WO2006090696A1 - Procédé de fabrication d’un amnion dénué d’épithélium - Google Patents

Procédé de fabrication d’un amnion dénué d’épithélium Download PDF

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Publication number
WO2006090696A1
WO2006090696A1 PCT/JP2006/303050 JP2006303050W WO2006090696A1 WO 2006090696 A1 WO2006090696 A1 WO 2006090696A1 JP 2006303050 W JP2006303050 W JP 2006303050W WO 2006090696 A1 WO2006090696 A1 WO 2006090696A1
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Prior art keywords
amniotic membrane
trypsin
epithelium
treatment
cells
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PCT/JP2006/303050
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English (en)
Japanese (ja)
Inventor
Eiji Kurihara
Junji Hamuro
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Arblast Co., Ltd.
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Publication of WO2006090696A1 publication Critical patent/WO2006090696A1/fr

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    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/005—Ingredients of undetermined constitution or reaction products thereof
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • freeze / thaw treatment is performed twice.
  • trypsin treatment is performed using a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v).
  • a trypsin solution containing a chelating agent selected from the group consisting of EDTA, NTA, DTPA, HEDTA, GLDA and any combination thereof can be used.
  • the chelating agent content can be set, for example, in the range of about O.lmM to about 0.6 mM.
  • the trypsin treatment is preferably performed under conditions where the trypsin solution contacts only the amnion epithelium side.
  • step (4) the following steps are performed after step (4).
  • a sheet-like construct comprising an amniotic membrane obtained by the method of the present invention and a cell layer constructed on the amniotic membrane.
  • a cell layer constructed on the amniotic membrane.
  • it has a cornea-like structure
  • a cell layer is constructed.
  • FIG. 1 is a diagram for explaining a method for fixing an amniotic membrane.
  • the amniotic membrane is sandwiched between a pair of frames, and in (b), the amniotic membrane is sandwiched between the frame and a flat plate member.
  • FIG. 2 is a flowchart showing a procedure for removing the amniotic epithelium.
  • FIG. 3 is a diagram showing a trypsin treatment method.
  • the frame is fixed in a trypsin solution with the upper side of the amniotic membrane facing down.
  • trypsin acts on the epithelial side of the amniotic membrane by placing a trypsin solution in the frame.
  • Fig. 4 shows HE-stained images of trypsin-treated amniotic membrane, raw amniotic membrane (with epithelium), and manually-treated amniotic membrane.
  • FIG. 5 is an immunostained image of trypsin-treated amniotic membrane.
  • FIG. 6 is an immunostained image of trypsin-treated amniotic membrane.
  • FIG. 7 is an immunostained image of live amniotic membrane (with epithelium).
  • FIG. 8 is an immunostained image of manually treated amniotic membrane.
  • FIG. 9 is a table summarizing the results of HE staining experiments and immunization experiments.
  • the present invention relates to a method for preparing an amniotic membrane from which the epithelium has been removed (hereinafter abbreviated as “amniotic membrane removing method”).
  • amniotic membrane preparation method of the present invention is characterized by including the following steps.
  • a step of preparing an amniotic membrane separated from a living body (1) A step of preparing an amniotic membrane separated from a living body.
  • step (1) prepare the amniotic membrane.
  • the amniotic membrane here is preferably human amniotic membrane.
  • human amnion can be collected by force such as human fetal membrane or placenta obtained as a postpartum at the time of delivery.
  • the human amniotic membrane can be prepared by treating and purifying an integral product such as human fetal membrane, placenta and umbilical cord obtained immediately after delivery.
  • Method for preparing such human amniotic membrane Can employ a known method such as the method described in JP-A-5-56987. Typically, this step is performed as follows.
  • a part of the placenta tissue is collected at the time of delivery, and then the amniotic tissue is manually detached from the placenta tissue. You may freeze once at this stage.
  • amniotic membrane from which blood cell components and chorion are removed at this stage, but after the freeze-thaw treatment (step 2) described later, removal of blood cell components and peeling of Z or chorion are performed. You can also.
