WO2015121683A1 - Procédé d'expansion - Google Patents

Procédé d'expansion Download PDF

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Publication number
WO2015121683A1
WO2015121683A1 PCT/GB2015/050434 GB2015050434W WO2015121683A1 WO 2015121683 A1 WO2015121683 A1 WO 2015121683A1 GB 2015050434 W GB2015050434 W GB 2015050434W WO 2015121683 A1 WO2015121683 A1 WO 2015121683A1
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limbal
epithelial
culture
cells
growth
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Majlinda Lako
Francisco Carlos Damorim DE FIGUEIREDO
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Newcastle University of Upon Tyne
Newcastle Upon Tyne Hospitals NHS Foundation Trust
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Newcastle University of Upon Tyne
Newcastle Upon Tyne Hospitals NHS Foundation Trust
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/40—Nucleotides, nucleosides or bases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/98—Xeno-free medium and culture conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/395—Thyroid hormones
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
    • C12N2533/92—Amnion; Decellularised dermis or mucosa

Definitions

  • the cornea is the clear dome-shaped window at the front of the eye, and its clarity and regular surface is vital for the transmission and focusing of light onto the retina, allowing accurate visual perception.
  • Corneal disease represents the second most common cause of world blindness after cataract [Chee KY et al., Clin Exp Opthalmol (2006); 34:64-73].
  • the cornea is covered by a stratified squamous epithelium which serves the dual function of protection of the ocular surface and also contributing to high quality vision.
  • the corneal epithelium is continuous with that of the surrounding conjunctiva, the transition between the two epithelia being formed by the limbal epithelium.
  • limbal stem cells also known as limbal stem cells (LSCs) [Ahmad S et al., Regen Med (2006); 1 :29-44; Chee KY et al., Clin Experiment Ophthalmol (2006); 34:64-73].
  • the population of LSCs are believed to be responsible for the regenerative function allowing the maintenance of the corneal epithelium and for the barrier function against the migration of conjunctival cells onto the cornea [Dua et al., Surv Ophthalmol
  • LSCD limbal stem cell deficiency
  • Corneal vascularisation and opacity have been estimated to cause blindness in eight million people worldwide each year (10% of total blindness), with various forms of LSCD contributing to this total.
  • LSCD LSCD
  • the treatment options depend on both the extent of the LSCD (i.e. partial or total) and whether the condition is unilateral or bilateral.
  • partial LSCD functioning LSCs are still present in limited numbers, and when the visual axis is covered with normal corneal epithelium and the patient is relatively asymptomatic, with good vision, LSCD is typically managed using medical treatment only.
  • partial LSCD where there is central corneal involvement, with consequent decreased l vision, significant irritation and persistent epithelial defect then surgical management, including sequential epithelial removal combined with amniotic membrane
  • LSCD total LSCD
  • stem cell therapy that allows for replacement of the damaged or absent LSC population.
  • the standard treatment for unilateral total LSCD involves transplanting large, whole tissue limbal grafts from the patient's healthy fellow eye [Kenyon and Tseng,
  • the small biopsies needed can be taken from the fellow eye of the patient with LSCD, even if the disease is bilateral to an extent, providing that there are remaining areas of healthy limbus in one eye [Sangwan et al., Cornea (2003); 22:478-481 ]. Additionally, using autologous donor tissue eliminates the requirement of systemic immunosuppression, compared to previously used whole tissue allografts [Holland et al., Opthalmology (2003); 1 10: 125-130]. Thirdly, if a biopsy does not grow in culture, it is safe and acceptable to harvest another biopsy from the fellow (donor) eye.
  • the ex vivo expansion of LSCs prior to transplantation typically uses heterologous, non- human animal-derived cells and products, such as a mouse 3T3 fibroblast cell feeder layer for co-culture and fetal calf serum (FCS) in the growth medium.
  • FCS fetal calf serum
  • the use of these non-human animal-derived products in LSC expansion has several drawbacks. Firstly, such a transplant would potentially be a xenograft and, as such, the patient may require immunosuppression to prevent rejection of the tissue. Secondly, the use of non-human animal-derived products in tissue destined for human transplantation has the potential to result in interspecies pathogen transfer. This latter risk would be further augmented on a background of immunosuppression.
  • the invention provides a method for expansion of limbal stem cells, comprising:
  • the method is free from non-human animal derived products.
  • the growth support is human amniotic membrane.
  • the human amniotic membrane is intact membrane.
  • a stromal surface of the limbal biopsy is in contact with the growth support.
  • the culture medium comprises at least one growth factor.
  • the at least one growth factor comprises insulin, hydrocortisone, tri-iodothyronine, adenine, epidermal growth factor.
  • the epithelial culture medium comprises cholera toxin.
  • the one or more antibiotics comprises two antibiotics, preferably penicillin and streptomycin.
  • the serum is derived from the same donor as the limbal biopsy.
  • the expanded limbal stem cells form an epithelial sheet on the growth support.
  • the limbal stem cells are expanded for a time sufficient to provide a composite structure comprising an epithelial sheet upon the growth support.
  • the epithelial sheet is at least (90% of 1.5cm 2 ), and preferably from about (90% of 1.5cm 2 ) to about (90% of 3.5cm 2 ).
  • the invention provides a method of treating limbal stem cell deficiency comprising:
  • step iv) substituting the epithelial culture medium of step iii) with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period for a time sufficient to provide a composite structure comprising an epithelial sheet upon the growth support;
  • the invention provides a method for expansion of limbal stem cells, comprising:
  • the invention provides a method of treating limbal stem cell deficiency comprising:
  • step iv) substituting the epithelial culture medium of step iii) with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period for a time sufficient to provide a composite structure comprising an epithelial sheet upon the growth support;
  • the invention provides a composite structure comprising an epithelial sheet upon the growth support obtainable by any one of the aforementioned methods.
  • Figure 1 provides a schematic representation of a Manufacturing method.
  • Figure 2 provides a schematic diagram to represent the limbal explant with human amniotic membrane (HAM) co-culture technique, (a) The limbal explant is placed stromal side down on the intact HAM. (b) According to current understanding, the limbal epithelium would be expected to proliferate and spread onto the HAM. The HAM would support the continuing proliferation and maintenance of the progenitor characteristics of the expanded limbal epithelium.
  • HAM human amniotic membrane
  • Figure 3 illustrates human limbal explant cultures on human amniotic membrane. A.
  • FIG. 1 Macroscopic picture of culture showing two inner rings indicating previous days growths and the present outgrowth as the outer ring.
  • Figure 4 illustrates explant outgrowths from limbal explants on human amniotic membrane using foetal calf serum and human serum. The outgrowth areas at weekly intervals were measured. The day of outgrowth is shown on the x-axis and the outgrowth area is shown on the y-axis.
  • Figure 5 illustrates outgrowth areas of four explant cultures.
  • Figure 6 illustrates outgrowth areas of explant cultures grown in medium previously stored at either 4°C or -20°C.
  • Figure 7 illustrates the effect of 4°C storage for 3 hours on explant outgrowth area.
  • Figure 8 provides a photograph of the limbal explant with 3T3 co-culture technique to show gross appearance (day 7).
  • a photograph of the culture well with the explant culture is shown in the left panel.
  • the corresponding greyscale image has been labelled to show the position of the explant (E) and the edge of the explant outgrowth (arrows) and the pre-plated 3T3 fibroblasts (3T3).
  • Figure 9 illustrates the microscopic appearance of limbal cultures using explant technique with inactivated 3T3 mouse fibroblast co-culture.
