CN102133427A - Method for preparing dried amnion - Google Patents
Method for preparing dried amnion Download PDFInfo
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- CN102133427A CN102133427A CN2010101139600A CN201010113960A CN102133427A CN 102133427 A CN102133427 A CN 102133427A CN 2010101139600 A CN2010101139600 A CN 2010101139600A CN 201010113960 A CN201010113960 A CN 201010113960A CN 102133427 A CN102133427 A CN 102133427A
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Abstract
The invention relates to a method for preparing dried amnion, which is characterized by comprising the following steps of: strictly sterilizing fresh amnion which carries no virus and is repeatedly rinsed, rebuilding and repairing ground substances at low temperature, unfolding the amnion, drying, sterilizing by cobalt 60 and preserving to reserve. The method for preparing the dried amnion has the advantages that the manufacturing process is simple and reliable and the cost is relatively low to ensure that the implementation and the popularization are easy; the prepared dried amnion can be preserved at normal temperature and conveniently transported; the cell phenotype and the basement membrane of the amnion are intact; fewer amnion ground substances are destroyed; the prepared dried amnion is superior to purely lyophilized amnion preserved at the same time and the like.
Description
Technical field
The present invention relates to a kind of preparation method of amniotic membrane, especially relate to a kind of preparation method of dry amniotic membrane.
Background technology
Amnion-derived in the embryo, be a kind of transparent, impassivity, blood vessel and lymphatic vessel, low antigenicity and have flexible tissue.This film is made up of epithelium layer, basement membrane, hypothallus, fibroblast layer, spongy layer, thick about 0.02～0.05mm.The amniotic membrane epithelial layer is a simple cuboidal epithelium, and there are a lot of microvilluss cell surface and side, and the ability of synthetic, secretion and deposition substrate film, extracellular matrix composition is arranged, and has the potential of differentiation, and can secrete multiple somatomedin; And amnion stroma contains the abundant collagen fiber and the protease inhibitor of various ways; The amniotic membrane basement membrane is the thickest basement membrane of human body (about 0.1 μ m), contains IV type collagen fiber net, and laminin and the proteoglycan that is rich in heparin sulfate can play barrier action, have lowered the permeability of amniotic membrane.
Along with patient's number of relevant diseases such as new vessels, inflammation and wound healing increases, the use of amniotic membrane is also more and more.The preparation of amniotic membrane, the especially preservation of amniotic membrane active component also just become a hot issue of present research.That adopts on the market at present mostly is the refrigerated dry amniotic membrane of direct process-biological lyophilizing amniotic membrane, it is that the scraps of paper with amniotic membrane are placed freeze dryer, vacuum condition is down with cobalt-60 radiative oven dry, and it is at room temperature vacuum-packed, lucifuge was placed two months under the room temperature, need place the normal saline that contains gentamycin to soak 3min before using.This method operation is simple relatively, and cost is lower, and be convenient to scale and produce in batches, but long preservation, convenient transportation, but need use special equipment, lyophilizing amnion tissue structure and epithelium ratio are easier to destroyed.Also have a kind of dry amniotic membrane-trehalose to soak amniotic membrane at present, it is to place 10% aqueous trehalose to soak isolated amniotic membrane, lyophilizing then, and vacuum packaging is kept in Dark Place after the x ray irradiation x sterilization.The amniotic membrane that this method is preserved is more approaching the nature fresh amnion aspect morphology, biology, but effect does not obtain more confirmations as yet.
Summary of the invention
The invention provides a kind of preparation method of dry amniotic membrane, dry amniotic membrane safety non-pollution, the performance that makes and fresh amnion is approaching, biological activity is high, collagen destroys less, be easy to operation technique, transportation and preserve convenient, substitute as fresh amnion, draw materials conveniently, practical, can be accepted and be applied to clinical by extensive patients.
The present invention adopts following technical scheme:
A kind of preparation method of dry amniotic membrane comprises the steps: the virus-free fresh amnion that carries with rinsing repeatedly through strict sterilization, and substrate is rebuild and repaired under the low temperature, the shop film, and drying, cobalt 60 sterilization backs are preserved standby.