  • the human amniotic membrane thus prepared can be stored frozen until the next treatment. Freezing of the human amniotic membrane can be performed, for example, at ⁇ 80 ° C. in a solution in which DMEM (Dulbecco's modified Eagle's medium) and glycerol are mixed in an equal volume ratio. Cryopreservation is expected to improve operability as well as decrease antigenicity.
  • DMEM Dulbecco's modified Eagle's medium
  • glycerol glycerol
  • amniotic membrane prepared by the above procedure is fixed to the frame and then subjected to the subsequent treatment. It becomes easy to handle by fixing the amniotic membrane with a frame.
  • Figure 1 shows a specific example of how to fix the amniotic membrane.
  • two frames (1, 2) of the same shape are used.
  • the amniotic membrane 10 is fixed by holding the edges of these two frames. Fix the amniotic membrane in an expanded state.
  • the amniotic membrane 10 is fixed using the frame 3 and the plate-like member 4.
  • the amniotic membrane 10 is spread on the plate-like member 4.
  • the epithelial side of the amniotic membrane 10 is turned up.
  • the upper force of the amniotic membrane 10 is also put on the frame 3, and the edge of the amniotic membrane 10 is sandwiched between the plate-like member 4 and the frame 3.
  • the amniotic membrane is once frozen and then thawed.
  • This freezing and thawing process makes it easier for the amniotic epithelial layer to peel off during subsequent trypsin treatment. This is thought to be due to the loosening of the adhesive state (bonded state) between the amniotic epithelial layer and the basement membrane.
  • a freezing temperature of about 20 ° C to about 80 ° C can be used. In consideration of the fact that a sufficient frozen state can be obtained and a general-purpose freezer can be used, it is preferable to freeze at about 80 ° C.
  • a melting temperature of about 4 ° C to about 50 ° C can be employed. Preferably the melting temperature is about 37 ° C.
  • freeze-thaw treatment it is preferable to repeat the freeze-thaw treatment.
  • the effect of the freeze-thaw treatment that the epithelium is easily detached in the subsequent trypsin treatment is enhanced.
  • the freeze-thaw treatment is preferably performed twice under the conditions of a freezing temperature of -80 ° C and a thawing temperature of 37 ° C.
  • the conditions (freezing temperature and thawing temperature) for each time when the freeze-thaw treatment is repeatedly performed may be the same, partly different, or different from each other. However, from the viewpoint of operability, it is preferable that the conditions are the same each time.
  • the amniotic membrane after the freeze-thaw treatment is treated with trypsin.
  • Trypsinization is performed by contacting the trypsin solution with the amniotic membrane.
  • a trypsin solution having a trypsin concentration of about 0.01% (w / v) to about 0.05% (w / v) can be used.
  • a trypsin solution having a trypsin concentration of about 0.02% (w / v) is used. If the trypsin concentration of the trypsin solution is too low, the action of trypsin will not be fully exerted.
  • trypsin concentration is too high, trypsin can act well on the amniotic epithelium, while trypsin also acts on the amnion dense layer and the basement membrane, which may damage the part.
  • trypsins are commercially available such as those derived from ushi, porcine, and human. For example
  • Trypsin-EDTA Invitrogen
  • trypsin 1 250 (Sigma) can be preferably used.
  • a chelating agent is usually added to the trypsin solution, but the chelating agent is not essential.
  • EDTA As a chelating agent, EDTA, NTA, DTPA, HEDTA, GLDA, etc. can be used. Any combination of these may be used.
  • the chelating agent is added, for example, to a concentration of about O.lmM to about 0.6 mM.
  • trypsin treatment under conditions where only the amnion epithelial side comes into contact with the trypsin solution. This is to protect the action force of trypsin on parts other than the amniotic epithelium. For example, immerse only the amnion epithelium side in a trypsin solution, do not add trypsin solution to the amnion epithelium side, apply it, and block the amnion chorion side to avoid contact with the solution. Thereafter, only the amnion epithelial side can be brought into contact with the trypsin solution by, for example, immersing it completely in a trypsin solution.
  • amniotic membrane (frame-fixed amniotic membrane) fixed in advance to the frame as shown in Fig. Lb is used, only the epithelial side of the amniotic membrane is exposed, so for example, the frame-fixed amniotic membrane is immersed in a trypsin solution. It is possible to contact only the amnion epithelium side with the trypsin solution.