  • the right column shows a corresponding high magnification view of the culture during the same time period illustrating the
  • Figure 10 illustrates the macroscopic and microscopic appearances of explant culture on
  • HAM Photograph of limbal epithelial outgrowths on HAM to show gross appearance (day 8).
  • (c) Low power photomicrograph of limbal epithelial outgrowth. The edge of the outgrowth is indicated by the arrows. Scale bar 200 ⁇ m
  • Figure 1 1 illustrates the fate of ex vivo expanded limbal epithelium using a limbal explant and HAM co-culture technique (in the absence of mouse 3T3 cells).
  • the top panel is a low power montage showing a cross section of a whole culture system including explant, HAM and outgrowth. 3 sections of the montage are shown at higher magnification below, (i) The surface of the limbal stroma is covered in a stratified epithelium as expected. However, this epithelium has proliferated to travel down the sides of the explant and onto the surface of the HAM.
  • the movement of the expanded epithelium is shown by the arrow, (ii) The majority of the HAM is covered by a uniform layer of small undifferentiated cells with high p63 expression, (iii) At the extreme periphery of the culture, the epithelium has begun to stratify and differentiate as seen by the lack of p63 staining in the superficial layers.
  • Figure 12 provides a panel showing the light microscopic and immunohistochemistry appearances for ex vivo expanded epithelium of patient 1.
  • H & E staining reveals an epithelium with a basal layer of tightly packed cuboidal cells which express high levels of p63, ABCG2, Vimentin & Ki67. Conversely, the basal layer does not express high levels of CK3.
  • Figure 13 illustrates TEM of cultured epithelium from patient 1.
  • the ex vivo cultured epithelium (EE) sits on the HAM.
  • the basal cells are much smaller and cuboidal (outer (larger) outline) with high N/C ratios (inner (smaller) outline) compared with the large columnar cells of adult corneal epithelium.
  • Areas of the expanded epithelium (shown by the dotted rectangles) were viewed at higher magnifications to reveal (b) the presence of microplicae (MP) on the superficial epithelial cells (c) the presence of desmosomes connecting the basal cells together and (d) hemidesmosomes connecting the basal cells to the basement membrane.
  • MP microplicae
  • Figure 14 illustrates the impact of cholera toxin addition to the culture media.
  • Two different explant pairs (R31 and R32 and R35 and R36) from one donor corneal ring were cultured in the absence (R31 and R35) and presence (R32 and R36) of cholera toxin. No significant changes were found in growth rate.
  • the inventors have investigated alternative methods for ex vivo expansion of LSCs.
  • the inventors have found a method for improving the quality and/or success and /or safety of a limbal stem cell transplant for direct transplantation into a patient.
  • the inventors have identified the minimal requirements necessary for successful ex vivo expansion of LSCs, enabling direct transplantation of the resultant cells into the eye of a patient.
  • the culture media and methods of the invention comprise minimal (preferably no) constituents that are detrimental to the overall success of subsequent transplantation procedure, or compromise safety of the product.
  • the inventors have developed a novel method that comprises culturing a limbal biopsy for a first time period in the presence of epithelial culture medium supplemented with serum and one or more antibiotics; and substituting the epithelial culture medium with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period.
  • the method of the invention therefore comprises at least one time period in which antibiotics are present in the culture medium and at least another (subsequent) time period in which the antibiotics are omitted from the culture medium.
  • the method of the invention provides an improved ex vivo LSC expansion method that provides cells suitable for direct transplantation into a patient and which does not suffer from the disadvantages of the prior art.
  • Culturing of the limbal biopsy for a first time period in the presence of epithelial culture medium supplemented with serum and one or more antibiotics removes the risk of contamination with pathogens present in the non- sterile biopsy environment, whereas substituting the epithelial culture medium with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period ensures that any (residual or new) pathogen contamination is not masked and can be detected by conventional screening techniques prior to transplantation.
  • the invention provides a method for expansion of limbal stem cells, comprising:
  • a limbal biopsy on a growth support; culturing for a first time period the limbal biopsy in the presence of epithelial culture medium supplemented with serum and one or more antibiotics; and substituting the epithelial culture medium with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period.
  • stem cell As used herein, the terms “culture” and “cell culture” are used interchangeably and refer to the process whereby cells, preferably stem cells, are grown (e.g. divide) under controlled conditions, preferably in vitro or ex vivo.
  • stem cell is used herein to refer to immature cells having the capacity to self- renew and to differentiate into more mature cells.
  • progenitor cells also have the capacity to self-renew and to differentiate into more mature cells, but are committed to a lineage (e.g., limbal progenitors are committed to the ocular lineage), whereas stem cells are not necessarily so limited.
  • progenitor cells can be interchangeably described as “stem cells” throughout the specification.
  • biopsy refers to the isolation of cells from a piece or pieces of tissue.
  • the term “biopsy” is used interchangeably with the term “explant” or "donor tissue”.
  • tissue pieces themselves are cultured, where cells are left in their surrounding extracellular matrix to more accurately mimic the in vivo environment.
  • the biopsy is from healthy donor tissue, and most preferably histocompatible with the recipient for the graft.
  • the explant is preferably from an adult.
  • the explant may be obtained from an animal (xenogenic cells).
  • the explant may be from a living donor or cadaver (allogenic cells).
  • the explant is from the proper patient / recipient and the method thus uses autologous cells as the explant.
  • the use of autologous explants is free from some of the disadvantages of the other methods of cell therapy, such as the lack of donors, need of immunosuppresive treatment to avoid rejection by the patient, as well as the ethical considerations linked to the use of embryonic cells.
  • the limbal biopsy is provided immersed in sodium
  • hyaluronate e.g. 14 mg/mL.
  • the donor tissue is mammalian, most preferably human.
  • the biopsy is placed on a growth support.
  • growth support refers to any material that allows for attachment and growth of cells.
  • Attachment refers to cells that adhere directly or indirectly to a substrate (e.g. a growth support) as well as to cells that adhere to other cells.
  • the growth support may be a two dimensional support or a three dimensional support.
  • the support may comprise any natural, recombinant or synthetic material which acts to support the viability, and /or proliferation the limbal stem cells of the biopsy.
  • the support may be naturally derived and contain cell derived materials e.g. extracellular matrix, (e.g.
  • the support may be synthetic, such as a polymeric scaffold, for example an electrospun scaffold or temperature responsive plastic, a hydrogel or contact lens.
  • the support may be a corneal disc, such as a thin denuded posterior corneal layer, taken from the patient or a donor from the eye bank.
  • the growth support is an amniotic membrane, preferably wherein the amniotic membrane has an extracellular matrix.
  • the amniotic membrane may be a denuded membrane or an intact membrane.
  • the amniotic membrane is derived from the amniotic membrane of a pregnant mammal, preferably a human (HAM).
  • the amniotic membrane has been maintained under sterile conditions and has been determined to be virus free, e.g., free from the hepatitis-B and C viruses and human immunodeficiency virus, and free from bacterial contamination.
  • the terms “medium”, “culture medium”, “culture media” and “media” are used interchangeably.
  • the cells are cultured in a defined epithelial culture media containing the minimum essential elements necessary to maintain the growth of mammalian stem cells, wherein the components of the media are both known and controlled.
  • a defined epithelial culture media containing the minimum essential elements necessary to maintain the growth of mammalian stem cells, wherein the components of the media are both known and controlled.
  • Such defined minimum essential media for epithelial culture are known in the art.
  • Chemically defined culture media for mammalian epithelial cell culture have been extensively developed and published over the last several decades. All components of defined media are well characterized.