As prepare coloured dry amniotic membrane, and need may further comprise the steps: the virus-free fresh amnion that carries of rinsing is through strict sterilization repeatedly, and substrate is rebuild and repaired under the low temperature, and amniotic membrane is painted, the shop film, and drying, cobalt 60 sterilization backs are preserved standby.
Described sterilization is that soak time was 5-10min after fresh amnion was soaked with the PBS that contains antibiotics, and PBS washes 3 times * 5min.Described antibiotics comprises 50,000 U/L penicillins, 80,000 U/L tobramycins, one or more combination in the 100mg/L streptomycin.
Described substrate is rebuild and repaired is that fresh amnion is soaked in the matrigel, and wherein temperature required is-20 ℃-4 ℃, and the time is 15min-30min, and matrigel comprises ECM glue, FN glue, Matrige glue, carrageenin, gelatin, a kind of in the IV Collagen Type VI.Adopt present technique can make the amniotic membrane outside have layer protecting film, make epithelium and substrate active ingredient can obtain most of the preservation, cell phenotype and basement membrane are complete, and substrate is destroyed few.
The painted coloured dyestuff of described amniotic membrane comprises fluorescein sodium, and tiger is red, trypan blue, and Gentian Violet, crystal violet, lissamine green, phenol red, methylene blue, sarranine, a kind of in methyl blue and the fuchsin, wherein temperature is 16 ℃-37 ℃, dyeing time 30s-5min.Adopt present technique can well help us in operation is used, to differentiate burst and the gauffer and the edge of amniotic membrane.
Described shop film comprises nitrocellulose filter, cellulose acetate membrane, microporous filter membrane, a kind of in the coverslip.Adopt present technique that amniotic membrane is fully flattened, keep certain degree of drawing can also distinguish the amniotic membrane epithelial surface simultaneously.
Described dry technology is treated to vacuum freeze-drying, and vacuum is dried, anhydrous Cacl
2Vacuum drying dries naturally, dries up, and the combination of one or more in 30-60 ℃ of incubator in the oven dry, take out the water content back between 0.1-5% that is dried to the amniotic membrane material.
Described amniotic membrane materials disinfection technology adopts cobalt 60 sterilizations.
The amniotic membrane material that we adopt technical solution of the present invention to make, be implanted in rabbit following 1 month, it has good organization's compatibility with the 1 monthly demonstration of the interior tiling of rabbit corneal substrate capsule bag postoperative in the lagophthalmos anterior chamber, do not see tangible inflammatory reaction and rejection, 2 weeks found that its amniotic membrane basement membrane was still complete after using it for amniotic membrane conjunctiva kposthesis simultaneously, there is the rabbit conjunctival epithelium to grow on the amniotic membrane, do not have tangible rejection and inflammatory reaction, illustrate that it can be used for conjunctiva as the cell based counterdie of conjunctival epithelium and rebuild.
Compare with existing amniotic membrane material, the present invention has the following advantages: (1) the present invention makes flow process simply, reliably, and easy to implement; (2) cost is relatively low, is easy to promote; (3) but room temperature is preserved down convenient transportation; (4) pathogen is cultivated, biological property detects and displaing microstructure observing finds that the amniotic membrane material of long period preservation (December) is pollution-free, be applied in people's conjunctiva kposthesis, postoperative finds that amniotic membrane plants sheet and can survive for 2 weeks-February, and can promote conjunctival epithelium in its superficial growth, the long-term effect ideal; (4) the present invention has added matrigel, has layer protecting film in the amniotic membrane outside, makes epithelium and substrate active ingredient can obtain most of the preservation, and cell phenotype and basement membrane are complete, and substrate is destroyed few, is better than the simple lyophilizing amniotic membrane of preserving with the time; (5) the present invention has added staining components, can well help us to differentiate burst and the gauffer and the edge of amniotic membrane in operation is used.(6) the present invention not only helps optimizing the amniotic membrane characteristic, can provide the important techniques support for the industrialization of organizational project amniotic membrane simultaneously.