  • This method also has the advantage that the trypsin treatment can be performed by a simple operation of immersing the frame-fixed amniotic membrane.
  • the method of immersing the entire frame in the trypsin solution involves immersing only the epithelial part of the amniotic membrane in the trypsin solution (for example, the epithelial side of the amniotic membrane). It may be soaked in a trypsin solution with the bottom facing down), the trypsin solution in the frame, or the epithelial side of the amniotic membrane only in contact with the trypsin solution by applying the trypsin solution to the amnion epithelium side.
  • the trypsin treatment time (contact time of the trypsin solution) is, for example, about 5 minutes to about 60 minutes. It is preferably about 10 minutes to about 20 minutes, more preferably about 15 minutes. If the treatment time is too short, trypsin cannot be sufficiently exerted, resulting in insufficient removal of the amniotic epithelium. On the other hand, if the treatment time is too long, trypsin may also act on the basement membrane and dense layer of the amniotic membrane to damage the part.
  • the temperature condition of trypsin treatment is, for example, about 25 ° C. to about 4 so that trypsin works well. Set to 2 ° C.
  • the trypsin treatment can be performed in a plurality of times.
  • the amniotic membrane is washed. This washing removes the attached trypsin solution and simultaneously removes the amniotic epithelium (epithelial cells). For example, leave it in a liquid with an appropriate flow (for example, flowing water), shake it in a suitable liquid (for example, shake up and down), or apply ultrasonic waves while immersed in a suitable liquid.
  • the amniotic membrane after trypsinization is washed by adding.
  • the liquid used for washing include physiological saline, phosphate buffer, pure water, and DMEM.
  • the washed amniotic membrane may be refrigerated or frozen until use.
  • it can be stored in a state of being immersed in a storage solution containing glyceride (for example, 50% glycerol-containing DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL)).
  • glyceride for example, 50% glycerol-containing DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL)
  • the washed amniotic membrane is subjected to a drying treatment.
  • a drying treatment By drying, a dried amniotic membrane that can be stored for a long time and is easy to handle can be obtained.
  • a freeze-drying process can be adopted as a method that satisfies the intensive conditions. Freeze-drying treatment is generally performed in a frozen state in a low pressure environment (vacuum) with a boiling point of about -20 ° C (107 Pa, 0.8 Torr) to about -50 ° C (4 Pa, 0.03 Torr).
  • freeze-drying treatment eg, frozen at about -40 ° C
  • Free Water is removed by sublimation.
  • freeze-drying treatment it can be dehydrated uniformly from the inside, and since a high degree of dryness is realized, it can be dried while maintaining its original function and form at a high level.
  • freeze-drying treatment is as follows: 1. Little deterioration during treatment 2. Easy sterilization 3. A dry product with excellent restitution can be obtained 4. A dry product with excellent storage stability can be obtained , Etc.
  • the lyophilization process can be performed by a lyophilizer equipped with a vacuum chamber, a cooling and heating device, and an exhaust device (cold trap and vacuum pump). Numerous freeze-drying equipment They are sold and can be dried using any of these.
  • the processing conditions can be set based on the instruction manual attached to the device to be used. In that case, it is possible to consider the size and dryness of the sample to be subjected to the drying process. The dryness can be set, for example, so that the water activity (AW) is less than 0.5.
  • a dried amniotic membrane having a desired size and shape By cutting or cutting the freeze-dried amniotic membrane, a dried amniotic membrane having a desired size and shape can be obtained.
  • the obtained dry amniotic membrane may be fixed to a support or a frame.
  • the dried amniotic membrane obtained by the drying treatment is stored in a suitable container so as to be substantially free from contact with oxygen.
  • a suitable container By being packaged in a state of being substantially shielded from oxygen, it becomes a dry epithelium containing no epithelium with excellent preservation properties.
  • the air in the container is sucked and removed to form a vacuum state, or the inside of the container is replaced with nitrogen. It can be packaged in a state where it is shut off. Alternatively, the remaining oxygen may be removed by enclosing a deoxidizing agent in the container. Note that any combination of these methods may be used.