  • Defined media typically consist of roughly fifty chemical entities at known concentrations in water.
  • the chemical components of the media fall into four broad categories: amino acids, vitamins, inorganic salts, trace elements.
  • the trace elements consist of a variety of inorganic salts included at micromolar or lower levels.
  • the four most commonly included trace elements present in almost all defined media are iron, zinc, selenium and copper.
  • Iron (ferrous or ferric salts) and zinc are typically added in micromolar concentrations, while the others are usually at nanomolar concentrations.
  • the numerous less common trace elements are usually added at nanomolar concentrations.
  • epithelial culture media comprising minimum essential elements necessary to maintain the growth of epithelial cells are well known in the art and include, by way of example only Minimum Essential Medium Eagle, Minimum Essential Medium Dulbecco, ADC-I, LPM (Bovine Serum Albumin-free), FIO(HAM), F12 (HAM), DCCMI, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM- without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E- with Earle's sale base), Medium M 199 (M 199H- with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM- H-with Hank's salt base
  • the defined epithelial culture media comprises D-MEM + GlutaMAXTM -1+ 1 g/L D-Glucose +Pyruvate (75%) and F-12 + GlutaMAXTM -1 Nutrient mixture (25%).
  • the defined epithelial culture media may be supplemented with additional
  • supplementary components may be added to the initial cell culture.
  • supplementary components may be added after the beginning of the cell culture.
  • the defined epithelial culture media is supplemented with serum.
  • serum is autologous and from the same donor as the tissue biopsy.
  • Autologous human serum if desired, can be subject to a conventional treatment, for example, thermal treatment, with the aim to inactivate complement.
  • the human autologous serum (HAS) can be obtained from the same patient object of the
  • the serum is any biologically- acceptable or biologically equivalent serum derivative or synthetic substitute thereof.
  • the biopsy is cultured in the absence of serum, i.e. in a culture medium that is essentially free from serum, or not supplemented with serum.
  • the defined epithelial culture media may also be supplemented with one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements, or any mixture and/or combination thereof.
  • the defined epithelial culture media may also be any suitable epithelial culture media. Additionally or alternatively, the defined epithelial culture media may also be any suitable epithelial culture media.
  • growth factor refers to any substance capable of maintaining or stimulating cellular growth, proliferation and/or cellular differentiation, including cytokines, steroids and hormones.
  • growth factors include, but are not limited to IGFs, such as IGF-I and IGF-II, VEGF, PDGF, EGF, fibroblast growth factor, bFGF, osteopontin, thrombospondin-1 , tenascin-C, PAI-1 , plasminogen, fibrinogen, fibrin, transferrin, Adenine, Adrenomedullin, Angiopoietin, Autocrine motility factor, Bone morphogenetic proteins, Brain-derived neurotrophic factor, Epidermal growth factor, Erythropoietin, Fibroblast growth factor, Glial cell line- derived neurotrophic factor, Granulocyte colony-stimulating factor, Granulocyte macrophage colony-stimulating factor, Growth differentiation factor-9, Hepatocyte growth
  • Transforming growth factor alpha Transforming growth factor beta, Tri-iodothryonine, Tumor necrosis factor-alpha, Vascular endothelial growth factor, Wnt Signaling Pathway, placental growth factor, Foetal Bovine Somatotrophin, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, or any biological equivalent, derivative or combination thereof.
  • the culture is supplemented with insulin, preferably from about 0.0001 to 0.1 M insulin, preferably from about 0.001 to 0.01 M insulin, most preferably about 0.008 M insulin.
  • the culture is supplemented with hydrocortisone, preferably from about 0.01 to 2 M hydrocortisone, preferably from about 0.1 to 1 M hydrocortisone, most preferably about 0.82 M hydrocortisone.
  • the culture is supplemented with tri-iodothyronine, preferably, from about 10 nM to 10 mM tri-iodothyronine, preferably from about 100 nM to 1 mM tri-iodothyronine, most preferably about 233 nM tri-iodothyronine.
  • the culture is supplemented with adenine, preferably, from about 0.01 to 10 mM adenine, preferably from about 0.10 to 1 mM adenine, most preferably about 0.13 mM adenine.
  • the culture is supplemented with a growth factor, such as epidermal growth factor, preferably from about 0.01 to 100 nM epidermal growth factor, preferably from about 0.1 to 10 nM epidermal growth factor, most preferably about 1.5nM epidermal growth factor.
  • a growth factor such as epidermal growth factor, preferably from about 0.01 to 100 nM epidermal growth factor, preferably from about 0.1 to 10 nM epidermal growth factor, most preferably about 1.5nM epidermal growth factor.
  • the defined epithelial culture media may also be any suitable epithelial culture media. Additionally or alternatively, the defined epithelial culture media may also be any suitable epithelial culture media.
  • antibiotics refers to any natural or synthetic substance that inhibits the growth of or destroys microorganisms.
  • the antibiotic may inhibit cell wall synthesis, protein synthesis, nucleic acid synthesis, or alter cell membrane function.
  • antibiotics include amoxycillin, ampicillin, penicillin, clavulanic acid, aztreonam, imipenem, streptomycin, gentamicin, vancomycin, clindamycin, ephalothin,
  • the media is supplemented with penicillin and/or streptomycin.
  • the antibiotic supplemented culture medium comprises from about 0.5 to about 1.5 % (v/v) antibiotic, more preferably about 1 % (v/v) antibiotic.
  • the defined epithelial culture media does not comprise, i.e. is free from antibiotics. Additionally or alternatively the defined epithelial culture media may be supplemented with a protein having ADP-riobosylation activity, for example a bacterial toxin.
  • Suitable bacterial toxins include cholera toxin (CT), Shiga toxin (ST1 , ST2, etc.), heat-labile toxin (LT, LT-lla, LT-llb, etc.) from E. coli.
  • CT cholera toxin
  • ST1 , ST2, etc. Shiga toxin
  • LT-lla heat-labile toxin
  • LT-llb heat-labile toxin
  • the toxins are modified to be non-toxigenic while remaining potent immunostimulatory molecules.
  • the culture is supplemented with from about 0.15 to 0.05 mM of cholera toxin, preferably about 0.096 mM of cholera toxin.
  • the culture does not comprise, i.e. is free from a protein having ADP-riobosylation activity.
  • the method is essentially free from non-human cells.
  • the method comprises culturing for a first time period the limbal biopsy in the presence of the above described epithelial culture medium supplemented with serum and one or more antibiotics.
  • the epithelial culture medium supplemented with serum and one or more antibiotics may be refreshed (i.e. removed and replaced with fresh epithelial culture medium supplemented with serum and one or more antibiotics) during the first time period.
  • the first time period is for at least a sufficient time for the limbal cells to undergo at least one division, for example the first time period is at least about 12 hours, more preferably at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.
  • the method additionally comprises substituting (i.e. removing and replacing) the epithelial culture medium used in the first time period with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period.
  • the epithelial culture medium supplemented with serum and free from antibiotics may be refreshed (i.e. removed and replaced with fresh epithelial culture medium supplemented with serum and free from antibiotics) during the second time period.
  • the second time period is for at least a sufficient time for the limbal cells to expand to form an epithelial sheet, for example the second time period is at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 days.
  • expand or “expanded” are used interchangeably to refer to increasing the cell number, e.g. the number of limbal stem cells, by cell division.
  • the cells may be expanded about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21 -fold, 22-fold, 23-fold, 24-fold or 25-fold, or more compared to a suitable control, for example an unexpanded biopsy tissue.
  • a suitable control for example an unexpanded biopsy tissue.