Description of drawings
The dry amniotic membrane that Fig. 1 makes for preparation method of the present invention (painted through fluorescein sodium) is preserved the slit lamp photo (100 *) of December.
The dry amniotic membrane that Fig. 2 makes for preparation method of the present invention (painted through Gentian Violet) is preserved the slit lamp photo (100 *) of December.
The dry amniotic membrane that Fig. 3 makes for preparation method of the present invention is preserved the amniotic membrane HE dyeing photo (200 *) of December.
Fig. 4 is for preserving the amniotic membrane HE dyeing photo (200 *) of December through simple lyophilizing.
The dry amniotic membrane that Fig. 5 makes for preparation method of the present invention is preserved the amniotic membrane Laminin 5 dyeing photos (200 *) of December.
The amniotic membrane Laminin 5 dyeing photos (200 *) of Fig. 6 for preserve December through simple lyophilizing.
The dry amniotic membrane that Fig. 7 makes for preparation method of the present invention is preserved the amniotic membrane Collagen IV dyeing photo (200 *) of December.
Fig. 8 is for preserving the amniotic membrane Collagen IV dyeing photo (200 *) of December through simple lyophilizing.
Amniotic membrane β-catenin that the dry amniotic membrane that Fig. 9 makes for preparation method of the present invention is preserved December photo (200 *) that dyes.
Figure 10 is the amniotic membrane β-catenin that preserves December through the simple lyophilizing photo (200 *) that dyes.
The dry amniotic membrane that Figure 11 makes for preparation method of the present invention is preserved the amniotic membrane MUC 16 dyeing photos (200 *) of December.
The amniotic membrane MUC 16 dyeing photos (200 *) of Figure 12 for preserve December through simple lyophilizing.
The rabbit corneal interlayer was implanted the back slit lamp photo in January after the dry amniotic membrane that Figure 13 makes for preparation method of the present invention was preserved December.
The rabbit corneal interlayer was implanted the back HE photo in January after the dry amniotic membrane that Figure 14 makes for preparation method of the present invention was preserved December.
The rabbit anterior chamber was implanted into the back slit lamp photo in January after the dry amniotic membrane that Figure 15 makes for preparation method of the present invention was preserved December.
The rabbit anterior chamber was implanted into the back HE photo in January after the dry amniotic membrane that Figure 16 makes for preparation method of the present invention was preserved December.
The dry amniotic membrane that Figure 17 makes for preparation method of the present invention is preserved behind the December slit lamp photo in 2 weeks behind rabbit amniotic membrane-conjunctiva kposthesis.
The dry amniotic membrane that Figure 18 makes for preparation method of the present invention is preserved behind the December field of operation conjunctiva K19 dyeing photo in 2 weeks behind rabbit amniotic membrane-conjunctiva kposthesis.
The average elasticity modulus contrasted situation after the dry amniotic membrane that Figure 19 makes for preparation method of the present invention (matrigel is an ECM glue) was preserved December with the normal freeze-drying amniotic membrane.
Average maximum was born tensile force contrast situation after the dry amniotic membrane that Figure 20 makes for preparation method of the present invention (matrigel is an ECM glue) was preserved December with normal freeze-drying amniotic membrane material.
Average tensile elongation contrasted situation after the dry amniotic membrane that Figure 21 makes for preparation method of the present invention (matrigel is an ECM glue) was preserved December with normal freeze-drying amniotic membrane material.
The specific embodiment
Embodiment 1
Preparation method of the present invention may further comprise the steps: donor is taken from the fetal membrane that obtains through cesarean section, and it is all negative that puerpera's art provirus is learned assay; The fetal membrane that obtains is cleaned its surperficial bloodstain with physiological saline solution in aseptic clean environment; Patient's both hands are worn sterile gloves, and amniotic membrane is separated gently from chorion, and aseptic BSS liquid is rinsed well, are tiled on the aseptic glass plate, strike off its spongy layer, fibroblast layer and serosity transudate etc. with the cell sleaker; With fresh amnion with containing 50,000 U/L penicillins, 80,000 U/L tobramycins, the 3 times * 5min of PBS soaking disinfection of 100mg/L streptomycin; Under-20 ℃ of-4 ℃ of temperature, then sterile amnion is soaked in 15min-30min in the ECM glue; In 16 ℃ of-37 ℃ of fume hoods, splash into the 0.25% aseptic Fluress 0.2ml 5min that on amniotic membrane, dyes, to dye amniotic membrane then back is tiled on the nitrocellulose membrane, in sterile hood, dry naturally, take out after when water content maintains 0.5% left and right sides approximately, plastics package then, cobalt 60 sterilizations are preserved standby.
