 4% formaldehyde and embedded in paraffin. Serial sections of 4 μm were routinely stained with haematoxylin and eosin (H&E). Immunohistochemistry was performed with a panel of antibodies for markers of human ocular surface epithelia (Table 5). Immunoreactivity was visualised following the use of a standard peroxidase technique (DAB detection kit) in a Ventana ES Immunohistochemistry Instrument (Tucson. AZ). Optimal antibody dilutions were determined by titration using the positive controls recommended by the manufacturers. . Histological evaluation and semiquantitative immunohistochemical localisation of the epithelial markers were carried out by two independent investigators using a microscope at x 400 magnification.

10

Results

Viability

The viability before and after storage of cultured conjunctival cells was at least 90% in 15 serum free media, but lower in the serum based medium (74%-81%). see Table 4 and Figure 2.

Table 4

20 Morphology

The morphology after storage for four days in both serum free and serum based media was equivalent to the morphology before storage. 7 days storage of cultured conjunctival cells appeared almost as good as the controls, see Figure 3.

Phenotype

A panel of immuohistochemical markers was used, which can be categorized into the following groups: 1) proposed stem cell markers (ABCG2 and deltaNP63alfa), 2) proliferation markers (Ki67 and PCNA), 3) corneal markers (K3). 4) adhesion markers (Integrin beta 4). and 5) mucin markers (Alcian). The phenotype was maintained following 4 and 7 days of storage in both serum based and serum free media, see Table 5.

Table 5

Example 2

Experimental design

Conjunctival biopsies were cultured on intact human amniotic membrane (HAM) in Dulbecco's modified Eagle's medium (DMEM) medium. The conjunctival epithelial equivalent explants were stored in a closed container in MEM-Hepes (serum free medium) at ambient temperature (23 ⁰C) for 40 hours, shaken at 80 rpm or 200 rpm for 6 hours, and subsequently stored for 12 hours. The storage medium was filled up to the maximum level of the plastic bottle, hence completely replacing air with medium.

Materials

As per Example 1.

Methods

Human conjunctival tissue was harvested and cultured on intact amniotic membranes as described in Example 1. with some minor modifications. The explants were cultured for 5 days in keratinocyte growth medium previously described by Ang et al.⁷Due to lack of growth, the protocol was changed to the conventional culture medium described above. Cultures were incubated for 22 days at 37⁰C under humidified 5% carbon dioxide, and the medium was changed every 2 to 3 days. The cultures were subject to eye bank storage in MEM-Hepes (pH 7.2) as described. The cultures were initially stored at ambient temperature (23 °C) for 40 hours. Next, four cultures were shaken at 80 rpm (n = 2) and 200 rpm (n = 2) for 6 hours using a IKA MTS 2/4 digital microtiter shaker (IKA Werke GmbH & Co. KG. Staufen. Germany), whereas two cultures were not subject to shaking. Finally, all cultures were stored for 12 hours. The cultures were analysed with respect to viability and morphology.

Results

Viability

The viability before and after transportation of cultured conjunctival cells in a full container was over 90%, as shown in Table 6. Table 6

Morphology

The morphology remained after transportation in a full container,, see Figure 4.

Example 3

Conjunctival biopsies were cultured on intact Human Amniotic Membrane (HAM) comparing three serum containing culturing media (culturing media 1 , 2 and 3, see Table 7). The conjunctival epithelial equivalent explants were stored in three different storage media, (two of them serum free and one serum containing as a control, see Table 8) at ambient temperature for 4 or 7 days. Table 7 - Culturing Media

Table 8 - Storage Media

Table 9: RPMI 1640 R0883 (Sigma-Aldrich)

Table 10: DMEM/F12 (1:1)

Harvesting of conjunctiva

Conjunctival biopsies were harvested from cadaveric donors at El centro de Oftalmologia Barraquer in Barecelona, Spain, and placed on a bottle filled with organ culture medium. The samples were stored and shipped in ambient temperature, from El centro de Oftalmologia Barraquer to the tissue culturing laboratory at Schepens Eye Research Institute in Boston, U.S.A.

Preparation of Human Amniotic Membrane (HAM)

HAMs were cryopreserved according to a previously reported method (Lee & Tseng 1997)³, at the tissue culturing laboratory at Ulleval University Hospital in Oslo,

Norway. The HAMs were shipped in dry ice from Norway to the tissue culturing laboratory at Schepens Eye Research Institute, and were still frozen when they arrived.