  • the second time period is for at least a time sufficient for the limbal stem cells to expand to form a defined area or size, for example, cells are cultured for a time sufficient so as to form an epithelial sheet that has an approximate area of about 0.5, 1 , 1.5, 2, 2.5, 3, or 3.5 cm 2 , or any range thereinbetween.
  • the second time provides that the cells are cultured for a time sufficient to achieve at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% confluence (or any range thereinbetween), most preferably 90% confluence on a defined surface of the growth support.
  • the epithelial sheet is at least 90% confluent over a defined surface having an area of 1.5cm 2 , and most preferably at least 90% confluent over a defined surface having an area of from about 1.5cm 2 to about 90% of 3.5cm 2 .
  • the method expands the limbal stem cells to form an epithelial sheet upon the growth support, resulting in a composite structure.
  • composite structure refers to a structure comprising two or more distinct layers, a support layer and an epithelial layer. Preferably the individual components remain separate and distinct within the composite structure.
  • the epithelial layer may comprise TAC (transient amplifying cells), TDC (terminally differentiated cells) and LSC (Limbal stem cells).
  • the method includes a step of histochemical staining or impression cytology to determine if the limbal stem cells have been expanded to the desired end point, i.e. to analyze the number and quality of cells in the epithelial sheet.
  • impression cytology refers to the application of cellulose acetate filter material to the surface of the epithelial sheet, so as to remove the superficial layers of the epithelium for analysis.
  • Impression cytology may be carried out to confirm the nature of LSCD (total or partial) and the presence of conjunctival cells on the central cornea.
  • a combination of CK13/CK12 or a combination of CK7/CK12 may be used as markers of the conjunctiva.
  • MUC5AC may be used as a marker of goblet cells in the conjunctiva.
  • the histological analysis is done to confirm both the multi-layered nature of the epithelium we are going to transplant/or have
  • histological staining refers to the identification of chemical compounds or biological molecules within and between biological cells using histological techniques. Histological techniques include (but are not limited to)
  • immunocytochemistry refers to the process of detecting antigens in cells of a tissue section through the use of antibodies binding to said antigens.
  • detection or visualisation of said antibody-antigen interactions can be accomplished using multiple techniques, for example conjugating an enzyme, such as peroxidase, to an antibody or tagging an antibody with a fluorophore.
  • the cells are cultured for a time sufficient to achieve an epithelial sheet that comprises at least about 2%, 3%, 4%, 5% or 10% limbal stem cells.
  • the invention provides a method of treating limbal stem cell deficiency comprising:
  • step iv) substituting the epithelial culture medium of step iii) with epithelial culture medium which is supplemented with serum and which is free from antibiotics, and culturing for a second time period for a time sufficient to provide a composite structure comprising an epithelial sheet upon the growth support;
  • the expanded epithelial stem cells of the invention are of particular use in various therapeutic settings.
  • the expanded cells are of particular use in various therapeutic settings.
  • the expanded cells are of particular use in various therapeutic settings.
  • transplantation and engraftment for example to treat disease, injury or wounding.
  • the grafts may be used to treat unilateral ocular disease, wounds or injury or bilateral ocular disease, wounds or injury.
  • the limbal stem cells may be used as grafts.
  • the expanded cells may be retained on a growth support (e.g. an amniotic membrane) and transplanted as a composite structure into a patient's eye.
  • the cells may be separated from the growth support (e.g. amniotic membrane) and transplanted onto a fresh support, suitable for implantation into an eye, to obtain a surgical graft.
  • the cultured or cultivated epithelial cells on the support are then transplanted to a patient's eye.
  • the invention provides the use of the expanded epithelial stem cells of the invention as a medicament.
  • the invention also provides a method of treating an ocular disease, wounds or injury comprising implanting a limbal stem cell graft of the invention into the eye of a mammalian subject in need thereof.
  • a method of corneal replacement comprising implanting an limbal stem cell graft of the invention into the eye of a mammalian subject in need thereof.
  • a limbal stem cell graft of the invention for use in corneal replacement.
  • the invention provides a limbal stem cell graft according to the invention for use in the treatment of ocular injury or ocular disease.
  • a method of treating ocular injury or ocular disease comprising administering an limbal stem cell graft of the invention into the eye of a mammalian subject in need thereof.
  • ocular injury refers to conditions resulting in an insufficient stromal micro-environment to support stem cell function, for example aniridia, keratitis, neurotrophic keratopathy, and chronic limbitis; or conditions that destroy limbal stem cells such as Partial limbal stem cell deficiency, Total stem cell deficiency, chemical or thermal injuries, Stevens- Johnson syndrome, ocular cicatricial pemphigoid, contact lens wear, or microbial infection.
  • the grafts of the invention are of particular use in the treatment of thermal or chemical injury's, such as burns.
  • the grafts of the invention are also of particular use in the treatment of ocular injury resulting from genetic disease.
  • the grafts are of particular use for the reconstruction of the ocular surface in patients with limbal stem cells deficiency, for reconstruction of the ocular surface in patients with corneal persistent epithelial defects and for the treatment of total and partial epithelial stem cell deficiency.
  • said limbal stem cell graft is derived from autologous cells, i.e. said cells are derived from the individual to be treated.
  • wound and “wounding” relate to damaged tissues, preferably damaged cornea, where the integrity of the cornea or tissue is disrupted as a result from i.e. external force, bad health status, aging, exposure to sunlight, heat or chemical reaction or as a result from damage by internal physiological processes.
  • Aim To evaluate the safety and effectiveness of human ex vivo expanded autologous limbal stem cells for the treatment of unilateral total limbal stem cell deficiency. Antibiotics removed from day 3 to reduce the risk of not detecting contamination organisms by BacT/ALERT testing Changes to timing of microbiology testing as agreed with QP.
  • the ex vivo expansion of autologous limbal stem cells (ALSC) of limbal epithelium is a method which involves taking a small biopsy of limbal epithelium and culturing it according to good manufacturing practice (GMP) to produce epithelial stem cells for transplantation to cure unilateral limbal stem cell deficiency (LSCD).
  • GMP good manufacturing practice
  • LSCD bilateral limbal stem cell deficiency
  • Autologous limbal stem cells are thus prepared from a small biopsy of limbal tissue over a feeder cell layer of human amniotic membrane (HAM). It is cultured for 12-22 days or until the outgrowth measures approximately 2 cm diameter. The cells are then ready for transplantation.
  • HAM human amniotic membrane
  • This final product is a sheet of autologous epithelial progenitor cells (ALSC), together with additional epithelial cells, such as TAC and TDC, which remains attached to the amniotic membrane and the composite structure is used for transplantation to the contralateral limbal stem cell deficient eye.
  • the final product is for immediate, autologous, single use and not stored or quarantined.
  • the minimum number of SC must equal or more than 3% of the total number of epithelial cells present in ex vivo expanded culture system
  • the corneal epithelium is maintained by a population of stem cells known as limbal stem cells (LSCs) due to their location in the basal layer of the outer border of the cornea known as the limbus.
  • LSCs limbal stem cells
  • Significant loss or damage to LSCs leads to limbal stem cell deficiency (LSCD) characterised by breakdown of the epithelium and conjunctivalisation of the corneal surface, which eventually leads to significant ocular pain and blindness (Ahmad et al., 2006).
  • the standard treatment of unilateral total LSCD involves transplanting large whole tissue limbal grafts from the patient's healthy fellow eye (putting that eye at potential risk of developing LSCD) or from living related or cadaveric donors (requiring high dose systemic immunosuppression with its associated health risks) (Kenyon and Tseng, 1989).