  • the container include a bag-like or tube-like product made of a plastic synthetic resin (two sheets may be overlapped and the periphery is sealed), or an inorganic material such as glass may be used. Can be used.
  • Another aspect of the invention relates to the use of non-epithelial-containing amniotic membranes prepared by the method of the invention.
  • the epithelium-free amniotic membrane itself can be used as a transplant material, it is preferably used as a substrate for cell culture. That is, the epithelium-free amniotic membrane of the present invention is a material useful as a substrate for constructing a cell layer (tissue) on the premise of transplantation.
  • Examples of cells grown on the epithelium-free amniotic membrane of the present invention include epithelial cells (corneal epithelial cells, skin epithelial cells, oral mucosal epithelial cells, etc.) and endothelial cells (corneal endothelial cells, etc.).
  • Oral mucosal epithelial cells include, for example, cells existing in the root of the tooth (oral mucosal epithelial cells), lip mucosal epithelial cells, palate mucosal epithelial cells, buttocks mucosal epithelial cells, etc. It is.
  • corneal epithelial cells or cells having the ability to separate into corneal epithelial cell-like cells are cultured on the epithelium-free amniotic membrane.
  • Corneal epithelial cells are obtained from the cornea of a suitable donor. Where available, it is preferable to use the recipient's own corneal epithelial cells.
  • corneal epithelial-like sheet that is not likely to have an immune rejection reaction when transplanted, thereby enabling transplantation without an immune rejection reaction. If the recipient's own corneal epithelial cells are not available or difficult to obtain, corneal epithelial cells other than the recipient can be used, but in this case, the donor should be selected in consideration of immunocompatibility. Is preferred.
  • the "cell having the ability to separate corneal epithelial cell-like cells” means a cell capable of constructing a corneal epithelial-like cell layer in vivo by in vitro culture or after transplantation. Taste.
  • the term “corneal epithelium-like cell layer” as used herein refers to various properties required for functioning as a corneal epithelium (for example, having high transparency and V ⁇ that is not keratinized in the uppermost layer). Cell layer with noria function).
  • oral mucosal epithelial cells have the ability to separate such corneal epithelial cell-like cells (see PCT / JP02 / 11857).
  • the presence of stem cells in the oral mucosal epithelium has been suggested, and it is thought that differentiation can be easily induced into cells forming an epithelial cell layer.
  • the use of oral mucosal epithelial cells means that it is easy to collect, that a large amount of cells can be collected, and that even when treating patients with binocular disease, it is possible to prepare transplanted materials using their own cells. And so on.
  • the advantage of being able to apply transplants derived from their own cells to patients who cannot collect corneal epithelial cells is expected to greatly eliminate clinically important rejection problems. .
  • Oral mucosal epithelial cells include cells present in the root of the tooth (oral mucosal epithelial cells), lip cells, palate cells, buttocks cells, and the like. Of these, the intraoral mucosal epithelial cells are particularly preferred because of their high proliferative ability and low antigenicity. Oral mucosal epithelial cells were excised with a scalpel or the like where the target cells exist It can be collected by retorting or retorting. In the oral marginal mucosal epithelial cells, the oral mucosal epithelium adhering to the tooth extraction can be separated from the enamel cement transition part and collected from the obtained tissue piece.
  • the collected oral mucosal epithelial cells can also be used, but considering immune rejection, it is preferable to collect oral mucosal epithelial cells from the patient's own oral cavity and subject them to culture.
  • the cells for corneal epithelium are cultured on the amniotic membrane-derived medical material of the present invention. That is, the collected corneal epithelial cells are seeded on an amnion-derived medical material and cultured under appropriate culture conditions.
  • cells for corneal epithelium have a cell density of about 1 ⁇ 10 3 Zcm 2 or more, preferably about 1 ⁇ 10 3 / cm 2 to about 1 ⁇ 10 7 / cm 2 , more preferably about 1 ⁇ 10 4 Seeds / cm 2 to about 1 ⁇ 10 6 cells / cm 2 can be seeded on non-epithelial-containing amnion.
  • Culture of corneal epithelial cells can also be performed in the presence of supporting cells.
  • the feeder cells are also called feeder cells and supply growth factors and the like into the culture medium.