Embodiment 2
Preparation method of the present invention may further comprise the steps: donor is taken from the fetal membrane that obtains through cesarean section, and it is all negative that puerpera's art provirus is learned assay; The fetal membrane that obtains is cleaned its surperficial bloodstain with physiological saline solution in aseptic clean environment; Patient's both hands are worn sterile gloves, and amniotic membrane is separated gently from chorion, and aseptic BSS liquid is rinsed well, are tiled on the aseptic glass plate, strike off its spongy layer, fibroblast layer and serosity transudate etc. with the cell sleaker; With fresh amnion with containing 50,000 U/L penicillins, 80,000 U/L tobramycins, the 3 times * 5min of PBS soaking disinfection of 100mg/L streptomycin; Under-20 ℃ of-4 ℃ of temperature, then sterile amnion is soaked in 15min-30min in the FN glue; In 16 ℃ of-37 ℃ of fume hoods, splash into the 0.1% aseptic trypan blue solution 0.2ml 5min that on amniotic membrane, dyes, to dye amniotic membrane then back is tiled on the nitrocellulose membrane, in sterile hood, dry naturally, take out after when water content maintains 0.5% left and right sides approximately, plastics package then, cobalt 60 sterilizations are preserved standby.
Embodiment 3
Preparation method of the present invention may further comprise the steps: donor is taken from the fetal membrane that obtains through cesarean section, and it is all negative that puerpera's art provirus is learned assay; The fetal membrane that obtains is cleaned its surperficial bloodstain with physiological saline solution in aseptic clean environment; Patient's both hands are worn sterile gloves, and amniotic membrane is separated gently from chorion, and aseptic BSS liquid is rinsed well, are tiled on the aseptic glass plate, strike off its spongy layer, fibroblast layer and serosity transudate etc. with the cell sleaker.With fresh amnion with containing 50,000 U/L penicillins, 80,000 U/L tobramycins, the 3 times * 5min of PBS soaking disinfection of 100mg/L streptomycin; Under-20 ℃ of-4 ℃ of temperature, then sterile amnion is soaked in 15min-30min in the Matrige glue; In 16 ℃ of-37 ℃ of fume hoods, splash into the red solution 0.2ml of the 0.5% aseptic tiger 5min that on amniotic membrane, dyes, to dye amniotic membrane then back is tiled on the nitrocellulose membrane, in sterile hood, dry naturally, take out after when water content maintains 0.5% left and right sides approximately, plastics package then, cobalt 60 sterilizations are preserved standby.
Referring to Fig. 1～12, use lyophilization amniotic membrane material commonly used at present, it is active that the preservation December can be kept the amniotic membrane part.The liquid state of the present clinical usefulness of state such as the U.S. is preserved amniotic membrane, preservation condition is had relatively high expectations, need before using to preserve after 6 months to detect once more, this section storage life tolerable staff carries out detailed inspection to the donor amniotic membrane, with eliminating malignant tumor, infectious disease, thereby provide healthy, fine membrane film, but this fluid preservation amniotic membrane cycle is longer for clinical, transportation inconvenience is difficult for preserving.The lyophilization amniotic membrane of domestic commercialization, most tissues is destroyed in the lyophilization processing procedure, cause keep amniotic membrane active aspect and biology the aspect have defective, amniotic membrane is torn easily in clinical operation, the surgical effect that influences in neat formation.Amniotic membrane material of the present invention easy to make, cost is lower, but room temperature preserve down, convenient transportation is easy to promote; Staining components (fluorescein sodium: Fig. 1, crystal violet: Fig. 2), can well help us to differentiate burst and the gauffer and the edge of amniotic membrane have been added simultaneously.Preserve December after HE dyeing (Fig. 3) and various fluorescence staining (Laminin 5 dyeing: Fig. 5, Collagen IV dyeing: Fig. 7, β-catenin dyeing: Fig. 9, MUC 16 dyeing: Figure 11) check that the effect (holding time, active component and basement membrane) that confirms its preservation is better than (the HE dyeing: Fig. 4 of the lyophilization amniotic membrane material of corresponding natural law, Laminin 5 dyeing: Fig. 6, Collagen IV dyeing: Fig. 8, β-catenin dyeing: Figure 10, MUC 16 dyeing: Figure 12).