  • the small biopsies needed can be taken from the fellow eye of the patient with LSCD, even if the disease is bilateral to an extent, providing there are remaining areas of healthy limbus in one eye (Sangwan et al., 2003a).
  • a biopsy does not grow in culture, it is safe and acceptable to harvest another biopsy from the fellow (donor) eye. This means the tissue is autologous and eliminates the requirement of systemic immune suppression compared to previously used whole tissue allografts (Holland et al. , 2003).
  • stem cell therapies are relatively new and as such, specific national and/or international protocols/guidance have yet to be established. Due to the lack of such specific guidance, the inventor has sought to minimise any risk to the patient by adopting certain modifications to the research methodologies in use at present. These include first, the replacement of all non-human animal products from the culture system and second, the conversion of all production procedures to current Good Manufacturing Practice (GMP) standards. In addition, for the first time, a strictly defined uniform group of patients with total unilateral LSCD and no other significant ocular conditions has been used to allow the success or failure of treating LSCD to be attributable directly to the proposed stem cell therapy (Kolli et al., 2010).
  • the conventional culture of limbal epithelium requires the use of animal cells (mouse 3T3 fibroblast feeder cells) or animal products (foetal calf serum) in the culture system (Daya et al. , 2005; Ramaesh and Dhillon, 2003; Koizumi et al. , 2001 ).
  • animal ingredients in the culture of biological materials destined for human purposes: one is the potential interspecies pathogen transfer and the second is the possible increased immune response that may require systemic immune suppression.
  • Impression cytology involves using a small filter paper to lift off the most superficial cells from the cornea. These are then stained to look for cytokeratin 19 (CK19). This is a marker on the cells which is specific to conjunctiva as opposed to corneal epithelium. Being CK19 positive is a hallmark of limbal stem cell deficiency, whereas normal cornea is CK19 negative (Sacchetti et al., 2005).
  • Epithelial defects occur in limbal stem cell deficiency. They lead to pain, inflammation and reduced vision. Improving this parameter is one of the main outcomes.
  • Corneal vascularisation is another hallmark of limbal stem cell deficiency. Normal corneal epithelium contains no blood vessels.
  • Corneal opacity occurs in stem cell deficiency due to scarring and is related to vision.
  • Opacity can occur in either the surface (epithelium) and/or deeper layers (stroma). It is difficult to appreciate the depth of scarring by normal examination and a simple plus score is recorded. Epithelial opacity will be removed by a successful limbal stem cell transplant, although deep stromal opacity will remain. However, if successful, a subsequent corneal graft can be performed to remove residual opacity. This was done for patients 1 and 2 with removal of opacity and marked vision improvement.
  • HAM Human amniotic membrane
  • HAM Human amniotic membrane
  • Recently human amniotic epithelial cells have been put forward as a suitable feeder layer for the culture of limbal stem cells; however an established cell line is not available as yet (Chen et al., 2007). To that effect, the inventors have developed over the last 8 years a culture system for expanding human limbal epithelium without using any animal cells or products which involves the culturing of a small piece of human limbal tissue on HAM using autologous human serum.
  • GMP Good Manufacturing Practice
  • the consented patient will be selected as an appropriate candidate for this procedure according to the criteria described in the clinical protocol (see Clinical Trial Protocol) and cells harvested for autologous use only and in accordance with the Human Tissues Act (HTact) and the Tissues and Cells Directive 2004/23/EC.
  • a 1.5 mm x 1.5 mm partial thickness (i.e. 100 ⁇ depth) limbal biopsy will be taken from the unaffected eye of an individual patient with total unilateral LSCD.
  • the limbal biopsy is obtained as a surgical procedure, if possible under local anaesthesia, in the ophthalmology theatre, at the Royal Victoria Infirmary (RVI) according to local current procedure.
  • NBCTF therapiess Facility
  • Acceptance criteria at BMF-HS Patient is subject to informed consent; Patient must be negative for mandatory infectious markers, i.e. HBsAg, anti-HBc/HBs, anti-HCV, anti-HTLV, syphilis and Anti-HIV1 &2;
  • the limbal tissue must be between 1-2mm x 1-2mm; The limbal tissue must be suspended/protected in a drop of 1.4% hyaluronic acid; the upper surface (epithelial side) of the limbal biopsy must be marked with a gentian violet surgical pen to indicate the orientation of the biopsy.
  • AS Human autologous serum
  • Blood is taken by venesection (no anticoagulant) from the patient during the work up and assessment visits following consent to the procedure.
  • the protocol specifies a volume of 60mls.
  • the tubes m ust be labelled with at least three points of identification of the patient (i.e. patient name, date of birth and RVI number); There should be a minimum of 30ml volume and the blood should show no signs of haemolysis.
  • HAM for clinical transplantation is obtained under a Third Party Agreement (TPA) from NHS BT Tissue Services, Liverpool (see Clinical Trial Protocol).
  • TPA Third Party Agreement
  • the amniotic membrane is prepared for culture using patient-specific growth medium 24-48 hours prior to the procurement of the limbal tissue to establish a stable base for culture of the limbal stem cells.
  • the HAM is supplied frozen (-80°C), attached to a nitrocellulose paper stored in nutrient medium in a sterile glass vial within sterile packaging.
  • the tissue must remain frozen (below -40°C) until required. Once thawed at ambient temperature 14-22°C, if not used immediately, must be kept at 4°C and used within 24 hours.
  • tissue vial At delivery the tissue vial must be intact, held securely within its primary packaging and still be frozen; Supplied tissue size is 3 cm x 3 cm; On thawing, the nutrient medium containing the tissue must show no sign of contamination i.e. be cloudy or opaque.
  • the final product is a sheet of autologous epithelial progenitor cells (ALSC), which remains attached to the amniotic membrane and the composite structure is used for transplantation to the contralateral limbal stem cell deficient eye of the same patient.
  • ALSC autologous epithelial progenitor cells
  • the final product is for immediate single use and not stored or quarantined.
  • the Qualified Person will release the tissue after ensuring that it has met the specific release criteria.
  • the final product is released for issue but remains in the GMP facility (in 37°C incubator) until the preparatory work on the patient is underway (same day).
  • the theatre team will liaise in a timely manner (i.e. 30 minutes in advance) with the GMP laboratory for the delivery of the tissue.
  • ALSC must be shown to be at least 90% confluent by visual assessment; ALSC must be morphologically sound (an evenly distributed layer of epithelial phenotype i.e. small and regular with primitive cytoplasm and large nuclei) both macro and microscopically; The donor m ust have negative results for infectious disease screening from a blood sample taken on the day of limbal biopsy. Day 0 medium must be negative for aerobes and anaerobes after 5 days BacT/ALERT culture. Day 0 medium mycology screen must be negative. A medium sample taken within 72 hours of release must be negative after 48 hours BacT/ALERT culture.
  • AS is a component of the epithelial growth medium (described below) used in the culture of the ALSC (see Annex 2 for validation). 40-60 mis of autologous blood is collected into sterile universal tubes [Sterilin] in the Department Ophthalmology and transported to BMF-HS by a member of staff from either Ophthalmology or BMF-HS.