At Schepens Eye Research Institute, the HAMs were thawed quickly by placing the sheets in a bottle and submerging the bottle in a 37 ⁰C water bath. The membranes were removed from the bottle and placed in a Petri Dish containing Phosphate Buffered

Saline (PBS). The membrane was rinsed twice with PBS solution containing 100 IU/ml penicillin, 100 μg/ml streptomycin and 5 μg /ml Amphotericin B for 10 minutes. The

HAMs were then sutured onto polyester membrane inserts sized 24 mm in diameter with Ethilon 6.0 sutures, with the epithelium facing up, and the wells were filled with medium in order to soak the HAMs in medium overnight before culturing.

Culturing of conjunctiva

The conjunctival biopsies were placed in a Petri dish filled with phosphate buffered saline, cut into 2 x 2 mm pieces with a scalpel and placed centrally on the HAM, conjunctival epithelium facing up. one piece in each insert. Culture media 1, 2, or 3 were carefully added with a micropipets tip slowly so that each explant was partially submerged. The cells were incubated at 37 ⁰C, under 5% CO2 and 95% air for 10 days.

Storing of conjunctival explants

On day 10 the inserts were removed from the culturing wells and placed in new wells filled with the Storage Medium Optisol GS, covered, sealed and left in a sterile bench to ensure sterile storage milieu. Some of the cultures were stored at ambient temperature for 4 days, and others were stored for 7 days. The cultures that were not stored, were harvested on day 10 to be used as controls.

Results

Viability

The viability before and after storage of cultured conjunctival cells was at least 90% in all media, as shown in Table 11 and Figures 5 and 8.

Table 11 - Viability following storage of cultured conjunctival cells.

Morphology

The morphology cultured conjunctival cells after storage of four days (see Figure 6) and seven days (see Figure 8) was equivalent to the morphology before storage.

Phenotype

The conjunctival marker K4 remained unchanged before and after storage, as shown in Figure 7.

Example 4

Transportation of stored cultured conjunctival cells

Human conjunctival cells were expanded in Quantum medium (control). Furthermore, human conjunctival cells were expanded in Quantum medium and stored at ambient temperature (23⁰C) for 4 days in MEM-HEPES (pH 7.2). During this period the culture was subject to shaking at 50 rpm for 7 hours. The storage medium was filled up to the maximum level of the plastic bottle, hence completely replacing air with medium.

Analyses performed

Viability was performed as previously described using a Leica TCS 4D confocal microscope. HE-sections were prepared as previously described. Phenotypic characterization was performed using a manual technique.

Results

Viability

The viability was over 97% before transportation/storage and 99% after transportation/storage, see Table 12.

Morphology The morphology was well maintained after transportation/storage, see Figure 9. Phenotvpe

The expression of K4, p75 and HPA were maintained after transportation/storage, see Figure 9.

Table 12

Reference List

1. Tanioka H, Kawasaki S. Yamasaki K et al. Establishment of a cultivated human conjunctiva] epithelium as an alternative tissue source for autologous corneal epithelial transplantation. Investigative Ophthalmology & Visual Science 2006;47:3820-7.

2. Ono K. Yokoo S, Mimura T et al. Autologous transplantation of conjunctival epithelial cells cultured on amniotic membrane in a rabbit model. Molecular Vision.13: 1138-43, 2007.

3. Lee SH, Tseng SC. Amniotic membrane transplantation for persistent epithelial defects with ulceration. American Journal of Ophthalmology 1997;123:303-12.

4. Utheim TP, Raeder S₅ Utheim OA et al. A novel method for preserving cultured limbal epithelial cells. BrJ. Ophthalmol. 2007,91:797-800.

5. Raeder S, Utheim TP₅ Utheim OA et al. Effects of Organ Culture and Optisol-GS Storage on Structural Integrity. Phenotypes, and Apoptosis in Cultured Corneal Epithelium. Invest Ophthalmol. Vis.Sci. 2007;48:5484-93.

6. Chen CC, Chang JH, Lee JB et al. Human corneal epithelial cell viability and morphology after dilute alcohol exposure. Invest Ophthalmol. Vis.Sci. 2002:43:2593-602.

7. Ang LP. Tan DT, Beuerman RW et al. Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system. Investigative Ophthalmology & Visual Science 45(6) 1789-95.

2004.

Claims

1. A method of storing cultured conjunctival cells comprising locating the cells in a liquid cell-storage medium at a temperature of between 3⁰C and 3O⁰C. and storing the cells for a storage period of at least 24 hours.