  • the proliferation efficiency of corneal epithelial cells can be expected to improve.
  • 3T3 cells Spiss mouse 3T3 cells, mouse NIH3T3 cells, 3T3J2 cells, etc.
  • mouse NIH3 T3 cells it is preferable to use mouse NIH3 T3 cells as supporting cells from the viewpoints of proliferation efficiency and ease of handling.
  • the feeder cells are previously inactivated using mitomycin C or the like. This is to prevent the growth of the corneal epithelial cells from being inhibited by the proliferation of the supporting cells themselves and increase the proliferation efficiency of the corneal epithelial cells. Such inactivation can also be achieved by radiation treatment.
  • the isolation membrane a known one having a pore size through which feeder cells cannot pass can be appropriately selected and used.
  • pore size is about 0.4 m to 3.
  • a 0 m film can be used.
  • the material of the separator is not particularly limited, and may be polyester or the like in addition to polycarbonate. Such separators are commercially available and can be easily manned.
  • inactivated support cells are seeded and cultured in a container such as a petri dish (first container) to form a support cell layer on the surface of the container.
  • a second container having a bottom surface formed of a separator is placed in the first container.
  • the position of the second container is adjusted so that the bottom surface of the second container is in the culture medium.
  • the epithelium-free amniotic membrane is placed on the bottom surface of the second container, that is, on the isolation membrane.
  • corneal epithelial cells are seeded on the amniotic membrane and cultured.
  • An amniotic membrane is placed in advance on the bottom surface of the second container (a drying treatment may be performed after placement), and this second container is placed in the first container seeded with feeder cells, Then, corneal epithelial cells may be seeded on the amniotic membrane and cultured.
  • the cell density of the support cells for example, about 1 X 10 2 or ZCM 2 or more, preferably about 1 X 10 2 pieces Zc m 2 ⁇ about 1 X 10 7 cells / cm 2, more preferably about 1 X 10 It can be from 3 pcs / cm 2 to about 1 X 10 5 pcs / cm 2 .
  • the number of supporting cells used is, for example, 1/10 3 times to 1 X 10 2 times, preferably 1/10 2 to 1 times the number of cells for corneal epithelium. Culturing can be performed under such conditions.
  • the proliferation rate of corneal epithelial cells decreases, and if the number is too small, satisfactory stratification of corneal epithelial cells cannot be obtained. On the other hand, when the number of supporting cells is too large, the proliferation rate of corneal epithelial cells is decreased, which is not preferable.
  • the culture solution used for culturing cells for corneal epithelium is not particularly limited as long as the cells can be grown and stratified.
  • DMEM Dynamic Eagle's medium
  • Ham's F12 medium at a predetermined ratio
  • FBS FBS
  • insulin 5 mg / ml
  • cholera toxin 0.1 nM
  • epidermal growth factor 10 ng / ml
  • penicillin streptomycin 50 IU / ml.
  • a mixed medium of DMEM and Ham F12 medium (mixed volume ratio 1: 1) is included. Also, triodothyronine (eg, 2 nM), glutamine (eg, 4 mM), transferrin (eg, 5 mg / ml), adenine (eg, 0.18 mM), and Z or Hyde mouth cortisone (eg, DMEMZ Ham F12 mixed medium supplemented with 0.4 mg / ml) can also be used.
  • triodothyronine eg, 2 nM
  • glutamine eg, 4 mM
  • transferrin eg, 5 mg / ml
  • adenine eg, 0.18 mM
  • Z or Hyde mouth cortisone eg, DMEMZ Ham F12 mixed medium supplemented with 0.4 mg / ml
  • the corneal epithelial cells proliferate and stratify. Subsequently, for the differentiation of the cells forming the cell layer and the induction of the Noria function, a step of contacting the surface layer of the layered cell layer with air (stratification step) is performed. This step is also referred to herein as air-lifting.
  • the surface of the culture solution is lowered by temporarily removing a part of the culture solution using a dropper, pipette, etc., so that the outermost layer of the cell layer is temporarily removed from the culture solution.
  • This can be done by exposing.
  • the cell layer can be lifted together with the amnion-derived medical material, and the outermost surface layer can be temporarily exposed to the culture medium surface force.