  • the serum is prepared after it has been left to clot for a minimum of 2 hours. Blood is centrifuged at 1600g for 10mins [Centra CL2 Bench top Centrifuge; Thermo Scientific, UK] and returned to the Class II cabinet. The serum is removed using a sterile pipette and transferred to new sterile tube(s). The serum is centrifuged again at 1600g for 10mins. On return to the Class I I cabinet the supernatant serum is transferred to sterile 20ml tubes and labelled with the patient's demographics and Trial No. If not used immediately for medium preparation then it is stored at -20°C in a controlled freezer. b) Epithelial Growth Medium Process Description
  • Basic and Complete Epithelial Growth Media are prepared according to current procedures. They are prepared 24-48hrs prior to the harvesting of the limbal tissue. All supplements are added according to the concentrations descri bed i n table 5 , havi ng been prepared accord i ng to manufacturer's instructions. The media is stored at 2-8°C in a monitored refrigerator.
  • Basic culture medium is prepared and filtered [0.22 ⁇ filter sterilising unit - Nalgene] using supplements and growth factors that will support the growth of limbal epithelial tissue in culture (see Table 5). It is then labelled with a Batch No; date of preparation and expiry date (22 days from date of preparation - see validation).
  • Complete medium is then prepared using 90% basic medium with the addition of 10% autologous serum (therefore specific to the patient tissue undergoing culture). This medium is filtered [0.22 ⁇ filter sterilising unit [Nalgene] and sufficient aliquots prepared in 15ml centrifuge tubes [TPP] to allow for single use at each medium change during the course of the cell expansion. Two 10ml aliquots are used on day -2 or -1 for plating the amniotic membrane and on day 0 of the culture to add the limbal explant. To these aliquots only, penicillin and streptomycin (or gentamicin in the case of penicillin allergy) is added in the concentrations below.
  • Penicillin & Streptomycin 1 50 units & 50 Mg/mi Invitrogen
  • amniotic membrane is prepared 24-48hrs prior to the harvesting of the limbal tissue to establish a stable base for culture of the limbal stem cells. This process produces a biological substrate for the growth of normal donor epithelial cells for clinical use.
  • the HAM is supplied attached to nitrocellulose paper within a nutrient medium in a glass vial in sterile packaging and is delivered on dry ice. After acceptance of the product at BMF-HS (checking Specification, Batch No. and expiry date) the HAM is stored in a -80°C controlled freezer if not used on the day of delivery.
  • the HAM is defrosted at ambient temperature 14-22°C immediately prior to use.
  • tissue is washed three times by submersing the tissue twice in 2mls of Dulbecco's Phosphate Buffered Saline [Sigma, UK] with 1 % Penicillin / streptomycin and then in 2mls Complete culture medium containing antibiotic. It is then placed on the lid of a six- well plate [TPP, Switzerland] to provide a sterile platform for manipulating the tissue.
  • the HAM is peeled from the nitrocellulose paper using sterile forceps and is placed over one sterile glass coverslip ensuring that the coverslip is fully covered and that there is an equal overhang of HAM at each side of the coverslip, trimming excess as necessary. Care is taken to ensure that the stromal side (sticky side) remains in contact with the coverslip and the epithelial side is facing up.
  • the overhanging HAM is folded over the underside of the coverslip and secured by placing the second sterile coverslip underneath as a sandwich. This procedure ensures that the HAM remains in situ throughout the culture process.
  • the HAM plus coverslips is then placed in a sterile 9.6 cm 2 culture well of a 6 well plate [TPP, Switzerland] and covered with 2 ml of the patient specific Complete culture medium with antibiotics.
  • the plate is labelled with patient demographics according to current procedure and must only be used for that patient. It is then placed in the tissue culture incubator at 37°C in a humidified atmosphere containing 5% CO2.
  • the prepared HAM is checked 24 hours later to ensure that it remains securely held on the coverslips and that the medium remains clear, negative for any bacterial contamination via visual inspection prior to commencing the limbal epithelial culture. d) Human autologous limbal stem cells explant
  • the Biomanufacturing Facility at the RVI provides a kit containing a receptacle for the tissue, labels, packaging material and documentation.
  • a member of BM F-HS will ensure a kit is received by ophthalmology theatre staff prior to limbal cell biopsy.
  • a member of theatre staff following the current procedures immediately transfers the tissue to the BMF-HS.
  • On arrival at the BMF-HS receipt of the tissue is documented accordingly using established procedures.
  • the limbal biopsy material is transferred into the BMF-HS clean room by receiving staff. Manufacturing Methods:
  • This process produces a single batch of ALSC sufficient to treat one individual once only.
  • the culture is initiated using the amniotic membrane that has been prepared specifically for that patient as it contains, in the culture medium, the patient's autologous serum.
  • the epithelium culture medium is removed from the culture well containing the HAM. Using sterile forceps, the limbal biopsy is then removed from the hyaluronic acid in which it has been suspended and placed on the centre of the HAM and gently pressed downward to promote attachment. 1.3 ml of epithelial medium is gently added to the culture well very slowly to ensure that the limbal biopsy does not become detached from the underlying HAM. Subsequently, the lid of the plate is replaced. The culture is then incubated at 37°C, 5% CO2 in a humidified atmosphere.
  • the culture medium is replaced every 2-3 days in the Class I I cabinet by carefully removing most of it with a sterile filtered pipette tip and replacing it with fresh medium, increasing the volume added to a minimum of 1.5ml.
  • the morphology of the tissue is observed at the same interval using the inverted microscope as described in section 2 and assessment is recorded on the worksheet.
  • the area of explant outgrowth is marked on the underside of the culture well at the time of each medium change to allow subsequent measurement of growth rate.
  • Phase 2 QC for sterility is taken at day 0 and within 72 hours of the transplant date, to allow for a 48 hour result to be available.
  • the product When outgrowing cells cover at least 90% of the total area of the HAM, the product is ready for transplant.
  • the ALSC product is transported to theatre (Ophthalmology Dept., RVI) in the plate in which it was cultured.
  • the culture plate with label attached is removed from the incubator and placed in a sterile overwrap and sealed.
  • the approved copy label is attached to this before leaving the cleanroom. Procedures are in place for packing and transporting the tissue at ambient temperature.
  • the BMF-HS have over twenty years experience of using these methods for assessing sterility of haematopoietic progenitor cell products.
  • the inventors have found that using BacT/ALERT [bioMerieux] paediatric blood culture bottles we are likely to detect relevant organisms when testing small sample volumes i.e. 1 ml.
  • Preparation of media A 2ml sample is taken from the prepared basic culture medium and transferred to a blood culture bottle [BacT/ALERTOPF Plus, bioMerieux] and sent to the Department of Microbiology, Newcastle Hospitals NHS Foundation Trust.
  • a 1 ml sample is taken from the prepared autologous serum and tested as above.
  • a 1 ml sample is taken from the prepared 'complete' culture medium containing autologous serum and tested as above.
  • the prepared HAM is checked after 24 hours to ensure that it remains
  • the plated amniotic membrane is removed from the incubator and the medium is removed before placing the limbal explant.
  • 0.5ml of the medium is collected in a cryo tube for mycology screening.
  • the remaining medium is put into a BacT/ALERTOPF Plus [bioMerieux] culture bottle for culture. Both samples are sent to the Department of Microbiology,
  • a macroscopic and microscopic inspection of the culture will be performed prior to the expected transplant date. This will ensure that the product meets the release criteria and also that there is no visual evidence of infection, such as cloudy medium. Tissue failing to meet the criteria for release will be discarded or used for Quality
  • a further 1 ml sample is removed from the media taken from the final product immediately prior to release for retrospective QA. This is sent to an external contract laboratory (Stockton Quality Control Laboratory, University Hospital of North Tees, Hardwick Stockton on Tees, TS19 8PE) for Eu. Ph sterility testing.