2. A method according to claim 1 wherein the liquid cell-storage medium is a serum free medium.

3. A method according to claim 2 wherein the serum free medium comprises Optisol-GS.

4. A method according to claim 2 wherein the serum free medium comprises: a buffering agent and minimal essential medium.

5. A method according to claim 4 wherein the minimal essential medium comprises amino acids, salts, glucose and vitamins.

6. A method according to claim 1 wherein the liquid cell-storage medium comprises serum.

7. A method according to claim 6 wherein the liquid cell-storage medium comprises PAA Quantum 286.

8. A method according to any one of the preceding claims wherein the cells are stored at a temperature of between 18 and 28⁰C, preferably 20 and 25⁰C, more preferably 22 and 24⁰C, more preferably 22 and 23⁰C.

9. A method according to any one of the preceding claims wherein the storage period is at least two days, preferably at least three days, more preferably at least four days, more preferably at least seven days.

10. A method according to any one of the preceding claims wherein the cells and the liquid cell-storage medium are stored in a sealed container, the cells and the liquid cell- storage medium filling the entire volume of the container such that substantially no gas is present above the surface of the liquid cell-storage medium.

11. A method according to any one of the preceding claims wherein the cells are located in the same liquid cell-storage medium throughout the storage period.

12. A method according to any one of the preceding claims wherein the volume of the liquid cell-storage medium is less than 25ml. preferably less than 10ml, more preferably less than 5ml.

13. A method according to any one of the preceding claims wherein the cultured conjunctival cells form part of a tissue.

14. A method according to any one of the preceding claims wherein there is substantially no increase in the differentiation of the conjunctival cells during the storage period.

15. A method according to any one of the preceding claims, further comprising the step of transporting the cells and the liquid cell-storage medium from a first location to a second location for a transportation period during the storage period.

16. A method according to claim 15 wherein the transportation period is at least 4 hours, preferably at least 6 hours, more preferably at least 4 days, more preferably at least 7 days.

17. A method of preparing conjunctival cells suitable for implantation comprising the steps of:

(i) providing a conjunctival cell explant obtained from a donor; (ii) culturing the conjunctival cells in the explant in a liquid medium suitable for cell culturing in order to expand the number of cells; and (iii) storing the cultured conjunctival cells in accordance with any one of the preceding claims.

18. A method according to claim 17 wherein step (ii) comprises the step of maintaining the conjunctival cells at a temperature of between 35⁰C and 39⁰C, preferably 37⁰C.

19. A method according to claim 17 or 18 wherein step (ii) comprises: maintaining the conjunctival cells submerged in the liquid medium suitable for cell culturing under an atmosphere comprising between 90% and 99% oxygen and between 10% and 1% carbon dioxide, preferably 95% oxygen and 5% carbon dioxide.

20. A method according to any one of claims 17 to 19, wherein the liquid medium suitable for cell culturing comprises pituitary extract.

21. A method according to any one of claims 17 to 20, wherein the liquid medium suitable for cell culturing comprises calcium ions, preferably at a concentration of between 0.03 and 0.3mM, preferably between 0.12 and 0.17 mM, more preferably 0.15mM.

22. A method according to any one of claims 17 to 21, wherein step (ii) is carried out for a period of at least 7 days.

23. A method according to any one of claims 17 to 22, wherein step (ii) comprises refreshing the liquid medium suitable for cell culturing. preferably between every 1 and 3 days.

24. A cultured conjunctival cell storage container comprising: a sealable container; and a liquid cell-storage medium, wherein the respective volumes of the interior of the sealable container and the liquid cell-storage medium are such that when the container is sealed, it is completely filled with the liquid cell-storage medium.

25. A cultured conjunctival cell storage container according to claim 24 wherein the interior volume of the sealable container is less than 25ml, preferably less than 10ml, more preferably less than 5ml.

26. A cultured conjunctival cell storage container according to claim 24 or 25 wherein the liquid cell-storage medium is a serum free medium.

27. A cultured conjunctival cell storage container according to claim 26 wherein the serum free medium comprises Optisol-GS.

28. A cultured conjunctival cell storage container according to claim 26 wherein the serum free medium comprises: a buffering agent and minimal essential medium.

29. A cultured conjunctival cell storage container according to claim 28 wherein the minimal essential medium comprises amino acids, salts, glucose and vitamins.

30. A cultured conjunctival cell storage container according to claim 24 or 25 wherein the liquid cell-storage medium comprises serum.

31. A cultured conjunctival cell storage container according to claim 30 wherein the liquid cell-storage medium comprises PAA Quantum 286.