  • the step may be performed by sending air into the culture solution using a tube or the like and bringing the air into contact with the uppermost layer of the cell layer. From the viewpoint of ease of operation, it is preferable to perform the method by lowering the surface of the culture solution and exposing the outermost layer of the cell layer.
  • the time for performing the stratification step that is, the time for contacting the outermost layer of the stratified cell layer with air varies depending on the state of the cell and the culture conditions, but is, for example, about 3 to 3 weeks, preferably 5 days. ⁇ 2 weeks, more preferably about 1 week.
  • a cell layer formed by stratifying cells derived from cells for corneal epithelium on the epithelium-free amniotic membrane is formed.
  • This corneal epithelial-like sheet can be used as a transplant material (substitute for corneal epithelium) for patients with damaged or deficient cornea, as well as non-epithelial amnion used as a culture substrate.
  • the epithelium-free amniotic layer is transplanted to the corneal epithelial defect portion so that it is on the eyeball side.
  • the graft is secured to the surrounding tissue using sutures to promote engraftment.
  • one obtained by removing a part of the amniotic membrane used as a culture substrate or one obtained by removing all of the amniotic membrane (that is, only the cell layer) can be used as a graft.
  • the amniotic membrane can be removed by an antibacterial treatment using tweezers or the like.
  • oral mucosal epithelial cells When oral mucosal epithelial cells are used as corneal epithelial cells, cells derived from oral mucosal epithelial cells are layered on the corneal epithelium-like sheet.
  • the cells derived from the oral mucosal epithelium can be confirmed by using as an index the expression of keratin 4 or keratin 13 specific to oral mucosal epithelial cells in the cells constituting the corneal epithelium-like sheet. .
  • the expression of keratin 3 can be used as an index.
  • This keratin 3 is known as one of corneal-specific keratins, but it has been confirmed that it is also expressed in oral mucosal epithelial cells.
  • amniotic membrane was treated by the procedure shown in Fig. 2 to prepare an amnion from which the epithelium had been removed (non-epithelial amnion).
  • operation method processing method
  • operation conditions processing conditions
  • Amniotic membranes were collected at the time of cesarean section in the operating room after giving sufficient informed consent with the obstetrician and gynecologist in advance for pregnant women scheduled for cesarean section without systemic complications. The operation was careful of cleanliness, and a special gown was worn after hand washing according to the surgical operation. Before delivery, a clean bat for collecting amnion and physiological saline for washing were prepared. After delivery, the placenta tissue was transferred to a vat and the amnion tissue was manually detached from the placenta. The adhesion between the amniotic membrane and the placenta Strong !, The part was excised with scissors.
  • the amniotic membrane was fixed by the method shown in Fig. Lb. First, the amniotic membrane was sterilized and placed on a Teflon (registered trademark) sheet so that the epithelial side was up. Thereafter, the amniotic membrane was stretched so that wrinkles and sagging were eliminated, and a sterilized Teflon seat frame was placed on the amniotic membrane. The Teflon seat and the Teflon seat frame were fixed using clips. As a result, the amniotic membrane is sandwiched between the Teflon seat and the Teflon seat frame (frame fixing). The extra amniotic membrane that protrudes to the outside is cut off. Observed under a microscope, it was confirmed that the epithelial side was up.
  • Teflon registered trademark
  • the frame-fixed amniotic membrane was transferred into a deep freezer at -80 ° C and left for about 30 minutes (freezing). After removing from the deep freezer, it was transferred into a 37 ° C incubator and allowed to stand for about 30 minutes (melting). The above operation was performed once again.
  • the epithelial side of the amniotic membrane was immersed in a trypsin solution (phosphate buffer containing 0.02% trypsin and 0.2 mM EDTA) and left for about 15 minutes (37 ° C).
  • a trypsin solution phosphate buffer containing 0.02% trypsin and 0.2 mM EDTA
  • the method shown in Fig. 3a was adopted as the dipping method. That is, the trypsin solution 5 was held in the Teflon seat frame 3 while holding the frame-fixed amniotic membrane 10 with the Teflon sheet 4 facing downward. As a result, only the epithelial portion of the amniotic membrane 10 is immersed in the trypsin solution 5.