  • a biopsy of residual IMP material not transplanted (removed when trimming the graft to size) will be sent for further testing to an exte rn a l l a b o ra to ry
  • phase 3 QC all microbiology and external testing results will be reviewed and recorded in the BMR. This includes the 5 day results from the samples taken 72 hours prerelease and the Eu.Ph sterility testing performed at the Stockton Quality Control Laboratory.
  • Necessary controls are in place to minimise any risk of cross contamination or compromising the integrity of each of the cultures being undertaken e.g. use of specific product labels; use of separate shelves in the incubator, processed separately in the Class II cabinet, use of patient specific culture medium and worksheet for each patient's LSC expansion culture.
  • Reagents and worksheets for each patient's LSC expansion culture are prepared in separate trays for transfer to the laboratory.
  • Product will be on a glass coverslip with 2 outer wrappers as shown in diagram below.
  • Each layer will contain a label to ensure all patient checks are possible at each stage of the procedure with the approved copy label attached to the second (sterile) bag such that the surgeon may make final patient demographic checks in theatre. All unused labels will be returned to the BMFHS for label reconciliation. Details will be kept in the batch manufacturing record.
  • Clinical trial end points include:
  • the cornea is anaesthetised using topical oxybuprocaine hydrochloride 0.4% eye drops
  • Corneal impressions are obtained using a single sterile Biopore membrane which is provided stretched over a short plastic tube [Millicell-CM 0.4 ⁇ , 13mm diameter, Millipore]. The patient is asked to look at a target placed in front of them and the lids are held apart. The Biopore membrane is pressed gently on the central cornea for up to 5 seconds to obtain a corneal impression. It is vital at this stage to ensure accurate placement of the membrane so that only a central corneal impression is made without inadvertent conjunctival contact which would produce a false positive result. The area of impression is easily identified as the white membrane becomes transparent when moist. This area is marked immediately so that the area of interest can be identified when dry.
  • the membranes can then be stained using histology or immunocytochemistry techniques. Finally, the areas of impression are cut out and mounted for light or fluorescent microscopy.
  • One impression is taken from each cornea and is sent to an Ophthalmic Pathologist at the Royal Hallamshire Hospital for independent assessment using PAS staining and cytokeratin markers.
  • Corrected visual acuity in Snellen equivalents are measured for each eye using a logarithmic visual acuity chart for testing at 4 metres.
  • the results from the manifest refraction may be placed in a trial frame and visual acuities measured.
  • the chart should be set at approximately eye level to the average height of a seated patient. A mark will be made on the floor (e.g., with tape) that is 4 metres away from the chart. If the patient reads no letters at 4 metres, the chart is then moved to 1 metre.
  • the test distance and illumination for the chart must be kept constant throughout the study. Begin by testing the right eye and the left eye must be covered. The patient is asked to read each letter, line by line, left to right, beginning with line 1 at the top.
  • the patient should be told that the chart has letters only, no numbers. If the patient reads a number, he or she should be reminded that the chart contains no numbers, and the examiner should then request a letter in lieu of the number. The patient is not to proceed to the next letter until s/he has given a finite answer. If the patient changes a response (e.g., that was a "C" not an "O") before s/he has read aloud the next letter, then the change must be accepted. If the patient changes a response, after having read the next letter, then the change is not accepted. The examiner must not point to specific letters on the chart during the test.
  • the pinhole method for measuring visual acuity is not acceptable.
  • the pain score is based on self- report.
  • the patient marks on a horizontal scale from 0 (No pain/discomfort) to 10 (severe pain).
  • the intent is to transplant the final product when cells are more than 90% confluent, over most of the area of the amniotic membrane on the 2.4cm x 2.4cm coverslip. They should not be allowed to remain in culture at full confluence otherwise epithelial cells will start to differentiate into corneal epithelial cells, forming cell layers and consequently lose progenitor cell capacity/stem cell phenotype.
  • Transplant of the limbal epithelium is scheduled to coincide with optimal growth of the limbal stem cells.
  • the ophthalmology theatre is provisionally booked for the transplant to take place 12-14 days after the initial limbal biopsy collection.
  • the limbal tissue remains in culture at 37°C until release procedure has been completed but is only sent to the ophthalmology theatre once the preparatory work on the patient's LSCD eye is almost completed as this may take 1-2 hours depending on the condition of the affected eye.
  • the plate containing the product (with the primary packaging label affixed) is transferred to a sterile plastic bag.
  • a secondary packaging annex 13 compliant label is attached to the outer bag.
  • the product is placed into another transparent bag with a further secondary packaging annex 13 compliant label before being placed flat in suitable/dedicated transport box using suitable packing material, e.g. 'bubble wrap' material to fill the space between the plate and box so that it will not move during transportation.
  • suitable packing material e.g. 'bubble wrap' material
  • the outer bag is opened before passing the product to the sterile area.
  • the amnion is washed in isotonic citrate solution to remove blood and treated with an antibiotic cocktail to reduce microbial load.
  • the antibiotic solution contains gentamicin sulphate (4g/l), imipenem (0.2g/l), nystatin (2.5x106 U/l), polymyxin B sulphate (0.2g/l), and vancomycin hydrochloride (0.05g/l) , prepared i n medi um 1 99 supplemented with 25mM HEPES.
  • Tissue is sourced from UK donors in compliance with rigorous ethical and clinical standards. The consent process is approved by the Human Tissue Authority. In house experts on tissue donor selection and medical history influence the standard across all donation programmes (blood, tissue and organ). The standard is written by UK blood services in compliance with MSBTO (advisory committee in the Microbiological Safety of Blood, Tissues and Organs). Much of the standard is above and beyond the minimum required by European/UK legislation and regulation. Tissue Services was previously licensed by the M H RA (Medicines and Healthcare product
  • This product is attached stromal side to nitrocellulose paper contained within a 7ml translucent polypropylene sterile bijou.
  • the secondary package is heat sealed in low density polyethylene bags compliant with EC Commission Directive 2002/72/EC.
  • the outer bag is labelled with graft type, unique donation number, expiry date, size, and storage requirements. Donation number, product type, status and expiry date are ISBT 128 barcoded.
  • Enclosed within the packaging is a transplant reporting form with a freepost envelope, which can be used for feedback. If an adverse event or reaction is suspected, telephone the tissue bank immediately.
  • Transportation protocols are validated to ensure that grafts arrive with the customer undamaged and in perfect condition.
  • Packaging materials are validated to ensure that the integrity of the graft is maintained up the point of use.
  • Delivery is in a disposable transport box containing dry ice (solid carbon dioxide) validated to keep the graft frozen until the time written on the box. It is delivered by either NHSBT Transport or via a courier, usually direct to the point of use e.g. eye ward/theatre at the RVI. Next working day delivery is included in the product price. More urgent delivery e.g. same day or by specified time can be arranged at additional cost. Where an operation is graft critical, the patient must not be taken to theatre before the graft has arrived and its condition checked.
  • This product must be stored below -40°C if it is not to be used within 48 hours. Freezers need to be designated for clinical use with 24/7 alarms and monitoring. Once thawed, this product must be used within 24hrs. Annex 2 - Validation of Culture Conditions.
  • HAM human limbal epithelium on human amniotic membrane
  • the inventors have shown, as have many others, the use of HAM is a suitable alternative to 3T3 fibroblasts (Burman et al. , 2004; Meller et al. , 2000). Details of the use of HAM are shown in section 3 and in Annex 1.
  • the HAM for the clinical study was obtained from N HS BT Tissue Services Liverpool under a Third Party agreement (TPA).
  • HAM provides a physical carrier for which to transplant the cells, even prior to culture confluence or sheet formation ( Figure 2).