Figure 13, the 14th, through the specific embodiment of amniotic membrane material of the present invention in clinical practice.The amniotic membrane of preserving December through room temperature of the present invention is applied to the rabbit corneal interlayer implant after through 1 month observation, membrane film is dissolving fully as yet, it is transparent to plant sheet, does not have tangible rejection and inflammatory reaction.
Figure 15, the 16th, through amniotic membrane material of the present invention another specific embodiment in clinical practice.After being applied to the rabbit anterior chamber and being implanted into, the amniotic membrane of preserving December through room temperature of the present invention, finds that its amniotic membrane is partly dissolved through 1 month observation, smaller volume, but basement membrane is still complete, plants sheet and bleaches, but do not have tangible rejection and inflammatory reaction.
Figure 17, the 18th, through amniotic membrane material of the present invention another specific embodiment in clinical practice.The amniotic membrane of preserving December through room temperature of the present invention is applied to the observation in 2 weeks behind rabbit amniotic membrane-conjunctiva kposthesis, find that its amniotic membrane basement membrane is still complete, there is the rabbit conjunctival epithelium to grow on the amniotic membrane, do not have tangible rejection and inflammatory reaction, illustrate that it can be used for conjunctiva as the cell based counterdie of conjunctival epithelium and rebuild.
Embodiment 4
The following stated is the specific embodiment of amniotic membrane material in clinical practice.
Purpose: contrast is through the hot strength and the elastic characteristic of various pretreated commercial amniotic membranes and experiment amniotic membrane.
Method: to comparing research through a kind of pretreated amniotic membrane and existing commercial amniotic membrane.Group 1 is the amniotic membrane (20 example) that lyophilizing is handled behind the ECM glue, and group 2 is normal freeze-drying amniotic membrane group (20 example).Amniotic membrane is cut into slice, in the load sensor of packing into, and progressively extends until fracture.The record and the elastic modelling quantity of two kinds of amniotic membranes when more finally breaking, displacement, maximum tolerance pulling force.
The result: at elastic modelling quantity, displacement, on the maximum tolerance pulling force, ECM glue group is obviously greater than lyophilization amniotic membrane group, and two groups of contrasts have significant difference (P＜0.05).
Conclusion: the lyophilizing amniotic membrane that ECM glue group is handled is keeping better resilient characteristic, and he will provide transplanted tissue's sheet that is easier to handle to be used for the eye table, and nerve and tendon are rebuild.
Figure 19,20,21st, through the specific embodiment of amniotic membrane material of the present invention on using.After the lyophilizing amniotic membrane room temperature that ECM glue is handled is preserved December, at elastic modelling quantity, all preserve the December group on the maximum tolerance pulling force significantly better than conventional lyophilizing amniotic membrane but on average stretching displacement difference do not have the significance meaning.A: lyophilization amniotic membrane group, b:ECM glue group.
Embodiment 5
The following stated is the specific embodiment of amniotic membrane material in clinical practice.
Purpose: contrast is through the varied in thickness of various pretreated commercial amniotic membranes and experiment amniotic membrane.
Method: to comparing research through a kind of pretreated amniotic membrane and existing commercial amniotic membrane.Group 1 is: be the amniotic membrane (20 example) that lyophilizing behind the ECM glue is handled, group 2 is normal freeze-drying amniotic membrane group (20 example).Amniotic membrane is cut into square, is soaked in 30min in the normal saline.Measure corresponding amniotic membrane thickness with corneal pachymeter then.Record also compares the thickness of two kinds of amniotic membrane rehydrations front and back.