  • FIG. 1 phosphate buffer containing 0.02% trypsin and 0.2 mM EDTA
  • the amniotic membrane 10 fixed to the frame can be immersed in the trypsin solution 5 by placing it in the container 6 with its epithelial side down.
  • the Teflon seat frame 3 is engaged with the protrusion 7 provided on the inner side of the container 6, thereby realizing a desired positional relationship between the liquid surface 5 a of the trypsin solution 5 and the amniotic membrane 10.
  • the washed amniotic membrane was sandwiched between a pair of sterilized plastic frames and fixed with clips. Transfer the entire frame into a deep freezer at 80 ° C, confirm that the amniotic membrane has frozen, and perform freeze-drying (-110 ° C, approximately 1 hour) using a vacuum freeze dryer (Yamato, NEOCOOL). It was. The conditions were set according to the instruction manual so that a sufficiently dry product could be obtained.
  • the amniotic membrane after freeze-drying is removed from the plastic frame, transferred to a double-layered bag with polyamide nylon on the outside and polyethylene on the inside, and vacuum packed using a household vacuum pack device (frame nova, magic pack). . Thus the amnion vacuum packed state obtained by by irradiating ⁇ line (approximately 25 kGy) and sterilized.
  • the sterilized amniotic membrane was stored at room temperature in a vacuum-packed state until just before use.
  • amniotic epithelial removal method was evaluated by the following method.
  • an epithelium-free amniotic membrane obtained by washing after trypsin treatment was used.
  • Non-epithelial-containing amniotic membrane was stained with HE by the following procedure. Remove the epithelium! / The amniotic membrane (raw amniotic membrane) and the amniotic membrane (manually treated amniotic membrane) from which the epithelium was manually removed were used for comparison.
  • non-epithelial-containing amniotic membrane trypsin-treated amniotic membrane
  • raw amniotic membrane raw amniotic membrane
  • amniotic membrane from which the epithelium has been manually removed manually treated amniotic membrane
  • the specimen was frozen and cut using a cryostat (CM1900 Leica) at a thickness of 8 m in a direction perpendicular to the amnion surface and mounted on a slide glass to prepare a frozen section.
  • the procedure and conditions for HE staining were as follows. 1.10% formalin solution 5 minutes, 2.flowing water wash 1 5 minutes, 3.hematoxin solution 10 seconds, 4.flowing water wash 15 minutes, 5.eosin solution 10 seconds, 6.flowing water wash 15 minutes, 7.70% ethanol 10 seconds, 8.90% ethanol 10 seconds, 9.95% ethanol 10 seconds, 10.100% ethano 10 seconds, 11.100% xylene 10 seconds, 12.100% xylene 30 minutes, 13. enclosed.
  • the encapsulated sample was observed under an optical microscope (Olympus BX50).
  • amnion When amnion is stained with hematoxin, amnion epithelial cells and dense nucleated cells are stained. On the other hand, when it is dyed with eosin, the dense layer is dyed. Therefore, according to HE staining, the presence or absence of epithelial cell detachment and the presence or absence of damage to the dense layer can be determined.
  • the basement membrane and the dense layer retain their original structure in addition to the complete removal of the epithelial layer. Whether the basement membrane and the dense layer maintain the structure can be evaluated by examining the presence or absence of characteristic components (remaining). Therefore, the basement membrane component and the dense layer component remain in the trypsin-treated amniotic membrane! / The ability to beat was examined by the immunostaining method shown below. As in the case of HE staining, the epithelium was removed, and the amniotic membrane (raw amniotic membrane) and the amniotic membrane (manually treated amniotic membrane) from which the epithelium had been manually removed were compared.
  • the method for preparing frozen sections of amniotic membrane is the same as that for HE staining. This section was used for immunostaining by the following procedure.
  • the encapsulated sample was observed under a fluorescence microscope (Leica DMIRB).
  • Collagen I Collagen I
  • Collagen III Collagen III
  • Collagen IV Collagen IV
  • Collagen V Collagen V
  • Collagen VII Chemicon MAB1345
  • Laminin-5 Chemicon MAB19562
  • fibronectin LSL LB-1021.