  • HAM and HS containing medium for the culture of human limbal epithelium results in the production of a culture system without non-human animal products and cells. It overcomes the following problems encountered with the other culture systems investigated in this chapter:
  • Phase I A prospective well-designed clinical translation study (Phase I) was carried out by the inventors in a strictly defined uniform group of patients with total unilateral LSCD and no other significant co-existing ocular pathology so that success or failure of reversal of total LSCD could be attributed solely to the LSC therapy (Kolli et al., 2010). Twelve consecutive patients with unilateral total LSCD were offered treatment with ex vivo expanded autologous LSCs on human amniotic membrane (HAM). Twelve of twelve patients had successful limbal stem cell cultures and went on to successful transplants with a mean follow-up of 19 months (Table 1). This failure rate in 3 cultures is in line with published data (Meller et al., 2002) where the majority of failures are due to underlying/undetected stem cell deficiency.
  • Mycoplasma testing on medium taken from 12 day old cultures was also performed. All cultures were negative for Mycoplasma arginini, Mycoplasma hyorhinis and Mycoplasma orale.
  • the validation results passed the acceptance criteria for culturing ALSC for transplant use.
  • the microscopic examination of the expanded tissue shows a stem cell like phenotype with small round tightly packed cells with large nuclei and little cytoplasm.
  • the culture shown in Figure 10 was placed in formalin after 1 4 d ays i n cu l tu re a n d th e n p a raffi n e m be d d ed a n d u s ed fo r immunohistochemistry.
  • Antibody against human p63 (which is a putative marker of SC cells and early TACs) was used to allow easy visualisation of the proliferating cells.
  • HAM has been consistently and successfully used as a co-culture substrate in the expansion of LSCs in studies by the inventors.
  • HAM is the preferred choice for co- culture substrate on which to grow LSCs for several reasons:
  • HAM allows the 3T3 mouse fibroblast layer to be totally excluded
  • HAM provides excellent and consistent expansion of LSC numbers
  • HAM has a physically resilient basement membrane on which the LSCs can grow and allows easy handling at surgery;
  • HAM contains many factors conducive to epithelial cell growth and may mimic the
  • HAM does not elicit an immune response (Dua et al., 2004).
  • FCS foetal calf serum
  • Approaches to resolving this issue would include using a serum free epithelial growth medium or replacing the FCS with human (autologous) serum (HAS).
  • HAS has been successfully used to replace the need for FCS in epithelial growth medium for the culture of a variety of epithelial cell types including skin, oral mucosa and cornea (Mazlyzam et al., 2008; Nakamura et al., 2006a; Nakamura et al., 2006b).
  • HAS has been used therapeutically for some time in the treatment of ocular surface disorders due to its many favourable properties, including support of epithelial growth (Rauz and Saw, 2010; Tsubota et al. , 1999). Therefore, the use of HAS to replace FCS in epithelial growth medium has many additional advantages as well as a means of eliminating animal products from the culture system.
  • the culture of human limbal epithelium has been successfully established with both 3T3 fibroblast co-culture and culture on HAM with epithelial growth medium containing HS by the inventors.
  • Epithelial outgrowths using an explant culture technique on HAM usi ng epithel ial growth medium supplemented with either FCS or HS were established under otherwise identical culture conditions.
  • the results consistently showed the equivalence or superiority of HS compared with FCS in terms of faster and greater epithelial outgrowths.
  • the consistent and reproducible growth of limbal epithelium using an explant technique on HAM relies on the specific and stable arrangement of the HAM being stretched flat across a coverslip while bathed in the HS supplemented growth medium.
  • the histology and immunohistochemical analysis of the cultured epithelium confirmed that an epithelium is formed on the HAM ( Figure 12) .
  • This epithelium is 2-3 cells layers thick although thicker at the edges of the cultures speci m en .
  • the basal layer of cel ls adj acent to the HAM showed characteristics indicating an actively expanding SC phenotype including: i. Small cuboidal cells with undifferentiated appearance (in contrast to the tall columnar cells with large amounts of cytoplasm typical of mature corneal epithelium).
  • TEM confirms the light microscopy findings demonstrating the formation of a primitive epithelium with a prominent basal layer of cuboidal cells with a high nucleus: cytoplasm ratio ( Figure 13).
  • the basal cells are attached to their basement mem brane via hem idesmosomes and to each other via desmosomes.
  • the superficial squamous cells which are not in contact with the HAM show early differentiation demonstrating primitive microplicae formation in keeping with a corneal epithelial phenotype.
  • keratopathy with keratolimbal allograft a limbal stem cell transplantation technique'
  • Kolli, S., Lako, M., Figueiredo, F., Mudhar, H. and Ahmad S. (2008) 'Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane', Regenerative Medicine, 3, (3), pp. 329-42.
  • mesenchymal and embryonic stem cell culture conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold!', Stem Cells, 25, (7), pp. 1603-9.

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Abstract

L'invention concerne un procédé d'expansion de cellules souches limbiques, consistant : (i) en la fourniture d'une biopsie limbique sur un support de croissance ; (ii) en la culture durant une première période de la biopsie limbique en présence d'un milieu de culture épithélial, auquel on ajoute du sérum et un ou plusieurs antibiotiques ; et (iii) en la substitution au milieu de culture épithélial de l'étape (ii) par un milieu de culture épithélial auquel on ajoute du sérum, et ne contenant aucun antibiotique, et en la culture durant une seconde période.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017100463A1 (fr) * 2015-12-08 2017-06-15 Youhealth Biotech, Limited Régénération du cristallin au moyen de cellules souches/progénitrices endogènes
CN121022744A (zh) * 2025-10-29 2025-11-28 丽水市绿谷生命健康研究院 一种基于人羊膜上皮干细胞构建组织工程化角膜的方法及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010133853A1 (fr) * 2009-05-22 2010-11-25 University Of Reading Greffe synthétique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010133853A1 (fr) * 2009-05-22 2010-11-25 University Of Reading Greffe synthétique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABOULGHASSEM SHAHDADFAR ET AL: "Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement", EXPERIMENTAL EYE RESEARCH, vol. 97, no. 1, 1 April 2012 (2012-04-01), pages 1 - 9, XP055179671, ISSN: 0014-4835, DOI: 10.1016/j.exer.2012.01.013 *
NADIA ZAKARIA ET AL: "Standardized Limbal Epithelial Stem Cell Graft Generation and Transplantation", TISSUE ENGINEERING PART C: METHODS, vol. 16, no. 5, 1 October 2010 (2010-10-01), pages 921 - 927, XP055153962, ISSN: 1937-3384, DOI: 10.1089/ten.tec.2009.0634 *
SAI KOLLI ET AL: "Successful Clinical Implementation of Corneal Epithelial Stem Cell Therapy for Treatment of Unilateral Limbal Stem Cell Deficiency", STEM CELLS, 1 January 2009 (2009-01-01), pages N/A - N/A, XP055179363, ISSN: 1066-5099, DOI: 10.1002/stem.276 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017100463A1 (fr) * 2015-12-08 2017-06-15 Youhealth Biotech, Limited Régénération du cristallin au moyen de cellules souches/progénitrices endogènes
CN108697500A (zh) * 2015-12-08 2018-10-23 优美佳生物技术有限公司 使用内源性干细胞/祖细胞的晶状体再生
CN121022744A (zh) * 2025-10-29 2025-11-28 丽水市绿谷生命健康研究院 一种基于人羊膜上皮干细胞构建组织工程化角膜的方法及应用

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