Result: almost do not increase amniotic membrane thickness after amniotic membrane soaks.Dry amniotic membrane average thickness be 19.68 ± 2.75 μ m, and corresponding moistening amniotic membrane average thickness is 19.84 ± 1.83 μ m (p＞0.05), yet the lyophilization amniotic membrane group (19.69 ± 1.97 μ m) of comparing the ECM glue group after the rehydration (20.64 ± 2.24 μ m) amniotic membrane more approaches the thickness (20.88 ± 2.33 μ m) of fresh amnion, and two groups of contrasts have significant difference (P＜0.05).
Conclusion: the lyophilizing amniotic membrane that ECM glue group is handled is keeping better thickness, and he will provide transplanted tissue's sheet that more approaches physiological situation to be used for the eye table, and nerve and tendon are rebuild.
Claims (8)
1. the preparation method of a dry amniotic membrane is characterized in that comprising the steps: the virus-free fresh amnion that carries with rinsing repeatedly through strict sterilization, and substrate is rebuild and repaired under the low temperature, the shop film, and drying, cobalt 60 sterilization backs are preserved standby.
2. the preparation method of a kind of dry amniotic membrane as claimed in claim 1 is characterized in that comprising the steps: that the virus-free fresh amnion that carries with rinsing repeatedly passes through strict sterilization, and substrate is rebuild and repaired under the low temperature, amniotic membrane is painted, the shop film, drying, cobalt 60 sterilization backs are preserved standby.
3. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: described sterilization is after fresh amnion is soaked with the PBS that contains antibiotics, soak time is 5-10min, PBS washes 3 times * 5min, described antibiotics comprises 50,000 U/L penicillins, 80,000 U/L tobramycins, one or more combination in the 100mg/L streptomycin.
4. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: described substrate is rebuild and repaired is that fresh amnion is soaked in the matrigel, wherein temperature required is-20 ℃-4 ℃, time is 15min-30min, and matrigel comprises ECM glue, FN glue, Matrige glue, carrageenin, gelatin, a kind of in the IV Collagen Type VI.
5. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: the painted coloured dyestuff of described amniotic membrane comprises fluorescein sodium, and tiger is red, trypan blue, Gentian Violet, crystal violet, lissamine green, phenol red, methylene blue, sarranine, a kind of in methyl blue and the fuchsin, wherein temperature is 16 ℃-37 ℃, dyeing time 30s-5min.
6. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: described shop film comprises nitrocellulose filter, cellulose acetate membrane, microporous filter membrane, a kind of in the coverslip.
7. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: described dry technology is treated to vacuum freeze-drying, and vacuum is dried, anhydrous Cacl
2Vacuum drying dries naturally, dries up, and the combination of one or more in 30-60 ℃ of incubator in the oven dry, take out the water content back between 0.1-5% that is dried to the amniotic membrane material.
8. as the preparation method of claim 1,2 described a kind of dry amniotic membranes, it is characterized in that: described amniotic membrane materials disinfection technology adopts cobalt 60 sterilizations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074295A (en) * | 2013-02-06 | 2013-05-01 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN105028386A (en) * | 2015-06-05 | 2015-11-11 | 热孜万·买买提明 | Preservation method of active amniotic membrane |
| CN105497984A (en) * | 2015-12-22 | 2016-04-20 | 马玉玲 | Preparation and preservation method of amniotic membrane biological agent |
-
2010
- 2010-01-21 CN CN2010101139600A patent/CN102133427A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074295A (en) * | 2013-02-06 | 2013-05-01 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN103074295B (en) * | 2013-02-06 | 2014-10-08 | 施萍 | Construction method of medical human amniotic membrane tissue reserve bank |
| CN105028386A (en) * | 2015-06-05 | 2015-11-11 | 热孜万·买买提明 | Preservation method of active amniotic membrane |
| CN105497984A (en) * | 2015-12-22 | 2016-04-20 | 马玉玲 | Preparation and preservation method of amniotic membrane biological agent |
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Application publication date: 20110727 |