  • Collagen IV, VII and laminin 5 are expressed in the amniotic basement membrane layer, and collagens I, III, V and fibronectin are expressed in the dense layer. Therefore, the remaining of the amnion basement membrane and dense layer can be observed by immunostaining with each antibody. In addition, in this experiment, PI staining is also performed, so that the presence or absence of amniotic epithelial cells can be simultaneously determined.
  • FIGS. Figures 5 and 6 are immunostained images of trypsin-treated amniotic membrane
  • Fig. 7 is an immunostained image of live amniotic membrane (with epithelium)
  • Fig. 8 is an immunostained image of manually-treated amniotic membrane.
  • a table summarizing the results of immunostaining is shown in FIG.
  • both the basement membrane component and the dense layer component remain to the same extent as the manually-treated amniotic membrane. That is, according to the above processing method, the basement membrane component and the dense layer component can remain as in the conventional manual processing method.
  • the method of the present invention it is possible to completely remove the epithelium while suppressing damage to the basement membrane, as in the conventional manual epithelial removal method. That is, according to the method of the present invention, an amniotic membrane having a basement membrane in which the epithelium is completely removed and the original structure is well maintained can be obtained. Such an amniotic membrane functions well as a substrate (base material) for cell culture.
  • the method of the present invention is very easy to operate and requires a shorter operation time than the conventional manual method. It is also easy to process a large number of amniotic membranes at the same time. Furthermore, since skilled techniques are not particularly required, it is easy to automate.

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Abstract

L’un des objets de l’invention est la réalisation de la dénudation complète d’épithélium amniotique par une opération simple causant le minimum de dégâts possible à la couche compact et à la membrane basale. Un autre objet est de proposer un procédé capable de traiter un certain nombre d’amnions simultanément. Le procédé de fabrication d’un amnion dénué d’épithélium comprend les phases suivantes : (1) préparation d’un amnion isolé d’un corps vivant ; (2) soumission de l’amnion à un traitement de congélation et de décongélation ; (3) traitement de l’amnion soumis au traitement de congélation et de décongélation avec de la trypsine ; et (4) lavage de l’amnion soumis au traitement par trypsine.
PCT/JP2006/303050 2005-02-22 2006-02-21 Procédé de fabrication d’un amnion dénué d’épithélium WO2006090696A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9433490B2 (en) 2010-12-22 2016-09-06 University Of Florida Research Foundation, Inc. Multilayered implant materials derived from amniotic membrane, methods of making the multilayered implant materials, and method of using multilayered implant materials
WO2020062350A1 (fr) * 2018-09-26 2020-04-02 成都清科生物科技有限公司 Amnios biologique acellulaire contenant une couche libre et son procédé de préparation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0833702A (ja) * 1994-02-24 1996-02-06 Res Dev Found 内臓の癒着または出血防止用羊膜移植片またはラップ
JP2001161353A (ja) * 1999-12-09 2001-06-19 Japan Ophthalmic Consultants:Kk 移植用細胞片及びその作成方法
JP2004024852A (ja) * 2002-04-30 2004-01-29 Amniotec:Kk 角膜内皮様シート、及びその作製方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0833702A (ja) * 1994-02-24 1996-02-06 Res Dev Found 内臓の癒着または出血防止用羊膜移植片またはラップ
JP2001161353A (ja) * 1999-12-09 2001-06-19 Japan Ophthalmic Consultants:Kk 移植用細胞片及びその作成方法
JP2004024852A (ja) * 2002-04-30 2004-01-29 Amniotec:Kk 角膜内皮様シート、及びその作製方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHWAB I.R. ET AL.: "Successful Transplantation of Bioengineering Tissue Replacements in Patiens with Ocular Surface Disease", CORNEA, vol. 19, no. 4, July 2000 (2000-07-01), pages 421 - 426, XP009010157 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9433490B2 (en) 2010-12-22 2016-09-06 University Of Florida Research Foundation, Inc. Multilayered implant materials derived from amniotic membrane, methods of making the multilayered implant materials, and method of using multilayered implant materials
WO2020062350A1 (fr) * 2018-09-26 2020-04-02 成都清科生物科技有限公司 Amnios biologique acellulaire contenant une couche libre et son procédé de préparation

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